Motion sensing superpixels (MOSES) is a systematic computational framework to quantify and discover cellular motion phenotypes

Version of Record

Accepted for publication after peer review and revision.

Download
Cite
Share
CommentOpen annotations (there are currently 0 annotations on this page).

Version of Record updated: July 2, 2019 (Go to version)
Version of Record published: February 26, 2019 (This version)
Accepted: January 11, 2019
Received: July 16, 2018

1. Of interest
Precise temporal control of neuroblast migration through combined regulation and feedback of a Wnt receptor

Erik S Schild, Shivam Gupta ... Hendrik C Korswagen

Research Article Updated Jun 7, 2023
Further reading

Article
Figures and data
Abstract
Introduction
Results
Discussion
Materials and methods
Data availability
References
Decision letter
Author response
Article and author information
Metrics

Abstract

Correct cell/cell interactions and motion dynamics are fundamental in tissue homeostasis, and defects in these cellular processes cause diseases. Therefore, there is strong interest in identifying factors, including drug candidates that affect cell/cell interactions and motion dynamics. However, existing quantitative tools for systematically interrogating complex motion phenotypes in timelapse datasets are limited. We present Motion Sensing Superpixels (MOSES), a computational framework that measures and characterises biological motion with a unique superpixel ‘mesh’ formulation. Using published datasets, MOSES demonstrates single-cell tracking capability and more advanced population quantification than Particle Image Velocimetry approaches. From > 190 co-culture videos, MOSES motion-mapped the interactions between human esophageal squamous epithelial and columnar cells mimicking the esophageal squamous-columnar junction, a site where Barrett’s esophagus and esophageal adenocarcinoma often arise clinically. MOSES is a powerful tool that will facilitate unbiased, systematic analysis of cellular dynamics from high-content time-lapse imaging screens with little prior knowledge and few assumptions.

https://doi.org/10.7554/eLife.40162.001

Introduction

During tissue development and homeostasis in multicellular organisms, different cell types expand and migrate to form defined organ structures. For example, during wound-healing, both immune and epithelial cells are required to proliferate and coordinately migrate (Schaffer and Nanney, 1996; Clark, 2013; Leoni et al., 2015). Aberrant cellular motion can be caused by deregulation of key signalling pathways in pathological conditions, including cancer, and may be critical for disease development and progression, for example leading to invasion and metastasis. Therefore, there is strong biological and clinical need for precise, quantitative characterisation of cellular motion behaviour in a tissue-relevant context to objectively compare the effects of drugs and genetic mutations.

When two cell populations adjoin in vivo, they often form a sharp, stable interface termed a ‘boundary’, with limited intermingling (Dahmann et al., 2011). In adult humans, sharp boundaries separate different types of epithelia, such as between the squamous and columnar epithelia in the esophagus, cervix and anus. Disruption of these boundaries can lead to disease. For example, disruption of the esophageal squamous columnar epithelial boundary is a feature of Barrett’s Esophagus (BE), a condition that confers a 30–50 fold increased risk of esophageal adenocarcinoma (EAC) (Gaddam et al., 2013). Understanding how complex tissue motion dynamics relate to pathological phenotypes, and how they can be affected by intrinsic and extrinsic factors, is therefore a key challenge in biomedical research. Live cell imaging over a long time (e.g. hours/days) is required to study the underlying complex cellular motion.

Recently, there has been a rising interest in scaling up live cell imaging for unbiased high-throughput screening and analysis to identify drug targets and develop therapies that can cause or prevent abnormal cellular motion (Schmitz et al., 2010; Held et al., 2010; Pau et al., 2013). Accordingly, a diverse range of co-culture experimental assays have been developed (Goers et al., 2014), amongst which wound-healing type assays are one of the simplest and most widely used. Attempts have been made to use these assays to study the regulation of complex biological/pathological processes such as the stable boundary formation between homo- and heterotypic cell populations using live cell imaging over a period of up to 6 days. However, a major barrier is how to analyse the resulting complex biological motion phenotypes and its variation between different tested conditions, particularly for multi-cell type populations (Goers et al., 2014) in high-content screens.

In general, there are two types of cell movements; ‘single-cell’ migration in which each cell migrates independently or ‘collective’ migration where a group of cells migrates together in a coordinated fashion. Single-cell migration has been well studied due to the availability of many single-cell tracking methods that can extract rich motion features even for unbiased high-throughput screens (Padfield et al., 2011; Meijering et al., 2012; Maška et al., 2014; Schiegg et al., 2015; Nketia et al., 2017). In contrast, collective migration is much less understood due to a lack of tools to extract equally rich quantitative motion features of cell populations in a high-throughput fashion with minimal prior knowledge.

Extension of existing single-cell tracking methods to analyse collective cell motion in general is non-trivial. Single-cell tracking methods all require accurate single-cell image segmentation, which is highly challenging when cells adjoin or overlap as they do frequently in tissue and confluent cell cultures. Moreover, there is a lack of systematic techniques to associate the motion of individual cells to the global moving collective. Alternatively, existing popular methods to analyse collective motion in cell populations such as Particle Image Velocimetry (PIV) (Szabó et al., 2006; Petitjean et al., 2010) and its variants (e.g. cell image velocimetry (CIV) (Milde et al., 2012)), do not require image segmentation. However, they only extract the global motion for a single video frame in the form of a velocity field, a velocity vector for each image pixel without assignment of pixels to individual cells. This lack of continuous tracking of cellular motion limits the application of PIV methods when characterising collective migration in complex biological phenomena such as boundary formation and chemoattraction. These biological processes often occur over a highly variable time period ranging from minutes to days, and identification of involved cell groups is often desired. Furthermore, PIV-extracted motion fields fail to exploit temporal continuity to reduce noise and avoid perturbations by imaging artefacts including visual clutter, image occlusion, and autofluorescence. Most prohibitively, PIV methods output only the velocities of image pixels. There is a lack of a systematic method to build motion ‘signatures’ to automatically and unbiasedly discriminate and predict biological motion phenotypes in large datasets. To this end, previous studies have attempted to extract additional motion parameters from PIV and appearance parameters from individual video frames (Neumann et al., 2006; Zaritsky et al., 2012; Zaritsky et al., 2014; Zaritsky et al., 2015; Zaritsky et al., 2017). These include applying velocity-based clustering in migrating monolayers, and handcrafted image features to describe local image appearance using, for example, local binary patterns (Zaritsky et al., 2012). However, these approaches are sensitive to the number of identified phenotypes and data outliers, requiring relatively high-quality imaging, restricting the experimental systems they can be applied to. Importantly, all the approaches (Neumann et al., 2006; Zaritsky et al., 2012; Zaritsky et al., 2014; Zaritsky et al., 2015; Zaritsky et al., 2017) assume prior knowledge of the motion behaviour. Notably, kymographs specifically exploit motion patterns known to be symmetrical around a given spatial axis such as wound healing and cannot be used to identify novel spatial motion features that could be relevant to normal tissue development or to a defined disease setting.

A suitable computational method for studying cellular motion therefore should be able to address the joint challenge of analysing single-cell and collective motion behaviour. Additionally, it must be: (i) scalable in a medium- or high-throughput manner; (ii) sufficiently robust to handle inevitable variations in image acquisition and experimental protocol; (iii) sensitive to detect motion differences resulting from small changes in environment or stimuli with minimal replicates; (iv) automatic, requiring no manual intervention except for initial setting of parameters; and (v) unbiased to enable motion characterisation (e.g. as a motion ‘signature’) with minimal prior assumptions of motion behaviour.

To address these analytical challenges, here we developed Motion Sensing Superpixels (MOSES), a computational framework that aims to provide a flexible and general approach for biological motion extraction, characterisation and phenotyping. We empowered PIV-type methods with a mesh formulation that enables systematic measurement and unbiased extraction of rich motion features for single and collective cell motion suitable for high-throughput phenotypic screens. We use the analysis of a multi-well plate-based in vitro assay to study the complex cell population dynamics between different epithelial cell types from the esophageal squamous-columnar junction (SCJ) to demonstrate the potential of MOSES. Our analysis illustrates how MOSES can be used to effectively ‘encode’ complex dynamic patterns in the form of a motion ‘signature’, which would not be possible using standard globally extracted velocity-based measures from PIV. Finally, a side-by-side comparison with PIV analysis on published datasets illustrates the biological relevance and the advanced features of MOSES. In particular, MOSES can highlight novel motion phenotypes in high-content comparative biological video analysis.

Results

In vitro model to study the spatio-temporal dynamics of boundary formation between different cell populations

To develop MOSES, we chose to investigate in vitro the boundary formation dynamics between squamous and columnar epithelia at the esophageal squamous-columnar junction (SCJ) (Figure 1A). To recapitulate features of the in vivo boundary formation, we used three epithelial cell lines in pairwise combinations and an experimental model system with similar characteristics to wound-healing and migration assays but with additional complexity. Together the resulting videos pose a number of analytical challenges that require the development of a more advanced method beyond the current capabilities of PIV and CIV.

Figure 1 with 5 supplements see all

Download asset Open asset

Temporary divider system to study interactions between cell populations.

(A) The squamous-columnar junction (SCJ) divides the stratified squamous epithelia of the esophagus and the columnar epithelia of the stomach. Barrett’s esophagus (BE) is characterised by squamous epithelia being replaced by columnar epithelial cells. The three cell lines derived from the indicated locations were used in the assays (EPC2, squamous esophagus epithelium, CP-A, Barrett’s esophagus and OE33, esophageal adenocarcinoma (EAC) cell line). (B) The three main epithelial interfaces that occur in BE to EAC progression. (C) Overview of the experimental procedure, described in steps 1–3. In our assay, cells were allowed to migrate and were filmed for 4–6 days after removal of the divider (step 4). (D) Cell density of red- vs green-dyed cells in the same culture, automatically counted from confocal images taken of fixed samples at 0, 1, 2, 3, and 4 days and co-plotted on the same axes. Each point is derived from a separate image. If a point lies on the identity line (black dashed), within the image, red- and green-dyed cells have the same cell density. (**E,F**) Top images: Snapshot at 96 h of three combinations of epithelial cell types, cultured in 0% or 5% serum as indicated. Bottom images: kymographs cut through the mid-height of the videos as marked by the dashed white line. All scale bars: 500 μm. (G) Displaced distance of the boundary following gap closure in (**E,F**) normalised by the image width. From left to right, n = 16, 16, 16, 17, 30, 17 videos.

https://doi.org/10.7554/eLife.40162.002

To model the relevant esophageal interfaces, we used three epithelial cell lines: EPC2, an immortalised squamous epithelial cell line from the normal esophagus (Harada et al., 2003); CP-A, an immortalised BE cell line with gastric columnar epithelial properties (Merlo et al., 2011); and OE33, derived from EAC (Boonstra et al., 2010). We co-cultured these lines in the following combinations: 1) EPC2:EPC2 (squamous:squamous, as a normal control); 2) EPC2:CP-A (squamous:columnar, as in Barrett’s esophagus); and 3) EPC2:OE33 (squamous:cancer, as in EAC) (Figure 1B). Two epithelial cell populations, each labelled with a different lipophilic membrane dye (Progatzky et al., 2013), were co-cultured in the same well of a 24-well plate, separated by a divider with width 500 µm. The divider was removed after 12 h and cells were allowed to migrate towards each other (Figure 1C).

We first compared the effects of the two dyes on proliferation and migration using monolayer combinations of the three cell types (Video 1). Proliferation was assessed by cell density, automatically counted from DAPI staining using convolutional neural networks (CNN; see Figure 1—figure supplement 1, Materials and methods). Migration (diffusive behaviour) was assessed by mean squared displacement (Park et al., 2015). Green and red fluorescent-labelled EPC2, CP-A, and OE33 cells proliferated at the same rate (Figure 1D, for EPC2 cells: slope = 0.976, Pearson correlation coefficient 0.978, Figure 1—figure supplement 1E), and migrated in a similar way (Figure 1—figure supplement 2). Compared to non-dyed cells, the mobility of CP-A cells were unaffected however both dyes equally reduced the mobility of EPC2 and OE33 cells. The diffusion modes of all cells were unaffected (Figure 1—figure supplement 2) and the boundary formation behaviour between dyed and non-dyed cells was identical.

Video 1

Download asset

posterframe for video — Dynamics of monolayer combinations of three oesophageal epithelial cell lines, EPC2:EPC2, CP-A:CP-A, OE33:OE33.

Bar: 500 μm.

https://doi.org/10.7554/eLife.40162.008

We next analysed boundary formation in the three different combinations of co-cultured epithelial cell lines (EPC2:EPC2, EPC2:CP-A, and EPC2:OE33), initially in serum-containing media (5% fetal bovine serum, FBS). In all three combinations, as expected, both populations moved as epithelial sheets (Video 2). Firstly, in the squamous combination using the same EPC2 cell line labelled with two different colours, the green- or red-labelled EPC2:EPC2 cells met and coalesced into a monolayer as expected. Secondly, in the squamous-columnar EPC2:CP-A combination, we observed boundary formation between the two populations after 72 h, following a short period of CP-A ‘pushing’ EPC2. Thirdly, in the squamous-cancer EPC2:OE33 combination, the cancer cell line OE33 expanded continuously, resulting in the disappearance of EPC2 from the field of view (Video 2, Figure 1E) as assessed by the motion field and confocal images (Figure 1—figure supplement 3). The forces that govern the behaviour of the two cell lines on contact are unknown and traction force microscopy is required to investigate the ‘retracting’ or ‘pushing’ behaviour of EPC2 or OE33 cells respectively in future studies. Nonetheless, the observed boundary formation in the EPC2:CP-A combination and disordered interactions between EPC2:OE33 cells suggest that the three chosen epithelial cell lines in the tested combinations may model certain motion dynamics of the in vivo cell-cell interactions. Interestingly, cell counting suggests the observed phenotypes were not due to differential proliferation rate between the individual cell lines in combination (Figure 1—figure supplement 4C,D).

Video 2

Download asset

Evidence from model systems, including Drosophila melanogaster embryonic parasegment (Monier et al., 2010) and anteroposterior and dorsoventral wing disc boundaries (Major and Irvine, 2005; Major and Irvine, 2006; Landsberg et al., 2009), suggests the importance of cell-cell interactions and collective migration for stable boundary formation between epithelial populations in vivo. Thus to create a dataset in which we predict that cell motion would be dramatically disrupted we repeated the assays in serum-free conditions. We used convolutional neural networks (Materials and methods) to determine the mean cell density and mean change in cell density from video frames over the first 48 h of cells grown in serum (5% FBS) and no serum (0% FBS) conditions. We observed that culturing of the same cell combinations in serum-free versus 5% serum medium had undetectable impact on cell density at the very confluent cell conditions investigated (Figure 1—figure supplement 4). However, as expected, serum-free condition induced large global changes in cell migration (Figure 1F, Figure 1—figure supplement 5A) with observed loss of cell contacts, collective sheet migration (Figure 1—figure supplement 5B,C) and the absence of boundary formation. We also observed reduced overall boundary displacement (Figure 1F,G) and all cell line combinations exhibited similar motion dynamics (Figure 1F,G, Video 2). These results illustrate that serum-free medium has a profound impact on cell motion dynamics and the generated video datasets in serum-free medium are ideal as an experimental condition for testing the ability of MOSES to detect motion dynamics under different experimental conditions. It is important to note that the serum-free condition is not used as a biological negative control of boundary formation but only as a computational negative control for the development of our method.

Development of MOSES to quantify cell motion dynamics

We next developed a computational workflow, MOtion SEnsing Superpixels (MOSES), to quantify cellular motion from video datasets. MOSES was formulated modularly with three components (Figure 2): 1) motion extraction; 2) construction of long-time motion tracks; and 3) capture of local dynamic context.

Figure 2 with 2 supplements see all

Download asset Open asset

Schematic diagram of MOtion SEnsing Superpixels (MOSES).

(A) High level overview of the three configurable components that define MOSES: motion extraction, construction of long-time motion tracks, and capture of local dynamic context. Long-time here indicates tracking of superpixels for longer than one frame or timepoint. (B) Example ways to practically implement each of the three high-level MOSES component concepts described in (A) in software. (C) Using long-time tracking of superpixels MOSES bridges single-cell tracking for sparse cell culture and PIV for confluent monolayers to extract global motion patterns for both scenarios, single-cell and collective motion within one computational framework.

https://doi.org/10.7554/eLife.40162.010

Component 1: Motion extraction

We use dense optical flow to avoid the shortcomings of using cell segmentation to track individual cells in confluent tissue. Optical flow is similar to particle image velocimetry (PIV) but yields higher resolution motion fields with a displacement vector for each pixel at every time point. PIV typically estimates a single velocity for an image patch or ‘window’ using spatial correlation. This corresponds to extracting only the superpixel movement in Figure 2A over one frame. Optical flow is also easier to modify to account for additional physical phenomena such as out-of-plane motion and large, discontinuous movements not captured within a PIV ‘window’ (Brox et al., 2004; Brox et al., 2009).

Component 2: Construct long-time motion tracks

To capture all spatial motion dynamics in the video, we construct long-time motion tracks that continuously track the cellular motion for any amount of time using superpixels. Local neighbouring image pixels are grouped into a pre-specified (see below) number of image regions or ‘superpixels’ to enable spatial averaging of the pixel-based motion fields (Figure 2A, Step 1). At each time point, the (x,y) position of a given superpixel is updated by averaging the optical flow (Farnebäck, 2003) over the image area covered by the superpixel to obtain the displaced direction and distance (Figure 2A, Steps 2-4). Subsequently, long-time superpixel motion tracks are generated by concatenating the positions of each superpixel at all time-points (Figure 2A, Step 5). All long-time superpixel motion tracks together encode the entire spatio-temporal motion history within the video. By default, the number of superpixels used are user-specified and fixed to sufficiently subsample only the field-of-view of the initial video frame. Where a video exhibits highly dynamic movement and coverage of the full spatio-temporal motion, including the movement of new cells not initially present but which later move into the field of view, is necessary for analysis, additional superpixels can be dynamically added during tracking. Tracks produced by the latter approach are known as dense trajectories (Wang et al., 2011). In our experiments, we used a fixed number of 1000 superpixels to cover an image size of 1344 x 1024 (with this setting an average superpixel is 37 pixels x 37 pixels covering $\approx$ 14,000 μm² (2x lens) or $\approx$ 3600 μm² (4x lens)) to monitor epithelial sheet dynamics. For multichannel images, each channel representing different coloured cell populations (i.e. red and green in our assay) was independently tracked in this manner (Figure 2B).

Depending on the specific application, background artefacts such as stage drift or floating debris, can contribute tracks that are biologically irrelevant and need to be filtered in post-processing (Figure 2B, middle panel). In this filtering step, superpixels are assigned to cover only the dynamic motion of all ‘objects’ of interest. The ‘object’ for single-cell tracking is each individual cell, and for epithelial sheets is the entire sheet within the video frame. To assign superpixels to each sheet, we use motion information (Figure 2—figure supplement 1, Materials and methods). This approach avoids relying on image intensity features whose variation commonly leads to segmentation errors. We validated our motion extraction and long-time track construction using published single-cell tracking datasets from the cell tracking challenge (Maška et al., 2014) (Video 3), and also experimentally, by spiking one of the dyed cell populations in the in vitro setup described above with a third, sparse population of blue-dyed cells. Our method produced very similar tracks (similarity score of 0.8) to an open software tool for single-particle tracking, TrackMate (Tinevez et al., 2017) (Figure 2—figure supplement 2, Video 4).

Video 3

Download asset

Video 4

Download asset

Component 3: Capture local dynamic context

A ‘mesh’ is constructed by ‘linking’ superpixels to capture how individual superpixels move with respect to their neighbours over time (Figure 2A, Step 6). The constructed dynamic mesh, inspired by work in human surveillance (Chang et al., 2011), naturally captures the local collective dynamics without explicit thresholding or clustering. Separate meshes are produced for each colour channel. Multiple different meshes can be defined and constructed for specific purposes (see Materials and methods). Because the mesh is a representation of interactions inferred from the local spatial relationships of superpixels, it can be used to derive robust local and global measures of motion relating to biological phenomena such as collective motion and tissue interactions (see below).

In summary, MOSES has been designed to describe the video motion in terms of the individual trajectories of moving ‘entities’ (defined by the image superpixels) and their spatiotemporal interactions (as captured by a dynamic mesh). Depending on the application, superpixels can be assigned to and biologically interpreted as part of an object, a single cell or multiple cells.

Quantitative measurement of squamous and columnar epithelial boundary formation using MOSES

To verify the enhanced ability of MOSES over standard PIV analysis to quantitatively assess relevant biological features of interest in tissue, we applied it to analyse the formation of boundary dynamics between squamous and columnar cells. In total, 125 videos (48 with 0% serum and 77 with 5% serum) were collected from four independent experiments and jointly analysed. These videos are highly heterogeneous, creating a challenging dataset for analysis (Supplementary file 1, Figure 3—figure supplement 1).

Standard velocity kymographs were able to firstly confirm our observations in Figure 1E–G of the differences in cell motion between 0% and 5% serum conditions. As shown in Figure 3A, all cell combinations grown in 0% serum exhibited a similar global motion pattern, minimal sheet-like motion and interface movement following gap closure. By contrast, in 5% serum the same three cell line combinations exhibited very different dynamics, amongst which we observed the formation of a stable equilibrium following contact only between EPC2 and CP-A cell sheets. However, subsequent computation of the mean sheet speed, as performed during routine PIV analysis, highlighted the analytical need for a computational framework to extract more descriptive motion measures. As shown in Figure 3B, aside from an indication of a global speed increase in 5% serum, speed was an insufficient metric to characterise the observed phenotypes, exhibiting high variance between replicates and failure to highlight the uniqueness of the EPC2:CP-A interaction. We thus used MOSES to construct more discriminative phenotypic measures to characterise the boundary formation of EPC2:CP-A.

Figure 3 with 10 supplements see all

Download asset Open asset

MOSES quantifies boundary formation and epithelial sheet interaction dynamics.

(A) Projected ‘x’-direction velocity kymograph of the dense optical flow for each cell line combination at the two serum concentrations used in the assay. The speed and direction of movement are indicated by the intensity and colour respectively (blue, moving left; red, moving right). (B) Average speed of each video for each cell combination, coloured green and yellow for the first and second cell line in the named combinations respectively. (**C,D**) Violin plots of boundary formation index (C) and mesh stability (D) for each video (black dot) for different cell combinations in 0% and 5% serum. Dashed line is the threshold, one standard deviation above the pooled mean value of all cell line combinations in 0% serum. Red solid line in violins = mean, black solid line in violins = median. (E) Maximum (Max.) velocity cross-correlation between the two sheets, before and after gap closure, left and right violins respectively for each cell line combination. Shaded region of all violins is the probability density of the data whose width is proportional to the number of videos at this value. (F) Boxplots showing median and interquartile range (IQR) of velocity order parameter and (G) mesh order for each cell line combination coloured green and yellow for the first and second cell line in the named combination, respectively. Whiskers show data within 1.5 x IQR of upper and lower quartiles.

https://doi.org/10.7554/eLife.40162.015

Using MOSES, we constructed a motion saliency map for visualising global motion patterns (Figure 3—figure supplement 2) and designed four different additional measures as summarised in Table 1 to quantitatively distinguish between different combinations of the three cell lines (EPC2, CP-A and OE33) used in our assay: i) boundary formation index, based on the degree that motion concentrates into a local spatial region (Figure 3—figure supplements 2 and 3); ii) mesh stability index, which measures the degree of movement between neighbouring cell groups locally within the sheet at the endpoint (Figure 3—figure supplement 4); iii) mesh order to measure the degree of collective motion of the whole sheet (Figure 3—figure supplement 5 , Video 5) and iv) the maximum velocity cross-correlation, which measures the average correlation in motion between the two epithelial sheets from their movement history (Figure 3—figure supplement 6). The first three are based on the mesh; the fourth uses the individual superpixel tracks. These measures represent examples of statistics derivable using MOSES and are not limited to wound-healing type assays. For technical details, see Materials and methods. Cells grown in 0% serum were used as a computational negative control to set appropriate cut-offs for detecting boundary formation in our computational analysis (see Materials and methods for details).

Video 5

Download asset

Table 1

Summary of MOSES-derived measurements for discriminating boundary formation phenotype in this paper and their biological interpretation and application.

https://doi.org/10.7554/eLife.40162.026

Proposed measures	Biological interpretation	Biological application
Motion Saliency Map	‘Heatmap’-like image that highlights spatial regions that attract or repel local cellular motion over a defined time period.	Quantitative spatio-temporal readout of scratch or wound-healing assays. Highlights areas of salient motion activity spatially such as the migration of macrophages locally to inflicted wound sites.
Boundary formation index (value from 0 to 1)	Quantifies the concentration of movement within one region of space. Multiple ‘hotspots’ decrease this index. Uniform distributed movement (no attraction) scores 0. Concentration of movement in a single region for example a line or point scores 1.	Quantification of the spatial uniformity or ‘spread’ of motion activity. Highlights for example if all migrating cells move uniformly to close the wound in a wound-healing assay.
Mesh stability index (value from $-$ ∞ to 1)	Measures the motion stability of local cell groups by measuring the change in relative spatial arrangement (topology) with respect to neighbouring cell groups at a given endpoint. Static epithelial sheets and epithelial sheets that move uniformly in a single direction preserve local cellular arrangement and scores 1. Motion that results in the local spatial rearrangement of cells such as neighbour exchange in embryos is unstable and scores < 1.	Assessing the final global movement stability of a collective such as an embryo or epithelial sheet based on the movement of the cells that compose it.
Mesh order (value > 0)	Measures the collectiveness of local cellular migration by the change in distance and direction of cells with respect to neighbouring cells. Hypothesizes that cells that are part of the same motion group exhibit the same ‘mesh’ force and move retaining the same local spatial arrangement.	Quantitative assessment of the extent of global sheet-like movement.

Computing the proposed indices for all videos, the boundary formation index was ranked on a continuous scale for all 5% serum videos (Figure 3—figure supplement 7) by MOSES. The boundary formation index was highest (median 0.74) for EPC2:CP-A grown in 5% serum (n = 30/77) (Figure 3C, Figure 3—figure supplement 8B), whilst EPC2:EPC2 and EPC2:OE33 in 5% serum were below the boundary formation cut-off. In 5% serum, EPC2:CP-A had the highest mesh stability index (median 0.90), compared to EPC2:EPC2 (0.76) and EPC2:OE33 (0.25) (Figure 3D, Figure 3—figure supplement 8C). Together these results illustrate that only the squamous-columnar combination (EPC2:CP-A) formed a final stable boundary.

We next measured sheet-sheet interactions using the maximum velocity cross-correlation (VCC) before and after gap closure for the three cell line combinations. Gap closure was automatically determined (Figure 3—figure supplement 9 , Materials and methods). For two initially diametrically opposed migrating sheets, a significant increase in VCC after gap closure compared to before gap closure is suggestive of increased sheet interaction. In 0% serum, no difference in velocity cross-correlation across all combinations was found before and after gap closure (Figure 3E ) - the two sheets do not move cohesively as a unit, as expected with minimal cell-cell contact. In serum, the difference for EPC2:CP-A (0.03 before and 0.20 after gap closure) was $~$ 3-6 times larger than for EPC2:EPC2 (0.01 to 0.08) and EPC2:OE33 (0.02 to 0.05) (c.f. left and right violin plots in Figure 3E ), suggesting potential physical interaction. Interestingly, CP-A:OE33 (Barrett’s:cancer, n = 6) also exhibited a substantial increase in VCC following gap closure (0.03 to 0.17) (Figure 3—figure supplement 8D,E) but no substantial increase was observed for CP-A:CP-A (0.01 to 0.06) (Figure 3—figure supplement 8D,E), confirming this observation was not simply CP-A cell line-specific. Both the velocity order parameter and our mesh order parameter (Figure 3F,G) indicate clear overall reduction in collective motion in cells grown in 0% serum compared to 5% serum.

Finally, we used MOSES with image segmentation and convolutional neural network (CNN) cell counting to assess morphological variations of the interface among all cell line combinations to check for ‘invasive fingers’ or boundary breakdowns that often exist in normal and tumour cell-formed boundaries (Figure 3—figure supplement 10A,B). Interestingly, we found non-invading boundaries in all cell line combinations except CP-A:OE33, where we observed clear infiltration with ‘finger-like’ protrusions of CP-A into the OE33 cell sheet grown in 5% serum (Figure 3—figure supplement 10B). No such ‘finger-like’ protrusions or significant intermixing of cells (Figure 3—figure supplement 10D,F–H) were detected in EPC2:CP-A under the same conditions. Altogether, our characterisation suggests that, among all tested cell-type combinations, we detect an ‘interacting’ final stable boundary uniquely in the squamous-columnar EPC2:CP-A combination grown in 5% serum (Video 6).

Video 6

Download asset

MOSES can measure subtle phenotype changes induced by external stimuli

The ability to assess subtle phenotype changes with a minimal number of replicates is critical for high-content screening applications. We thus tested the ability of MOSES to detect changes in EPC2:CP-A boundary formation using an external perturbation. The epidermal growth factor receptor (EGFR) is frequently mutated in EAC, sometimes overexpressed in BE and is activated by bile acid reflux, the main observed cause of BE clinically (Dixon et al., 2001; Souza, 2010). In our experiments we thus used epidermal growth factor (EGF), the ligand of EGFR, as a biologically relevant external perturbation. Increasing amounts of EGF (0 ng/ml to 20 ng/ml) were added to the culture medium to assess incremental effects on cellular motion and boundary formation in the EPC2:CP-A combination. A total of 40 videos (each 144 h, one frame per h) were collected from three independent experiments, in a 24-well plate medium-throughput screen (Supplementary file 2). With increasing EGF, the boundary position was displaced a distance farther from the initial point of contact between the two cell populations, with slightly enhanced cell speed and decreased boundary coherence (Figure 4A–D). This is quantitatively reflected in the shape of the mean normalised strain curve (Figure 4E) which measures the average distance between neighbouring cell groups (Materials and methods): at 0 ng/ml EGF, the curve linearly increases before plateauing around 72 h; as EGF concentration increases, the curve becomes more linear and the plateau is lost above 5 ng/ml.

Figure 4 with 2 supplements see all

Download asset Open asset

EGF titration at physiological levels disrupts boundary formation.

(A) Destabilisation of the junction with EGF addition. All in 5% serum with snapshots of endpoint (144 h). Shown also is the green channel CP-A MOSES mesh. The closeness of the lines indicates impeded motion leading to a local aggregation of superpixels in the vicinity and is suggestive of a boundary. The less lattice-like the mesh, the less ordered the motion. Blue triangles mark the boundary position in the image and its corresponding inferred position in the CP-A mesh. All scale bars: 500 μm. (B) Top: maximum projected video kymograph. Bottom: x-direction velocity kymograph computed from optical flow for the representative videos in (A). (C) Grouped boxplot of the average speed for the different cell lines in the combination in 5% serum with increasing EGF concentration. (D) Mean displaced distance of the boundary normalised by image width following gap closure with increasing EGF concentration in 5% serum. Mean displaced distance of EPC2:CP-A and EPC2:OE33 cultured in 5% serum from Figure 1G are also plotted for comparison. T-test was used with * indicating p = < 0.05, ** p = < 0.01, *** p = < 0.001. Error bars are plotted for ± one standard deviation of the mean. (E) Mean normalised strain curves for EPC2:CP-A in 5% serum for each concentration of EGF. The mean curve for EPC2:OE33 videos in 5% serum without EGF in Figure 3 is shown for comparison (black curve). (**F–H**) Violin plots of boundary formation index (F), mesh stability index (G) and maximum velocity cross-correlation (H) for each concentration of EGF and cells in 5% serum. Red solid line = mean, Black solid line = median. Dots are individual videos, total n = 40. Shaded region is the probability density of the data whose width is proportional to the number of videos at this value. Violins of respective measures for EPC2:OE33 in 5% serum without EGF with thresholds (horizontal black line) from Figure 3 is shown for comparison. (**I,J**) Boxplots of velocity order (I) and mesh order (J) for individual cell lines (left) and pooled across the two cell lines in the combination (right). Values for EPC2:OE33 in 5% serum without EGF and threshold from Figure 3 are shown for comparison.

https://doi.org/10.7554/eLife.40162.028

The boundary formation index decreased with increasing EGF (0.74 at 0 ng/ml to 0.46 at 20 ng/ml, comparable to EPC2:OE33 without EGF (0.46)), indicating loss of the boundary (i.e. index below the 0.69 cut-off) (Figure 4F). The mesh stability index decreased from 0.94 (stable, 0 ng/ml EGF) to 0.72 (unstable, 20 ng/ml) (Figure 4G), suggesting increased movement between neighbouring cells and the loss of interaction between the two cell populations since the maximum VCC difference before and after gap closure decreased from 0.16 (0 ng/ml EGF) to 0.04 (20 ng/ml EGF) (Figure 4H). The maximum VCC after gap closure was similar to that for EPC2:OE33 (0.02) (Figure 4H), but the mesh stability index remained higher (Figure 4G). Together these measures show that above 5 ng/ml EGF, the phenotype of EPC2:CP-A becomes similar to that between EPC2 and the EAC cell line, OE33 (Video 7). Cell counting and quantification of fluorescence decay suggest cell division is not the primary factor influencing the boundary at the cell density used in our experiments in 5% serum (Figure 4—figure supplement 1). Of note, titrating EGF did not rescue the effect of serum absence, with non-significant changes in the boundary formation index (0 ng/ml: 0.60 ± 0.07, 20 ng/ml: 0.65 ± 0.03) and maximum velocity cross-correlation, although the mesh stability index decreased, likely due to increased cell movement (Figure 4—figure supplement 2 (n = 25)).

Video 7

Download asset

Using both the velocity order parameter and mesh order index to characterise collective motion, we found little change in collective motion upon EGF addition in 0% serum (Figure 4—figure supplement 2J,K), which might explain the lack of boundary formation. However, in 5% serum plus EGF, the two measures exhibited opposite results: raising EGF concentration increased velocity order but decreased mesh order, Figure 4I,J. We note however that the mesh order, by explicitly accounting for the motion of neighbouring cells, better reflects human observation of motion in videos (Videos 5–7). This highlights the pitfalls of only quantifying the individual alignment of velocity vectors computed from one video frame. Cell counting for 0% serum again suggested minimal influence of cell proliferation (Figure 4—figure supplement 2L,M). In summary, this example with EGF demonstrates that MOSES enables robust continuous-scale quantification of motion phenotype following systematic perturbation.

MOSES generates motion signatures and 2D motion maps for unbiased characterisation of cellular motion phenotypes

High-content imaging screens are often explorative, with the aim of screening for unknown differences in complex cellular motions from a large number of videos in an unbiased manner (Zaritsky et al., 2017). In general it is therefore not known a priori the behaviour of the imaged cells. MOSES addresses this need for an unbiased phenotyping approach by enabling the systematic generation of unique ‘motion signatures’ for individual videos in a manner similar to the relatively automatic generation of geometric features for cell shape quantification in high-content image screens (Boutros et al., 2015; Bray et al., 2016). Below we demonstrate that unsupervised machine learning techniques requiring no manual user annotation can be applied to MOSES generated signatures to visualise all videos onto a 2D motion phenotype map. This advanced feature of MOSES enables easy visual assessment of motion phenotype and the generation of hypotheses without the need to individually interrogate each video.

The general process for the motion map generation is illustrated in Figure 5A. To position each video on a 2D map, we applied principal component analysis (PCA) to the normalised mesh strain curves of the 77 videos of all cell line combinations cultured in 5% serum conditions to learn the principal component vectors that define the x-y axis of the 2D map. The normalised mesh strain curve for each video was used here as an example 1D motion signature to summarise the entire video motion (see Materials and methods for constructing more descriptive signatures). The constructed PCA map of the 77 videos from cells cultured in 5% serum (Figure 5B) shows that this unbiased approach automatically clusters all videos corresponding to each cell line combination. Furthermore, the videos were ordered in a continuous manner, as shown by the increasingly linear shape of the mean normalised strain curve when looking left to right across the plot in Figure 5B from CP-A:CP-A to EPC2:OE33. This clustering was not achieved with root mean squared displacement (RMSD), the non-mesh version of the MOSES-normalised mesh strain curve (Figure 5—figure supplement 1). Moreover, it appears independent of the particular dimensionality reduction technique used (Figure 5—figure supplement 2), indicating that the signatures constructed using MOSES are intrinsically informative. Finally, the 1D MOSES-based motion signatures trained a machine learning classifier with no further processing to predict cell combination identity better than RMSD (Figure 5—figure supplement 2).

Figure 5 with 2 supplements see all

Download asset Open asset

MOSES generates motion signatures to produce a 2D motion map for unbiased characterisation of cellular motion phenotypes.

In all panels, each point represents a video (see legends for colour code). The position of each video on the 2D plot is based on the normalised mesh strain curves, analysed by PCA. (A) The mapping process for a single video. (B) The 5% serum videos (n = 77) were used to set the PCA that maps a strain curve to a point in the 2D motion map. (C) The 0% serum videos (n = 48) were plotted onto the same map defined by the 5% serum videos using the learnt PCA. In (B) and (C), the mean mesh strain curves for each cell combination are shown in the insets. Light blue region marks the two standard deviations with respect to the mean curve (solid black line). (D) Same map as in (C) with points coloured according to 0% or 5% serum. (E) The normalised mean strain curves for 0–20 ng/ml EGF addition to EPC2:CP-A from Figure 4E plotted onto the same map defined by the 5% serum videos.

https://doi.org/10.7554/eLife.40162.033

To demonstrate how such 2D motion phenotype maps can be used to compare videos, we next mapped the 48 videos from 0% serum cultures on the same axes as the videos from 5% serum cultures (Figure 5C,D). The videos from 0% serum mapped to a different area of the 2D plot, whilst preserving the continuous ordering of the previous videos. Therefore, without having watched the videos, it is easy to predict that the cells have markedly different motion dynamics in 0% serum compared to 5% serum. Furthermore, since the points for the 5% serum videos cover a larger area of the 2D plot than the 0% serum videos, one can predict more diversity of motion in 5% serum (Figure 5D).

The motion map can also capture subtle changes in dynamic behaviour. This is demonstrated by mapping the mean video motion for each concentration of EGF from 0 to 20 ng/ml (represented by the respective mean normalised strain curves for each concentration (one per concentration from a total n = 40 videos, Figure 4E) onto the same axis as the 5% serum videos in the absence of EGF (square points in Figure 5E). With increasing EGF, the squamous-columnar EPC2:CP-A motion dynamics become increasingly similar to squamous-cancer EPC2:OE33 above 5 ng/ml, as evidenced by the square points moving from the area of blue circular EPC2:CP-A points into the area of orange circular EPC2:OE33 points. Thus our motion map is consistent with the result using the specific derived measures (above). These results illustrate the ability to detect biological and technical variability across independent experiments and that MOSES possesses the required features for an algorithm to be used in an unbiased manner in high-content screens with minimal prior knowledge.

Comparison between MOSES and PIV

Finally, to further illustrate the full potential of MOSES and to demonstrate its application, we compared MOSES with the widely used PIV method on two published timelapse microscopy datasets of epithelial monolayers. In the original publication, Malinverno et al. (2017) used PIV to describe the induction of large-scale coordinated motility in MCF-10A RAB5A expressing cells compared to MCF-10A control cells. In the publication associated with the second dataset, Rodríguez-Franco et al. (2017) used PIV to show the detection of deformation waves that propagate away from the cell boundary between two epithelial monolayers. The MDCK cell monolayers expressed EphB2 and its ligand ephrinB1, respectively.

Reanalysing the datasets with MOSES, we found that motion fields inferred from optical flow by MOSES were similar to those from PIV, yielding both similar speed curves and velocity kymographs. However, MOSES exhibited greater sensitivity to salient motion events (indicated in Figure 6A). Compared to PIV velocity vectors, MOSES superpixel tracks are a more data-efficient (see Discussion) encoding of the spatio-temporal velocity distribution that naturally enhances and preserves the salient motion. Reconstructed velocity kymographs from the MOSES motion trajectories capture not only the pattern of the full velocity kymograph but further selectively enhanced the detection of the deformation wave signature formed at the interface between EphB2/ephrinB1 epithelial monolayers (as indicated in Figure 6A, right panel). Thus, all velocity-based statistics that can be derived from PIV, such as the velocity order parameter, are fully preserved in MOSES. Yet, MOSES offers additional advanced possibilities. Firstly, instantaneous velocity-based measures from single videos commonly derived from PIV are noisy. For example, the velocity order parameter variation for the slower MCF-10A control cells is non-smooth and highly variable between consecutive time points (Figure 6B). This leads to the misinterpretation that at certain time points, MCF-10A control cell motion is more collective than MCF-10A RAB5A expressing cells following induction. In contrast, the MOSES mesh order exploits long-time continuity and neighbourhood relations to robustly capture collective motion in a manner consistent with human observation (Video 5). Secondly, long-time MOSES tracks and superpixel mesh strain curves can unbiasedly cluster the global motion pattern into small spatio-temporal groups (Figure 6C, Video 8). This provides a systematic approach to the interrogation of motion sources (Figure 6D, Video 9). The computation of motion saliency maps from forward and backward tracked frames effectively highlight the spatial concentration of motion, and the boundary formation index attests to the efficacy of the MOSES-enabled measures across independent datasets. Finally, and uniquely, MOSES’ meshes and tracks present a systematic framework for users to define extensive sets of custom measures to comprehensively characterise complex motion phenotypes, such as the investigated boundary formation behaviour between epithelial cells. By exploiting this, we successfully constructed feature descriptors to generate interpretable motion maps for our large video dataset. Figure 6E summarises the key points of comparison between MOSES and PIV.

Video 8

Download asset

Video 9

Download asset

Figure 6

Download asset Open asset

Comparison between MOSES and PIV.

(A) Left: average speed curves of MCF-10A control (CTRL) and doxycycline inducible RAB5A-expressing (RAB5A) monolayer cell migration after doxycycline addition (Supp. Movie 3 of Malinverno et al., 2017) computed using PIV and MOSES (optical flow). Green triangles indicate notable events in the movie; E1 (4 h): addition of doxycycline, E2 (16 h): first bright ‘flash’ in movie followed by accelerated movement of RAB5A expressing cells, E3 (25 h): timepoint at which RAB5A cells moved fastest. Right: velocity kymographs of Videos 1 and 2 (c.f. Figures 3 and 4 respectively in Rodríguez-Franco et al. (2017)) computed from PIV and MOSES motion fields showing the presence of deformation waves (black dash-dot line) due to cell jamming following initial gap closure (green dashed line). The corresponding velocity kymographs reconstructed from a fixed number of 1000 MOSES superpixel tracks and a ‘dense’ number of superpixel tracks (starting with 1000 superpixels) is shown for comparison. The speed and direction of movement are indicated by the intensity and colour, respectively (blue, moving right; red, moving left). (B) Top and middle: velocity order parameter curves as defined in Malinverno et al. (2017) for the MCF-10A control and RAB5A expressing cell lines was computed for the same movies as (A) using PIV and MOSES. Bottom: corresponding MOSES mesh order curve. Black vertical dashed line mark the addition of doxycycline. (C) MOSES superpixel tracks computed for wound-healing assay of MCF-10A and RAB5A cells (Supp. Video 19 of Malinverno et al., 2017) were automatically clustered into distinct groupings and coloured uniquely according to their mesh strain curve using GMM with BIC model selection (Materials and methods, Video 8 of this paper). (D) (i,ii) Automatic clustered superpixels and associated motion saliency maps (backward tracking to identify ‘initial’ motion, forward tracking to identify ‘final’ motion) for different videos of MCF10A-control (CTRL) and RAB5A monolayer migration in Malinverno et al. (2017), (see also Video 9). (iii) Left to right: snapshots of initial and final frames, MOSES superpixel tracks (1000 superpixels) overlaid on final frames, associated ‘final’ motion saliency map and boundary formation index for CTRL and RAB5A cells for same videos in (C). (iv) Snapshot of final frame, overlaid MOSES superpixel tracks (1000 superpixels) and associated ‘final’ motion saliency map and boundary formation index for the boundary formation between EphB2 and ephrinB1 expressing MDCK monolayers (Rodríguez-Franco et al., 2017). (E) Summary of the comparison between MOSES and PIV. Long-time refers to the tracking of movement beyond one timepoint.

https://doi.org/10.7554/eLife.40162.036

Discussion

We have shown that MOSES combines the advantages of existing PIV and single-cell tracking methods to provide a single systematic approach for the analysis of complex motion and interaction patterns. Its operating principle builds upon two established and highly successful approaches, long-time trajectories and graphs/meshes. In computer vision, spatio-temporal ‘signatures’ constructed using long-time trajectories have proven superior to ‘signatures’ derived from PIV-type motion fields for encoding complex spatio-temporal events such as human actions in very large datasets (Wang et al., 2011). Meshes/graphs are ubiquitously regarded as one of the best approaches to capture relationships between ‘objects’ across many disciplines, from Google search to protein-protein interaction networks (Szklarczyk et al., 2015), flocking analyses (Ballerini et al., 2008; Zhou et al., 2013; Shishika et al., 2014), to detection of cell jamming in biological physics (Lačević et al., 2003; Park et al., 2015). MOSES uniquely brings together these disparate uses of long-time trajectories and meshes into one general analysis framework. As a result, MOSES satisfies the four criteria (robust, sensitive, automatic, and unbiased) necessary for characterising and establishing new phenotypes from live cell imaging. The analysis of datasets that include variable quality videos and experiments with a small number of replicates demonstrates the potential of the proposed computational framework.

Importantly, MOSES is progress towards overcoming the individual limitations of single-cell tracking and PIV-type velocity methods. Single-cell tracks are notoriously problematic over long times; the track of a single cell may be lost or broken into many separate tracks. MOSES superpixel tracks avoids this and recovers the global motion patterns (c.f. motion saliency maps, derived measures and motion signatures). Side-by-side comparison of MOSES and the standard PIV method using published datasets demonstrates that MOSES not only enables all the measurements of PIV, but by further exploiting long-time tracks and neighbourhood relationships, delivers greater physical and biological insights. Complex salient spatio-temporal motion patterns and events such as boundary formation, deformation waves due to cell jamming between two cell populations and cell death can all be quantitatively captured by MOSES. Critically, the ability of MOSES to perform long-time tracking (up to 6 days demonstrated in this study) enabled spatial localisation of the cell populations involved in a particular motion phenotype.

MOSES does not require complex user settings to facilitate reproducibility in analyses because it does not aim to threshold or cluster out the moving objects or phenotypes during analysis, which would introduce intermediate processing errors. Rather its philosophy is to facilitate systematic generation of many motion-related measurements based on trajectory and mesh statistics sufficient for applying machine learning methods for data-driven object segmentation, video classification and phenotype detection in large video collections (e.g. Figure 5, motion map) with minimum prior information. The main parameter the user specifies is the number of initial superpixels, which determines the spatial resolution of analysis. No complicated fitting of complex models and no special hardware such as GPUs are required. MOSES is modular and its components can be readily adapted to suit specific applications, for example non-square superpixel shapes to better capture cells that undergo large shape changes. Analysis of 96 videos with 1344 × 1024 pixel resolution and 145 frames by tracking 1000 superpixels takes under 4 h on an unoptimized code implementation running on a single PC (3.2 GHz, 16 GB RAM). Results are stored efficiently (~1–2 MB per video) compared to ~0.1–1 GB per video, depending on the subsampling used to save the full spatio-temporal PIV/optical flow motion fields. Altogether our study illustrates the potential of MOSES as a powerful and systematic computational framework. It is particularly useful for unbiased explorative high-content screening with an aim to discover fundamental principles of cellular motion dynamics in biology and to identify factors or drugs that alter cellular motion dynamics in disease aetiology and treatment.

Materials and methods

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Cell line (H. sapiens)	EPC2	https://www.med.upenn.edu/molecular/documents/EPCcellprotocol032008.pdf		Prof. Hiroshi Nakagawa (University of Pennsylvania)
Cell line (H. sapiens)	CP-A	CP-A (KR-42421) (ATCC )	ATCC:CRL-4027; RRID:CVCL_C451
Cell line (H. sapiens)	OE33	ECACC	ECACC:96070808; RRID: CVCL_0471
Cell line (H. sapiens)	AGS	AGS (ATCC CRL-1739)	ATCC:CRL-1739; RRID:CVCL_0139
Chemical compound, drug	KSFM	Gibco/Thermo Fisher	Cat#:17005042
Chemical compound, drug	RPMI 1640 medium	Gibco/Thermo Fisher	Cat#:21875–034
Chemical compound, drug	Human recombinant EGF	Gibco/Thermo Fisher	Cat#:PHG0313
Chemical compound, drug	Celltracker Orange (CMRA)	Life Technologies/ Thermo Fisher	Cat#:C34551
Chemical compound, drug	Celltracker Green (CMFDA)	Life Technologies/ Thermo Fisher	Cat#:C7025
Chemical compound, drug	Celltracker DeepRed	Life Technologies/ Thermo Fisher	Cat#:C34565
Chemical compound, drug	Image-iTTM FX Signal Enhancer	Thermo Fisher	Cat#:I36933
Chemical compound, drug	Antibody diluent, background reducing	Agilent Dako	Cat#:S3022
Chemical compound, drug	Antibody dilutent	Agilent Dako	Cat#:S0809
Chemical compound, drug	Fluoromount-G	SouthernBiotech	Cat#:0100–01
Chemical compound, drug	DAPI (1 mg/ml)	Thermo Fisher	Cat#:62248	1:1000 dilution
Antibody	Goat polyclonal anti-mouse Alexa Fluor 488	Thermo Fisher	Cat#:A-11001; RRID:AB_2534069	1:400 dilution
Antibody	Phalloidin Alexa Fluor 647	Thermo Fisher	Cat#:A22287; RRID:AB_2620155	1:400 dilution
Antibody	Mouse monoclonal anti E-cadherin	Becton, Dickinson U.K Ltd.	Cat#:610181; RRID:AB_397580	1:400 dilution
Other	25 culture-inserts 2-Well for self-insertion	Ibidi	Cat#:80209
Software	Fiji ImageJ	https://imagej.net/Fiji	RRID:SCR_002285	TrackMate Plugin
Software /Algorithm	Motion Sensing Superpixels	This paper	RRID:SCR_016839	https://github.com/fyz11/MOSES

Mean squared displacement (MSD)

Request a detailed protocol

As a measure of cellular motions, MSD was computed as a function of time interval, $Δ t$ as in (Park et al., 2015).

M S D (Δ t) = ⟨ {| r_{i} (t + Δ t) - r_{i} (t) |}^{2} ⟩

where $r_{i} (t)$ is the position of the superpixel $i$ at time $t$ and $⟨ \cdot ⟩$ is the average over all time $t$ and all superpixels. For small $Δ t$ , the MSD increases as a power law $Δ t^{α}$ , where the exponent $α$ is determined empirically by fitting. For unity exponent ( $α = 1$ ), the movement is uncorrelated random Brownian motion and cellular motion is diffusive. When $α > 1$ , cellular motions are super-diffusive, and when $α = 2$ , motions are ‘ballistic’.

Root Mean Squared Displacement (RMSD)

Request a detailed protocol

As a summary of the whole video motion and a measure of movement, the root mean squared displacement was computed as a function of time relative to the initial time $t_{0}$ , $RMSD (t) = \sqrt{⟨ {| r_{i} (t) - r_{i} (t_{0}) |}^{2} ⟩}$ where $r_{i} (t)$ is the position of the superpixel $i$ at time $t$ and $⟨ \cdot ⟩$ is the average over all time $t$ and all superpixels. For multi-channel videos, the average RMSD was used to describe video motion. Unless otherwise stated in the text, the normalised RMSD (here division by maximum value within the common time window of comparison) was plotted to permit comparison across different conditions. To compare across videos of different duration, in Figure 1—figure supplement 4B we instead compute the RMSD divided by its value at 96 h, the maximum timepoint shared by both our 96 h and 144 h videos.

Normalised spatial correlation

Request a detailed protocol

For each superpixel $i$ we define its neighbourhood, $N_{i}$ as all superpixels $j$ which lie within a specified distance, $r$ . Given the time-dependent velocity function $V \equiv V (t) = r (t + 1) - r (t)$ where $r (t)$ is the track (all (x,y) positions) up to time $t$ , the normalised spatial correlation of a video with a total of $n$ superpixel tracks is defined as

Request a detailed protocol

The boundary formation index (Figure 3—figure supplement 2) quantifies the extent to which motion concentrates into localised spatial regions in the motion saliency map as a signal-to-noise ratio with value from 0 to 1 suitable for global comparison across video datasets. For example a boundary concentrates motion along a ‘line’ whilst cell death may generate multiple spot-like concentrations (Figure 3—figure supplement 3). The higher the index, the more that motion is concentrated into a single spatial region. To compute the boundary formation index from the visual motion saliency image, the motion saliency image was segmented into ‘high’ and ‘low’ intensity using Otsu thresholding and the normalised signal-to-noise ratio was computed, defined by $\frac{m e a n (high) - m e a n (low)}{m e a n (high)}$

(Figure 3—figure supplement 2). The mean was used as the motion saliency map was computed from a sparse set of points given by the number of superpixels. Individual pixel statistics such as the maximum intensity are therefore noisy and not robust measures. The denominator was set to be the mean of the ‘high’ intensity region in order to give a numerical value bounded between 0–1 for standardised comparison. As this measure captures the ‘peakiness’ of the spatial distribution, it can also be used to quantify other localised spatial processes with adaptation for example cell death.

Normalised mesh strain curves

Request a detailed protocol

For a superpixel, $i$ at time $t$ we define the mesh strain, $ε_{i} (t)$ of the local neighbourhood, $N_{i}$ with $n$ neighbours as the mean of the absolute difference in the distance between superpixel $i$ and superpixel $j$ in its neighbourhood at time $t$ , $r_{i j} (t)$ and at the start at $t = 0$ , $r_{i j} (0)$ so that $ε_{i} (t) = \frac{1}{n} Σ_{j \in N_{i}} | r_{i j} (t) - r_{i j} (0) |$ where $| \cdot |$ is the absolute value or L1-norm. The time-dependent mesh strain for one mesh is the mean local neighbourhood mesh strain over all superpixels for each time frame. The result is a vector the same length as the number of frames in the video. For multi-channels, the average vector is used to describe video motion. The absolute value of the strain curve is susceptible to the image acquisition conditions and geometry whilst the motion information is primarily encoded by the shape of the resulting curve. To permit comparison across different conditions, the normalised strain curve (here division by maximum strain within the common time window of comparison, 0-96 h here) up to 96 h (the maximum timepoint shared between 96 h and 144 h videos in this paper) was used as a simple signature to describe the global video motion pattern when computing motion maps.

Normalised mesh strain, L1-norm and robustness

Request a detailed protocol

Here we provide more details as to why we chose the L1-norm for computing the mesh strain. When computing the ‘distance’ between two vectors, $x_{1}, x_{2}$ both of length $n$ one can define the notion of distance in different ways. A popular family of distances or norms to use is the $L^{p}$ -norm denoted ${| | x_{1} - x_{2} | |}_{p}$ defined mathematically as follows:

{| | x_{1} - x_{2} | |}_{p} = {(\sum_{i = 1}^{n} {| x_{1}^{i} - x_{2}^{i} |}^{p})}^{\frac{1}{p}} w h e r e i = 1, \dots, n i s t h e i t h e l e m e n t o f t h e v e c t o r

$| \cdot |$ denotes the absolute difference. Within this family the most popular is the L1 $(p = 1)$ and L2 $(p = 2)$ norms. The L1-norm is also called the mean absolute deviation and the L2-norm the Euclidean or mean squared distance. The quadratic function of L2 amplifies large differences (> 1) and reduces the effect of smaller differences close to zero thus where differentiability is not a concern L1 is the more robust choice. In our case where most of the distances are larger than 1 (as all superpixels are initially seeded at a distance > 1), we chose to use L1 which is more resistant to the effect of extremal values as a result of errors in motion extraction.

Mesh stability index

Request a detailed protocol

The mesh stability index attempts to quantify the global stationarity (Figure 3—figure supplement 4) across a moving collective such as an epithelial sheet by measuring the change in the average distance between each superpixel and its neighbours. Specifically, it measures the flatness of the normalised mesh strain curve as defined above. Note whilst a curve is flat only if its gradient is flat that is 0, it is more visually appealing to report increasing stability with increasing values therefore we define the mesh stability index as 1 minus the end gradient. The end gradient is most stable when it has a value of 0 therefore this index is upper bounded by 1. To standardise the value of the gradient for comparison, we normalise also across time such that for any length video, time is from 0 to 1. To compute the end gradient stably without curve fitting procedures, we assume the end point is locally linear with respect to time and average the first-order differences over the last few frames. For 96 h videos, the period of stability given by the curve plateau is shorter, therefore the last 10 frames (10 h) were used for computing the gradient. For 144 h videos, the last 24 frames (24 h) were used.

Mesh order

Request a detailed protocol

Inspired by the definition of the velocity order parameter, the mesh order is identically defined but uses the local resultant mesh strain vector instead of instantaneous velocity vectors for computation (Figure 3—figure supplement 5). In this paper, we used the MOSES mesh but any similar mesh construction is equally valid. Given a mesh, the mesh strain vector for a superpixel is the sum of the displacement vector of the superpixel relative to each neighbouring superpixel (Figure 3—figure supplement 5). The mesh order is computed for each frame accordingly for a video. The mean value over all frames was reported for statistical comparison.

Normalised velocity cross-correlation

Request a detailed protocol

Cross-correlation measures the similarity between two signals taking into account time delay, (Figure 3—figure supplement 6). As ‘signals’, we use the time-dependent velocity $V (t)$ computed from the spatial displacement between the location $r (t + 1)$ at time $t + 1$ and location $r (t)$ at time $t$ , $V (t) = r (t + 1) - r (t)$ which is spatially location-independent. Velocity is a vector quantity with both $x$ and $y$ components. Letting $\cdot$ denote the scalar product, the normalised velocity cross-correlation (VCC) of the tracks of red superpixel $i$ and green superpixel $j$ at time $t$ and time lag $m$ such that the extremas are bound by [-1,1] is

Request a detailed protocol

The MOSES code is available open-source under a Ludwig non-commercial and academic license at GitHub, https://github.com/fyz11/MOSES.git (Zhou, 2019; copy archived at https://github.com/elifesciences-publications/MOSES) where it is maintained and updated. Example data for testing can be downloaded from Google Drive, https://drive.google.com/open?id=0BwFVL6r9ww5BaTh6NExLR1JMUXM. The full video dataset can be found under DOIs, https://dx.doi.org/10.17632/j8yrmntc7x.1, https://dx.doi.org/10.17632/vrhtsdhprr.1 for videos with and without EGF addition, respectively.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source code files for MOSES are available and maintained on GitHub (copy archived at https://github.com/elifesciences-publications/MOSES). Analysed videos are made available online with unique DOIs ( http://dx.doi.org/10.17632/j8yrmntc7x.1, http://dx.doi.org/10.17632/vrhtsdhprr.1) through Mendeley datasets.

The following data sets were generated

1. Zhou FY
(2018) Mendeley Data
Normal 2 Cell Population Migration Dataset.
https://doi.org/10.17632/j8yrmntc7x.1
1. Zhou FY
2. Puig CR
(2018) Mendeley Data
EGF Addition to EPC2:CP-A.
https://doi.org/10.17632/vrhtsdhprr.1

References

1. Achanta R
2. Shaji A
3. Smith K
4. Lucchi A
5. Fua P
6. Süsstrunk S
(2012) SLIC superpixels compared to state-of-the-art superpixel methods
IEEE Transactions on Pattern Analysis and Machine Intelligence 34:2274–2282.
https://doi.org/10.1109/TPAMI.2012.120
- PubMed
- Google Scholar
Conference
1. Ali S
2. Shah M
(2007) A lagrangian particle dynamics approach for crowd flow segmentation and stability analysis
IEEE Conference On Computer Vision and Pattern Recognition.
https://doi.org/10.1109/CVPR.2007.382977
- Google Scholar
1. Ballerini M
2. Cabibbo N
3. Candelier R
4. Cavagna A
5. Cisbani E
6. Giardina I
7. Lecomte V
8. Orlandi A
9. Parisi G
10. Procaccini A
11. Viale M
12. Zdravkovic V
(2008) Interaction ruling animal collective behavior depends on topological rather than metric distance: evidence from a field study
PNAS 105:1232–1237.
https://doi.org/10.1073/pnas.0711437105
- PubMed
- Google Scholar
1. Boonstra JJ
2. van Marion R
3. Beer DG
4. Lin L
5. Chaves P
6. Ribeiro C
7. Pereira AD
8. Roque L
9. Darnton SJ
10. Altorki NK
11. Schrump DS
12. Klimstra DS
13. Tang LH
14. Eshleman JR
15. Alvarez H
16. Shimada Y
17. van Dekken H
18. Tilanus HW
19. Dinjens WNM
(2010) Verification and unmasking of widely used human esophageal adenocarcinoma cell lines
JNCI: Journal of the National Cancer Institute 102:271–274.
https://doi.org/10.1093/jnci/djp499
- Google Scholar
(2015) Microscopy-based high-content screening
Cell 163:1314–1325.
https://doi.org/10.1016/j.cell.2015.11.007
- PubMed
- Google Scholar
1. Bray MA
2. Singh S
3. Han H
4. Davis CT
5. Borgeson B
6. Hartland C
7. Kost-Alimova M
8. Gustafsdottir SM
9. Gibson CC
10. Carpenter AE
(2016) Cell painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes
Nature Protocols 11:1757–1774.
https://doi.org/10.1038/nprot.2016.105
- PubMed
- Google Scholar
1. Brox T
2. Bruhn A
3. Papenberg N
4. Weickert J
(2004)
High accuracy optical flow estimation based on a theory for warping

European Conference on Computer Vision 3024:25–36.
- Google Scholar
Conference
1. Brox T
2. Bregler C
3. Malik J
(2009)
Large displacement optical flow

IEEE International Conference on Computer Vision and Pattern Recognition. pp. 41–48.
- Google Scholar
Conference
(2011) Probabilistic group-level motion analysis and scenario recognition
IEEE International Conference On Computer Vision. pp. 747–754.
https://doi.org/10.1109/ICCV.2011.6126312
- Google Scholar
Book
1. Clark R
(2013)
The Molecular and Cellular Biology of Wound Repair

Springer Science & Business Media.
- Google Scholar
(2011) Boundary formation and maintenance in tissue development
Nature Reviews Genetics 12:43–55.
https://doi.org/10.1038/nrg2902
- PubMed
- Google Scholar
(2001) Bile reflux gastritis and Barrett's oesophagus: further evidence of a role for duodenogastro-oesophageal reflux?
Gut 49:359–363.
https://doi.org/10.1136/gut.49.3.359
- PubMed
- Google Scholar
Report
1. Eilers PH
2. Boelens HF
(2005)
Baseline Correction with Asymmetric Least Squares Smoothing

Leiden University Medical Centre.
- Google Scholar
Book
1. Farnebäck G
(2003)
Two-Frame Motion Estimation Based on Polynomial Expansion

Springer.
- Google Scholar
1. Fraley C
(1998) How many clusters? which clustering method? answers via model-based cluster analysis
The Computer Journal 41:578–588.
https://doi.org/10.1093/comjnl/41.8.578
- Google Scholar
1. Gaddam S
2. Singh M
3. Balasubramanian G
4. Thota P
5. Gupta N
6. Wani S
7. Higbee AD
8. Mathur SC
9. Horwhat JD
10. Rastogi A
11. Young PE
12. Cash BD
13. Bansal A
14. Vargo JJ
15. Falk GW
16. Lieberman DA
17. Sampliner RE
18. Sharma P
(2013) Persistence of Nondysplastic Barrett's esophagus identifies patients at lower risk for esophageal adenocarcinoma: results from a large multicenter cohort
Gastroenterology 145:548–553.
https://doi.org/10.1053/j.gastro.2013.05.040
- PubMed
- Google Scholar
(2014) Co-culture systems and technologies: taking synthetic biology to the next level
Journal of the Royal Society Interface 11:20140065.
https://doi.org/10.1098/rsif.2014.0065
- Google Scholar
1. Harada H
2. Nakagawa H
3. Oyama K
4. Takaoka M
5. Andl CD
6. Jacobmeier B
7. von Werder A
8. Enders GH
9. Opitz OG
10. Rustgi AK
(2003)
Telomerase induces immortalization of human esophageal keratinocytes without p16INK4a inactivation

Molecular Cancer Research : MCR 1:729–738.
- PubMed
- Google Scholar
1. Held M
2. Schmitz MH
3. Fischer B
4. Walter T
5. Neumann B
6. Olma MH
7. Peter M
8. Ellenberg J
9. Gerlich DW
(2010) CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging
Nature Methods 7:747–754.
https://doi.org/10.1038/nmeth.1486
- PubMed
- Google Scholar
1. Landsberg KP
2. Farhadifar R
3. Ranft J
4. Umetsu D
5. Widmann TJ
6. Bittig T
7. Said A
8. Jülicher F
9. Dahmann C
(2009) Increased cell bond tension governs cell sorting at the Drosophila anteroposterior compartment boundary
Current Biology 19:1950–1955.
https://doi.org/10.1016/j.cub.2009.10.021
- PubMed
- Google Scholar
(2003) Spatially heterogeneous dynamics investigated via a time-dependent four-point density correlation function
The Journal of Chemical Physics 119:7372–7387.
https://doi.org/10.1063/1.1605094
- Google Scholar
1. Leoni G
2. Neumann PA
3. Sumagin R
4. Denning TL
5. Nusrat A
(2015) Wound repair: role of immune-epithelial interactions
Mucosal Immunology 8:959–968.
https://doi.org/10.1038/mi.2015.63
- PubMed
- Google Scholar
1. Major RJ
2. Irvine KD
(2005) Influence of Notch on dorsoventral compartmentalization and actin organization in the Drosophila wing
Development 132:3823–3833.
https://doi.org/10.1242/dev.01957
- PubMed
- Google Scholar
1. Major RJ
2. Irvine KD
(2006) Localization and requirement for myosin II at the dorsal-ventral compartment boundary of the Drosophila wing
Developmental Dynamics 235:3051–3058.
https://doi.org/10.1002/dvdy.20966
- PubMed
- Google Scholar
1. Malinverno C
2. Corallino S
3. Giavazzi F
4. Bergert M
5. Li Q
6. Leoni M
7. Disanza A
8. Frittoli E
9. Oldani A
10. Martini E
11. Lendenmann T
12. Deflorian G
13. Beznoussenko GV
14. Poulikakos D
15. Haur OK
16. Uroz M
17. Trepat X
18. Parazzoli D
19. Maiuri P
20. Yu W
21. Ferrari A
22. Cerbino R
23. Scita G
(2017) Endocytic reawakening of motility in jammed epithelia
Nature Materials 16:587–596.
https://doi.org/10.1038/nmat4848
- PubMed
- Google Scholar
1. Maška M
2. Ulman V
3. Svoboda D
4. Matula P
5. Matula P
6. Ederra C
7. Urbiola A
8. España T
9. Venkatesan S
10. Balak DM
11. Karas P
12. Bolcková T
13. Streitová M
14. Carthel C
15. Coraluppi S
16. Harder N
17. Rohr K
18. Magnusson KE
19. Jaldén J
20. Blau HM
21. Dzyubachyk O
22. Křížek P
23. Hagen GM
24. Pastor-Escuredo D
25. Jimenez-Carretero D
26. Ledesma-Carbayo MJ
27. Muñoz-Barrutia A
28. Meijering E
29. Kozubek M
30. Ortiz-de-Solorzano C
(2014) A benchmark for comparison of cell tracking algorithms
Bioinformatics 30:1609–1617.
https://doi.org/10.1093/bioinformatics/btu080
- PubMed
- Google Scholar
(2012) Methods for cell and particle tracking
Methods in Enzymology 504:183–200.
https://doi.org/10.1016/B978-0-12-391857-4.00009-4
- PubMed
- Google Scholar
(2011) An in vitro co-culture model of esophageal cells identifies ascorbic acid as a modulator of cell competition
BMC cancer 11:461.
https://doi.org/10.1186/1471-2407-11-461
- PubMed
- Google Scholar
(2012) Cell image velocimetry (CIV): boosting the automated quantification of cell migration in wound healing assays
Integrative Biology 4:1437–1447.
https://doi.org/10.1039/c2ib20113e
- PubMed
- Google Scholar
(2010) An actomyosin-based barrier inhibits cell mixing at compartmental boundaries in Drosophila embryos
Nature cell biology 12:60–65.
https://doi.org/10.1038/ncb2005
- PubMed
- Google Scholar
1. Neumann B
2. Held M
3. Liebel U
4. Erfle H
5. Rogers P
6. Pepperkok R
7. Ellenberg J
(2006) High-throughput RNAi screening by time-lapse imaging of live human cells
Nature Methods 3:385–390.
https://doi.org/10.1038/nmeth876
- PubMed
- Google Scholar
(2017) Analysis of live cell images: methods, tools and opportunities
Methods 115:65–79.
https://doi.org/10.1016/j.ymeth.2017.02.007
- PubMed
- Google Scholar
(2011) Coupled minimum-cost flow cell tracking for high-throughput quantitative analysis
Medical image analysis 15:650–668.
https://doi.org/10.1016/j.media.2010.07.006
- PubMed
- Google Scholar
1. Park JA
2. Kim JH
3. Bi D
4. Mitchel JA
5. Qazvini NT
6. Tantisira K
7. Park CY
8. McGill M
9. Kim SH
10. Gweon B
11. Notbohm J
12. Steward R
13. Burger S
14. Randell SH
15. Kho AT
16. Tambe DT
17. Hardin C
18. Shore SA
19. Israel E
20. Weitz DA
21. Tschumperlin DJ
22. Henske EP
23. Weiss ST
24. Manning ML
25. Butler JP
26. Drazen JM
27. Fredberg JJ
(2015) Unjamming and cell shape in the asthmatic airway epithelium
Nature Materials 14:1040–1048.
https://doi.org/10.1038/nmat4357
- PubMed
- Google Scholar
1. Pau G
2. Walter T
3. Neumann B
4. Hériché JK
5. Ellenberg J
6. Huber W
(2013) Dynamical modelling of phenotypes in a genome-wide RNAi live-cell imaging assay
BMC Bioinformatics 14:308.
https://doi.org/10.1186/1471-2105-14-308
- PubMed
- Google Scholar
1. Peng J
2. Peng S
3. Jiang A
4. Wei J
5. Li C
6. Tan J
(2010) Asymmetric least squares for multiple spectra baseline correction
Analytica Chimica Acta 683:63–68.
https://doi.org/10.1016/j.aca.2010.08.033
- PubMed
- Google Scholar
Book
(1994)
Geometry-Driven Diffusion in Computer Vision

Springer.
- Google Scholar
(2010) Velocity fields in a collectively migrating epithelium
Biophysical Journal 98:1790–1800.
https://doi.org/10.1016/j.bpj.2010.01.030
- PubMed
- Google Scholar
(2013) From seeing to believing: labelling strategies for in vivo cell-tracking experiments
Interface Focus 3:20130001.
https://doi.org/10.1098/rsfs.2013.0001
- PubMed
- Google Scholar
(2017) Long-lived force patterns and deformation waves at repulsive epithelial boundaries
Nature Materials 16:1029–1037.
https://doi.org/10.1038/nmat4972
- PubMed
- Google Scholar
1. Schaffer CJ
2. Nanney LB
(1996)
Cell biology of wound healing

International Review of Cytology 169:151–181.
- PubMed
- Google Scholar
(2015) Graphical model for joint segmentation and tracking of multiple dividing cells
Bioinformatics 31:948–956.
https://doi.org/10.1093/bioinformatics/btu764
- PubMed
- Google Scholar
1. Schmitz MH
2. Held M
3. Janssens V
4. Hutchins JR
5. Hudecz O
6. Ivanova E
7. Goris J
8. Trinkle-Mulcahy L
9. Lamond AI
10. Poser I
11. Hyman AA
12. Mechtler K
13. Peters JM
14. Gerlich DW
(2010) Live-cell imaging RNAi screen identifies PP2A-B55alpha and importin-beta1 as key mitotic exit regulators in human cells
Nature Cell Biology 12:886–893.
https://doi.org/10.1038/ncb2092
- PubMed
- Google Scholar
(2005) Definition and properties of Lagrangian coherent structures from finite-time Lyapunov exponents in two-dimensional aperiodic flows
Physica D: Nonlinear Phenomena 212:271–304.
https://doi.org/10.1016/j.physd.2005.10.007
- Google Scholar
(2014) Male motion coordination in anopheline mating swarms
Scientific reports 4:6318.
https://doi.org/10.1038/srep06318
- PubMed
- Google Scholar
1. Souza RF
(2010) The role of acid and bile reflux in oesophagitis and Barrett's metaplasia
Biochemical Society Transactions 38:348–352.
https://doi.org/10.1042/BST0380348
- PubMed
- Google Scholar
(2006) Phase transition in the collective migration of tissue cells: experiment and model
Physical Review E 74:061908.
https://doi.org/10.1103/PhysRevE.74.061908
- PubMed
- Google Scholar
1. Szklarczyk D
2. Franceschini A
3. Wyder S
4. Forslund K
5. Heller D
6. Huerta-Cepas J
7. Simonovic M
8. Roth A
9. Santos A
10. Tsafou KP
11. Kuhn M
12. Bork P
13. Jensen LJ
14. von Mering C
(2015) STRING v10: protein-protein interaction networks, integrated over the tree of life
Nucleic Acids Research 43:D447–D452.
https://doi.org/10.1093/nar/gku1003
- PubMed
- Google Scholar
(2017) TrackMate: an open and extensible platform for single-particle tracking
Methods 115:80–90.
https://doi.org/10.1016/j.ymeth.2016.09.016
- PubMed
- Google Scholar
Conference
1. Wang H
2. Kläser A
3. Schmid C
4. Liu C-L
(2011) Action recognition by dense trajectories
IEEE Conference on Computer Vision and Pattern Recognition. pp. 3169–3176.
https://doi.org/10.1109/CVPR.2011.5995407
- Google Scholar
1. Wang B
2. Mezlini AM
3. Demir F
4. Fiume M
5. Tu Z
6. Brudno M
7. Haibe-Kains B
8. Goldenberg A
(2014) Similarity network fusion for aggregating data types on a genomic scale
Nature Methods 11:333–337.
https://doi.org/10.1038/nmeth.2810
- PubMed
- Google Scholar
(2016)
Microscopy cell counting and detection with fully convolutional regression networks

Computer Methods in Biomechanics and Biomedical Engineering: Imaging & Visualization 6:283–292.
- Google Scholar
(2012) Emergence of HGF/SF-induced coordinated cellular motility
PLOS ONE 7:e44671.
https://doi.org/10.1371/journal.pone.0044671
- PubMed
- Google Scholar
1. Zaritsky A
2. Kaplan D
3. Hecht I
4. Natan S
5. Wolf L
6. Gov NS
7. Ben-Jacob E
8. Tsarfaty I
(2014) Propagating waves of directionality and coordination orchestrate collective cell migration
PLOS Computational Biology 10:e1003747.
https://doi.org/10.1371/journal.pcbi.1003747
- PubMed
- Google Scholar
(2015) Seeds of locally aligned motion and stress coordinate a collective cell migration
Biophysical Journal 109:2492–2500.
https://doi.org/10.1016/j.bpj.2015.11.001
- PubMed
- Google Scholar
1. Zaritsky A
2. Tseng YY
3. Rabadán MA
4. Krishna S
5. Overholtzer M
6. Danuser G
7. Hall A
(2017) Diverse roles of guanine nucleotide exchange factors in regulating collective cell migration
The Journal of Cell Biology 216:1543–1556.
https://doi.org/10.1083/jcb.201609095
- PubMed
- Google Scholar
Conference
1. Zhou B
2. Tang X
3. Wang X
(2013)
Measuring crowd collectiveness

Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition. pp. 3049–3056.
- Google Scholar
Software
1. Zhou FY
(2019) MOSES (Motion Sensing Superpixels), version 9ab910f
GitHub.

https://github.com/fyz11/MOSES

Decision letter

Hamid Mohammadi

Reviewing Editor; Francis Crick Institute, United Kingdom
Didier Y Stainier

Senior Editor; Max Planck Institute for Heart and Lung Research, Germany

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Motion Sensing Superpixels (MOSES) is a systematic framework to quantify and discover cellular motion phenotypes" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Roberto Cerbino (Reviewer #3).

As you will note, the three expert reviewers find some merit in the MOSES method but raise many issues that preclude publication. As we feel the additional work needed to address the issues raised by the reviewers would take more than two months to complete, we are returning your submission to you now in case you wish to submit elsewhere for speedy publication. Further, the number and complexity of these issues make the editorial team reluctant to encourage a resubmission unless a very substantial amount of additional work was performed. In this case, eLife would be willing to look at a revised paper. Please note that it would be treated as a new submission with no guarantees of acceptance. The two most critical factors from an editorial perspective are:

i) Demonstrating the utility of the method on a diverse range of imaging inputs (reviewer #3 point #5 – there is also a reference to some suggested data) and;

ii) Increased biological measurements, including analysis of proliferation and/or cell density. Given that this study is focused on a new technique, the final decision on this work will be highly dependent on convincingly demonstrating the broad utility of the methodology to the cell migration and tissue morphogenesis community. In addition, it will be important to address the technical issues raised, including out of plane movement or loss of cells, further analysis of the 'boundary', and improving the readability of the study.

Our decision has been reached after consultation between the reviewers.

Reviewer #1:

In the manuscript "Motion Sensing Superpixels (MOSES): A systematic framework to quantify and discover cellular motion phenotypes", Zhou et al. present a new method for measuring cell motility in epithelial sheets and apply this to boundary formation in Barrett's Esophagus.

The approach presented fills the technical gap between PIV/Optical flow measurements and single-cell tracking and provides a method to characterise, and through PCA, discover, motion phenotypes within 2D epithelia. This is certainly a technically interesting concept that is relatively simple to implement and provides insight into motion phenotypes within a tissue. This method will be broadly applicable to many problems in characterising cellular motion. In general I find the manuscript well written and comprehensive in its description of the technique, but not particularly easy to read.

One of the difficulties is that it is unclear to me whether the authors intend to reveal new insight into the fundamental phenomena or simply provide a framework for automating the analysis for e.g. drug discovery. I would argue that cell state (sub cellular protein levels, cell cycle, cell death/extrusion, as examined in Schmitz et al., 2010, Pau et al., 2013, Held et al., 2010) are equally important and are not addressed by the current method.

In the present manuscript, only the cell motion is considered and yet cell proliferation/density is likely to be very important also. Can the authors provide cell density measurements (a proxy for cell proliferation) as a function of time, perhaps using the CNN counting approach, to give some estimate of motion versus proliferation?

This would be particularly interesting in light of the experiments involving titration of EGF. These are very interesting and provide some insight into how collective dynamics/cell adhesion are important in the tissue mechanics in general.

Another consideration is that of axial/upward motion at the interface between the two epithelial monolayers as they meet. Presumably the MOSES approach would register very little motion in a region where cells are being extruded upward (as in Figure 1B). How do the authors envisage addressing this?

Overall, I think that this is a technically interesting manuscript, which provides a set of tools for measuring and characterising cell motion phenotypes from high-throughput time-lapse imaging. This is likely to be a broadly useful tool for the community and addresses a long-standing problem of characterising mesoscale behaviour with close to single-cell accuracy. In my opinion, a revised manuscript would need to provide some additional information regarding the proliferation/density/state of cells within the tissue also.

Reviewer #2:

In this study, the authors utilized their newly developed mesh-based computational framework, MOSES, to analyze the collective motion of the cells and interactions between epithelial monolayers before and after their lateral collision. They have examined three main epithelial interactions that occur in normal esophagus to esophageal adenocarcinoma progression. These interactions (Video 2) lead to formation of a stable boundary with highly dynamic motion of the two cell populations after collision (in squamous-squamous interactions), a stable boundary with less dynamic motion of cell populations after collision and having squamous monolayer pushed by columnar monolayer (squamous-columnar), and no boundary formation with retraction of squamous cell population after collision (squamous-cancer). Authors mainly define two measurements (boundary formation index and motion stability index) to quantify the differences of these boundaries. Both measurements result in selection of squamous-columnar as stable boundary while leave the other two cell combinations in the "unstable boundary" category. Further, authors investigate the effect of activation of EGFR signaling pathway in boundary formation using MOSES. While the approach is appropriate, the current level of analyses does not provide substantial enough improvement relative to other works to justify publication in eLife. There are a number of critiques that authors should address before considering this manuscript for publication.

1) Although the nature of boundaries between squamous-squamous and squamous-cancer are distinct, both are considered as "no boundary" based on boundary index analysis and "unstable boundary" based on stability index analysis. Authors should define new measurements to extract the differences between these two boundaries.

2) As stated by the authors, PIV can be also used for motion extraction in dense monolayers. Authors should clearly state how potential users might benefit using MOSES that otherwise would not be possible using PIV.

3) How would the mesh analysis be affected in case that there would be no continuity in monolayers after collision (i.e. occurrence of detached patches of cells after collision)? Could it be captured in disorder index analysis?

4) Authors have used no serum condition in order to disrupt the cell-cell contact within the monolayers. As serum may affect numerous cell functions, it is not clear if loss of collective sheet migration is because of the reduced cell-cell contacts. Authors are encouraged to use cells that have deleted intercellular adhesion (e.g. α catenin).

5) It would be beneficial to measure migratory behavior of each superpixel with respect to its distance from the boundary before and after collision.

6) How may the analyses change in cases that a combination of stable and unstable boundaries exist in the same field of view?

7) Authors state that in squamous-squamous interactions when cells are exposed to 20 ng/ml of EGF the values of boundary and stability indices are similar to that of squamous-cancer interactions. However, there is no retraction observed in squamous-squamous interactions (Video 7). Again, this needs to be resolved.

Reviewer #3:

The manuscript under review describes a method (MOSES) to quantify cell dynamics. The method is tested with epithelial monolayers made of different cell types. MOSES appears to be an interesting approach to the quantification of cell dynamics. However, for the reasons outlined below, I cannot recommend publication of the manuscript in the present form.

1) The paper is too technical to be really useful for potential users. In particular:

1a) The description of the track filtering and mesh formulation steps are so involved that in the end, I did not understand how a mesh is generated and used.

1b) In a similar manner, the boundary formation index (and to some extent also the motion stability index) is introduced in a way that requires a bona fide effort from the reader to believe that it really measures what is claimed. In my view, a methodological paper like this one, does not benefit from having all the important definitions in the Materials and methods section. Also, it would be greatly appreciated to be able to understand why the proposed parameters for the quantification of the cell dynamics outperform other possible choices.

1c) There are parts that are incomprehensible, such as for instance the paragraph entitled Automated cell counting with convolutional neural networks. This is a pity because the principal component analysis represents one of the most promising features of the method.

1d) Some quantities are not sufficiently defined. For instance, how is TrackMate similarity in Figure 2—figure supplement 2C defined? How is the distance defined in the vertical axis of Figure 1 - figure supplement 5B?

1e) Some choices are not clearly explained: why the cut-off for boundary formation and stability are defined by using the standard deviation? This choice seems to me to be an ambiguous one. For instance, a cut-off for boundary formation is "defined statistically as one standard deviation higher than the pooled mean of all three combinations (Figure 3C). Above this cut-off, cell combinations are categorised as forming a boundary". Surprisingly, a few lines below the authors write "Similarly, experiments in 0% serum were used to set the global motion stability threshold (0.87), one standard deviation below the pooled mean" (Figure 3D). Can the authors explain this asymmetry? The use of standard deviations for fixing thresholds would need in my view that a band exists (pooled mean +/- standard deviation) where the behavior is not determined. Only below mean + sd and above mean - sd a clear behavior could be attributed.

I suggest that the authors consider seriously revising the structure of their paper to make it understandable to a wide audience and to enable the potential reader to properly evaluate the powerfulness and the limitations of the proposed approach.

2) The literature review presented in the Introduction is also not clear, being a puzzling mix of methods to quantify the cell dynamics (such as tracking and PIV) and models (such as the vertex model). To me, the authors fail in merging successfully these two branches of literature and, in particular, in explaining where does MOSES fit and why the previously available tools, are not as good as MOSES in terms of robustness, sensitivity, automatization, and unbiasedness.

3) There are some claims that seem not to be supported by the experimental results. Typical examples are:

3a) Subsection “In-vitro model to study the spatio-temporal dynamics of boundary formation between different cell populations”, first paragraph: Figure 1—figure supplement 2 is cited in support of the fact that cells labeled with different color dye move in a similar way. Inspection of SF2(Figure 1 - figure supplement 2) shows that in some cases the MSD can differ by about one order of magnitude. How is it that the authors consider this a proof of similarity?

3b) The method is allegedly working perfectly in an automatic and unbiased fashion. How comes that the boundary in Figure 3—figure supplement 9A does not seem to be adequately determined?

3c) The statement "The mesh disorder index showed statistically significant increases with EGF concentration" does not seem to be supported by the data in Supplementary Figure 9, where a non-monotonic behavior would also be compatible with the data.

4) There are some claims whose general validity (i.e. in other experiments) is rather dubious.

4a) The Authors claim that "individual cells behave similarly to their neighbors so global motion patterns can be used as a proxy to study single cell behavior". I doubt that this is always true. For instance, for the liquid states found in [Nature Materials 16, 587-596 (2017)] I believe that this claim wouldn't be true.

4b) Given that the L1-norm at is not defined, what is the general validity of the statement "We use L1 for robustness as this value is > 1"?

5) The authors propose MOSES as a robust, sensitive, automatic and unbiased method and I trust them that this might be true. However, the current version of the manuscript proves that MOSES works fairly well in the few cases selected by the authors and it is not clear to me that it could be really used successfully in all other cases. Maybe, the authors could comment on this by suggesting cases in which they expect MOSES to work and cases where they think it would not. For instance, do the authors think that MOSES would work for the experiments in [Nature Materials 16, 1029 (2017)]?

In summary, I do see some potential in MOSES but the current version is not making a good job in explaining clearly what the method does, why is it better than other approaches and when it should be applicable. I hope that the authors will be able to address the above issues.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for submitting your article "Motion Sensing Superpixels (MOSES): A systematic framework to quantify and discover cellular motion phenotypes" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Didier Stainier as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Roberto Cerbino (Reviewer #2); Alison McGuigan (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

This manuscript is well improved and of high scientific value; however two of the reviewers raised several significant points that need clarification. These should be addressed in detail to resolve their concerns. The issue of serum-free condition as negative control may require additional experimentation for validation.

Reviewer #1:

The second version of the manuscript has been improved extensively and reads better than the first one. However, there are a few criticisms the authors should address before considering this manuscript for publication.

An important feature of the tissue boundaries is their shape. It would be beneficial if authors could also incorporate the analysis of boundary roughness in their computational framework.

Authors stated that the "cancer cell line OE33 pushed EPC2 out of the field of view". Since the traction forces applied at the interface between the two sheets is not presented in the manuscript, it is unclear whether the EPC2 cells retracted upon contact with OE33 cells or are continuously pushed by application of physical forces exerted by OE33 cells at the interface.

Authors have conducted new experiments, where the impact of depletion of Ca²⁺ on boundary formation is examined, to elucidate how the absent of cell-cell contact may disrupt the collective motion of the epithelial sheets. However, it is still unclear how serum depletion impacts the proliferation rate of the cells and that affects the collective motion of the sheets.

In conclusion, authors should present how much their computational framework can potentially improve our knowledge in biological processes and how it could be used to tackle new challenges.

Reviewer #2:

The authors have done an extensive amount of work to try to address all the reviewers' comments. As a result, the manuscript is now more clear and convincing in describing the proposed MOSES approach for quantifying the motility of cells belonging to a collective. I thus recommend publication in eLife.

Reviewer #3:

This paper presents a potentially useful tool to enable quantification of cell movement from large numbers of movies to better identify molecules that modulate collective cell migration dynamics. The scale of the data collected is impressive but I found this paper incredibly challenging to read and to understand what the data measurements physically represented and what these measurements meant biologically for our understanding of cell cooperation during boundary formation. Furthermore, I did not really understand the argument made by the authors that serum free represents no boundary formation versus for example delayed boundary formation since cells will move slower and proliferate less (and have a gap to fill before a boundary can form). This manuscript is interesting but is not currently accessible to a broad readership in my opinion.

The major comment I had that I think could significantly improve the impact of the work is can the authors better highlight the functional importance of the metrics they are quantifying in terms of the biological behaviours. For example is a boundary the best thing to be describing here or is wound healing a better example to be able to extract out different cell movement regimes to highlight the power of the tool? I could not understand how the metrics highlighted here could give me new insight into how a boundary is formed therefore it was not clear what I could learn using the tool.

The use of serum free medium to prevent boundary formation does not seem the most robust approach as this also likely impacts cell movement and proliferation, which will impact the timing required to form the boundary in the model being used (since the cells have to proliferate to fill the open space between the two cell domains. I am not sure of the logic behind specifying that doing things in serum free media gives a negative control that corresponds to a no-boundary formation case.

https://doi.org/10.7554/eLife.40162.047

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

Reviewer #1:

In the manuscript "Motion Sensing Superpixels (MOSES): A systematic framework to quantify and discover cellular motion phenotypes", Zhou et al. present a new method for measuring cell motility in epithelial sheets and apply this to boundary formation in Barrett's Esophagus.

The approach presented fills the technical gap between PIV/Optical flow measurements and single-cell tracking and provides a method to characterise, and through PCA, discover, motion phenotypes within 2D epithelia. This is certainly a technically interesting concept that is relatively simple to implement and provides insight into motion phenotypes within a tissue. This method will be broadly applicable to many problems in characterising cellular motion. In general I find the manuscript well written and comprehensive in its description of the technique, but not particularly easy to read.

One of the difficulties is that it is unclear to me whether the authors intend to reveal new insight into the fundamental phenomena or simply provide a framework for automating the analysis for e.g. drug discovery.

MOSES is a computational framework that was developed with an aim to reveal new insights into fundamental phenomena of cellular motion dynamics. However, the biological data generated in this study are best suited to demonstrate the potential of MOSES rather than to conclude any new insight into the fundamental biological principles of cellular motion dynamics. In the revised manuscript, we have carried out a comprehensive comparison between MOSES and a widely used biological motion analysis framework, PIV (Particle Image Velocimetry). We illustrated a number of advanced features of MOSES over PIV and additionally have now emphasised the potential of MOSES in automated high-throughput screening applications. The ability of MOSES to employ a systematic analysis pipeline to generate motion maps in an unbiased way that captures local and global motion patterns through a dynamic mesh construction, will enable future study of novel biological insights in cellular motion dynamics. These points have now been clarified in the revised manuscript.

I would argue that cell state (sub cellular protein levels, cell cycle, cell death/extrusion, as examined in Schmitz et al., 2010, Pau et al., 2013, Held et al., 2010) are equally important and are not addressed by the current method.

General methods for quantifying cell states are indeed important and pattern recognition methods are being applied to analyse protein levels. When cells are sparse, existing published software such as TrackMate, CellProfiler, CIV can be used for single cell or particle tracking. Our work here, however, primarily focuses on the extraction and analysis of collective biological motion and complex phenotypes regardless of cellular density for which software is rarely available and current analysis capabilities are limited. We have now mentioned this in the Discussion.

In the present manuscript, only the cell motion is considered and yet cell proliferation/density is likely to be very important also. Can the authors provide cell density measurements (a proxy for cell proliferation) as a function of time, perhaps using the CNN counting approach, to give some estimate of motion versus proliferation?

This would be particularly interesting in light of the experiments involving titration of EGF. These are very interesting and provide some insight into how collective dynamics/cell adhesion are important in the tissue mechanics in general.

Thank you for the suggestion. We considered that for cell counting to accurately reflect cell proliferation rate, the whole field of view should be covered or the cell population should be largely static (not migratory). In our videos of migrating epithelial sheets, the cells are both moving and proliferating. In these circumstances we propose two approaches to estimate cell proliferation independent of migration.

i) Cell density counting using CNNs (Materials and methods). To avoid issues of moving areas, cells were counted from randomly sampled equal sized image window patches of 64x64 pixels with full cell coverage inside. The average cell density for each cell population was reported as the average over 100 random such windows per time point (Materials and methods).

ii) Quantification of the rate in fluorescence decay. As fluorescently stained cells divide, over time the dye intensity exponentially decays. The faster the division rate, the faster the decay rate. (Materials and methods).

As requested, we applied this to the EGF experiments. Results are shown in new Figure 4—figure supplement 1. Both methods show no differences in the cell proliferation rates of a confluent epithelial sheet with increasing EGF concentration in 5% serum. This is different from a sparse culture in which EGF concentration is more likely to influence cell proliferation rate.

Another consideration is that of axial/upward motion at the interface between the two epithelial monolayers as they meet. Presumably the MOSES approach would register very little motion in a region where cells are being extruded upward (as in Figure 1B). How do the authors envisage addressing this?

Our primary interest in developing a scalable approach for high-throughput screening led us to focus our work on monolayers of cells and 2D timelapse videos, which are the most readily available. However we note that Boquet-Pujadas et al. (BioFlow: a non-invasive, image-based method to measure speed, pressure and forces inside living cells. Scientific reports 7.1 (2017): 9178) have shown already that the optical flow method of motion extraction can be extended with additional mathematical constraints to capture out-of-plane motion. Although the current version of MOSES does not contain such a feature however it can be extended and applied on 3D+time imaging to capture such extrusion processes in the future. This possibility has been discussed in our revised manuscript (see revised main text).

Reviewer #2:

[…] 1) Although the nature of boundaries between squamous-squamous and squamous-cancer are distinct, both are considered as "no boundary" based on boundary index analysis and "unstable boundary" based on stability index analysis. Authors should define new measurements to extract the differences between these two boundaries.

To address this reviewer’s comment, we quantified the observed differences between the squamous-squamous and squamous-cancer boundaries displaced distance and the results is now shown in the new Figure 1G.

2) As stated by the authors, PIV can be also used for motion extraction in dense monolayers. Authors should clearly state how potential users might benefit using MOSES that otherwise would not be possible using PIV.

We include a new section and a summary table in the revised manuscript that explicitly compares the benefits of MOSES with respect to PIV using two published datasets. Please see Results section “Comparison between MOSES and PIV” and new Figure 6.

3) How would the mesh analysis be affected in case that there would be no continuity in monolayers after collision (i.e. occurrence of detached patches of cells after collision)? Could it be captured in disorder index analysis?

To address this reviewer’s comments to perform the mesh analysis when there is no continuity in monolayers after collision we used a video of EPC2 (R):OE33 (G) cells with 5ng/ml of Lapatnib (EGF inhibitor). At this concentration, after the two sheets collide, EPC2 cells rapidly die and shrivel up, creating numerous ‘gaps’ in the epithelial sheet. The results are shown in the new Figure 3—figure supplement 3, where these irregular movements in EPC2 are clearly captured in snapshots of the mesh (Figure 3—figure supplement 3A), and registered in the corresponding mesh strain curve (Figure 3—figure supplement 3B).

4) Authors have used no serum condition in order to disrupt the cell-cell contact within the monolayers. As serum may affect numerous cell functions, it is not clear if loss of collective sheet migration is because of the reduced cell-cell contacts. Authors are encouraged to use cells that have deleted intercellular adhesion (e.g. α catenin).

Although we agree with this reviewer that depleting α catenin will disrupt cell/cell contact, to be able to disrupt and re-establish cell-cell contacts with high efficiency and reproducibility is crucial to test the robustness of MOSES. This is the reason why we did not use genetic manipulation to disrupt cell-cell contacts because the efficacy of RNAi or CRISPR mediated depletion of any given gene can be highly variable among different experimental conditions and it is not easily reversible.

Depletion of Ca²⁺ is very well known to disrupt the cell-cell contacts in epithelial cells and addition of Ca+ can also induce the re-establishment of the cell/cell contacts. Thus we measured the corresponding boundary formation index in cells with Ca+ depletion or with various concentrations of Ca+ added back to culture mediums as indicated. Additionally we performed the experiment with different FBS concentrations and different media; KSFM, KSFM+RPMI and KSFM+RPMI+FBS, to control for potential media effects. To measure motion collectiveness we used the standard velocity order parameter. The results are shown in the Author response image 1. Across all samples a significant correlation between the motion collectiveness as measured by the velocity order parameter and the boundary formation index (Pearson correlation coefficient, r = 0.41) was found when all n = 33 samples were pooled together (right hand scatter plot in Author response image 1).

Author response image 1

Download asset Open asset

All numerical concentrations without units in the figure refer to ng/ml, thus Cal:1000 denotes a 1000ng/ml concentration of Ca²⁺.

5) It would be beneficial to measure migratory behavior of each superpixel with respect to its distance from the boundary before and after collision.

Due to the difficulty of representing this information for all the superpixels (1000) across 144 frames and in 2D with a moving boundary, we approximated the interface between the two cells as a vertical line and constructed velocity kymographs from the extracted dense optical flow which the superpixels use to update their (x,y) positions, as in the paper of Trepat (Rodríguez-Franco et al., 2017). These are included as the new Figure 3A for the three principal cell combinations EPC2:EPC2, EPC2:CP-A and EPC2:OE33 in 0% and 5% serum, and as the new Figure 4B for EGF addition to EPC2:CP-A in serum.

6) How may the analyses change in cases that a combination of stable and unstable boundaries exist in the same field of view?

Unfortunately we do not have a video dataset where we have exclusively studied the coexistence of stable and unstable boundaries in the same field of view. However our experiments with EGF inhibition when applied to the cell combinations of EPC2:CP-A (squamous-columnar) and EPC2:OE33 (squamous-cancer) contain a small set of examples for illustration of the required changes and we report our results in Author response image 2.

In Author response image 2A, we observed partial rescue of the EPC2:OE33 phenotype upon EGF inhibition with motion behaviour more similar to EPC2:CP-A however at these concentrations the EGF inhibitor (Lapatnib) is toxic to the cells and leads to death. This effectively induces the formation of ‘multiple unstable boundaries’ in the EPC2 cells with a moving OE33 front. The boundary formation index cut-offs established in the paper in new Figure 3C implicitly assume the presence of one boundary. Applied here, whilst the boundary formation index still captures the motion concentration in the motion saliency map, as stated in the new main text, it will no longer be specific in only quantifying the interface (the boundary) between the two cell populations (Author response image 2B). To handle the presence of multiple boundaries, one can segment the different unique boundaries by thresholding on the motion saliency map and deriving more specific measures such as counting the number of ‘spot-like’ sites.

Author response image 2

Download asset Open asset

7) Authors state that in squamous-squamous interactions when cells are exposed to 20 ng/ml of EGF the values of boundary and stability indices are similar to that of squamous-cancer interactions. However, there is no retraction observed in squamous-squamous interactions (Video 7). Again, this needs to be resolved.

We assume by retraction the reviewer is referring to the squamous-columnar (EPC2:CP-A) combination where the CP-A cells push the squamous EPC2 cells out of the field of view. We have quantified this displaced distance relative to the size of the image in the new Figure 4D with the EPC2:OE33 videos as reference. Grouping the displaced distance in terms of <5ng/ml and >=5ng/ml, the difference in displaced distance is highly significant, and there was no statistically significant difference in the displaced distance between EPC2:CP-A with 20ng/ml EGF and EPC2:OE33.

Reviewer #3:

The manuscript under review describes a method (MOSES) to quantify cell dynamics. The method is tested with epithelial monolayers made of different cell types. MOSES appears to be an interesting approach to the quantification of cell dynamics. However, for the reasons outlined below, I cannot recommend publication of the manuscript in the present form.

1) The paper is too technical to be really useful for potential users. In particular:

1a) The description of the track filtering and mesh formulation steps are so involved that in the end, I did not understand how a mesh is generated and used.

We have simplified the definitions and abstracted the details more in the main text and the Materials and methods section, and refer interested/more specialised users to the Materials and methods and source code. We have added a schematic diagram of MOSES as a new Figure 2, and the new Figure 2—figure supplement 1 illustrates the technical explanation of the track filtering, which is detailed in the Materials and methods.

Track Filtering:

Our regular spaced superpixels across the whole image act like mini-cameras that assess the local velocity. In the initial frames, only those superpixels that cover the epithelial sheet will move. The rest will remain static as they cover the background image pixels that are black. The objective of the track filtering is to only identify those ‘wanted’ superpixels that cover the movement of the epithelial sheet. Using the above observation, briefly the wanted superpixels can be captured by thresholding on the observed velocities of superpixels in the initial few frames. We have added new text on this in the Materials and methods section of the main text.

Mesh formulation:

The mesh formulation aims to capture the movement of individual superpixels with respect to its local neighbouring superpixels. This involves taking each individual superpixels and connecting it to its neighbours. What constitutes a neighbour? Mathematically this is user defined based on the particular application. For example the nearest k superpixels based on distance gives rise to the popular k-NN neighbour graph when considering flocking behaviour, or one can choose all superpixels within a cutoff distance d_c as we have done here. The definition of neighbour can be recalculated at every time frame but here we only compute the neighbour at the initial timepoint, frame = 0 and assume the same neighbours are retained in all subsequent frames. By doing so if the distance between the neighbours increases dramatically this suggests disruptive motion (a group of cells that started close do not end up in the same spatial area). We have added new text also on this in the Materials and methods section of the main text.

1b) In a similar manner, the boundary formation index (and to some extent also the motion stability index) is introduced in a way that requires a bona fide effort from the reader to believe that it really measures what is claimed.

We apologise for being too complex in our descriptions, and have worked to improve them throughout the manuscript. The boundary formation index is derived from the motion saliency map which is theoretically motivated by Lagrangian mechanics (subsection “Quantitative measurement of

squamous and columnar epithelial boundary formation using MOSES”, second paragraph) in the new main text and above discussion regarding divergence of a motion field).

The mesh stability index (called motion stability index in the original manuscript) is introduced to measure the rate of movement of neighbouring superpixels relative to each other. If the distances between adjacent superpixels are constantly moving then this leads to an unstable mesh and a low stability index. On the other hand if the distance between adjacent cell groups do not change with respect to their initial configuration then the stability index is high. We highlight this property with simulated data (see new Figure 3—figure supplement 4), and have endeavoured to make all the descriptions easier for readers outside the field to follow.

In my view, a methodological paper like this one, does not benefit from having all the important definitions in the Materials and methods section. Also, it would be greatly appreciated to be able to understand why the proposed parameters for the quantification of the cell dynamics outperform other possible choices.

We have now ensured that the important definitions are explained in the main text and refer only to the very detailed definitions and methods in the supplementary material. We also now include a table (new Table 1) whereby each statistical indices in the paper has a biological definition/interpretation (e.g., mesh order measures the collectiveness of local cellular migration) and example biological applications. Finally a number of new figures have been introduced to illustrate these more complicated concepts (see new Figure 3—figure supplements 2-5).

1c) There are parts that are incomprehensible, such as for instance the paragraph entitled Automated cell counting with convolutional neural networks. This is a pity because the principal component analysis represents one of the most promising features of the method.

We apologise for the confusion. The paragraph entitled ‘automated cell counting with convolutional neural networks’ in the Materials and methods has now been edited for clarity. A new supplementary figure has been added (see new Figure 1—figure supplement 1) that clarifies better the training and application procedure of the convolutional neural network.

1d) Some quantities are not sufficiently defined. For instance, how is TrackMate similarity in Figure 2—figure supplement 2C defined? How is the distance defined in the vertical axis of Supplementary Figure 9D?

Similarity of MOSES tracks with TrackMate tracks are measured using the normalised cross-correlation between the paired tracks (see new Figure 2—figure supplement 2 legend). The potentially confusing original Supplementary Figure 9 showing the mesh disorder index is now removed and no longer part of the manuscript. The distance was in number of superpixels as in the spatial correlation computation of Figure 1—figure supplement 5B.

1e) Some choices are not clearly explained: why the cut-off for boundary formation and stability are defined by using the standard deviation? This choice seems to me to be an ambiguous one.

The metrics exhibit a unimodal distribution (see violin plots of new Figure 3C-E). Correspondingly it is statistically justified to use the standard deviation and the mean to set cut-offs. This practice is equivalent to using a t-distribution/normal distribution and in line with the standard practice of one-tailed t-tests. We have added a new section in the Materials and methods to explain this more clearly.

For instance, a cut-off for boundary formation is "defined statistically as one standard deviation higher than the pooled mean of all three combinations (Figure 3B). Above this cut-off, cell combinations are categorised as forming a boundary".

The higher the boundary formation index, the greater the probability of forming a boundary. Thus we set one standard deviation higher than the pooled mean. This corresponds to a one-tailed t-test where we need to observe values at least as high as this threshold to confidently predict a boundary. The null hypothesis is the absence or insufficient evidence for boundary formation with the pooled combinations.

Surprisingly, a few lines below the authors write "Similarly, experiments in 0% serum were used to set the global motion stability threshold (0.87), one standard deviation below the pooled mean". Can the authors explain this asymmetry?

Similarly the lower the mesh stability the more the epithelial sheet moves as a whole. We set one standard deviation lower than the pooled mean corresponding to the one-tailed test where we need to observe values at least as low as this to predict instability. The null hypothesis is that with the pooled combinations we already see mesh stability. Therefore the interesting phenomena to test for here is instability.

By choosing the cut-offs in this way we are being more stringent with what constitutes ‘boundary-forming’ and ‘unstable’ which are the two ‘interesting’ phenotypes contrary to the norm.

The use of standard deviations for fixing thresholds would need in my view that a band exists (pooled mean +/- standard deviation) where the behavior is not determined. Only below mean+ sd and above mean-sd a clear behavior could be attributed.

Our threshold choices are equivalent to fitting the data with a 2-class classifier. The reviewer’s suggestion of +/- cutoffs corresponds to a 3-class classifier is also a plausible model where the extra class is used as a ‘miscellaneous’ class where we are unsure of the phenotype. We believe that both models are justified. We chose to use the former which is more fitting in the context of drug screening.

I suggest that the authors consider seriously revising the structure of their paper to make it understandable to a wide audience and to enable the potential reader to properly evaluate the powerfulness and the limitations of the proposed approach.

We have improved the readability of the manuscript during our revision and the revised manuscript have been read by various colleagues including a former journal editor outside the field. We hope the revised manuscript is now more understandable to the broader reader.

2) The literature review presented in the Introduction is also not clear, being a puzzling mix of methods to quantify the cell dynamics (such as tracking and PIV) and models (such as the vertex model). To me, the authors fail in merging successfully these two branches of literature and, in particular, in explaining where does MOSES fit and why the previously available tools, are not as good as MOSES in terms of robustness, sensitivity, automatization, and unbiasedness.

We have carried out extensive modifications to the Introduction by providing more technical context for our work with respect to the existing literature. We have also included a new comprehensive comparison between MOSES and PIV, the most commonly used method and demonstrate the more advanced features of MOSES over PIV in the main text and Discussion.

3) There are some claims that seem not to be supported by the experimental results. Typical examples are:

3a) Subsection “In-vitro model to study the spatio-temporal dynamics of boundary formation between different cell populations”, first paragraph: Figure 1—figure supplement 2 is cited in support of the fact that cells labeled with different color dye move in a similar way. Inspection of SF2 (Figure 1—figure supplement 2) shows that in some cases the MSD can differ by about one order of magnitude. How is it that the authors consider this a proof of similarity?

Thank you for the comments. We have investigated the issue and found a batch effect inherent in the MSD calculation. In one batch of videos, one cell type was plated more asymmetrically (occupying ~70% of the image) as opposed to the normal 50% and we didn’t filter these tracks out in the MSD computation. The corrected quantification is presented in the new Figure 1—figure supplement 2 and statistical tests have been carried out on the extracted MSD components.

3b) The method is allegedly working perfectly in an automatic and unbiased fashion. How comes that the boundary in Figure 3—figure supplement 9A does not seem to be adequately determined?

Figure 3—figure supplement 9A illustrates the process by which we infer the gap closure frame starting from image segmentation of the fluorescent images. The objective was not to delineate perfectly the sheet boundaries but to accurately infer the time of gap closure for downstream analysis. Comparison of the inferred gap closure times over n = 246 videos with the corresponding consensus manual annotations of 3 independent researchers demonstrate high correlation (0.902 Pearson correlation) and an agreement of 94% within 5 frames, (new Figure 3—figure supplement 9E) which is very comparable to the 97% accuracy between individual humans. This justifies the initial quality of image segmentation used for the intended purpose of inferring the frame in which the gap between the two sheets are closed.

3c) The statement "The mesh disorder index showed statistically significant increases with EGF concentration" does not seem to be supported by the data in Supplementary Figure 9, where a non-monotonic behavior would also be compatible with the data.

During the revision, we carried out extensive testing using our own and published datasets and concluded that the newly proposed mesh order in the revised manuscript is more general than the initially proposed mesh order index as a measure of collective motion based on our MOSES mesh construction. The previously proposed mesh disorder index was only applicable between videos with identical initial configuration i.e. same type of cells and plating as in the case of the EGF titration videos. The results shown in Supplementary Figure 9 (original manuscript) was intended to highlight that the mesh disorder index deviates from uniformity when EGF concentrations > 5ng/ml. Statistical tests were carried out to identify this deviation from a constant line model which is reasonable for 0-5ng/ml. Thus overall the behaviour is non-monotonic. With our new improved mesh order measurement, Supplementary Figure 9 of the original manuscript is now removed as we no longer use the mesh disorder index.

4) There are some claims whose general validity (i.e. in other experiments) is rather dubious.

4a) The Authors claim that "individual cells behave similarly to their neighbors so global motion patterns can be used as a proxy to study single cell behavior". I doubt that this is always true. For instance, for the liquid states found in [Nature Materials 16, 587-596 (2017)] I believe that this claim wouldn't be true.

We address this point together with point 5 (see below).

4b) Given that the L1-norm at is not defined, what is the general validity of the statement "We use L1 for robustness as this value is > 1"?

We apologise for neglecting to define this norm in our previous version of the manuscript. We have added a new section “Normalised Mesh Strain, L1-norm and robustness” in the Materials and methods explaining our design choice.

5) The authors propose MOSES as a robust, sensitive, automatic and unbiased method and I trust them that this might be true. However, the current version of the manuscript proves that MOSES works fairly well in the few cases selected by the authors and it is not clear to me that it could be really used successfully in all other cases. Maybe, the authors could comment on this by suggesting cases in which they expect MOSES to work and cases where they think it would not. For instance, do the authors think that MOSES would work for the experiments in [Nature Materials 16, 1029 (2017)]?

We thank the reviewer for their help in making available the raw videos of [Nature Materials 16, 587-596 (2017)] available. Together with the videos in [Nature Materials 16, 1029 (2017)] as our validation datasets we have refined our claims in the revised manuscript. In addition we profiled the similarity and differences between MOSES and PIV. The new results are included in the revised manuscript as a new section with an additional Figure 6 and accompanying summary table.

[Editors' note: the author responses to the re-review follow.]

Reviewer #1:

The second version of the manuscript has been improved extensively and reads better than the first one. However, there are a few criticisms the authors should address before considering this manuscript for publication.

An important feature of the tissue boundaries is their shape. It would be beneficial if authors could also incorporate the analysis of boundary roughness in their computational framework.

We quantified the ‘boundary roughness’ according to the deviation of the geometrical boundary shape from a straight line, L/L₀ where L is the length of the boundary and L₀ is the length of the straight line joining the endpoints of the boundary as in Javaherian, Sahar, et al (Modulation of cellular polarization and migration by ephrin/Eph signal-mediated boundary formation." Integrative Biology 9.12 (2017): 934-946), (Figure 3—figure supplement 10). No significant departures from a straight line (L/L₀=1) were found. A new paragraph for extracting the boundary to calculate boundary shape has been added to the Materials and methods.

Authors stated that the "cancer cell line OE33 pushed EPC2 out of the field of view". Since the traction forces applied at the interface between the two sheets is not presented in the manuscript, it is unclear whether the EPC2 cells retracted upon contact with OE33 cells or are continuously pushed by application of physical forces exerted by OE33 cells at the interface.

We acknowledge that without traction force measurements we can’t draw a clear conclusion about the forces at play in our assay, whether it was EPC2 cells which retracted or was continuously pushed by OE33 cells. The wording in our previous manuscript, “cancer cell line OE33 pushed EPC2 out of the field of view”, was used to describe the phenomenon of OE33 cells expanding into EPC2 cells resulting in their eventual disappearance from the field-of-view. This is supported by the derived motion field (Figure 1—figure supplement 3). Just before gap closure, EPC2 and OE33 cells are moving in opposite directions. After gap closure EPC2 cells near the interface continue to move in opposite directions to OE33 cells initially before they appear to be dominated by OE33 cells. This is suggestive of ‘pushing’. Unfortunately due to the large fluorescence decay in OE33 cells near the interface it is not fully conclusive whether this ‘pushing’ is continuously maintained for the full duration of the timelapse video. Confocal image of cell junctions in fixed samples at 72 h together with evidence of motion suggest continual application of physical forces. However further experimentation is required to confirm this under timelapse microscopy study. In the main we now state “in the squamous-cancer EPC2:OE33 combination, the cancer cell line OE33 expanded continuously, resulting in the disappearance of EPC2 from the field of view (Video 2, Figure 1E) as assessed by the motion field and confocal images (Figure 1—figure supplement 3). The forces that govern the behaviour of the two cell lines on contact are unknown and traction force microscopy is required to investigate the ‘retracting’ or ‘pushing’ behaviour of EPC2 or OE33 cells respectively in future studies”.

Authors have conducted new experiments, where the impact of depletion of Ca²⁺ on boundary formation is examined, to elucidate how the absence of cell-cell contact may disrupt the collective motion of the epithelial sheets. However, it is still unclear how serum depletion impacts the proliferation rate of the cells and that affects the collective motion of the sheets.

To address this question, we used cell density as an indicator of cell proliferation. The mean cell density and mean change in cell density over the first 48 h (≈ 16 h before and 32 h after gap closure) were estimated from cells grown in serum (5% FBS) and no serum (0% FBS) conditions from individual video frames using the established convolutional neural network approach (Materials and methods). Our results, shown in the new Figure 1—figure supplement 4, suggest that under the experimental conditions used in these studies, the lack of serum has undetectable impact on cell density. This is in complete contrast to the observed increase in collective cell migration and presence of boundary formation in 5% serum. Together these results illustrate that serum-free medium has a profound impact on cell motion dynamics. We therefore used 0% serum in the paper as a computational negative control for the detection of boundary formation. We have modified wording in the text to provide explicit clarification to avoid confusion (see subsection “In-vitro model to study the spatio-temporal dynamics of boundary formation between different cell populations”, last paragraph).

In conclusion, authors should present how much their computational framework can potentially improve our knowledge in biological processes and how it could be used to tackle new challenges.

We thank the reviewer for the constructive comment. To address this we have rewritten the Introduction to provide additional rationale for the need of developing a computational framework like MOSES. In particular we highlight the advantage of MOSES over single-cell tracking and existing PIV/CIV type methods, “we developed Motion Sensing Superpixels (MOSES), a computational framework that aims to provide a flexible and general approach for biological motion extraction, characterisation and phenotyping. We empowered PIV-type methods with a mesh formulation that enables systematic measurement and unbiased extraction of rich motion features for single and collective cell motion suitable for high-throughput phenotypic screens.”

In the revised Discussion, the potential of MOSES to advance biological knowledge and its ability to tackle new challenges is stated as the following.

1) Single-cell tracks are notoriously problematic over long times; the track of a single cell may be lost or broken into many separate tracks. MOSES superpixel tracks avoids this and recovers the global motion patterns (c.f. motion saliency maps, derived measures and motion signatures). Whilst side-by-side comparison of MOSES and the standard PIV method using published datasets demonstrates that MOSES not only enables all the measurements of PIV, but by further exploiting long-time tracks and neighbourhood relationships, delivers greater physical and biological insights. (Discussion, second paragraph).

2) Complex salient spatio-temporal motion patterns and events such as boundary formation, deformation waves due to cell jamming between two cell populations and cell death can all be quantitatively captured by MOSES. Critically, the ability of MOSES to perform long-time tracking (up to 6 days demonstrated in this study) enabled spatial localisation of the cell populations involved in a particular motion phenotype. (Discussion, second paragraph).

3) MOSES does not require complex user settings to facilitate reproducibility in analyses because it does not aim to threshold or cluster out the moving objects or phenotypes during analysis, which would introduce intermediate processing errors. Rather its philosophy is to facilitate systematic generation of many motion-related measurements based on trajectory and mesh statistics sufficient for applying machine learning methods for data-driven object segmentation, video classification and phenotype detection in large video collections (e.g. Figure 5, motion map) with minimum prior information. The main parameter the user specifies is the number of initial superpixels, which determines the spatial resolution of analysis. No complicated fitting of complex models and no special hardware such as GPUs are required. (Discussion, last paragraph).

All of these illustrate the potential of MOSES as a powerful and systematic computational framework that is particularly useful for unbiased explorative high-content screening with an aim to discover fundamental principles of cellular motion dynamics in biology and to identify factors or drugs that can alter cellular motion dynamics in disease aetiology and treatment.

Reviewer #3:

This paper presents a potentially useful tool to enable quantification of cell movement from large numbers of movies to better identify molecules that modulate collective cell migration dynamics. The scale of the data collected is impressive but I found this paper incredibly challenging to read and to understand what the data measurements physically represented and what these measurements meant biologically for our understanding of cell cooperation during boundary formation. Furthermore, I did not really understand the argument made by the authors that serum free represents no boundary formation versus for example delayed boundary formation since cells will move slower and proliferate less (and have a gap to fill before a boundary can form). This manuscript is interesting but is not currently accessible to a broad readership in my opinion.

The major comment I had that I think could significantly improve the impact of the work is can the authors better highlight the functional importance of the metrics they are quantifying in terms of the biological behaviours. For example is a boundary the best thing to be describing here or is wound healing a better example to be able to extract out different cell movement regimes to highlight the power of the tool? I could not understand how the metrics highlighted here could give me new insight into how a boundary is formed therefore it was not clear what I could learn using the tool.

The aim of developing MOSES is as “a computational framework that aims to provide a flexible and general approach for biological motion extraction, characterisation and phenotyping. We empowered PIV-type methods with a mesh formulation that enables systematic measurement and unbiased extraction of rich motion features for single and collective cell motion suitable for high-throughput phenotypic screens.”. We have provided additional rationale for the need of developing such a computational framework in the revised Introduction. Accordingly in this paper the metrics are presented to highlight how MOSES offers a more flexible platform to define customized motion measures related to a specific biological phenomena compared to existing methods that better discriminates between the resulting motion phenotypes. Here we did not aim to specifically shed insight into how a boundary is formed. However it is important to note that further studies using our method to conduct a high-content screen following genetic or other experimental manipulations would enable us to reveal new mechanistic insights into boundary formation in an unbiased manner.

The use of serum free medium to prevent boundary formation does not seem the most robust approach as this also likely impacts cell movement and proliferation, which will impact the timing required to form the boundary in the model being used (since the cells have to proliferate to fill the open space between the two cell domains. I am not sure of the logic behind specifying that doing things in serum free media gives a negative control that corresponds to a no-boundary formation case.

This is a miscommunication on our part. In this paper our ‘negative’ control serves only to demonstrate the sensitivity of MOSES to distinguish between the clear boundary-like behaviour of EPC2:CP-A in 5% serum and the lack of in the corresponding ‘negative’ no-serum case. We do not attempt to claim any experimental conditions used in this study are biologically important in boundary formation as some of the conditions such as serum-free condition cannot be used as a rigorous biological control. Here we used serum-free medium as a condition to test the sensitivity of MOSES because it has a profound impact on cell motion dynamics with absence of boundary formation. We have now clearly stated this in the revised manuscript (subsection “In vitro model to study the spatio-temporal dynamics of boundary formation between different cell populations”, last paragraph, subsection “Quantitative measurement of squamous and columnar epithelial boundary formation using MOSES”, third paragraph).

To address this reviewer’s specific comments about the impact of serum on the timing and speed of boundary formation, we carried out further measurement during the revision and our results (new Figure 1—figure supplement 4, Figure 4—figure supplement 2) showed that:

1) The presence of serum causes variation in gap closure time (within +/- 5 h) but is not significant and consistent across all cell-line combinations (Figure 1—figure supplement 4A,B). This is in contrast to the notable average increased speed in serum across all cell combinations shown in Figure 3B.

2) The presence of serum does not have significant impact on the cell movement at the assay endpoint. All videos (96 h and 144 h) have the same normalized RMSD curves at late timepoints, (Figure 1—figure supplement 4B).

3) Consistent with our findings, serum has profound impact on cell movement. In 5% serum, cells moved more collectively (Figure 3F,G, Figure 1—figure supplement 5C) and moved quicker (Figure 3A,B). However increased cell movement speed alone does not contribute to boundary formation. Addition of EGF to EPC2:CP-A cells in no-serum conditions caused notable increase in cell movement speed but had minimal impact on collective migration and boundary formation (Figure 4—figure supplement 2C,G,J,K).

https://doi.org/10.7554/eLife.40162.048

Article and author information

Author details

Felix Y Zhou

Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom

Contribution
Conceptualization, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing, Conceived and implemented all of MOSES and analysed all data

Contributed equally with
Carlos Ruiz-Puig

For correspondence
felix.zhou@ludwig.ox.ac.uk

Competing interests
A patent is pending for MOSES (UK application no. GB1716893.1, International application no. PCT/GB2018/052935)). The code is available open-source and free for academic and non-profit users under a Ludwig academic and non-profit license.

"This ORCID iD identifies the author of this article:" 0000-0003-4463-1165
Carlos Ruiz-Puig

Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom

Contribution
Conceptualization, Resources, Data curation, Validation, Investigation, Methodology, Writing—review and editing, Performed all live cell imaging videos. Established the 24-well plate based co-culture system.

Contributed equally with
Felix Y Zhou

Competing interests
A patent is pending for MOSES (UK application no. GB1716893.1, International application no. PCT/GB2018/052935)). The code is available open-source and free for academic and non-profit users under a Ludwig academic and non-profit license.

"This ORCID iD identifies the author of this article:" 0000-0002-9214-3804
Richard P Owen

Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom

Contribution
Resources, Methodology, Writing—review and editing, Aided initial prototyping of MOSES with mixed co-culture timelapse videos

Competing interests
No competing interests declared
Michael J White

Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom

Contribution
Resources, Methodology, Writing—review and editing, Aided initial prototyping of MOSES with mixed co-culture timelapse videos

Competing interests
No competing interests declared
Jens Rittscher
1. Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom
2. Institute of Biomedical Engineering, Department of Engineering, University of Oxford, Oxford, United Kingdom
3. Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford, United Kingdom
Contribution
Conceptualization, Supervision, Methodology, Project administration, Writing—review and editing, Provided computational supervision

For correspondence
jens.rittscher@eng.ox.ac.uk

Competing interests
A patent is pending for MOSES (UK application no. GB1716893.1, International application no. PCT/GB2018/052935)). The code is available open-source and free for academic and non-profit users under a Ludwig academic and non-profit license.
Xin Lu

Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom

Contribution
Conceptualization, Supervision, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing, Provided overall project supervision

For correspondence
xin.lu@ludwig.ox.ac.uk

Competing interests
A patent is pending for MOSES (UK application no. GB1716893.1, International application no. PCT/GB2018/052935)). The code is available open-source and free for academic and non-profit users under a Ludwig academic and non-profit license.

"This ORCID iD identifies the author of this article:" 0000-0002-6587-1152

Funding

Ludwig Institute for Cancer Research

Felix Y Zhou
Carlos Ruiz-Puig
Richard P Owen
Jens Rittscher
Xin Lu

Engineering and Physical Sciences Research Council (EP/F500394/1)

Felix Y Zhou

Oxford Health Services Research Committee

Richard P Owen

Oxford University Clinical Academic Graduate School

Richard P Owen

Cancer Research UK (C5255/A19498)

Michael J White

Engineering and Physical Sciences Research Council (EP/M013774/1)

Jens Rittscher

Cancer Research UK (C9720/A18513)

Xin Lu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Prof. Hiroshi Nakagawa for generous donation of EPC2 cells and Profs. Roberto Cerbino and Xavier Trepat for making available the raw videos in their publications. We thank Dr. Mary Muers, Dr. Françoise Howe, Profs. Sebastian Nijman, Francis Szele, Colin Goding and Shankar Srinivas for critical reading of the manuscript; Mark Shipman for technical assistance with timelapse microscopy. This work is mainly funded by the Ludwig Institute for Cancer Research (LICR) with additional support from a CRUK grant to XL (C9720/A18513). FYZ is mainly funded through the EPSRC Life Sciences Interface Doctoral Training Centre EP/F500394/1, CRP and XL are funded by LICR, RPO is supported by LICR, the Oxford Health Services Research Committee and Oxford University Clinical Academic Graduate School supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at the Oxford University Hospitals Trust, Oxford, MJW is supported by CRUK (C5255/A19498, through an Oxford Cancer Research Centre Clinical Research Training Fellowship), and JR is funded by LICR and the EPSRC SeeBiByte Programme Grant (EP/M013774/1). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

Senior Editor

Didier Y Stainier, Max Planck Institute for Heart and Lung Research, Germany

Reviewing Editor

Hamid Mohammadi, Francis Crick Institute, United Kingdom

Publication history

Received: July 16, 2018
Accepted: January 11, 2019
Version of Record published: February 26, 2019 (version 1)
Version of Record updated: July 2, 2019 (version 2)

Copyright

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

1,641

Page views
138

Downloads
6

Citations

Article citation count generated by polling the highest count across the following sources: PubMed Central, Crossref, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Mendeley

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

Felix Y Zhou
Carlos Ruiz-Puig
Richard P Owen
Michael J White
Jens Rittscher
Xin Lu

(2019)

Motion sensing superpixels (MOSES) is a systematic computational framework to quantify and discover cellular motion phenotypes

eLife 8:e40162.

https://doi.org/10.7554/eLife.40162

Categories and tags

Research organism

Human

Share this article

Cite this article

Temporary divider system to study interactions between cell populations.

Dynamics of monolayer combinations of three oesophageal epithelial cell lines, EPC2:EPC2, CP-A:CP-A, OE33:OE33.

Sheet migration in EPC2:EPC2, EPC2:CP-A and EPC2:OE33 in 0% and 5% FBS.

Schematic diagram of MOtion SEnsing Superpixels (MOSES).

Single-cell motion extraction using MOSES workflow compared to manual annotations used in the cell tracking challenge dataset (Maška et al., 2014).

Assessment of individual cell motion in confluent cell sheets using spiked-in fluorescent cell populations and MOSES.

MOSES quantifies boundary formation and epithelial sheet interaction dynamics.

Comparison of the temporal stability of velocity and mesh strain vectors for measuring collective motion.

Summary of MOSES-derived measurements for discriminating boundary formation phenotype in this paper and their biological interpretation and application.

Representative videos of the sheet migration of all tested cell combinations in 5% FBS. (R) and (G) denote red and green dyed cells respectively.

EGF titration at physiological levels disrupts boundary formation.

Motion dynamics of EPC2(R):CP-A(G) under increasing EGF addition (0–20 ng/ml).

MOSES generates motion signatures to produce a 2D motion map for unbiased characterisation of cellular motion phenotypes.

Long-time superpixel track extraction and unbiased track clustering using the mesh strain curve of each superpixel as the feature vector for Supp.

Long-time superpixel track extraction and unbiased track clustering using the mesh strain curve for Supp. Movie 3 and 7 of Malinverno et al. (2017).

Comparison between MOSES and PIV.

Author details

Felix Y Zhou

Contribution

Contributed equally with

For correspondence

Competing interests

Carlos Ruiz-Puig

Contribution

Contributed equally with

Competing interests

Richard P Owen

Contribution

Competing interests

Michael J White

Contribution

Competing interests

Jens Rittscher

Contribution

For correspondence

Competing interests

Xin Lu

Contribution

For correspondence

Competing interests

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

Categories and tags

Research organism

Further reading