Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary:
In this study, Bu et al examined the dynamics of TRPV4 channel in cell overcrowding in carcinoma conditions. They investigated how cell crowding (or high cell confluence) triggers a mechano-transduction pathway involving TRPV4 channels in high-grade ductal carcinoma in situ (DCIS) cells that leads to large cell volume reduction (or cell volume plasticity) and proinvasive phenotype.
In vitro, this pathway is highly selective for highly malignant invasive cell lines derived from a normal breast epithelial cell line (MCF10CA) compared to the parent cell line, but not present in another triple-negative invasive breast epithelial cell line (MDA-MB-231). The authors convincingly showed that enhanced TRPV4 plasma membrane localization correlates with highgrade DCIS cells in patient tissue samples.
Specifically in invasive MCF10DCIS.com cells, they showed that overcrowding or overconfluence leads to a decrease in cell volume and intracellular calcium levels. This condition also triggers the trafficking of TRPV4 channels from intracellular stores (nucleus and potentially endosomes), to the plasma membrane (PM). When these over-confluent cells are incubated with a TRPV4 activator, there is an acute and substantial influx of calcium, attesting to the fact that there are a high number of TRPV4 channels present on the PM. Long-term incubation of these over-confluent cells with the TRPV4 activator results in the internalization of the PMlocalized TRPV4 channels.
In contrast, cells plated at lower confluence primarily have TRPV4 channels localized in the nucleus and cytosol. Long-term incubation of these cells at lower confluence with a TRPV4 inhibitor leads to the relocation of TRPV4 channels to the plasma membrane from intracellular stores and a subsequent reduction in cell volume. Similarly, incubation of these cells at low confluence with PEG 3000 (a hyperosmotic agent) promotes the trafficking of TRPV4 channels from intracellular stores to the plasma membrane.
Strengths:
The study is elegantly designed and the findings are novel. Their findings on this mechanotransduction pathway involving TRPV4 channels, calcium homeostasis, cell volume plasticity, motility, and invasiveness will have a great impact in the cancer field and are potentially applicable to other fields as well. Experiments are well-planned and executed, and the data is convincing. The authors investigated TRVP4 dynamics using multiple different strategies- overcrowding, hyperosmotic stress, and pharmacological means, and showed a good correlation between different phenomena.
Weaknesses:
A major emphasis in the study is on pharmacological means to relate TRPV4 channel function to the phenotype. I believe the use of genetic means would greatly enhance the impact and provide compelling proof for the involvement of TRPV4 channels in the associated phenotype.
In this regard, I wonder if siRNA-mediated knockdown of TRPV4 in over-confluent cells (or knockout) would lead to an increase in cell volume and normalize the intracellular calcium levels back to normal, thus ultimately leading to a decrease in cell invasiveness.
We greatly appreciate the positive feedback regarding the design of our study and the novelty of our findings. We also acknowledge the valuable suggestion to complement our pharmacological approaches with genetic manipulation of TRPV4.
In response to the comment regarding siRNA-mediated knockdown or knockout of TRPV4, we fully agree that this would further substantiate our findings. In the revised manuscript, we implemented shRNA targeting TRPV4 to investigate its functional effects on intracellular calcium level changes, cell volume plasticity, and invasiveness phenotypes, assessed through singlecell motility assays under cell crowding or hyperosmotic stress. These results have been incorporated into the revised manuscript, and detailed descriptions of these findings are included below.
Using the shRNA approach that resulted in ~50% reduction of TRPV4 expression
(Supplementary Figure 6A and 6B show TRPV4 expression levels via IF and immunoblots, respectively), we examined the effect of reduced TRPV4 on intracellular calcium levels in MCF10DCIS.com cells under normal density (ND) and stress conditions (confluent; Con and hyperosmotic; PEG) using Fluo-4 AM imaging (Fig. 4S-X). We found that shRNA TRPV4 slightly decreased calcium levels in ND cells, likely due to fewer active calcium channels at the plasma membrane resulting from lower TRPV4 expression (as shown in the summary plot in Fig. 4W). With fewer active calcium channels, cells treated with shRNA TRPV4 exhibited less reduction in intracellular calcium levels under cell crowding conditions compared to control cells. Additionally, hyperosmotic stress using PEG 300 induced smaller calcium spikes in shRNA cells compared to the significant spike observed in control cells. This reduced calcium response to Con and hyperosmotic stress in shRNA cells was reflected in the decreased cell volume reduction by PEG 300 shown in Fig. 4Y. Consequently, shRNA-mediated TRPV4 reduction impaired cell volume plasticity in MCF10DCIS.com cells and abolished the pro-invasive mechanotransduction capability involving cell volume reduction, as evidenced by no increase in cell motility (both cell diffusivity and directionality) under hyperosmotic conditions (Fig. 5H-J). These findings demonstrate the critical role of TRPV4 in conferring pro-invasive
mechanotransduction capability to MCF10DCIS.com cells through cell volume reduction.
Reviewer #2 (Public review):
Summary:
The metastasis poses a significant challenge in cancer treatment. During the transition from non-invasive cells to invasive metastasis cells, cancer cells usually experience mechanical stress due to a crowded cellular environment. The molecular mechanisms underlying mechanical signaling during this transition remain largely elusive. In this work, the authors utilize an in vitro cell culture system and advanced imaging techniques to investigate how non-invasive and invasive cells respond to cell crowding, respectively.
Strengths:
The results clearly show that pre-malignant cells exhibit a more pronounced reduction in cell volume and are more prone to spreading compared to non-invasive cells. Furthermore, the study identifies that TRPV4, a calcium channel, relocates to the plasma membrane both in vitro and in vivo (patient samples). Activation and inhibition of the TRPV4 channel can modulate the cell volume and cell mobility. These results unveil a novel mechanism of mechanical sensing in cancer cells, potentially offering new avenues for therapeutic intervention targeting cancer metastasis by modulating TRPV4 activity. This is a very comprehensive study, and the data presented in the paper are clear and convincing. The study represents a very important advance in our understanding of the mechanical biology of cancer.
Weaknesses:
However, I do think that there are several additional experiments that could strengthen the conclusions of this work. A critical limitation is the absence of genetic ablation of the TRPV4 gene to confirm its essential role in the response to cell crowding.
We are deeply grateful for the positive assessment of our study and its contribution to advancing our understanding of mechanical signaling in cancer progression. We also greatly appreciate the suggestion to incorporate genetic ablation experiments to further validate the role of TRPV4 in cell crowding responses.
As noted in our response to Reviewer #1, we employed an shRNA approach to investigate the functional effects of TRPV4 knockdown on intracellular calcium level changes, cell volume plasticity, and invasiveness phenotypes. We assessed these effects using Fluo-4 AM calcium assay, single-cell volume measurements, and single-cell motility assays under cell crowding or hyperosmotic stress. These results have been incorporated into the revised manuscript and are described in detail in our response to Reviewer #1's "weaknesses" comment.
Reducing TRPV4 expression levels by shRNA diminished mechanosensing intracellular calcium changes under cell crowding and hyperosmotic conditions using PEG 300 treatment. Furthermore, a significantly reduced cell volume plasticity was observed under hyperosmotic conditions in shRNA treated cells compared to control cells (Fig. 4S-X). This diminished mechanosensing capability abolished the pro-invasive mechanotransduction effect, as assessed by single cell motility under hyperosmotic conditions (Fig. 5H-J). These findings demonstrate the critical role of TRPV4 in conferring pro-invasive mechanotransduction capability to MCF10DCIS.com cells through cell volume reduction.
Reviewer #1 (Recommendations for the authors):
The way the results or discussion section is written. It was a little confusing for me to relate to some phenomena. For example, it is not clear how TRPV4 inhibition (due to overcrowding) leads to a decrease in intercellular calcium levels, especially when TRPV4 channels were intercellular (not on the PM) to begin with (in normal density (ND) conditions). Along the same lines, how GSK219 causes a dip in calcium levels in ND cells when TRPV4 channels are primarily intercellular (Figure 4E). If most of the TRPV4 channels that are translocated to the PM in response to cell crowding are in an inactive state, how do they confer enhanced cell volume plasticity relative to non-invasive cell lines?
Thank you very much for raising this important point. We fully agree with your concern and have significantly revised the manuscript to clarify this aspect. Specifically, we have emphasized that a modest level of TRPV4 channels are constitutively active at the plasma membrane in normal density (ND) cells. This is now discussed in detail in the context of Fig. 4:
Page 14: “Considering these factors, we hypothesized that cell crowding might inhibit calcium-permeant ion channels that are constitutively active at the plasma membrane, including TRPV4, which would then lower intracellular calcium levels and subsequently reduce cell volume via osmotic water movement.”
Page 16-17: “… However, the temporal profile of Fluo-4 intensity in Fig. 4E, which corresponds to the time points marked in Fig. 4D (t1: baseline and t2: dip), clearly shows the dip at t2, indicated by ΔCa (the vertical dashed line between the dip and baseline). This modest Fluo-4 dip at t2 represents the inhibition of activity by GSK219 on a small population of constitutively active TRPV4 channels at the plasma membrane under ND conditions.
In Con cells, 1 nM GSK219 caused a smaller dip in Fluo-4 intensity compared to the one observed in ND cells, with no subsequent changes. This is likely due to fewer constitutively active TRPV4 at the plasma membrane in Con cells than in ND cells. …These findings suggest that a substantial portion of TRPV4 channels relocated to the plasma membrane under cell crowding was inactive, and some constitutively active TRPV4 channels already present in the membrane became inactive as a result of cell crowding.”
'Internalization' might be a better word than 'uptake' in the following line in the results section
"...activating TRPV4 under cell crowding conditions triggered channel uptake, indicating that TRPV4 trafficking depended on the channel's activation status."
Thank you very much for this suggestion. As recommended, we replaced ‘uptake’ with internalization’ on page 18:
“However, in Con cells, where a large number of inactive TRPV4 channels are likely located at the plasma membrane, GSK101 treatment notably reduced plasma membrane-associated TRPV4 in a dose-dependent manner through internalization (Fig. 4O, 4Q), consistent with previous findings65. These data suggest that plasma membrane TRPV4 levels were largely
regulated by the channel activity status. Specifically, channel activation led to the internalization of TRPV4, while channel inhibition promoted the relocation of TRPV4 to the plasma membrane.”
Out of curiosity:
Is there any information on what the intercellular TRPV4 channels are doing in the cytosol and in the nucleus? Is there any role of intercellular calcium stores in the proposed pathway?
We greatly appreciate this insightful question. Although we were unable to find studies specifically exploring the roles of cytosolic TRPV4, a recent study (Reference 74) identified a role for nuclear TRPV4 in regulating calcium within the nucleus. We speculate that when TRPV4 activity is severely impaired, such as with additional TRPV4 inhibition under cell crowding conditions, some TRPV4 channels may be redirected to the nucleus. This redistribution could help maintain nuclear calcium homeostasis.
This discussion is included on page 18 of the manuscript:
“These findings suggest that further TRPV4 inhibition under crowding conditions triggers a distinct trafficking alteration. Recent studies have implicated nuclear TRPV4 in regulating nuclear Ca2+ homeostasis and Ca2+-regulated transcription74. In light of this study and our findings, TRPV4 may relocate to the nucleus as a compensatory mechanism to maintain nuclear calcium regulation. This relocation could reflect an adaptive response to preserve calcium-dependent transcriptional programs or other nuclear processes essential for cell survival under mechanical stress.”
One recommendation is to add some explanation or some minor details for the convenience of the reader. For example:
At normal or lower confluence, cells show an acute large dip in intercellular calcium when an inhibitor is applied implying that there are a few TRPV4 channels on the PM and they are constitutively active.
Thank you very much for highlighting this important point and for the helpful suggestion to improve clarity. We have significantly revised the text associated with Fig. 4 to ensure this point is clear. Specifically, we have added the following explanation on page 16:
"This modest Fluo-4 dip at t2 represents the inhibition of activity by GSK219 on a small population of constitutively active TRPV4 channels at the plasma membrane under ND conditions."
Reviewer #2 (Recommendations for the authors):
(1) Figure 1. The authors frequently change the medium to prevent acidification in overconfluent cultures. A cell viability assay should be performed to ensure that the over-confluent cells remain healthy and viable during the experiments. There are commercial kits that can be easily used to quantify the number of viable cells and the extent of cell toxicity. The number of viable cells would provide a more reliable basis for comparison between normal density and overconfluent conditions.
Thank you very much for raising this important point. We have consistently observed that cell crowding does not induce significant cell death in MCF10DCIS.com cells. To address your recommendation, we performed a viability assay using propidium iodide (PI) to selectively stain dead cells and WGA-488 to stain all live cells. Cell death was quantified under normal density (ND) conditions and at 1, 3, 5, 7, and 10 days post-confluence.
Our results indicate that cells remain similarly viable post-confluence, with minimal cell death
(~1.5%) compared to ND cells (~0.75%). These findings are summarized in Supplementary Figure 2, demonstrating that over-confluent cultures remain healthy and viable during the experiments.
(2) Figure 2. To determine whether the reduction in cell volume is reversible, over-confluent cells can be further diluted back to normal density. Additionally, the reversibility of TRPV4 channel trafficking to the plasma membrane should be assessed under these conditions in IF experiments and cell surface biotinylation.
Thank you for this suggestion. We reseeded the previously overcrowded (OC) cells at normal density and observed that their TRPV4 distribution predominantly returned to being intracellular, with only modest plasma membrane localization, as shown by line analysis (Supplementary Figure 10A-C, page 13). Furthermore, their invasiveness decreased to levels comparable to the original normal density (ND) cells (Supplementary Figure 3C and 3E, page 6). These results demonstrate the reversibility of TRPV4 trafficking changes and the increase in invasiveness under mechanical stress.
Page 6. "The enhanced invasiveness of MCF10DCIS.com cells under cell crowding was largely reversible. When OC cells were reseeded at normal density for invasion assays, their invasive cell fraction decreased to approximately 15%, slightly lower (p = 0.012) than the initial value of around 24% (Suppl. Fig. 3C, 3E)."
Page 13. “We investigated whether TRPV4 relocation to the plasma membrane induced by cell crowding is reversible, as suggested by its impact on invasiveness (Suppl. Fig. 3E). To test this, previously OC MCF10DCIS.com cells were reseeded under ND conditions. We then assessed TRPV4 localization via immunofluorescence (IF) imaging to determine if most channels returned to the cytoplasm and could be relocated to the plasma membrane under mechanical stress, such as hyperosmotic conditions. Consistent with their initial ND state, reseeded ND MCF10DCIS.com cells displayed intracellular TRPV4 distribution (Suppl. Fig. 10A). Upon exposure to hyperosmotic stress (74.4 mOsm/Kg PEG300), TRPV4 was again relocated to the plasma membrane (Suppl. Fig. 10B). These findings, quantified through line analysis (Suppl. Fig. 10C), demonstrate that the mechanosensing response of MCF10DCIS.com cells is reversible.”
(3) Figure 3B. A control using intracellular proteins such as GAPDH or Tubulin is missing. Including this control would help exclude the possibility of cell rupture or compromised cell membranes in crowded environments, which is very common in a cell crowding environment.
Thank you very much for pointing this out. The control lanes (GAPDH) were already included in the full gel results shown in Supplementary Figure 5. For the immunoprecipitation and immunoblotting of surface-biotinylated cell lysates, we did not expect to detect GAPDH; however, some GAPDH signals were still observed. As shown for MCF10DCIS.com cells, less GAPDH was detected under OC conditions, but the immunoprecipitated samples displayed significantly higher levels of TRPV4 on the cell surface compared to ND cells (Supplementary Figure 5A). For the whole cell lysates, TRPV4 protein levels were comparable across different cell lines based on the immunoblot results, with consistent GAPDH signals serving as a loading control (Supplementary Figure 5B).
(4) Figure 4. To convincingly demonstrate TRPV4 relocation to the plasma membrane, IF should be performed under non-permeable conditions (i.e., without detergents like saponin). This approach ensures that only plasma membrane proteins are accessible to antibodies, reducing intracellular background. The same approach should be applied to Piezo1 and TfR.
Thank you for this suggestion. We observed that under non-permeable conditions, primary antibodies could still access intracellular proteins. To address this issue, we employed extracellular-binding TRPV4 antibodies to selectively detect TRPV4 relocation to the plasma membrane under hyperosmotic conditions (74.4 mOsm/kg PEG 300) in live MCF10DCIS.com cells, as shown in Supplementary Figure 9. These results clearly demonstrate the plasma membrane relocation of TRPV4 under hyperosmotic conditions, distinguishing it from control conditions. Unfortunately, we were unable to identify high-affinity extracellular-binding antibodies for Piezo1 and TfR. Nevertheless, our findings strongly support the mechanosensing plasma membrane relocation of TRPV4.
Essential Weakness:
Throughout the study, only TRPV4 inhibitors and activators were used to show that TRPV4 relocation is associated with intracellular calcium concentration and cell size changes. It is crucial to use TRPV4 KO or KD cells to confirm that the observed effects are specific to TRPV4 and not due to off-target effects on other proteins. Additionally, fusing a plasma membrane targeting sequence to TRPV4 to make a constitutive plasma membrane-localized construct could demonstrate the opposite effect.
Thank you very much for this important comment. As noted in our response to Reviewer #1, we employed an shRNA approach to investigate the functional effects of TRPV4 knockdown on intracellular calcium level changes, cell volume plasticity, and invasiveness phenotypes. We assessed these effects using Fluo-4 AM calcium assay, single-cell volume measurements, and single-cell motility assays under cell crowding or hyperosmotic stress. These results have been incorporated into the revised manuscript and are described in detail in our response to Reviewer #1's "weaknesses" comment.
Reducing TRPV4 expression levels by shRNA diminished mechanosensing intracellular calcium changes under cell crowding and hyperosmotic conditions using PEG 300 treatment. Furthermore, a significantly reduced cell volume plasticity was observed under hyperosmotic conditions in shRNA treated cells compared to control cells (Fig. 4S-X). This diminished mechanosensing capability abolished the pro-invasive mechanotransduction effect, as assessed by single cell motility under hyperosmotic conditions (Fig. 5H-J). These findings demonstrate the critical role of TRPV4 in conferring pro-invasive mechanotransduction capability to MCF10DCIS.com cells through cell volume reduction.
Minor Points:
The introduction section is poorly written; many results currently included in the introduction would be more appropriately placed in the discussion section. The long redundant introduction makes the article hard to read through.
Thank you very much for pointing this out. In the revised introduction, we have significantly reduced references to the results, streamlining the section to make it more concise and focused. This adjustment ensures the introduction is clearer and avoids redundancy, improving the readability of the manuscript.
