Author Response:
Reviewer #1 (Public Review):
The study investigates the nature of "trailblazer" cells in distinct tumor models, including luminal B (MMTV/PyMT) and triple negative (TNBC) tumors (C3-TAg). The authors note that the trail-blazer phenotypes in the TNBC model are more complex relative to the Luminal B model and represent distinct EMT programs associated with the expression of distinct EMT-TFs (Zeb1, Zeb2 and Fra-1). They demonstrated that of numerous EMT-TFs, Zeb1 and Fra-1 were required for increased cancer cell migration and invasion. They reveal that TGF-beta and EGF-mediated signaling are required for the diverse EMT states that are required for trailblazer cell activity and increased cell migration/invasion. TGF-beta signaling engaged Zeb 1 and Zeb2 while EGF sig-naling activated Fra-1. Indeed, inhibitors of either TGF-beta or EGF signaling could impair cell migration/invasion. While both pathways contributed to trailblazer phenotypes, EGF signaling was shown to interfere with certain TGF-beta induced transcriptional response, including the ex-pression of genes encoding extracellular matrix proteins.
One concern was the heavy reliance of the C3-TAg as the sole TNBC model in which the dis-tinct trailblazer phenotypes were described. The data in Fig. 3 of the submission reveals that the phenotypes observed in the C3-TAg model could be recapitulated in a TNBC patient-derived xenograft model (PDX). Using this PDX, the authors were able to show vimentin expression in lung metastatic TNBC cells that were intravascular, those that had extravasated and clusters of cancer cells fully within the lung parenchyma. This was an important addition to the manuscript. The additional experiments to investigate the role of Zeb1 and Zeb1 more fully, beyond the focus on Fra-1 in the initial submission was an additional strength of the new submission. Additional clarifications to the discussion also clarified the concepts articulated in the study. The study em-ploys multiple breast cancer models, utilizes numerous in vitro and in vivo assessments of the trailblazer phenotypes, and the experimental design is rigorous and the interpretation of the data is sound. The manuscript will be of general interest to the research community.
Thank you for the supportive comments. We are glad that the revisions addressed your prior concerns.
Reviewer #2 (Public Review):
This represents an important study that demonstrates a high degree of heterogeneity within trailblazer cells in clusters that participate in collective migration. Solid methods highlight this het-erogeneity and show that in TNBC cancers, trailblazer cells are defined by vimentin (and not Keratin 14) and are dependent on both TGFbeta and EGFR signaling. Additional, single cell stud-ies would further support this work.
Thank you for the suggestion. Our current data establishes that trailblazer cells are heterogene-ous using FACS, immunostaining and functional studies of fresh tumor organoids and estab-lished tumor organoid lines. In addition, our RNA-seq experiments provided deep insight into the nature of gene expression changes that corresponded with the evolution of new trailblazer states. This discovery of trailblazer cell heterogeneity was one of multiple key new discoveries in this manuscript, along with revealing a Krt14-independent invasion mechanism, the regulation of trailblazer cells by Tgfβ and Egfr signaling and a new compromise mode of signal integration. We agree that our results support further investigation of the nature and function of basal-like breast cancer heterogeneity during the progression to metastasis. However, a comprehensive implementation of scRNA-seq is mostly likely required to further unravel new aspects of hetero-geneity that substantially advance upon the conclusions supported by our current data. Such an undertaking is beyond the scope of this investigation.
We agree that scRNA-seq would be confirmatory of trailblazer cell heterogeneity that has been demonstrated with multiple approaches rather than a new discovery of heterogeneity.
Strengths:
The paper highlights that collective migration, and the nature of trailblazer cells can be highly heterogeneous. This is important as it suggests that the ability to move between states may su-persede a singular phenotype.
The paper uses animal models and organoids and in several areas attempts to correlate find-ings to human tissues.
The experiments are logically described.
Reviewer #3 (Public Review):
Cancer is a disease of many faces and in particular, the ability of cancers cells to change their phenotypes and cell behaviors - cancer cell plasticity - is a major contributor to cancer lethality and therapeutic challenge of treating this disease. In this study, Nasir, Pearson et al., investigate tumor cell plasticity through the lens of invasive heterogeneity, and in particular in models of tri-ple-negative breast cancer (TNBC), a subtype of breast cancer with particularly poor clinical prognosis and more limited treatment modalities. Using organoid models in a variety of matrix systems, microscopy, and signaling pathway inhibitors, they find that invading TNBC breast tu-mors, primarily in the C31-Tag genetically engineered mouse model of TNBC, are composed of heterogeneous invasive/"trailblazer" type tumor cells that in many cases express vimentin, a classical intermediate filament marker of epithelial-mesenchymal transition, and reduced keratin-14, another filament marker of basal epithelial cells associated with collective invasion in differ-ent breast cancer models. Supportive genetic and pharmacologic evidence is provided that gen-eration of these cells is TGF-beta signaling pathway driven, likely in vivo from the surrounding tumor microenvironment, in accord with published studies in this space. Another important as-pect of this study is the good transcriptional evidence for multiple migratory states showing dif-fering degrees of partial overlap with canonical EMT programs, dependent on TGF-beta, and suggestive but at present incomplete understanding of a parallel program involving Egfr/Fra-1 mediated effects on invasion. When taken in context with other recent studies (Grasset et al. Science Translational Medicine 2022), these data are broadly supportive of concept of targeting vimentin-dependent invasion programs in TNBC tumors.
The core conclusions of this paper are generally supported by the data, but there are some conceptual and technical considerations that should be taken into account when interpreting this study. Specific comments:
- The contribution of the different vimentin-positive trailblazer cells to distant metastasis was not directly confirmed in vivo in this study. Given the limited proliferative potential of many fully EMT'd cells and in light of recent studies indicating that invasion can be uncoupled from meta-static potential, it seems important to directly test whether the different C31-tag isolates, varying in invasive potential in this study, produce metastases and if so do metastases abundance corre-late with the invasive potential in 3D culture. The collection of lungs at 34 days post injection de-scribed in methods is too short to evaluate metastatic frequency.
We agree that it is important to determine the contribution of trailblazer cells towards metastatic dissemination. In this manuscript, we show that Vimentin expressing cells in a triple negative breast cancer (TNBC) PDX model disseminate to the lungs (Figure 3F). We have also shown that Vimentin expressing SUM159 breast cancer (BC) trailblazer cells spontaneously metasta-size to the lungs in previous publications (Fig. 2–figure supplement 1C) and (Westcott et al, J Clin Invest, 2015, 10.1172/JCI77767 and Maine et al, Oncotarget, 2016, 10.18632/oncotarget.7408). Notably, the depletion of genes specifically expressed in trailblazer cells reduced spontaneous metastasis without significantly impinging on primary tumor growth (Westcott et al, J Clin Invest, 2015, 10.1172/JCI77767 and Maine et al, Oncotarget, 2016, 10.18632/oncotarget.7408). Our new results in Figure 5D show that Tgfβ activates genes that define the trailblazer state in the metastatic SUM159 trailblazer cell model. Thus, features of the Tgfβ regulated trailblazer program in the C3-TAg cells is active in the SUM159 trailblazer model of spontaneous metastasis. In addition, commonly employed BC cell line metastasis models, such as MDAMB231 derivatives are highly mesenchymal (Fig. 2–figure supplement 1C) and (Kang et al, Cell, 2003, 10.1016/S1535-6108(03)00132-6 and Minn et al, Nature, 2005, 10.1038/nature03799, as examples).
It is not technically feasible to establish a correlation between the relative invasion of The C3-TAg GEMM primary tumors and spontaneous metastasis. C3-TAg GEMM primary tumors de-velop rapidly and the mice must be euthanized prior to the detection of metastasis. This limitation of the model is mentioned in the Results section “Trailblazer cells are specified by Vimentin ex-pression in basal-like breast cancer patient tumors”. The aggressive primary tumor growth and limited spontaneous metastasis of the the C3-TAg model has also been previously reported by others (Green et al, Oncogene, 2000, 10.1038/sj.onc.1203280). Surgical resection of the original primary tumor is not feasible option to allow metastases to form since additional tumors develop in multiple mammary glands.
In response to reviewer requests, we initiated the growth of orthotopic primary tumors from con-trol or Tgfβ treated 1339-org cells to address the relationship between induction of the trailblazer state and primary tumor cell dissemination. We had to euthanize the mice at day 34 (d34) be-cause tumors within both cohorts had reached the maximum permitted diameter of 2 cm. This will be indicated in the Methods section with revised text. We detected CTCs from the mice bearing control and Tgfβ treated 1339-org cell tumors. However, no micrometastases were de-tected, which is indicated in the text describing Figure 4–figure supplement 3A-B. Thus, per-forming surgical resection in new experiments would not be expected to allow the later detection of metastasis, as there did not appear to be DTCs in the lungs that could initiate colonization. In addition, we would have to resect the tumors prior to d34 to successfully and humanely remove the primary tumors, further reducing the odds of metastases developing. We will continue our work to identify an experimental balance that permits sufficient primary tumor growth to initiate spontaneous metastasis. However, the time scale of resolving this technical challenge is uncer-tain and we believe that our published analysis of trailblazer cell metastasis and new findings here showing the dissemination of Vimentin expressing cells in a PDX model addresses the question of whether Vimentin expressing trailblazer cells metastasize.
We agree that certain cell states induced by EMT programs can limit the proliferative potential of tumor cells. As described in the Introduction, we previously found that the induction of a trailblaz-er state in a subset of breast cancer cell line models triggers a collateral cost in fitness that limits the ability of trailblazer cells to initiate tumor growth (Westcott et al, Cancer Res, 2020, 10.1158/0008-5472.CAN-20-0014). The traits that distinguish trailblazer cells which are capable of tumor initiation and metastasis versus trailblazer cells with reduced fitness have begun to be delineated. Our prior report suggested that cells that were dependent on p63 for growth lost their proliferative capacity when converting to a trailblazer state (Westcott et al, Cancer Res, 2020, 10.1158/0008-5472.CAN-20-0014). C3-TAg cells are not dependent on p63 for growth, which is indicated by the vast majority of the tumor cells lacking p63 expression in primary tumors and primary tumor organoids (Westcott et al, Cancer Res, 2020, 10.1158/0008-5472.CAN-20-0014), similar to the metastatic SUM159 breast cancer cell line model. We were also able to derive clonal trailblazer cell lines that lacked detectable p63 expression from a C3-TAg tumor (Figure 2—figure supplement 1B) and grow organoids even when the limited extent of p63 expression was further reduced by Tgfβ (Figure 5C). Additionally, the persistent Tgfβ treated 1339-org cells, which were enriched for trailblazer cells and had reduced p63 expression, were capable of initiating primary tumor growth (Figure 4F). Together, these results indicate that C3-TAg trail-blazer cells are capable of initiating metastatic colonization. However, given the heterogeneity in trailblazer states that we discovered, it is possible that a subset of trailblazer cell states have re-duced proliferative capacity. Our analysis approach in this manuscript would not necessarily de-tect these low fitness trailblazer cells if they were a relatively small fraction of the total trailblazer population. We will clarify this point in the Discussion section in the revised manuscript. Our re-sults have begun to reveal mechanisms for the transcriptional regulation of trailblazer cell heter-ogeneity. We plan to continue delineating the regulatory programs conferring specific transcrip-tion state, defining approaches for the prospective isolation of distinct trailblazer subpopulations and determining trailblazer subpopulation specific biomarkers to understand the specific contri-bution of distinct trailblazer subpopulations towards metastasis. Given the scope of this analysis, it is not feasible to incorporate these future studies into this manuscript.
- The invasion of cancer cells is dependent on 3D matrix composition. In other studies, collec-tive cancer invasion is performed in exclusively collagen type 1 gels or in other instances entirely in 3D reconstituted basement membrane gel, e.g. lung cancer invasion studies. In this study, the authors use a mixture composed of both matrices. Given the invasion suppressive effects of matrigel, particularly for epithelial type cells, further studies would be important to determine whether the invasion phenotypes seen in this study are generalizable across matrix environ-ments.
The invasion of C3-TAg and PyMT organoids embedded in a 100% pure reconstituted base-ment is shown in Fig. 1–figure supplement 1G. We will emphasize that trailblazer invasion was evaluated in multiple ECM compositions with revised text and figure graphic. We also provide images for the reviewer showing that C3-TAg organoids collectively invade in a pure Collagen I ECM. Importantly, these findings are consistent with our results showing that Vimentin express-ing cells are associated with basal-like mammary tumor cell invasion in the complex ECM of C3-TAg GEMM primary tumors (Figure 2G) and patient primary tumors (Figure 3D). Moreover, Vimentin expressing cells disseminated to the lungs in the TNBC PDX that we evaluated (Figure 3F).
The ECM composition selected for experiments is dictated by the experimental question(s) being addressed. It is unlikely that mammary tumor cells would only ever collectively invade through an ECM that is either pure Collagen I or pure reconstituted basement membrane (BM). Indeed, it has been proposed that mixtures of Collagen I and BM proteins best reconstitute the complexity of primary tumor ECM (Hooper et al, Methods Enzymol, 2006, 10.1016/S0076-6879(06)06049-6). In line this observation, mixtures of Collagen I and BM proteins have been routinely used for the past 20 years to define mechanisms of 3D invasion; Xiang and Muthuswamy, Methods En-zymol, 2006, 10.1016/S0076-6879(06)06054-X; Calvo et al, Nat Cell Biol, 2013 10.1038/ncb2756; and Kato et al, eLife, 2023, 10.7554/eLife.76520, as examples).
Consistent with the known complexity of the ECM in the tumor microenvironment (TME), we detect Collagen I and Collagen IV (a key component of experimental BM) in the TME of primary breast cancer tumor models (Westcott et al, J Clin Invest, 2015, 10.1172/JCI77767). Important-ly, we have found that a mixture of collagen I and experimentally derived BM proteins reliably reveals breast cancer trailblazer cell invasion mechanisms that promote the malignant progres-sion and metastasis of primary tumors and whose expression correlates with poor patient out-come (Westcott et al, J Clin Invest, 2015, 10.1172/JCI77767 and Westcott et al, Cancer Res, 2020, 10.1158/0008-5472.CAN-20-0014, as examples). Notably, the relative differences in trail-blazer and opportunist cell invasive phenotypes are not dictated by the ECM composition used in our 3D assays. We have previously tested the invasion of trailblazer and opportunist subpopula-tions in different ECM compositions using both spheroid vertical invasion assays (Westcott et al, J Clin Invest, 2015, 10.1172/JCI77767). Increasing collagen I concentration enhanced the rela-tive rate of trailblazer cell invasion, with trailblazer cells always showing a significantly enhanced invasion relative to opportunist cells.
The relationship between trailblazer and opportunist cells that we have detected in primary tu-mors is recapitulated when using mixtures of Collagen I and BM proteins in our past publications and in this manuscript. The clonal opportunist cell lines derived from a C3-TAg tumor expressed high levels of the transcription factor p63 (Figure 2–figure supplement 1A-B). We previously showed that p63 restricts induction of a trailblazer state in human breast cancer trailblazer cell lines (Westcott et al, Cancer Res, 2020, 10.1158/0008-5472.CAN-20-0014). Notably, we showed that p63 expressing C3-TAg cells were not able to initiate collective invasion in the same ECM composition used in our current manuscript. Moreover, p63 cells in primary C3-TAg tumors were noninvasive opportunist cells that were limited to trailing p63-low trailblazer cells when collective-ly invading in primary tumors and in organoids (Westcott et al, Cancer Res, 2020). We now show that p63 expressing opportunist cell lines are limited to invading behind primary C3-TAg trailblazer cells and trailblazer cell lines in our 3D invasion assays (Figure 1B and Figure 1–figure supplement 1D-E). Together, these results indicate that the ECM employed in our 3D assays reveals the mechanistic underpinnings of both trailblazer and opportunist cell invasion in primary tumors.
With respect to lung cancer invasion, leader cells that we would classify as trailblazer cells have been isolated from 2 non-small cell lung cancer cell line spheroid models grown in pure reconsti-tuted BM extract (Konen et al, Nat Comm, 2017, 10.1038/ncomms15078). However, it unclear whether these cell line derived NSCLC trailblazer cells are more intrinsically invasive than non-trailblazer siblings in primary NCSCLC tumors or if the traits associated cell line NSCLC trail-blazer cells are required for metastasis. These tests have never been reported to the best of our knowledge. Similarly, it is not clear whether these NSCLC cell line derived trailblazer cells reflect features of primary NSLC primary tumor cells, as we are unaware of any such comparisons be-ing reported. Thus, there is no reason to consider pure reconstituted BM to be an equivalent or preferred experimental option to define trailblazer cell features. Nevertheless, as we mentioned before, our discovery approach identifies trailblazer cells that are intrinsically more invasive than opportunist siblings across multiple ECM conditions, including pure reconstituted BM and, im-portantly, in primary tumors.
- TGF-beta is well known to induce EMT. Although this study identifies potential transcriptional mediators of the invasion/trailblazer program, is this program reversible?
We have previously shown the breast cancer trailblazer cells can convert to an opportunist state, demonstrating that trailblazer states are reversible (Westcott et al, J Clin Invest, 2015, 10.1172/JCI77767). In this manuscript. we show that C3-TAg organoid lines derived in the Tgfbr1 inhibitor A83-01 have few if any cells with a trailblazer phenotype relative to C3-TAg pri-mary tumors, suggesting a reversion of the trailblazer state (Fig. 4C and Figure 4–figure sup-plement 2A-C). However, our results do not entirely rule out the possibility that only non-trailblazer cells grew to establish the organoid lines. Indeed, the problem of tracing phenotypic conversions when evaluating heterogeneous populations is a systemic challenge that extends beyond our analysis of trailblazer cells. Clearly defining the conversion rates for trailblazer cells will require multiple genetic markers to distinguish the different trailblazer states we have now identified, in addition to phenotypic and molecular analysis over multiple days, or possibly weeks. Thus, further definition of the rate of reversion of different trailblazer cells is worthy line of future investigation rather than a feasible objective of this study.
