Weakly migratory metastatic breast cancer cells activate fibroblasts via microvesicle-Tg2 to facilitate dissemination and metastasis
Abstract
Cancer cell migration is highly heterogeneous, and the migratory capability of cancer cells is thought to be an indicator of metastatic potential. It is becoming clear that a cancer cell does not have to be inherently migratory to metastasize, with weakly migratory cancer cells often found to be highly metastatic. However, the mechanism through which weakly migratory cells escape from the primary tumor remains unclear. Here, utilizing phenotypically sorted highly and weakly migratory breast cancer cells, we demonstrate that weakly migratory metastatic cells disseminate from the primary tumor via communication with stromal cells. While highly migratory cells are capable of single cell migration, weakly migratory cells rely on cell-cell signaling with fibroblasts to escape the primary tumor. Weakly migratory cells release microvesicles rich in tissue transglutaminase 2 (Tg2) which activate fibroblasts and lead weakly migratory cancer cell migration in vitro . These microvesicles also induce tumor stiffening and fibroblast activation in vivo and enhance the metastasis of weakly migratory cells. Our results identify microvesicles and Tg2 as potential therapeutic targets for metastasis and reveal a novel aspect of the metastatic cascade in which weakly migratory cells release microvesicles which activate fibroblasts to enhance cancer cell dissemination.
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Author Response
Reviewer #2 (Public Review):
This fascinating study describes a possible effect of cancer-generated microvesicles on fibroblasts. Microvesicles from a particularly metastatic line promote more contractile and proliferative fibroblasts, and there is a key role for at least one microvesicle factor - the crosslinking enzyme Transglutaminase-2. A wide range of studies help identify and elucidate these effects, but a few aspects remain unclear.
- MV- has more crosslinking TGM2 but also less MMP14 degradation, and so ECM is more stable either way. The authors should describe any other factors that would give a similar effect as these. The authors should address: do other genes change with TGM2 knockdown; does MMP14 change? If the latter changes, does it have a more important role than TGM2?
We included a more thorough …
Author Response
Reviewer #2 (Public Review):
This fascinating study describes a possible effect of cancer-generated microvesicles on fibroblasts. Microvesicles from a particularly metastatic line promote more contractile and proliferative fibroblasts, and there is a key role for at least one microvesicle factor - the crosslinking enzyme Transglutaminase-2. A wide range of studies help identify and elucidate these effects, but a few aspects remain unclear.
- MV- has more crosslinking TGM2 but also less MMP14 degradation, and so ECM is more stable either way. The authors should describe any other factors that would give a similar effect as these. The authors should address: do other genes change with TGM2 knockdown; does MMP14 change? If the latter changes, does it have a more important role than TGM2?
We included a more thorough investigation into the proteomics data to determine what other factors in the MVs may induce fibroblast activation or matrix remodeling. Lists of “fibroblast-activating proteins” and “matrix remodeling proteins” were generated based on online datasets. All fibroblast-activating proteins tested were more highly expressed in MV- compared to MV+, but TGM2 was the only protein on this list with significantly increased expression (Figure 3b-d).
A large variety of matrix-remodeling proteins were detected in the MV proteomics, including matrix ligands, proteases, protease inhibitors, and crosslinking enzymes. Interestingly, MV+ had significantly higher levels of the matrix remodeling proteins TIMP3, FN1, and COL8A1 (Figure 3d). MV- had significantly higher levels of the crosslinking enzymes PLOD1 and PLOD3, the matrix ligand COL12A1, and TGM2 (Figure 3d). As TGM2 can be categorized as both a matrix remodeling and fibroblast-activating protein and was significantly greater in the MV- compared to MV+, we believe this addition to the paper reinforces our focus on TGM2 (Figure 3).
- Perhaps the cleanest and important study of MV effects is in Fig.6j,k, but it shows in vivo differences that are barely significant or not significant, and compares to 'SF' serum free media as a control. Are serum components detected in Mass Spec? If so, wouldn't this suggest a serum supplemented media is a better control? The serum is usually from another species, which is a further (xenogeneic) concern that motivates care and discussion about dose -- especially given the high frequency of injection. Also, is there a survival difference for the mice?
Thank you for bringing this concern to our attention. We realize that our wording was not clear. MVs are isolated under serum-free conditions and after isolation are resuspended in serum-free media. For this experiment, our mice were injected with either MVs suspended in serum-free media or serum-free media alone. We have revised the text to explain this more thoroughly.
Additionally, we were unable to assess survival differences as our IACUC protocol requires sacrificing mice upon a certain percentage of weight loss.
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eLife assessment
The interesting manuscript shows that breast cancer cells that are poorly migratory in culture can be more metastatic in mice. This is due, at least in part, to the secretion of extracellular vesicles containing the the crosslinking enzyme Transglutaminase-2, which can activate fibroblasts in the tumours. These fibroblasts can then promote metastatic phenotypes. This study demonstrates how cancer cells can manipulate the cells around them in order to disseminate.
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Reviewer #1 (Public Review):
The current manuscript by Schwager and colleagues describes a mechanism by which poorly migratory MDA-MB-231 cells can be metastatic. This study follows a recent paper from the same group (published in January) demonstrating that these poorly migratory cells are more metastatic than their highly migratory counterparts, and that this is due at least in part to E-Cadherin expression and the ability to form circulating tumour cell (CTC) clusters. In the current study, the authors show that the low migratory cells secrete unique EVs that can activate fibroblasts, concomitant with metastatic progression, and that this function is dependent on the presence of Tg-2. The novelty of this work is in the phenotypic heterogeneity of tumour cells, even within cell lines, and the importance the microenvironment in …
Reviewer #1 (Public Review):
The current manuscript by Schwager and colleagues describes a mechanism by which poorly migratory MDA-MB-231 cells can be metastatic. This study follows a recent paper from the same group (published in January) demonstrating that these poorly migratory cells are more metastatic than their highly migratory counterparts, and that this is due at least in part to E-Cadherin expression and the ability to form circulating tumour cell (CTC) clusters. In the current study, the authors show that the low migratory cells secrete unique EVs that can activate fibroblasts, concomitant with metastatic progression, and that this function is dependent on the presence of Tg-2. The novelty of this work is in the phenotypic heterogeneity of tumour cells, even within cell lines, and the importance the microenvironment in mediating metastasis associated with this diversity. While interesting, this work uses only one model, which was very recently published. The study, I think, would require repetition within additional models, as well as the inclusion of mechanistic studies designed to determine why the EV cargo differs between the highly and poorly migratory subclones.
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Reviewer #2 (Public Review):
This fascinating study describes a possible effect of cancer-generated microvesicles on fibroblasts. Microvesicles from a particularly metastatic line promote more contractile and proliferative fibroblasts, and there is a key role for at least one microvesicle factor - the crosslinking enzyme Transglutaminase-2. A wide range of studies help identify and elucidate these effects, but a few aspects remain unclear.
1. MV- has more crosslinking TGM2 but also less MMP14 degradation, and so ECM is more stable either way. The authors should describe any other factors that would give a similar effect as these. The authors should address: do other genes change with TGM2 knockdown; does MMP14 change? If the latter changes, does it have a more important role than TGM2?
2. Perhaps the cleanest and important study of MV …
Reviewer #2 (Public Review):
This fascinating study describes a possible effect of cancer-generated microvesicles on fibroblasts. Microvesicles from a particularly metastatic line promote more contractile and proliferative fibroblasts, and there is a key role for at least one microvesicle factor - the crosslinking enzyme Transglutaminase-2. A wide range of studies help identify and elucidate these effects, but a few aspects remain unclear.
1. MV- has more crosslinking TGM2 but also less MMP14 degradation, and so ECM is more stable either way. The authors should describe any other factors that would give a similar effect as these. The authors should address: do other genes change with TGM2 knockdown; does MMP14 change? If the latter changes, does it have a more important role than TGM2?
2. Perhaps the cleanest and important study of MV effects is in Fig.6j,k, but it shows in vivo differences that are barely significant or not significant, and compares to 'SF' serum free media as a control. Are serum components detected in Mass Spec? If so, wouldn't this suggest a serum supplemented media is a better control? The serum is usually from another species, which is a further (xenogenic) concern that motivates care and discussion about dose -- especially given the high frequency of injection. Also, is there a survival difference for the mice?
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