We show that direct contact with MSCs is necessary
We show that direct contact with MSCs is necessary and sufficient to induce DDR2 upregulation in breast cancer cells. DDR2 expression in MSC is required for collagen deposition and leads to increased DDR2 expression and activation in breast cancer cells. This paracrine-autocrine MSC-cancer cell axis results in breast cancer alignment with collagen fibers facilitating migration, invasion, and metastasis. Homozygous slie mice form fewer and smaller breast cancer metastasis that heterozygous and wild-type mice, and this finding provides strong evidence for a critical function of DDR2 in metastasis.
The ability to upregulate DDR2 in response to MSC-initiated signals appears to be a specific property of cancer cells, but not of benign breast epithelial cells. LN-, BM-, or AD-MSCs were unable to induce DDR2 expression, proliferation, migration, or invasion (data not shown) in nontumorigenic MCF10A kinesin 5 or patient-derived primary breast epithelial cells, in contrast to the robust DDR2 upregulation induced in all breast cancer cells tested. These data are in line with our previous report that DDR2 is not expressed in normal breast epithelium from patient’s samples and is only overexpressed in cancer (Toy et al., 2015). These results are also in agreement with a study showing that DDR2 upregulation in breast cancer cells does not initiate, but is induced during EMT and participates in collagen type I-mediated stabilization of Snail1, promoting breast cancer cell invasion and metastasis (Zhang et al., 2013). Collectively, these data lead us to postulate that MSC-derived DDR2 in the primary tumor endows cancer cells that have already initiated the metastatic process with growth and migratory advantage through alignment with collagen. Contact with fibrillar collagen induces DDR2 upregulation and signaling activation in breast cancer cells, thereby stabilizing EMT transcription factors and contributing to metastatic growth and dissemination.
There is substantial evidence that breast cancer cells are highly responsive to signals from MSCs through cytokine networks, resulting in increased tumor initiating cells (TICs), tumor growth, metastasis, and MSC homing to the primary tumor site. Li et al. (2012) report that MSCs induce IL-1 dependent tumor initiation, but this effect has not been observed in MDA-MB-231 and -453 cells, neither of which secretes IL-1 (Li et al., 2012). Our tumor initiation studies (data not shown) support these observations. Here, we demonstrate that MSCs induce breast cancer growth and metastasis that requires direct cell-cell contact and deposition of insoluble collagen I fibrils and is independent of their effect on TICs. These mechanisms may be interdependent, as contact between MSCs and breast cancer cells is shown to induce activation of cytokines conducive to metastatic dissemination (Karnoub et al., 2007). The crosstalk between MSC-derived DDR2 and the cytokine networks in the tumor microenvironment warrants further investigation.
Author Contributions M.E.G. designed and performed experiments, analyzed data, and co-wrote the paper. E.E.M. performed and analyzed 3D cultures. T.A. analyzed the human breast cancer metastasis tissue and assisted with animal experiments. C.A-G. analyzed data from mammosphere formation assays and 3D cultures. N.M. performed and analyzed collagen knockdown assays. A.L. performed and analyzed co-culture live-imaging studies. Y-C.C. developed, performed, and analyzed high-throughput single-cell migration assays. K.S. contributed with collagen analysis in human tissues and hanging drop assays. E.Y. contributed with microfluidics migration assays. K.K. performed statistical analyses. C.G. bred and genotyped slie mice. C.G.K. conceived the study, designed experimental strategies, analyzed data, and co-wrote paper.
Acknowledgments We thank members of the Kleer lab and Dr. Hernan Roca for discussions during the execution of this project, Dr. Gabriel Nunez for critical reading of the manuscript, and Dr. S. Takayama for guidance with the hanging drop spheroid assays. This work was supported by NIH grants (R01CA125577 and R01CA107469 to C.G.K.; F30CA196084 to T.A.; and R25GM086262 [PREP program,] to C.A-G.) and the University of Michigan Cancer Center support grant (P30CA046592).