(G-254) Studying the Crosstalk between MSCs and Osteotropic Cancer Cells

Introduction:: Mesenchymal Stem cells (MSCs) have been identified in vivo as perivascular cells (pMSCs) in the bone marrow sinusoids and in all vascularized tissues. In the bone marrow’s sinusoids, pMSCs provide trophic support to hematopoietic stem cells (HSCs) by the secreting of specific factors such as CXCL12 and KITL. We have shown that melanoma extravasation mechanism is also mediated by pMSCs² through similar trophic and cell-cell contact mechanisms². In this context, these studies aim to study the crosstalk between osteotropic breast, melanoma, and prostate cancer cells and MSCs by: i) exposing MSCs to cancer cell conditioned medium (CM) and ii) testing direct communication (DC) of cancer cells on MSCs by co-culturing cells in transwells. The MSCs that are influenced by cancer cells CM and directly (co-culture) are then used in a transmigration assay to test their effect on osteotropic cancer cells transmigration. We hypothesize that cancer influenced MSCs have different secretion profiles, proliferation rates, and enhanced potential to increase the invasiveness of osteotropic cancer cell lines.

Materials and Methods:: Human MSCs from different donors are cultured on the bottom chamber of the transwell system with a 3 or 0.45 uM pore membrane. Cancer cells (melanoma, prostate, and breast cancer cell lines) are added on the upper chamber at different densities. To allow for the cell populations to communicate through soluble factors, both populations are cultured in 5% FBS medium and allowed to interact for variable time frames (24-120h). We propose that the MSCs and cancer cells will cross-talk and induce the MSCs to promote increased invasion of cancer cells. MSC population are then recovered and used as inductors of cancer cell transmigration. For this, hMSCs are seeded on the bottom surface of an 8-um pore diameter insert membrane. After 48h, cancer cells that reach the lower chamber are counted. Membranes without hMSCs are used as controls. Cells that reach the lower chamber are counted with the Image-J software. For each cancer cell line, each assay was performed in triplicate. Basic statistical analyses (t-test and ANOVA) were performed using Microsoft Excel software. We expect that the cancer cell induced MSCs will enhance cancer cell migration.


Results, Conclusions, and Discussions::

Our results showed that MSC-cancer crosstalk resulted in a 2.5 fold average increase in migration levels for melanoma and prostate cancer cell lines after 24 hours of stimulation. For prostate cancer, the CM group had higher migration levels, while DC resulted in higher melanoma migration. When stimulation time was increased to 120 hours, migration levels for CM groups significantly decreased, while DC migration levels remained elevated. This is partly due to the degradation of the soluble factors present in the CM groups after 120 hours (which does not occur in 24-hour stimulation). We hypothesize that the enhancement on cancer cell transmigration is mediated by epigenetic changes on MSCs that results in higher production of soluble factors such as CXCL12, KITL and PDGF-BB and cell-cell adhesive molecules such as MCAM. To test this hypothesis, we performed ELISA of such soluble factors and observed significant changes on the production of these cytokines on MSCs that were previously stimulated by cancer cell lines. We didn’t observe significant changes on the expression of MCAM, which suggest that the mechanism is mostly mediated by an increase on the production of soluble factors. We also observed an increased proliferation rate of MSCs exposed to cancer cells.








Conclusion

: Exposing MSCs to soluble factors produced by cancer cells resulted in a reciprocal higher cancer transmigration level. We show that MSC-cancer cross talk can alter MSC expression profiles of molecules involved in cell migration, potentially increasing responsiveness of cancer cells to the presence of MSCs.






Acknowledgements (Optional): :
We thank Margie Harris for the assistance with hMSC preparation. This work was supported by a grant from National Institutes of Health/National Cancer Institute (R01CA251612) and from the L. David and E. Virginia Baldwin




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