Adhesion of MM cells to the BM microenvironment can lead to cell adhesion mediated drug resistance (CAM-DR). We have previously shown that treatment with Dexamethasone (a drug commonly used against MM alone or in combination with new therapeutic agents) induces in MM cells increased assembly of CD138, and F-actin containing membrane extensions named podia. Using this 2D co-culture model we found that podia from MM cells from patients or MM cell lines elongate on the surface of BM stromal cells and also interconnect distant MM cells correlating with CAM-DR. Formation of podia was observed after at least 6 days of co-culture when many soluble factors and extracellular matrix proteins may have been secreted and remodelled. We also showed that preventing SDF1-alpha binding to the CXCR4 receptor in MM cells inhibits podia formation. In order to investigate the possible role of ECM proteins and their ligands in podia assembly we have now tested the formation of podia in 3D co-cultures of MM and stromal cells embedded in gels composed of polymerised extracellular matrix proteins found in the BM including collagen I and collagen IV. We now found that MM cells incubated overnight in 3D matrix lattices assembled podia that elongated along fibres of polymerised extracellular matrix that put them in contact with stromal cells or other distant MM cells. We also found that culture of MM cells in 3D matrices in the absence of stromal cells did not result in podia formation. These results suggest that stromal cells promote podia formation in contact with the extracellular matrix. It has been shown by Neri et al1that beta7 integrins are involved in MM cell adhesion to BM cells and extracellular matrix during recruitment to the BM and they are involved in CAM-DR. We found that integrin beta7 KD MM cells failed to form podia to the same extent as the parental cell lines in 2D and 3D cultures. Podia assembled by beta7 KD cells were shorter and highly irregular in shape in comparison to long and straight podia formed by parental MM cells. Using immunostaining, we found that podia in beta7 KD cells lacked internal F-actin as well as vimentin filaments that we identified in podia of parental MM cells. However, total levels of these cytoskeletal proteins remained unchanged as detected by western blot. Dexamethasone-induced increased formation of podia was also dependent on the expression of beta7 integrins by MM cells and the presence of BM stromal cells in 2D and 3D culture conditions.
We conclude that podia promote the formation of cellular networks of distant MM cells as well as the contact with BM stromal cells in 3D and this process is involved in CAM-DR against Dexamethasone. Podia assemble in contact with stromal cells and along extracellular matrix cables and require polymerisation of actin and vimentin cytoskeletal filaments downstream of beta7 integrins.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.