The Role of Tetraspanin 5 Expressing Macrophages in Breast Cancer

Tetraspanins (TSPANs) are a family of transmembrane proteins that are highly conserved and found on the cell membrane and within extracellular vesicles such as exosomes. To date 33 members of this family have been discovered in mammalian cells and some of these have been implicated in supporting tumour growth, invasion, tumour engraftment and metastasis. Macrophages are the dominant leukocyte population found in the tumour microenvironment. These tumour-associated macrophages (TAMs) actively promote all aspects of tumour initiation, growth, and development, however very little is known regarding the role of TSPANs in TAMs and this will form the basis of this study. In this study, we characterized the expression of all TSPAN members at the gene level using real time qPCR in freshly isolated human monocytes, human monocyte-derived macrophages (MDMs), MDMs cultured in tumour conditioned medium derived from a human breast cancer cell line (to mimic in vitro macrophages exposed to tumour factors), MDMs activated by either LPS or IL-4. After that, we determined the expression of some TSPAN members (TSPAN5, CD9 and CD63) at the protein level using flow cytometry and western blotting. Our data showed that all 33 TSPAN members were differentially expressed by different macrophages subsets; however, following exposure to tumour-derived factors we see a significant increase in TSPAN5. We therefore hypothesise that TSPAN5 plays a role in supporting the pro-tumour activities of TAMs. To test this hypothesis, we confirmed the expression of TSPAN5 at the protein level by immunohistochemistry in breast cancer tissue and used siRNA knockdown to determine the role of TSPAN5 in human MDMs. NextSeq500 was performed to identify how TSPAN5 influences gene expression in BMDMs prepared from C57Bl/6J wild-type (WT) and mice with a genetic deletion of TSPAN5. The role of TSPAN5 in a mammary carcinoma (E0771) model in C57BL/6 WT and TSPAN5KO mice was also determined. Our data showed that knockdown of TSPAN5 increased human MDM migration and NextSeq500 (n=3) revealed significant changes in genes involved in cell adhesion (Fn1, Cdh11, Lamb1, Cyr61, Postn and Wisp2) and immune regulation (PTGS2, GPX8 and Crip2) in BMDMs from TSPAN5KO mice compared to WT mice. Moreover, both genetic and antibody ablation of TSPAN5 in C57BL/6 significantly reduced primary EO771 tumour growth and enhanced the survival of tumour bearing mice. The importance of TSPAN5 expressing TAMS in the promotion of tumour growth was further confirmed by clodronate liposome depletion of macrophages, where loss of these cells promoted tumour growth in the TSPAN5KO mice. Our study demonstrates for the first-time that TAMs are the main source of TSPAN5 in breast cancer and targeting TSPAN5 in cancer may be a novel therapeutic approac