Philippe Gasque

The complement system plays a crucial role in cancer development. Our study investigated the role of C3a anaphylatoxin on the tumor microenvironment. Our models consisted of mesenchymal stem cells (MSC-like, 3T3-L1), macrophages (Raw 264.7 Blue, (RB)) and tumor cells (melanoma B16/F0). Recombinant mouse (Mo) C3a (rC3a) was produced in CHO cells transfected with a Mo-IL10-signal peptide-Mo C3a plasmid construct. The effects of rC3a, IFN-γ, TGF-β1, and LPS were tested on the expression of C3, C3aR, PI3K, cytokines, chemokines, transcription factors, antioxidant defense mechanisms, angiogenesis and macrophage polarization (M1/M2). 3T3-L1 expressed the highest levels of C3, while C3aR was expressed more by RB. Interestingly, expression of C3/3T3-L1 and C3aR/RB was markedly upregulated by IFN-γ. rC3a was found to upregulate the expression of anti-inflammatory cytokines (IL-10) on 3T3-L1 and TGF-β1 on RB. rC3a also upregulated the expression of pro-inflammatory cytokines in RB. The expression of CCL-5 increased in 3T3-L1 in response to rC3a. On RB, rC3a did not alter M1/M2 polarization but upregulated the expression of antioxidant defense genes, HO-1, and VEGF. C3/C3a produced mainly by MSC may play a critical role in TME remodeling by stimulating both anti-inflammatory and proangiogenic activities of tumor stromal cells.

CD248 (endosialin) belongs to a glycoprotein family that also includes thrombomodulin (CD141), CLEC14A, and CD93 (AA4) stem cell markers. We analyzed the regulated expression of CD248 in vitro using skin (HFFF) and synovial (FLS) mesenchymal stem cell lines, and in fluid and tissue samples of rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Cells were incubated with either rhVEGF165, bFGF, TGF-β1, IL1-β, TNF-α, TGFβ1, IFN-γ, or PMA (Phorbol ester). There was no statistically significant change in membrane expression. A soluble (s) form of cleaved CD248 (sCD248) was detected after cell treatment with IL1-β and PMA. Matrix metalloprotease (MMP) MMP-1 and MMP-3 mRNAs were significantly up-regulated by IL1-β and PMA. A broad MMP inhibitor blocked the release of soluble CD248. In RA synovial tissue, we identified CD90+ perivascular MSCs double-stained for CD248 and VEGF. High sCD248 levels were detected in synovial fluid from RA. In culture, subpopulations of CD90+ CD14− RA MSCs were either identified as CD248+ or CD141+ cells but CD93−. CD248 is abundantly expressed by inflammatory MSCs and shed in an MMP-dependent manner in response to cytokines and pro-angiogenic growth factors. Both membrane-bound and soluble CD248 (acting as a decoy receptor) may contribute to RA pathogenesis.