Necroptosis induced by anti-EMMPRIN antibody and complement shifts macrophage polarization

Tumors grow and metastasize by reprogramming the immune system to secrete mediators that promote angiogenesis and suppress the anti-tumoral functions of existing and recruited immune cells. Thus, disrupting this reprogramming and restoring the ability of the immune system to attack the tumor is a major therapeutic goal. We have previously identified a novel epitope in EMMPRIN, a pro-angiogenic protein, and used a polyclonal rabbit-anti-mouse antibody (161-pAb) to attack it in three different mouse tumor models. 161-pAb significantly reduced tumor growth and number of metastatic foci, reduced angiogenesis, alleviated immunosuppression in the tumor microenvironment (TME) by reducing TGFβ levels, and increased macrophage and CD8 T cell infiltration into the tumors. Investigating the mechanisms involved, we now show that 161-pAb together with complement, induced necroptotic cell death in vitro in both mouse (CT26, RENCA) and human (Skov3, A498) cell lines. This was corroborated by the co-existence of necrotic (release of LDH) and apoptotic (reduced caspase 3 activity) features, as well as unique necroptosis features such as reduced caspase-8 activity, increased phosphorylation of MLKL, and release of dsRNA. Incubating the mouse RAW 264.7 macrophages or the human U937 monocytic-like cells with supernatants obtained from tumor cells subjected to the antibody and complement, resulted in a significant elevation in IL-10 (which stimulate CD8 T cell cytotoxicity), IL-1β, and TNFα levels, that did not occur in cells subjected to necrosis (H₂O₂) or apoptosis (doxorubicin or etoposide). Thus, necroptosis initiates the re-polarization of macrophages, which may lead to the alleviation of immune suppression in the TME.