FIGHTING FIRE WITH FIRE: alleviating immune suppression in the tumor microenvironment by targeting complement inhbiition.

Since the discovery of immune checkpoint inhibitors, cancer immunotherapy took its rightful place as an important pillar in the battle against cancer. Despite being one of the oldest and most extensively characterized immunologic pathways in various pathophysiological conditions, the complement system is often overlooked in the context of tumor immunology. Overall, there is no consensus about its role within the tumor microenvironment, since both tumor-promoting and tumor-suppressing properties have been described, depending on the tumor type, stage and complement factor(s) involved. To protect host cells against complement-mediated lysis, the complement system is tightly regulated at different stages by a series of complement regulatory proteins (CRPs). However, several reports have described that overexpression of CRPs on cancer cells correlates with worse prognosis. The effect of blocking or downregulating CRPs on tumor growth and survival has been explored in a limited number of cancer cell lines of which B-cell lymphoma cell lines are by far the most extensively studied. As far as we know, the functional role of CRPs has never been studied in the context of melanoma. Therefore, we aim to investigate the effect of knocking-out (KO) CD59 in melanoma cell lines on complement mediated tumor cell death and subsequent anti-tumor immune responses. The expression of CD46, CD55 and CD59 on human melanoma cell lines and the Raji cell line was analyzed using flow cytometry. CD59 was successfully knocked-out via the CRISPR-Cas9 technology in the 624-mel and 1087-mel cell lines. To assess complement-dependent cell lysis (CDC), the 624-mel and 1087 mel (both KO and wildtype (WT)) were treated with normal human serum, eculizumab, an anti-C5 monoclonal antibody (mAb) served as a negative control. The effect of the complement system was monitored using the IncuCyte Live Cell Analysis system. AnnexinV staining was used to measure the phosphatidylserine (PS) flip flop as an indication of regulated cell death. To assess whether the type of cell death is immunogenic, calreticulin (CRT) exposure was measured using an anti-CRT mAb; vesicular ATP-release was detected with quinacrine by flow cytometry. The tested melanoma cell lines were all positive for CD46, CD55 and CD59, but displayed variable expression levels on a per cell basis. Our findings demonstrate that even low concentrations of NHS induce cell death in CD59 KO cell lines, in contrast to WT counterparts. Moreover, we observed PS flip-flop, CRT exposure and ATP release preceding cell membrane disruption, suggesting that melanoma cells succumb to the complement system in an immunogenic manner. Overall, our findings underscore the critical role of CRPs, particularly CD59, in shielding tumor cells from complement-mediated lysis and highlight the potential of targeting CD59 as a therapeutic strategy in melanoma treatment.