Abstract A143: Melanoma cell resistance to phagocytosis is unrelated to expression of conventional “eat-me/don't eat-me” signals
Authors: Anderson Katie L. , Lins Debra , Volkmer Jens-Peter , Shimizu Yoji , Weissman Irving L. , Mescher Matthew , Modiano Jaime 
Published: May 2016

Abstract
Blockade of CD47 to disable “don't eat me” signals and upregulation of calreticulin and phosphatidylserine to enhance “eat-me” signals increase phagocytosis and anti-tumor immune responses in various hematopoietic and solid tumors. Melanomas are notoriously resistant to apoptotic signals and to other cytolytic mechanisms used by immune effector cells; however, the effects of modulating pro- and anti-phagocytic pathways in this disease are unknown. For this study, we hypothesized that melanoma cells would display unique resistance to phagocytosis. Furthermore, we hypothesized that this resistance would be mitigated by CD47 blockade, which in turn would promote adaptive anti-tumor, T cell responses. To test these hypotheses, we utilized a multi-species approach and examined the phagocytosis of human, mouse, and dog melanoma cells in vitro. We blocked the interaction between CD47 on tumor cells and SIRPα on macrophages using anti-CD47 monoclonal antibodies, SIRPα mimotopes, and a soluble fusion protein of the SIRPα extracellular domain. In all cases, we confirmed that these reagents blocked &gt;85% of the maximum binding of CD47 to recombinant SIRPα. We observed that the susceptibility of lymphoma and melanoma cells to phagocytosis was dramatically different in all three species: whereas lymphoma cells were readily phagocytosed by mouse macrophages, melanoma cells were resistant to phagocytosis. In addition, significant increases in lymphoma cell phagocytosis were observed upon CD47 blockade while we noted only small increases in melanoma cell phagocytosis. The conservation of this resistance across the three species tested suggests this mechanism is essential for tumor survival. To determine if the small increases in melanoma phagocytosis following CD47 blockade improved the anti-tumor immune response, we treated tumor-bearing (B16) mice with the anti-CD47 monoclonal antibody, MIAP301. This led to a slight increase in antigen-specific CD8+ and CD4+ T cells in tumor draining lymph nodes in vivo, albeit without evidence of tumor regression or improved survival. Next, we tested whether we could overcome melanoma cell resistance to phagocytosis by increasing expression of “eat me” signals. We showed that treatment with doxorubicin increased phosphatidylserine and calreticulin on the outer plasma membrane of melanoma cells without altering membrane integrity. Once again, we noted a small increase in melanoma cell phagocytosis when we combined chemotherapy and CD47 blockade. However, this did not consistently promote enhanced activation of antigen-specific T cells in vitro or in vivo, and the combined chemo-immunotherapy did not reduce tumor burden or increase survival. Overall, we conclude that melanoma cells display an evolutionarily conserved resistance to phagocytosis, which cannot be fully mitigated by CD47 blockade or by chemotherapeutic upregulation of “eat me” signals. Additional work will be needed to define whether resistance of melanoma cells to chemo-immunotherapy is mediated through secreted soluble factors, through direct cell-to-cell contacts, or through modulation of the tumor environment.
Citation Format: Katie L. Anderson, Debra Lins, Jens-Peter Volkmer, Yoji Shimizu, Irving L. Weissman, Matthew Mescher, Jaime Modiano. Melanoma cell resistance to phagocytosis is unrelated to expression of conventional “eat-me/don't eat-me” signals. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A143.