Abstract
Therapeutic activation of macrophage phagocytosis has the ability to restrain tumour growth through phagocytic clearance of tumour cells and activation of the adaptive immune response. Our objective for this study was to evaluate the effects of modulating pro- and anti-phagocytic pathways in malignant melanoma. In order to identify evolutionarily conserved mechanisms of resistance that may be important for melanoma cell survival, we utilized a multi-species approach and examined the phagocytosis of human, mouse, and dog melanoma cells. We observed that melanoma cells from all three species displayed unexpected resistance to phagocytosis that could not be fully mitigated by blockade of the 'don't eat me' signal CD47 or by chemotherapeutic enhancement of known 'eat me' signals. Additionally, CD47 blockade failed to promote anti-melanoma immune responses or tumour regression in vivo. This melanoma resistance to phagocytosis was not mediated by soluble factors, and it was unaffected by siRNA-mediated knockdown of 47 prospective 'don't eat me' signals or by CRISPR-Cas-mediated CD47 knockout. Unexpectedly, CD47 knockout also did not enhance phagocytosis of lymphoma cells, but it eliminated the pro-phagocytic effect of CD47 blockade, suggesting that the pro-phagocytic effects of CD47 blockade are due in part to Fc receptor engagement. From this study, we conclude that melanoma cells possess an evolutionarily conserved resistance to macrophage phagocytosis. Further investigation will be needed to overcome the mechanisms that mediate melanoma cell resistance to innate immunity.
Highlights
Activated macrophages have the potential to mediate robust anti-tumour immunity through phagocytic clearance of tumour cells and antigen presentation to the adaptive immune system [1]
Mouse melanoma displays resistance to phagocytosis that is not mitigated by CD47 blockade. (a) CD47 expression: labelling of mouse melanoma (B16-OVA) and lymphoma (A20) cells with the anti-CD47 mAb MIAP301 (PE) as compared to an isotype control was detected by flow cytometry. (b) Efficacy of CD47 blockade: cells were incubated with CV1-G4 at the indicated concentrations for 15 minutes, followed by labelling with MIAP301 (PE) to detect unoccupied CD47 binding sites. (c) Phagocytosis assay: carboxyfluorescein succinimidyl ester (CFSE)-labelled tumour cells were incubated with J774 macrophages in the presence of control IgG4 or CV1-G4
Phagocytosis was quantified as the percent of F4/80+ J774 cells that engulfed CFSE+ tumour cells per total F4/80+ population
Summary
Activated macrophages have the potential to mediate robust anti-tumour immunity through phagocytic clearance of tumour cells and antigen presentation to the adaptive immune system [1]. This macrophage function relies on a balance of pro-phagocytic ( ‘eat me’) and anti-phagocytic (‘don’t eat me’) signals expressed on the surface of target cells [2]
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