Antagonists of CD39 and CD73 potentiate doxycycline repositioning to induce a potent antitumor immune response

Abstract

Studies have reported that cellular metabolism at the tumor-immune microenvironment (TiME) serves as a critical checkpoint and perturbs/supports anti-cancer immunity. Extra cellular ATP (eATP) may mediate anti-cancer immune response; however, its catabolism by ectonucleotidase generates immunosuppressive adenosine. In the presented work, we have tried to repurpose doxycycline with or without an antagonist of ectonucleotidase for mitigating ATP metabolism and immunosuppression. In this methodology eATP and adenosine levels were quantified. Bone marrow-derived M1 and M2 polarized macrophages were maintained in tumor mimicking condition (TMC). Total/CD4⁺Tcells were co-cultured with macrophages to understand the impact of doxycycline and/or antagonist of ectonucleotidase on T cell/subset differentiation. Preclinical efficacy of doxycycline and/or ectonucleotidase antagonist and their synergy was scored in 4T1-induced breast carcinoma. We found that Doxycycline manipulated macrophage polarization by decreasing the frequency of CD206⁺M2 macrophages, which resulted in enhanced CD4⁺ directed CD8⁺ T cell response. Doxycycline alleviated the expression of CD39 and CD73, rescuing ATP catabolism. Doxycycline delayed tumor growth by enhancing F4/80⁺ CD86⁺ M1 macrophages and subsequently anti-tumor Tbet⁺ CD4⁺Tcells, attenuating the frequency of FOXP3⁺ regulatory T cells, which was cooperatively supported by ARL67156 and AMPCP (CD39 and CD73 antagonist).A synergy was observed with ARL67156 and AMPC Pensuring a possibility of using doxycycline alone or in combination with an antagonist of ectonucleotidase to present adenosine-mediated immunosuppression. Subsequently, our finding indicated that prospective usage of doxycycline as a novel metabolic checkpoint blocker (IMB) against ectonucleotidase and may be modified/delivered appropriately as a monotherapy or in combination with antagonists of ectonucleotidases as an IMB.