Abstract 4053: Targeting oxygen consumption with metformin and phenformin have differential effects on immune cells in the tumor microenvironment
Authors: Budhu Sadna , Assouvie Anais , Bah Mamadou , Verma Svena , Serganova Inna , Mane Mayuresh , Zurita Juan , Koutcher Jason , Ponomarev Vladimir , Wolchok Jedd D. , Merghoub Taha 
Published: March 2024

Abstract
T cell immune checkpoint blockade (ICB) has shown remarkable promise in melanoma and other cancers. However, most patients do not show clinical benefit. This is because tumors can activate multiple checkpoints and immunosuppressive pathways to evade anti-tumor immune responses. Inhibition of these immune suppressive mechanisms and immune checkpoints or repolarizing the tumor microenvironment (TME) to become more accessible to the immune system may be necessary for maximal therapeutic efficacy of immunotherapies. There is increasing evidence that tumor hypoxia can attenuate the antitumor immune response by promoting immune suppression and inhibiting direct killing by cytotoxic immune cells. We propose that blocking tumor oxygen consumption using drugs that target the mitochondrial complex I (phenformin and metformin) will enhance the efficacy of immunotherapies such as ICB in preclinical models such as B16 melanoma. In this study, we examined how low oxygen levels (hypoxia) influence T cell effector function such as cytotoxic activity and cytokine production in vitro. We co-cultured B16 melanoma cells in the presence of tumor specific T cells (Pmels) and incubated at 37ºC in either normoxia (~21% O2) or hypoxia (3% O2) and assessed killing and intracellular cytokine production 24 - 48 hours later. We show that the ability of tumor antigen-specific T cells to kill tumor cells or make effector cytokine such as IFNγ and TNFα is significantly reduced in hypoxia settings. We also examined the effects of hypoxia targeting drugs (metformin and phenformin) on T cell priming and activation. When T cells were activated in the presence of each drug in vitro, there was enhanced expression of T cell activation markers (CD25, Granzyme B) as well as an increase in their differentiation into central memory T cells. We next determined whether these drugs could alter the TME in vivo by treating C56BL/6J mice bearing established B16 tumors with phenformin and metformin and then examine the effects on the immune system in the tumor, tumor draining lymph nodes and spleen by flow cytometry. We found that these three drugs have differential effects on both the innate and adaptive the immune system in the tumors and periphery. Finally, we tested whether these drugs could delay B16 melanoma growth in vivo as a monotherapy or in combination with anti-PD1. We found that while both drugs can significantly delay tumor growth as monotherapies, only metformin showed improved anti-tumor efficacy when combined with anti-PD-1 in vivo. These studies show that while both drugs target tumor oxygen consumption (via inhibition of complex I), and can delay B16 tumor growth in vivo, they have differential effects on the TME and will need further considerations when combining with immune based therapies such as ICBs.
Citation Format: Sadna Budhu, Anais Assouvie, Mamadou Bah, Svena Verma, Inna Serganova, Mayuresh Mane, Juan Zurita, Jason Koutcher, Vladimir Ponomarev, Jedd D. Wolchok, Taha Merghoub. Targeting oxygen consumption with metformin and phenformin have differential effects on immune cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4053.