Reduction of tumor hypoxia by anti-PD-1 therapy assessed using pimonidazole and [18F]FMISO

Abstract

Introduction: Hypoxia is common in solid tumors and creates an immunosuppressive environment that leads to resistance to immunotherapy, such as an anti-programmed death receptor-1 (PD-1) therapy. It has been suggested that anti-PD-1 therapy may reduce tumor hypoxia by remodeling the tumor vasculature; however, it is unclear how anti-PD-1 therapy reduces hypoxia over time. Therefore, we investigated the relationship between hypoxia and immune activation by anti-PD-1 therapy in murine cancer models.

Methods: Anti-PD-1 antibody was injected to CT26- and MC38-tumor-bearing mice on days 0 and 5. Tumor hypoxia was non-invasively evaluated using positron emission tomography (PET) with [¹⁸F]fluoromisonidazole ([¹⁸F]FMISO) on days 3 and 7. Histological analysis was conducted to investigate the infiltration of immune cells in [¹⁸F]FMISO-accumulated hypoxic area. In addition, the immune cell population in tumors and the percentages of cancer and immune cells under hypoxic conditions were analyzed at single-cell level using flow cytometry.

Results: Flow cytometric analysis of CT26 tumors on day 3 showed that anti-PD-1 therapy reduced hypoxia without inhibition of tumor growth. In addition, the infiltration of CD8⁺ T cells was increased in treated tumors. In contrast to CT26 tumors, the percentage of hypoxic cells in MC38 tumors did not change on days 3 and 7, and there was minimal immune activation induced by anti-PD-1 antibody. Changes in hypoxia in CT26 tumors were not detected by [¹⁸F]FMISO-PET, but autoradiogram showed that [¹⁸F]FMISO accumulated in immunosuppressed areas, where the infiltration of immune cells was relatively low.

Conclusion: Reduction of hypoxia was induced in CT26 tumor, in which adequate immune response to anti-PD-1 therapy was exhibited, at an early time point before suppression of tumor growth. Our findings suggest that anti-PD-1 therapy can create a tumor microenvironment that facilitates immune activation by reducing hypoxia.

Keywords: Cancer imaging; Hypoxia; Immune checkpoint blockade; PD-1; [(18)F]FMISO.