The magnitude of the host immune response can be regulated by either stimulatory or inhibitory immune checkpoint molecules. Receptor-ligand binding between inhibitory molecules is often exploited by tumours to suppress anti-tumour immune responses. Immune checkpoint inhibitors that block these inhibitory interactions can relieve T-cells from negative regulation, and have yielded remarkable activity in the clinic. Despite this success, clinical data reveal that durable responses are limited to a minority of patients and malignancies, indicating the presence of underlying resistance mechanisms. Accumulating evidence suggests that tumour hypoxia, a pervasive feature of many solid cancers, is a critical phenomenon involved in suppressing the anti-tumour immune response generated by checkpoint inhibitors. In this review, we discuss the mechanisms associated with hypoxia-mediate immunosuppression and focus on modulating tumour hypoxia as an approach to improve immunotherapy responsiveness.

Hypoxia-activated prodrugs (HAP) are a promising class of antineoplastic agents that can selectively eliminate hypoxic tumor cells. This study evaluates the hypoxia-selectivity and antitumor activity of CP-506, a DNA alkylating HAP with favorable pharmacologic properties. Stoichiometry of reduction, one-electron affinity, and back-oxidation rate of CP-506 were characterized by fast-reaction radiolytic methods with observed parameters fulfilling requirements for oxygen-sensitive bioactivation. Net reduction, metabolism, and cytotoxicity of CP-506 were maximally inhibited at oxygen concentrations above 1 μmol/L (0.1% O2). CP-506 demonstrated cytotoxicity selectively in hypoxic 2D and 3D cell cultures with normoxic/anoxic IC50 ratios up to 203. Complete resistance to aerobic (two-electron) metabolism by aldo-keto reductase 1C3 was confirmed through gain-of-function studies while retention of hypoxic (one-electron) bioactivation by various diflavin oxidoreductases was also demonstrated. In vivo, the antitumor effects of CP-506 were selective for hypoxic tumor cells and causally related to tumor oxygenation. CP-506 effectively decreased the hypoxic fraction and inhibited growth of a wide range of hypoxic xenografts. A multivariate regression analysis revealed baseline tumor hypoxia and in vitro sensitivity to CP-506 were significantly correlated with treatment response. Our results demonstrate that CP-506 selectively targets hypoxic tumor cells and has broad antitumor activity. Our data indicate that tumor hypoxia and cellular sensitivity to CP-506 are strong determinants of the antitumor effects of CP-506.

Background: Tumor-targeted therapy causes impressive tumor regression, but the emergence of resistance limits long-term survival benefits in patients. Little information is available on the role of the myeloid cell network, especially dendritic cells (DC) during tumor-targeted therapy. Methods: Here, we investigated therapy-mediated immunological alterations in the tumor microenvironment (TME) and tumor-draining lymph nodes (LN) in the D4M.3A preclinical melanoma mouse model (harboring the V-Raf murine sarcoma viral oncogene homolog B (BRAF)^V600E mutation) by using high-dimensional multicolor flow cytometry in combination with multiplex immunohistochemistry. This was complemented with RNA sequencing and cytokine quantification to characterize the immune status of the tumors. The importance of T cells during tumor-targeted therapy was investigated by depleting CD4⁺ or CD8⁺ T cells in tumor-bearing mice. Tumor antigen-specific T-cell responses were characterized by performing in vivo T-cell proliferation assays and the contribution of conventional type 1 DC (cDC1) to T-cell immunity during tumor-targeted therapy was assessed using Batf3^−/− mice lacking cDC1. Results: Our findings reveal that BRAF-inhibitor therapy increased tumor immunogenicity, reflected by an upregulation of genes associated with immune activation. The T cell-inflamed TME contained higher numbers of activated cDC1 and cDC2 but also inflammatory CCR2-expressing monocytes. At the same time, tumor-targeted therapy enhanced the frequency of migratory, activated DC subsets in tumor-draining LN. Even more, we identified a cDC2 population expressing the Fc gamma receptor I (FcγRI)/CD64 in tumors and LN that displayed high levels of CD40 and CCR7 indicating involvement in T cell-mediated tumor immunity. The importance of cDC2 is underlined by just a partial loss of therapy response in a cDC1-deficient mouse model. Both CD4⁺ and CD8⁺ T cells were essential for therapy response as their respective depletion impaired therapy success. On resistance development, the tumors reverted to an immunologically inert state with a loss of DC and inflammatory monocytes together with the accumulation of regulatory T cells. Moreover, tumor antigen-specific CD8⁺ T cells were compromised in proliferation and interferon-γ-production. Conclusion: Our results give novel insights into the remodeling of the myeloid landscape by tumor-targeted therapy. We demonstrate that the transient immunogenic tumor milieu contains more activated DC. This knowledge has important implications for the development of future combinatorial therapies.