Effect of Hypoxia on the Expression of Immune Checkpoint Molecules in Non-Muscle Invasive Bladder Cancer

Abstract

Bladder cancer is a major global health challenge, particularly due to the high prevalence and recurrence rates associated with non-muscle invasive bladder cancer (NMIBC). Hypoxia within the tumor microenvironment (TME) is a driver of immune evasion and is a potential prognostic marker for NMIBC. One key mechanism of immune evasion involves the upregulation of immune checkpoint proteins (ICPs) on tumour cells, which suppress adaptive immune responses. In this study, we investigated the effect of hypoxia on ICP expression in bladder cancer cells and resected NMIBC tissues. We hypothesized that hypoxic conditions promote the expression of ICPs in NMIBC, thereby facilitating immune escape. Two human NMIBC cell lines, RT112 and SW780, were cultured under hypoxic (0.5% O₂) or standard (20% O₂) conditions for 24 or 48 hours and analyzed using polychromatic flow cytometry. To determine whether these changes were mediated by the HIF-1α pathway, we employed two methods of HIF-1α inhibition followed by flow cytometric analysis. In parallel, NMIBC tissue microarrays were assessed by immunohistochemistry (IHC) for PD-L1, CEACAM-1, and carbonic anhydrase IX (CAIX). Staining intensity of positive cells were scored and used to explore correlations between ICP expression and hypoxic regions within tumor cores. Under hypoxic conditions, expression of PD-L1 and CEACAM-1 was upregulated in RT112 and SW780 cells, while CD155 expression was upregulated in SW780 cells. Interestingly, SW780 cells exhibited a time-dependent response to hypoxia, with checkpoint expression upregulated at 24 hours and diminishing by 48 hours. HIF-1α inhibition using a selective small molecule inhibitor abrogated the hypoxia-induced upregulation of PD-L1 and CEACAM-1, implicating this pathway in their regulation. In contrast, IHC analysis of NMIBC tissues revealed no significant correlations between hypoxia (as indicated by CAIX) and ICP expression. Additionally, PD-L1, CEACAM-1, and CAIX showed limited potential as predictive markers for clinical outcomes. In summary, our findings indicate that hypoxia can induce ICP expression in NMIBC cell lines via HIF-1α-dependent mechanisms in vitro. Elucidating the mechanisms by which the TME regulates ICPs may support the development of personalized immunotherapeutic strategies, ultimately improving patient outcomes and standards of care.