In solid tumors, the tumor microenvironment (TME) is often characterized by inflammation, hypoxia, and acidosis, which significantly impact cancer progression. These attributes of the TME substantially contribute to forming an immunosuppressive environment and influence cytokine production, immune cell recruitment, and their function. Carbonic anhydrase IX (CAIX), a hypoxia-induced enzyme, plays a crucial role in tumor cell adaptation to low oxygen conditions and generating an acidic tumor microenvironment. However, the role of CAIX in regulating anti-tumor immune responses has not been extensively investigated, and thus, it was a goal of this study.

Spheroids of pancreatic ductal adenocarcinoma cells (PDAC) were established and treated with anti-CAIX combinatorial therapy using an inhibitor of CAIX enzymatic activity, SLC0111, and CAIX-specific antibody, M75. The infiltration of spheroids with immune cells and spheroid cell viability were evaluated using a Cytation instrument. RNA samples of PDAC cells were analyzed for cytokine expression by RT2 Profiler Array. RNA samples of CAIX+ and CAIX-silenced fibrosarcoma cells were analyzed by Clariom followed by unsupervised clustering. Gene Set Enrichment Analysis (GSEA) was used to analyze gene expression. The relationship between CAIX levels and gene sets identified by GSEA was also analyzed in TCGA sarcoma samples and pancreatic cancer. Human T cell responses were evaluated in the presence of culture media collected from CAIX+ and CAIX- cells by flow cytometry.

We found that combination therapy against CAIX, or silencing CAIX expression, led to enhanced infiltration of immune cells into spheroids. CAIX-suppressed spheroids exhibited a higher degree of cancer cell death. Differential gene expression analysis of PDAC cells treated with combinatorial therapy and CAIX-silenced fibrosarcoma cells revealed the upregulation of genes involved in anti-tumor immune responses. T cell activation assays showed that conditioned media from CAIX+ tumor cells dramatically inhibited T cell responses, while conditioned media from CAIX-silenced cells increased activation of CD8 T cells. Furthermore, GSEA analysis identified that attenuation of CAIX expression is associated with the upregulation of genes involved in IFN-γ, IFN-α, and inflammatory responses. A negative correlation between CAIX levels and these immune pathways, which predicted better overall survival, was confirmed by in silico analysis of cancer patient samples.

We found that besides the known effects of CAIX on the survival of tumor cells in hypoxia, the activity of this enzyme plays an important role in the suppression of anti-tumor immune responses. Thus, targeting CAIX may represent a new opportunity to create a favorable milieu for anti-tumor immune responses in the TME. Funding: The Slovak Research and Development Agency 20-0480.