Aryl Hydrocarbon Receptor Modulation Alters Immune Checkpoint Expression and Inflammatory Mediators in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, partly because the cancer cells can evade the immune system, also the pancreatic cancer cells are highly heterogeneous, and this tumor has a late onset of symptoms, which become visible only in advanced stages. For this study, we wanted to investigate the role of the aryl hydrocarbon receptor (AhR), a potential regulator of immune response and inflammatory pathways in PDAC. Using patient-derived peripheral blood mononuclear cells (PBMCs), we sought to determine how modulation of AhR activity affects the expression and production of immune checkpoint molecules and inflammatory mediators. To reflect the subtle effects of AhR activity on immunity, we divided PBMCs from 22 PDAC patients into four groups: unmodulated control, AhR stimulated by carbidopa, AhR stimulated by tapinarof, and AhR suppressed by bay. After 24 hours of modulation, the PBMCs were frozen for gene expression analysis using RT-PCR. The culture supernatants were also frozen for target protein analysis using ELISA. After experimental analysis all patient results were separated into two parts based on the AhR expression level of the unmodulated group: low AhR and high AhR. The Mann-Whitney test was used to determine the statistical significance in GraphPad software. Our study demonstrated different patterns of gene regulation in response to AhR modulation. In the high AhR expression group, carbidopa significantly downregulated AhR expression (p < 0.05), while in the low AhR expression group, tapinarof induced a significant upregulation of AhR (p < 0.05). Regardless of baseline AhR levels, tapinarof stimulation consistently increased CYP1A1 transcription (p < 0.05) across both groups. Conversely, AhR inhibition with bay resulted in a decreasing trend of CYP1A1 expression in both AhR groups. Interestingly, PTGS2 (encoding COX-2) remained downregulated in AhR-stimulated groups, but no reliable changes were observed. AhR modulation also affected immune checkpoint gene expression. Tapinarof treatment showed an upregulating trend in CD274 (PD-L1) transcription, independent of the initial AhR levels, while CD279 (PD-1) expression remained relatively stable across all conditions. At the protein level, carbidopa treatment was associated with a decreasing trend in soluble PD-L1 concentrations across both AhR expression groups. In contrast, soluble PD-1 levels remained elevated following AhR stimulation with carbidopa and tapinarof. Notably, soluble PGE2 levels showed a decreasing trend in the low AhR group after suppression with bay. These findings suggest the unique immunological role of AhR primary expression, functional activity, and different modulators in PDAC patients' blood. AhR activation enhances CYP1A1 expression, slightly reduces PD-L1 protein production with carbidopa, and affects inflammation-related genes in a context-dependent manner. AhR inhibition attenuates these responses, particularly in patients with low baseline AhR expression. However, these results suggest that AhR may be one of the main targets as a key regulator of immune responses in PDAC, and present new strategies for improving the efficacy of immunotherapeutic interventions.