Baseline AHR Expression Shapes Immune Response to Pharmacological Modulation in PBMCs From Pancreatic Cancer Patients

Arenida Bartkeviciene^1*Aldona Jasukaitiene¹Inga Zievyte¹Sandra Ivanauskiene¹Gabija Stachneviciute¹Kornelija Jenceviciute¹Gabriele Karvelyte¹Darius Stukas¹Agne Sikarske²Daiva Urboniene³Toivo Maimets⁴Kristaps Jaudzems⁵Astra Vitkauskiene³Jason Matthews^6,7Antanas Gulbinas¹Zilvinas Dambrauskas¹

• ¹Laboratory of Surgical Gastroenterology, Institute for Digestive Research, Lithuanian University of Health Sciences, Eiveniu 4, 50103, Kaunas, Lithuania
• ²Department of Surgery, Medical Academy, Lithuanian University of Health Sciences, 44307, Kaunas, Lithuania
• ³Department of Laboratory Medicine, Lithuanian University of Health Sciences, Eiveniu 4, 50103, Kaunas, Lithuania
• ⁴Department of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010, Tartu, Estonia
• ⁵Department of Physical Organic Chemistry, Latvian Institute of Organic Synthesis, Aizkraukles 21, LV-1006, Riga, Latvia
• ⁶Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, 1046 Blindern, 0317, Oslo, Norway
• ⁷Department of Pharmacology and Toxicology, University of Toronto, ON M5S 1A8, Toronto, Canada

Background: Pancreatic ductal adenocarcinoma (PDAC) remains largely unresponsive to immunotherapy because of its highly immunosuppressive tumor microenvironment. Aryl hydrocarbon receptor (AHR), a ligand-dependent transcription factor, has emerged as a key regulator of immune homeostasis and inflammation. However, its systemic immunomodulatory role in PDAC, particularly outside the tumor microenvironment, remains poorly understood. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with PDAC and healthy donors were isolated and treated ex vivo with two AHR agonists (Carbidopa and Tapinarof) and one antagonist (BAY 2416964). The samples were stratified into Low and High/Medium AHR expression groups. Flow cytometry (FC), qPCR, ELISA, Luminex assays, and immunofluorescence imaging were used to evaluate immune checkpoint expression, cytokine secretion, monocyte polarization, and subcellular AHR localization. Overall survival analysis was performed based on the baseline AHR expression levels. Results: Baseline AHR expression strongly influenced the immunological effects of AHR modulators. In High/Medium AHR PBMCs, Carbidopa increased PD-L1 and soluble PD-1 (sPD-1) levels, while IL10 expression was suppressed. In contrast, BAY significantly reduced PD-1 and sPD-1 levels in Low AHR PBMCs, whereas Tapinarof induced the highest IL10 expression. All modulators reduced the proportion of M2-like monocytes, indicating a shift toward less immunosuppressive phenotypes. Nuclear translocation of AHR protein varied across treatments and expression levels. Kaplan–Meier analysis revealed a non-significant trend toward improved overall survival in the High/Medium AHR group (log-rank p = 0.276). Conclusion: Baseline AHR expression critically shapes the immune response to pharmacological modulation in PBMCs from PDAC patients. These findings suggest that AHR profiling may serve as a clinically relevant biomarker for stratifying patients and guiding personalized immunotherapy approaches for PDAC.