Multiplexed Immune Profiling and 3D Co-culture Assays to Assess the Individual Checkpoint Therapy Response in Head and Neck Squamous Cell Carcinoma

Verena Schweihofer¹Daniela Schulz²Raquel Blazquez³Gero Brockhoff¹Tobias Ettl²Mathias Fiedler²Sina Franziska Heimer⁴Julia Schikora⁵Richard Bauer^2*Anja Kathrin Wege¹

• ¹Bavarian Cancer Research Center (BZKF), Department of Gynecology and Obstetrics, University Medical Center Regensburg, Regensburg, Germany
• ²Department of Oral and Maxillofacial Surgery, Experimental Oral and Maxillofacial Surgery, Center for Medical Biotechnology, Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, Germany
• ³Bavarian Cancer Research Center (BZKF), Department of Internal Medicine III, Hematology and Medical Oncology, University Hospital Regensburg, Regensburg, Germany
• ⁴Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, Germany
• ⁵Experimental Ophthalmology, University Marburg, Marburg, Germany

Background: Immune checkpoint inhibitors (ICIs) have become an integral part of cancer therapy, but only a minority of patients experience durable responsiveness. Response rates vary greatly and are often unpredictable, highlighting the urgent need for predictive biomarkers to guide treatment decisions.Methods: We investigated immune- and tumor-specific expression and secretion profiles in peripheral blood and tumor samples derived from patients with head and neck squamous cell carcinoma (HNSCC). We combined flow cytometry, LEGENDplex™ immune profiling, and preoperative/postoperative serum cytokine analyses to determine checkpoint molecules (e.g., PD-1, TIM-3, LAG-3), immune cell profiles, as well as key markers on tumor cells (CD44, PD-L1, MHC class I/II). In addition, a 3D co-culture model using tumor slices and autologous mononuclear cells from selected HNSCC patients were analyzed upon atezolizumab and pembrolizumab treatment. Results: Co-expression of PD-1 and TIM-3 on a subset of CD8+ tumor-infiltrating T cells was frequently observed, alongside a pronounced infiltration of myeloid cells in the tumor microenvironment. In the peripheral blood, we detected elevated levels of soluble CD27 in patients compared to control and distinct preoperative cytokine profiles (e.g., reduced IFN-γ, CCL3, CCL20; elevated IL-15/IL-16). Postoperatively, most cytokines showed lower cytokine levels compared to healthy controls but significantly higher CCL2 levels. Furthermore, tumor–immune co-cultures from selected patients showed a stronger apoptotic response and phenotypic differences (e.g., increased PD-1 and CD137 expression) upon atezolizumab treatment. Individual changes in soluble factor release (e.g., Gal-9, sPD-L1, sCD25, and sTIM-3) was noticeable upon co-culture under checkpoint therapy. Conclusions: This study provides proof-of-principle data suggesting that a combined multiplexed marker profiling and a functional 3D co-culture assay may help to explore ICI response for HNSCC patients in the future. However, extensive studies with larger cohorts are warranted to validate and refine this approach.