Abstract 3928: Analyzing treatment effects of chemo- and immunotherapy in the context of the TME in an <i>ex vivo</i> model of HNSC

Abstract Head and neck squamous cell carcinoma (HNSC) accounts for 450.000 deaths worldwide each year. Rising cases of HNSC have been predicted. Understanding the tumor biology in patients is crucial to developing more efficient treatment strategies against HNSC. Especially in the context of immunotherapy, the interplay of immune, cancer and stroma cells in the tumor microenvironment (TME) strongly influences the biological characteristics and behavior of the tumor and its response to therapy. However, most HNSC in vitro models such as patient-derived cell lines and organoids do not recapitulate TME complexity. Patient-derived tumor fragments (PDTFs) are a novel pre-clinical model to bridge this gap. Isolated directly from patients, they preserve the spatial and cellular tumor composition, allowing analysis of tumor behavior and reaction to treatment in the context of the TME. In this study, we adapted the PDTF methodology to study the effects of combined chemo- and immunotherapy on HNSC. To generate PDTFs, tumors from patients with HPV-negative HNSC were extracted in routine surgery and immediately transferred to the lab. The tumors were mechanically dissected into 1 mm3 fragments, termed PDTFs. Several PDTFs per tumor were distributed across wells of a cell culture plate and subjected to different treatments with cisplatin, nivolumab or caspase inhibition as well as combinations. After short-term ex vivo culture of max. 72h, we performed gene expression analysis via qPCR and RNA-seq as well as histological analysis of PDTFs. In total, PDTFs from 14 different HNSCs could be subjected to treatment, including 2 longitudinal biopsies from the same patient. Histological analysis of freshly extracted PDTFs showed the intact TME ex vivo. Transcriptomic analysis revealed a significant reaction to treatment that was consistent with previous in vitro results. HALLMARK gene sets involved in cell cycle progression (Mitotic spindle, G2M Checkpoint, E2F Targets), EMT as well as glycolysis were downregulated, while inflammatory signaling-related gene sets were enriched, especially in the combined treatment with immunotherapy. Additionally, increased IFNγ expression indicates specific effect of the immunotherapy on leucocytes in the TME. Matched PDTFs from the same patient showed strong correlation of transcriptomic behavior. Overall, we could show treatment-specific reaction of the PDTFs to both chemotherapy and immunotherapy, that included effects on cell cycle, metabolism and inflammation. The measurable reaction of the leucocytes within the TME sets this model apart from other ex vivo models. Together with the patient-specific differences in transcriptomic behaviour, evident in the matched PDTFs, this makes PDTFs an interesting ex vivo model whose application for patient-specific treatment prognosis will be the subject of further studies. Citation Format: Mareike S. Haarmann, Philipp Zimmermann, Malte Suchan, Dominik Funken, Jens Peter Klußmann, Johannes Brägelmann. Analyzing treatment effects of chemo- and immunotherapy in the context of the TME in an ex vivo model of HNSC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3928.