S2555
Radiobiology - Microenvironment
ESTRO 2026
Results: Sex chromosome dosage defined three major TME profiles: female (XX) tumors showed increased immune infiltration and cytotoxic states, defining a more immune-active phenotype, XØ tumors displayed immune-deserted features and XY tumors were enriched in fibroblasts and regulatory/exhausted immune phenotypes. Within XY tumors, inflammatory CAFs (iCAFs) were strongly enriched for pathways related to epithelial–mesenchymal transition and extracellular matrix organization, supporting their role in shaping a fibrotic and immunosuppressive microenvironment. These results position fibroblasts, particularly in XY tumors, as key modulators of the immune-stromal-tumor interface. Conclusion: Altogether, this data establishes sex chromosome dosage as a critical variable in HNSCC, shaping tumor- stroma interactions and the immune landscape. Moving beyond binary sex classifications may uncover novel mechanisms of tumor progression and inform sex-aware therapeutic strategies. References: Hollows, R. et al. Association between loss of Y chromosome and poor prognosis in male head and neck squamous cell carcinoma. Head Neck 41, 993– 1006 (2019).Hu, C., Zhang, Y., Wu, C. & Huang, Q. Heterogeneity of cancer-associated fibroblasts in head and neck squamous cell carcinoma: opportunities and challenges. Cell Death Discov 9, 124 (2023).Obradovic, A. et al. Immunostimulatory Cancer-Associated Fibroblast Subpopulations Can Predict Immunotherapy Response in Head and Neck Cancer. Clin. Cancer Res. 28, 2094–2109 (2022).Abdel-Hafiz, H. A. et al. Y chromosome loss in cancer drives growth by evasion of adaptive immunity. Nature 619, 624–631 (2023). Keywords: sex, HNSCC, TME Digital Poster 3953 miR-19b-5p Mediates Invasion after Intraoperative Radiotherapy in the Luminal B Breast Cancer Oliwia Piwocka 1,2 , Wiktoria Suchorska 1,2 , Katarzyna Kulcenty 2 1 Department of Electroradiology, Poznan University of Medical Sciences, Poznan, Poland. 2 Department of Medical Physics, Greater Poland Cancer Center, Poznan, Poland Purpose/Objective: Breast-conserving surgery (BCS) followed by intraoperative radiotherapy (IORT) is a standard treatment for breast cancer (BC), however, local recurrence remains a significant challenge [1]. One hypothesis proposes that fibroblast activation during
wound healing promotes tumour recurrence. Surgical wound fluid (SWF) from BC patients contains inflammatory factors that can significantly influence the tumour microenvironment, particularly cancer- associated fibroblasts (CAFs) [2]. This study investigates how SWF collected after IORT affects invasion in heterogeneous 3D culture mimicking BC TME. Material/Methods: Primary CAFs isolated from three patients with Luminal B HER2+ BC were co-cultured with BT474 cells to generate heterogeneous spheroids. After five days in culture, spheroids were treated with 10% SWFs collected from patients following IORT or BCS and incubated for 48 hours. Spheroid invasion was monitored for seven days in 30% Matrigel. To investigate the mechanism underlying the increased invasion observed in the IORT-SWF group, small RNA sequencing was performed on exosomes isolated from SWFs. Differentially expressed miRNAs were identified, and miR-19b-3p and miR-20a-5p were selected for functional validation. Spheroids were subsequently transfected with specific miRNA inhibitors, and invasion was reassessed under identical conditions. Predicted targets and signalling pathways regulated by miR-19b-3p were explored using the KEGG database and miRBase. The expression of selected genes was analysed by RT- qPCR. Results: Invasion analysis of BC spheroids treated with SWF from patients after IORT or BCS revealed significantly higher invasion in the IORT-SWF condition. Small RNA sequencing identified several differentially expressed miRNAs in IORT-SWF, with miR-19b-3p and miR-20a-5p showing the most significant changes. Functional assays demonstrated that silencing miR-19b-3p, but not miR-20a-5p, reduced spheroid invasion. Bioinformatic analysis and RT-qPCR validation indicate that miR-19b-3p modulates invasion through activation of the TGF- β pathway and regulation of genes involved in filopodia formation and extracellular matrix (ECM) remodelling. Conclusion: IORT-derived SWF enhanced BC spheroid invasion compared to BCS. Among the differentially expressed miRNAs, miR-19b-3p was identified as a key regulator of this effect. Its inhibition reduced invasion and indicated activation of TGF- β signalling and genes linked to filopodia formation, as well as ECM remodelling, highlighting miR-19b-3p as a potential driver of post-IORT tumour invasiveness. References: 1.Vaidya, J. S. et al. (2020). Long term survival and local control outcomes from single dose targeted intraoperative radiotherapy during lumpectomy (TARGIT-IORT) for early breast cancer: TARGIT-A
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