ESTRO 2025 - Abstract Book

S3902

Radiobiology - Microenvironment

ESTRO 2025

3913

Digital Poster Breast-Conserving Surgery and IORT: Modulating Cancer-Associated Fibroblast Subtypes and EMT in a 3D Breast Cancer Microenvironment Oliwia Piwócka 1,2,3 , Wiktoria Suchorska 1,3 , Katarzyna Kulcenty 3 1 Department of Electroradiology, Poznan University of Medical Sciences, Poznan, Poland. 2 Doctoral School, Poznan University of Medical Sciences, Poznan, Poland. 3 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. One emerging explanation involves the activation of fibroblasts within the wound-healing response [1]. Surgical wound fluid (SWF) from breast cancer (BC) patients contains inflammatory factors that can significantly influence the tumor microenvironment, particularly cancer-associated fibroblasts (CAFs) [2]. This study investigates how SWF collected after breast-conserving surgery (BCS) and intraoperative radiotherapy (IORT) affects CAF phenotype in a 3D co-culture model with primary CAFs from Luminal A, Luminal B, and HER2+ BC subtypes, enabling detailed analysis of CAF subgroups (mCAFs, meCAFs, pCAFs) and their phenotype in a complex in vitro environment. Material/Methods: Cell culture: A 3D coculture model was established from breast cancer cell lines of Luminal A, Luminal B and HER2+ subtypes and primary CAFs isolated from breast cancer patients. Cells were seeded on ultra-low attachment plates in ratio 4000 breast cancer cells to 1000 CAFs. Phenotype analysis: CAFs phenotype was evaluated with flow cytometry (markers: CD90, CD324, CD325, PDGFRb, vimentin) and immunofluorescence (markers associated with CAFs phenotype: FAP, aSMA and their subgroups: MMP11, CXCL12, VEGF, SDF-1). Invasion: Cells were seeded as described above and their invasion was assessed with a use of Matrigel in the Incucyte SX3. Results: Using immunofluorescence and flow cytometry, we observed that exposure to SWF enhanced fibroblast activation, marked by increased expression of fibroblast activation protein (FAP) and alpha-smooth muscle actin (αSMA). Additionally, SWF stimulates epithelial-to-mesenchymal transition (EMT) processes, as evidenced by E-cadherin, N cadherin and vimentin shifts and invasion assay results. These findings suggest that SWF-derived cytokines and growth factors can reprogram CAFs, promoting aggressive phenotypes and possibly facilitating tumor recurrence post-treatment. Conclusion: This study provides novel insights into how IORT-induced changes in the stromal environment may drive recurrence and underscores targeted interventions' potential role in modulating CAF activity in the tumor microenvironment. References: 1. Kulcenty, K., Piotrowski, I., Rucinski, M., Wroblewska, J. P., Jopek, K., Murawa, D., & Suchorska, W. M. (2020). Surgical Wound Fluids from Patients with Breast Cancer Reveal Similarities in the Biological Response Induced by Intraoperative Radiation Therapy and the Radiation-Induced Bystander Effect-Transcriptomic Approach. International journal of molecular sciences, 21(3), 1159. 2. Kulcenty, K., Piotrowski, I., Wróblewska, J. P., Wasiewicz, J., & Suchorska, A. W. M. (2019). The Composition of Surgical Wound Fluids from Breast Cancer Patients is Affected by Intraoperative Radiotherapy Treatment and Depends on the Molecular Subtype of Breast Cancer. Cancers, 12(1), 11. Keywords: CAFs, breast cancer, 3D model