ESTRO 2025 - Abstract Book

S3906

Radiobiology - Microenvironment

ESTRO 2025

4038

Poster Discussion Spatial microenvironment remodulation by preoperative chemo- and chemoradiotherapy in non-small-cell lung cancer Zuzanna Nowicka 1 , Julia Sołek 2 , Bartłomiej Tomasik 3 , Maria Wołyniak 1 , Barbara Łochowska 4 , Robert Dziedzic 5 , Witold Rzyman 5 , Michał Bieńkowski 6 , Mariusz Łochowski 7 , Marcin Braun 2 , Wojciech Fendler 1 1 Department of Biostatistics and Translational Medicine, Medical University of Łódź, Łódź, Poland. 2 Department of Pathology, Chair of Oncology, Medical University of Łódź, Łódź, Poland. 3 Department of Oncology and Radiotherapy, Faculty of Medicine, Medical University of Gdańsk, Gdańsk, Poland. 4 Department of Radiotherapy and General Oncology, Regional Multi-Specialist Center for Oncology and Traumatology of the Nicolaus Copernicus Memorial Hospital in Łódź, Łódź, Poland. 5 Department of Thoracic Surgery, Faculty of Medicine, , Medical University of Gdańsk, Gdańsk, Poland. 6 Department of Pathomorphology, , Medical University of Gdańsk, Gdańsk, Poland. 7 Department of Thoracic Surgery and Respiratory Rehabilitation, Medical University of Łódź, Łódź, Poland Purpose/Objective: Radiation and chemotherapy have both immunosuppressive and immunostimulatory effects. Although radiotherapy was proposed as a primer for immunotherapy based on preclinical data, the results of clinical trials evaluting radiotherapy-immunotherapy cominations are unequivocal. This could be explained by our incomplete understanding of the direct treatment effects on the tumor microenvironment (TME). To better characterize the impact of radiotherapy and chemotherapy on the cancer-immune interaction, we evaluated treatment-induced changes in non-small lung cancer (NSCLC) TME using spatial transcriptomics. Material/Methods: We prepared tissue microaarays (TMA) from 18 formalin-fixed, paraffin-embedded (FFPE) NSCLC samples. These samples were obtained after a median of 41 days following the completion of preoperative neoadjuvant chemotherapy (CHT), neoadjuvant chemoradiotherapy (CRT) or primary resection (PR; 6 samples in each group). Median patient age at surgery was 66, 67 and 67 years in CHT, CRT and PR groups respectively and adenocarcinomas constituted 33%, 50% and 50% of each respective group. After whole-slide H&E examination, experienced pathologist prepared two 1-mm wide cores/sample from each invasive tumor margin, avoiding areas of necrosis. TMA sections of 5 μm were cut onto Visium slides (10X Genomics) and dual indexed libraries were prepared and sequenced. Alignment was performed with Space Ranger software v 2.0.1 and downstream analysis was done using Seurat package. Louvain clustering was performed on integrated gene expression from all slides and clusters were annotated based on canonical marker genes. Results: After filtering to exclude cores with insufficient material quality, ~4000 spots from 31 cores from all 18 patients were available for downstream analysis. Gene expression profiles aligned well with pathologic tissue assessment based on H&E images and histologic tumor subtype. Comparison of gene expression-based clusters between subgroups showed a cluster that was present in both CRT and CHT, but absent in PR group that was characterized by increased expression of markers related to neutrophil presence and activity ( S100A8 ) and genes involved in antigen presentation ( CD74, B2M ). Gene set enrichment analysis based on genes differentially expressed in CRT in comparison to PR samples in cluster annotated as comprising stromal and immune cells showed downregulation of epithelial to mesenchymal transition (EMT), TNF alpha signaling and hypoxia, while similar analysis comparing CHT and PR showed downregulation of EMT and TNF alpha signaling pathways. Conclusion: Pre-operative CHT and CTT induce changes in the TME that involve infiltration by immune cells, antigen presentation and EMT. Spatial transcriptome profiling enables novel insights into therapy-induced changes of cancer microenvironment.