O17 Dissecting the epigenetic and transcriptional evolution of cancer associated fibroblasts through cutaneous squamous cell carcinoma disease progression

Abstract Introduction and aims Cancer-associated fibroblasts (CAFs) play an important role in the microenvironment of tumour growth. Single-cell transcriptomic analysis of cutaneous squamous cell carcinoma (cSCC) and other cancers revealed two major CAFs subtypes: matrix-producing contractile myofibroblastic CAFs (myCAFs) and immunomodulating inflammatory CAFs (iCAFs). While major research has focused on cancer cells, the epigenetic and transcriptional evolution of CAFs from primary lesion through recurrence to metastasis in cSCC is currently unknown. Methods We developed a screening pipeline to functionally characterize primary CAFs and patient-matched control fibroblasts from different cSCC stages. We investigated cell morphology, extracellular matrix (ECM) remodelling, transcriptome and cytokine secretion and correlated their CAF phenotype to disease severity. We performed in-depth bulk RNA sequencing (RNAseq) and Assay for Transposase-Accessible Chromatin sequencing (ATACseq) analysis of CAFs established from a severe (T3) cSCC progressing from primary lesion, recurrence to metastasis formation. Results We isolated 21 CAFs from patients’ biopsies with early and advanced cSCC (T1–T3). With increased squamous cell carcinoma (SCC) severity, our screening pipeline revealed a clear correlation with an increased iCAF phenotype, proangiogenic gene expression, ECM alignment and a loss of cell contractility. Interestingly, CAFs isolated from the progressing T3 SCC at different disease stages showed a similar trend, indicating that CAFs actively evolve with disease severity in a tumour. Furthermore, we observed a strong decrease in H3K27 acetylation with the lowest in metastatic CAFs. We performed ATACseq and RNAseq of primary, recurrence and metastatic CAFs revealing epigenetic and transcriptional changes with disease progression. Conclusions Comparing CAFs from different cSCC stages revealed a pronounced correlation of an iCAF signature with epigenetic changes and increased disease severity. Similar changes were observed within a severe (T3) SCC progressing from primary lesion to metastasis, providing unique insights how CAFs actively evolve with disease advancement.