Table_1_Isolation and characterization of head and neck cancer-derived peritumoral and cancer-associated fibroblasts.docx

IntroductionHead and neck squamous cell carcinomas (HNSCC) are characterized by strong cellular and molecular heterogeneity and treatment resistance entailing poor survival. Besides cell-intrinsic properties, carcinoma cells receive important cues from non-malignant cells within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are a major component of the TME that impact on the molecular make-up of malignant cells and have a decisive function in tumor progression. However, the potential functionality of fibroblasts within tumor-adjacent, macroscopically normal tissue remains poorly explored.MethodsHere, we isolated primary peritumoral fibroblasts (PtFs) from macroscopically normal tissue in vicinity of primary human papillomavirus-negative and -positive oropharyngeal HNSCC and compared their phenotype and functionality with matched CAFs (n = 5 pairs) and with human oral fibroblasts (hOFs).ResultsExpression patterns of CD90, CD73, CD105, smooth muscle actin, Vimentin, and S100A4 were comparable in PtFs, CAFs, and hOFs. Cell proliferation and doubling times of CAFs and PtFs were heterogeneous across patients (n =2 PtF>CAF; n = 1 CAF>PtF; n = 2 CAF=PtF) and reflected inferior growth than hOFs. Furthermore, PtFs displayed an reduced heterogeneity in cell size compared to matched CAFs, which were characterized by the presence of single large cells. Overall, conditioned supernatants from CAFs had more frequently growth-promoting effects on a panel of carcinoma cell lines of the upper aerodigestive tract carcinoma cell lines (Cal27, Cal33, FaDu, and Kyse30), whereas significant differences in migration-inducing effects demonstrated a higher potential of PtFs. Except for Kyse30, CAFs were significantly superior to hOFs in promoting proliferation, while PtFs induced stronger migration than hOFs in all carcinoma lines tested. Analysis of soluble factors demonstrated significantly increased VEGF-A production in CAFs (except in pat.8), and significantly increased PDGF-BB production in PtFs of two patients. Tube formation assays confirmed a significantly enhanced angiogenic potential of conditioned supernatants from CAFs compared to hOFs on human umbilical vascular endothelial cells (HUVECs) in vitro.DiscussionHence, matched CAFs and PtFs present in HNSCC patients are heterogeneous in their proliferation-, migration-, and angiogenesis-promoting capacity. Despite this heterogeneity, CAFs induced stronger carcinoma cell proliferation and HUVEC tube formation overall, whereas PtFs promoted migration of tumor cells more strongly.</p

Additional file 1 of A transcriptomic map of EGFR-induced epithelial-to-mesenchymal transition identifies prognostic and therapeutic targets for head and neck cancer

Additional file 1: SupplementaryFigure 1. Copy number variation and expression of EGFR in Kyse30and FaDu cells. Supplementary Figure 2. GSEA of EGF- and EpEX-treated Kyse30and FaDu cells. Supplementary Figure 3. Over-representation analysis of genesof the EGFR-mediated EMT signature. SupplementaryFigure 4. Comparison ofEGFR-mediated EMT, pEMT, and EMT signatures. Supplementary Figure 5. Comparisonof EMT signatures for prognostic purposes.Supplementary Figure 6. ITGB4,ITGA6, LAMA3, LAMB3, and LAMC2 expression in HNSCC. Supplementary Figure 7. ITGB4expression in malignant and non-malignant single cells in different cancerentities. Supplementary Figure 8. ITGA6 expression in malignant andnon-malignant single cells in different cancer entities. Supplementary Figure 9. LAMA3expression in malignant and non-malignant single cells in different cancerentities. Supplementary Figure 10. LAMB3 expression in malignant andnon-malignant single cells in different cancer entities. Supplementary Figure 11. LAMC2expression in malignant and non-malignant single cells in different cancerentities. Supplementary Figure 12. ITGB4 expression in knockdown clonesof Kyse30 and FaDu cells. Supplementary Figure 13. Wound healing capacity of control andITGB4-knockdown cell lines. SupplementaryFigure 14. Tumor buddingintensities in HNSCC

Additional file 4 of A transcriptomic map of EGFR-induced epithelial-to-mesenchymal transition identifies prognostic and therapeutic targets for head and neck cancer

Additional file 4: Supplementary Table 3. Gene expression correlation with ITGB4 in the HPV-negativeTCGA cohort. Batch correlation analysis identified correlations of geneexpression with integrin beta 4 (ITGB4). Gene ID, Spearman correlation, andp-value are indicated for the top ten positively (co-regulated) and negativelycorrelated genes (counterregulated)

Additional file 2 of A transcriptomic map of EGFR-induced epithelial-to-mesenchymal transition identifies prognostic and therapeutic targets for head and neck cancer

Additional file 2: Supplementary Table 1. EGFR-mediated EMT

Additional file 3 of A transcriptomic map of EGFR-induced epithelial-to-mesenchymal transition identifies prognostic and therapeutic targets for head and neck cancer

Additional file 3: Supplementary Table 2. TCGA HPV- HNSC cohort

Additional file 5 of A transcriptomic map of EGFR-induced epithelial-to-mesenchymal transition identifies prognostic and therapeutic targets for head and neck cancer

Additional file 5: Supplementary Table 4. SCC1 cell line: DEGs overlapping with EGFR-mediated EMT signature