Integrated single-cell and bulk RNA sequencing reveals prognostic and immunotherapy-associated myofibroblastic cancer-associated fibroblast subtypes in head and neck squamous cell carcinoma.

Background: Cancer-associated fibroblasts (CAFs) are involved in some critical aspects of the pathogenesis of head and neck squamous cell carcinoma (HNSCC), including the formation of a tumor-permissive extracellular matrix (ECM) structure, angiogenesis, and the immune and metabolic reprogramming of the tumor microenvironment (TME). This study aimed to examine the plasticity and metabolic heterogeneity of CAFs in HNSCC patients before and after immunotherapy. Methods: An integrated single-cell and bulk RNA sequencing (RNA-seq) analysis based on data from the GSE195832, GSE65868, and The Cancer Genome Atlas (TCGA)-HNSCC datasets was conducted. The functional plasticity and transcriptome diversity of the categorized CAF subtypes were analyzed using the DoRothEA tool and scMetabolism package. The relationship between the genes specifically expressed in the myofibroblast-like cancer-associated fibroblast (myCAF) subtype and prognosis was then examined by univariate and multivariate analyses. The influence of myCAF on immune cell modulation within the TME was analyzed using the Seurat and clusterProfiler packages. The therapeutic strategies for HNSCC were explored using the Cancer Therapeutics Response Portal (CTRP) and PRISM Repurposing datasets. Results: In total, seven types of cells were annotated based on 11 clusters. The CAFs were then re-categorized into the following three subtypes: inflammatory cancer-associated fibroblasts (iCAFs), proliferating cancer-associated fibroblasts (pCAFs), and myCAFs. The percentage of myCAFs was reduced in HNSCC following the immunotherapy. The functional plasticity of the CAFs was further confirmed by the diverse enriched pathways. Notably, the myCAFs were closely associated with DNA repair, oxidative phosphorylation, and transcription factor E2F targets. Further, the myCAFs were found to be the subtype most relevant to the prognosis of HNSCC and were found to be involved in modulating the immune cells in the TME of HNSCC. Additionally, a higher myCAF score was related to the higher half-maximal inhibitory concentration (IC50) values of D-4476, GW-583340, spautin-1, and VER-155008, and the lower IC50 values of JTE-607, TG100-115, ML320, and TGX-221. Moreover, patients with lower myCAF scores responded better to immunotherapy. Conclusions: This study, which was based on single-cell and bulk RNA-seq analyses, showed the plasticity and metabolic heterogeneity of CAFs in HNSCC. Our findings may contribute to understandings of the immunotherapy response in HNSCC.