# Cancer-Associated Fibroblast Heterogeneity Shapes Prognosis and Immune Landscapes in Head and Neck Squamous Cell Carcinoma

## TL;DR

This study shows that different types of cancer-associated fibroblasts in head and neck cancer affect patient survival and immune responses, offering new ways to predict outcomes.

## Contribution

The study identifies distinct fibroblast subtypes and demonstrates their reproducible association with patient survival and immune landscapes in head and neck cancer.

## Key findings

- Inflammatory fibroblast programs are strongly linked to patient survival and immune profiles in head and neck cancer.
- Fibroblast subtypes identified through single-cell RNA sequencing predict outcomes across multiple patient cohorts.
- Validation with cultured fibroblasts confirms the preservation of transcriptional programs ex vivo.

## Abstract

Cancer progression is influenced not only by cancer cells themselves but also by surrounding supportive cells in the tumor environment. Among these, cancer-associated fibroblasts play important roles in shaping tumor behavior and immune responses, but their diversity and clinical significance in head and neck cancer are not fully understood. In this study, we analyzed single-cell gene expression data to identify distinct fibroblast subtypes in head and neck squamous cell carcinoma. We further examined whether gene signatures derived from these fibroblast subtypes could predict patient outcomes using large clinical datasets. We found that specific inflammatory fibroblast programs were strongly associated with patient survival and distinct immune environments. Importantly, these fibroblast-related patterns were reproducible across independent patient cohorts and were supported by additional experiments using cultured fibroblasts. Our findings provide new insights into the biological and clinical relevance of fibroblast diversity and suggest that stromal features may complement existing biomarkers to improve risk stratification in head and neck cancer.

Background/Objectives: Head and neck squamous cell carcinoma (HNSCC) is a biologically heterogeneous malignancy with poor outcomes in advanced disease. Increasing evidence indicates that the tumor microenvironment, particularly cancer-associated fibroblasts (CAFs), plays an important role in tumor progression and immune regulation. However, the diversity of CAF subsets and their clinical relevance in HNSCC remain incompletely understood. This study aimed to characterize CAF heterogeneity and assess the prognostic significance of CAF subset-specific transcriptional programs. Methods: Single-cell RNA sequencing data from HNSCC tumors were analyzed to identify CAF subsets based on differentially expressed genes. CAF subset-specific gene signatures were used to construct prognostic risk models for overall survival (OS) and progression-free survival (PFS) in The Cancer Genome Atlas HNSCC cohort, with validation in an independent dataset. CAF-driven prognostic groups were defined, and their immune landscapes and biological pathways were evaluated. Bulk RNA sequencing of primary CAF cultures was performed for validation. Results: Six CAF subsets were identified, including myofibroblastic (myCAF), inflammatory (iCAF), antigen-presenting, and extracellular matrix-related CAFs. Risk scores derived from inflammatory CAF subsets consistently predicted shorter OS across independent cohorts, whereas PFS prediction showed greater cohort dependency. CAF-based stratification identified patient subgroups with distinct immune profiles and pathway enrichment patterns. These results were supported by validation analyses and by bulk RNA sequencing of primary CAFs, demonstrating preservation of myCAF- and iCAF-like transcriptional programs ex vivo. Conclusions: CAF heterogeneity has important prognostic and immunological implications in HNSCC. Inflammatory CAF-related transcriptional programs represent robust markers of patient survival and may complement tumor-intrinsic biomarkers.