Single-cell RNA sequencing reveals pro-invasive cancer-associated fibroblasts in hypopharyngeal squamous cell carcinoma

Abstract Background Hypopharyngeal squamous cell carcinoma (HPSCC) has the worst prognosis among all head-and-neck cancers, and treatment options are limited. Tumor microenvironment (TME) analysis can help identify new therapeutic targets and combined treatment strategies. Methods Six primary HPSCC tissues and two adjacent normal mucosae from six treatment-naïve patients with HPSCC were analyzed using scRNA-seq. Cell types were curated in detail, ecosystemic landscapes were mapped, and cell–cell interactions were inferred. Key results were validated with The Cancer Genome Atlas and cell biology experiments. Results Malignant HPSCC epithelial cells showed significant intratumor heterogeneity. Different subtypes exhibited distinct histological features, biological behaviors, and spatial localization, all affecting treatment selection and prognosis. Extracellular matrix cancer-associated fibroblasts (mCAFs) expressing fibroblast activation protein were the dominant CAFs in HPSCC tumors. mCAFs, constituting an aggressive CAF subset, promoted tumor cell invasion, activated endothelial cells to trigger angiogenesis, and synergized with SPP1 + tumor associated macrophages to induce tumor progression, ultimately decreasing the overall survival of patients with HPSCC. Moreover, the LAMP3 + dendritic cell subset was identified in HPSCC and formed an immunosuppressive TME by recruiting Tregs and suppressing CD8+ T cell function. Conclusions mCAFs, acting as the communication center of the HPSCC TME, enhance the invasion ability of HPSCC cells, mobilizing surrounding cells to construct a tumor-favorable microenvironment. Inhibiting mCAF activation offers a new anti-HPSCC therapeutic strategy. Graphical Abstract