Single‐Cell and Spatial Transcriptomic Profiling of Penile Squamous Cell Carcinoma Reveals Dynamics of Tumor Differentiation and Immune Microenvironment

Penile squamous cell carcinoma (PSCC) is a highly aggressive malignancy without effective treatment due to limited knowledge of its development and tumor microenvironment (TME). In this study, single-nucleus RNA sequencing (snRNA-seq) and high-resolution spatial transcriptomics are employed to comprehensively investigate the development trajectories and the TME. The results revealed that PSCC cells mimicked the differentiation and tissue organization of normal penile epithelium, independent of the human papillomavirus (HPV) infection status. Notably, a spatial subtype, Tum_1, appeared at early stage of tumor differentiation and in tumor-normal boundary regions. This subtype exhibited enhanced basal-like and stemness features and showed high LAMC2 expression, which activated laminin-integrin signaling via ITGA6/ITGB4, promoting tumor invasiveness. Furthermore, the results indicated that HPV-positive basal stem-like neoplasms dampened the immune function of T cells and macrophages, promoting an immunosuppressive environment that facilitates tumor progression. Supporting this, the patients with head and neck squamous cell carcinoma and lung squamous cell carcinoma who have high expression of HPV-positive Tum_1 signatures derived greater benefit from PD-1 blockade therapy. In summary, the findings provide a comprehensive spatial landscape of the PSCC TME and suggest potential treatment approaches targeting laminin-integrin interaction and immunotherapy, especially in HPV-positive patients.