High resolution spatial transcriptomics decodes the microenvironmental determinants of response to Nr-CWS therapy in cervical precancerous lesions

Immunotherapy with Nocardia rubra cell wall skeleton (Nr-CWS) can clear human papillomavirus (HPV) and induce regression of cervical precancerous lesions, yet many patients do not respond. The cellular and molecular basis for this heterogeneous clinical outcome remains unknown. Using high-resolution spatial transcriptomics, we profiled squamous intraepithelial lesions (SIL) from patients stratified by their subsequent response to Nr-CWS therapy. We discovered that non-responders were characterized by an immunosuppressive epithelial phenotype, defined by aberrant expression of the pro-inflammatory alarmin S100A9 in squamous epithelial cells (SECs), which was associated with poorer overall survival in cervical cancer. Non-responding lesions were enriched in proliferative (MKI67+) and pro-angiogenic (VEGFA+) SEC subpopulations and spatially organized into a pro-tumorigenic cellular neighborhood (CN-10) overexpressing S100A9 and ANXA1. In contrast, responders exhibited protective glandular-cell-enriched neighborhoods (CN-13) and supportive spatial interactions between lymphatic endothelial cells and fibroblasts. The unfavorable microenvironment was further defined by an immunosuppressive cloak of VCAN+ stromal cells and CD163+ M2 macrophages encircling SECs. Thus, response to Nr-CWS is determined by a pre-existing spatial network of epithelial-stromal-immune interactions, providing a roadmap for patient stratification and rational combination therapies.