Defining the Transcriptional Landscapes of the Tumor Microenvironment of Cervical and Vaginal Cancers at Single-Cell Resolution

Abstract

Malignancies found within vaginal tissue are often diagnosed as cancers of the cervix, vulva, or urethra and are clinically treated with similar modalities. However, the rarity of vaginal cancer may be an artifice of categorization; current treatment paradigms do not take into account tissue-specific mutations and differences in mechanistic pathways intracellularly. Understanding the shared and distinctly different transcriptional profiles of vaginal and cervical tumors at a single-cell resolution will provide insights in vaginal tumor biology and will open avenues for future clinical interventions. Biopsies of tumor and adjacent normal tissue from 9 patients (3 adenocarcinomas (ADC), 3 squamous cell carcinomas (SCC) from the cervix, and 3 vaginal SCC) were collected and analyzed by single-cell RNA sequencing (scRNA-seq) to compare the tumor, immune, and stromal features of cervical and vaginal cancers. Collectively, over 50,000 cells were analyzed by scRNA-seq in this study. We performed dimensionality reduction and clustering analysis of the single-cell transcriptomes to identify the major cell types composing the vaginal and cervical tumor tissues. Compared to Cervical SCC, Vaginal SCC tissues showed reduced fractions of macrophages (-2.7 log2-fold; padj < 0.02) and T cells (-3.7 log2-fold; padj < 0.02) by differential cell proportion analysis (RAISIN). Likewise, the vaginal SCC epithelial cell compartments showed downregulation of inflammatory pathways including TNF signaling via NFKB (NES = -5.7, padj = 5.0 × 10-19), IL2 STAT5 signaling (NES = -4.5, padj = 1.6 × 10-12), and interferon gamma response (NES = -4.3, padj = 9.4 × 10-12), among the Hallmark pathway collection. On the other hand, vaginal SCC epithelial cells showed significant upregulation of oxidative phosphorylation (NES = 4.8, padj = 1.7 × 10-17), p53 pathway (NES = 4.2, padj = 1.8 × 10-13), mTORC1 signaling (NES = 4.2, padj = 1.9 × 10-13), and estrogen early and late response (NES = 4.0, padj < 7.5 × 10-12) compared to cervical SCC. These results highlight distinct differences in the cell type composition and cancer epithelial pathways in vaginal vs. cervical SCC. Among upregulated pathways in vaginal SCC, ER and mTORC1 pathway activation may represent targets for therapeutic intervention worthy of further investigation.