Single-cell profiling identifies STAT3/NF-κB regulatory hubs and cytokine crosstalk in the tumor microenvironment

Tumor progression is driven by dynamic interactions between malignant cells and the tumor microenvironment (TME), yet the regulatory mechanisms governing cellular heterogeneity and intercellular communication remain incompletely characterized. Here, we performed integrative single-cell RNA sequencing (scRNA-seq) analysis of publicly available datasets from non-small cell lung cancer and breast cancer to systematically map transcriptional heterogeneity and regulatory networks within the TME.

Using a unified computational pipeline with Seurat v5, SCENIC, and ligand–receptor modeling, we resolved major cellular populations, including malignant epithelial cells, immune subsets, cancer- associated fibroblasts, and endothelial cells, and their transcriptional states. Malignant cells displayed pronounced intratumoral heterogeneity, occupying a continuum of proliferative, metabolic, and invasive phenotypes linked by pseudotime trajectories.

Gene regulatory network inference identified STAT3, NF-κB, MYC, and HIF-1α as central hubs coordinating tumor-associated programs. Notably, we uncovered a cytokine-mediated immunoregulatory axis between malignant cells and tumor-associated macrophages, driven by IL6– IL6R and CCL2–CCR2 signaling. Cell–cell communication analysis further revealed coordinated networks supporting immune suppression, inflammation, and angiogenesis.

These findings provide a systems-level framework of TME organization and highlight key transcriptional circuits and signaling pathways as promising targets for disrupting tumor– microenvironment crosstalk in precision oncology.