Single cell analysis of early metastasis identifies targetable tumor subpopulation and mechanisms of immune evasion in squamous cell cancers

Abstract Profiling tumors at single-cell resolution provides an opportunity to understand complexities underpinning lymph-node metastases in head and neck squamous-cell carcinoma. Single-cell RNAseq (scRNAseq) analysis of cancer-cell trajectories identified a sub-population of pre-metastatic cells, driven by actionable pathways including AXL and AURK. Blocking these two proteins blunted tumor invasion in patient-derived cultures. Furthermore, scRNAseq analyses of tumor-infiltrating CD8+ T-lymphocytes showed two distinct trajectories to T-cell dysfunction, corroborated by their clonal architecture based on single-cell T-cell receptor sequencing. By determining key modulators of these trajectories, followed by validation using external datasets and functional experiments, we uncovered a novel role for SOX4 in mediating T-cell exhaustion. Finally, interactome-analyses between pre-metastatic tumor-cells and CD8+ T-lymphocytes uncovered a putative role for the Midkine pathway in immune-modulation; this was confirmed by scRNAseq of tumors from humanized mice. Aside from specific findings, this study demonstrates the importance of tumor heterogeneity analyses in identifying key vulnerabilities during early metastasis