Vlad Constantin Sandulache

Inactivating NOTCH1 mutations in head and neck squamous cell carcinoma (HNSCC) were described over a decade ago, suggesting a tumor suppressor function — unlike its oncogenic role in other tumors. Today, much debate persists regarding a putative oncogenic role in HNSCC as well, with reports that NOTCH1 signaling drives tumor growth and a cancer stem cell (CSC) phenotype. In this work, comprehensive experiments unequivocally demonstrate that NOTCH1 is a tumor suppressor in HNSCC regardless of mutation or activation status and that it reduces CSC frequency. We developed a signature of NOTCH1 activation showing the pathway is associated with very early differentiation, an altered tumor microenvironment, and better prognosis. Clarifying whether NOTCH1 occasionally functions as an oncogenic driver in HNSCC is crucial to prognosis and personalized therapy. The results presented unify the field, reconcile conflicting data, and provide critical insights into the biological and clinical significance of NOTCH1 , with broader implications in other squamous carcinomas with NOTCH1 mutations.

Current spatial T cell receptor (TCR) profiling approaches lack the resolution needed to link clonal identity, transcriptional state, and spatial positioning of individual T cells in the tumor microenvironment. Here, we introduce a spatial TCR profiling strategy that resolves individual T cell clones together with their transcriptional states at single-cell resolution and applied the method to human head and neck squamous cell carcinoma. Presumed tumor-specific T cells were broadly dispersed throughout the tumor microenvironment, and cells of the same clone occupied distinct transcriptional states in different locations: Immune-rich regions contained more plastic or progenitor cells, whereas tumor-dense regions were enriched for exhausted states. Patients exhibited notably different spatial architectures of antitumor T cell responses, revealing variation that was not captured by high-resolution, spatially agnostic methods such as spectral flow cytometry and single-cell RNA sequencing. These results provide a blueprint for dissecting antigen-specific T cell states in human tumors and reveal how T cell states are spatially coordinated with local cues across the tumor microenvironment.

NRF2 targeted suppression or restoration of wild type KEAP1 decreases distant metastasis in cisplatin resistant HNSCC cells.

In vivo expression of cisplatin transporters in CDDP resistant HNSCC tumors.

Supplementary Table S5A. Bulk-level single cell ATAC sequencing analysis showing immediate (short-term) chromatin openings (peaks) in HN30-P cells following CDDP treatment; Supplementary Table S5B. Bulk-level single cell ATAC sequencing analysis showing long-term chromatin openings (peaks) gained in cisplatin-resistant HN30-R8 cells following CDDP treatment.

Supplementary Table S4A. The KNC genes enriched in cluster 1 in HNSCC cells; Supplementary Table S4B. Gene activity score of cell surface markers in cluster 1 in HNSCC cells.

Relationship between TP53 status and the NRF2/KEAP1/CUL3 pathway in OCSCC TCGA samples.

Cisplatin sensitivity following knockdown of NRF2 or restoration of wildtype KEAP1 in cisplatin resistant HNSCC cells is not mediated through apoptosis.

Supplementary Materials and Methods