De-Chen Lin

Head and neck squamous cell carcinoma (HNSCC) development is accompanied by metabolic remodeling of the tumor microenvironment, yet how metabolic programs evolve across specific cell populations during tumor progression remains poorly understood. We integrated single-cell RNA-sequencing data from five HNSCC cohorts comprising 645,351 cells from 114 patients and inferred metabolic flux using single-cell Flux Estimation Analysis (scFEA). Myeloid cells showed the highest and most consistent metabolic allocation to lactate export among major cell types. In ten patients with paired normal mucosa and carcinoma, myeloid lactate-export allocation increased from 1.72% to 3.31% of total inferred metabolic flux, with an increase in every patient. SPP1+ macrophages showed the highest lactate-export allocation in carcinoma. Lactate export was predominantly associated with MCT4/ SLC16A3 . In carcinoma, myeloid SLC16A3 expression exceeded SLC16A1 by 17-fold, and CPTAC-HNSCC confirmed tumor enrichment of MCT4 protein. Together, these findings identify increased MCT4-associated lactate export as a prominent feature of myeloid metabolic reprogramming during HNSCC progression.

Differential H3K27ac peak analysis.

Transcriptional intratumoral heterogeneity (ITH) is a hallmark of aggressive cancers. Investigation into the ITH programs that drive tumor metastasis and immune evasion could help identify potential treatment and prevention approaches. Through single-cell RNA sequencing analysis of upper aerodigestive squamous cell carcinoma (UASCC) cells and patient tumors, we uncovered a hybrid epithelial–mesenchymal transition (hEMT) ITH program linked to metastatic dissemination. The transcription factor ETS1 was identified as a master regulator of the hEMT program, directly activating prometastatic genes and promoting distant spread in vivo. Unexpectedly, ETS1 also orchestrated an immune-cold tumor microenvironment by transcriptionally activating STAT1 and CD274 (PD-L1), suppressing T lymphocyte infiltration, and elevating immune checkpoint molecules. Clinically, high ETS1 expression in tumors strongly correlated with poor survival and resistance to immune checkpoint blockade across multiple cohorts. Drug screening demonstrated that ETS1-high cancers were vulnerable to HSP90 inhibitors (e.g., alvespimycin), which suppress ETS1 by disrupting hypoxia-inducible factor 1 alpha–mediated transcriptional activation. Together, this work reveals ETS1 as a dual driver of tumor distal metastasis and immune evasion in UASCC, while nominating HSP90 inhibition as a tailored treatment strategy for ETS1-driven tumors. These findings provide a roadmap for targeting aggressive ITH subsets and overcoming immunotherapy resistance. Significance:Transcriptional intratumoral heterogeneity drives metastasis and immune evasion via an ETS1-regulated hybrid EMT program, which can be targeted with HSP90 inhibitors to treat aggressive, immunotherapy-resistant tumors.

Overlapped genes between ETS1 ChIP-Seq data.

Functional validation of ETS1 in UASCC cell lines.

Immune profiling in ETS1-driven tumor models.

HSP90 inhibition suppresses ETS1-driven cell migration in UASCC cells.

Correlation between ETS1 expression and protein levels of its target genes.

ETS1 modulates sensitivity to HSP90 inhibition in UASCC cells.

ETS1 expression is associated with poor immunotherapy response.