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Gallbladder cancer (GBC) is the most common malignancy of the biliary tract, with high metastasis incidence and extremely low survival rate. The tumor endothelial cells (TECs) are fundamental components in the tumor microenvironment and significantly contribute to various tumor progression; however, the roles of TECs in GBC are poorly understood. Here, using single-cell RNA sequencing, we identify a GBC-enriched endothelial population-CD34+CD90+ ECs (SAEndo2). The CD34+CD90+ endothelial subset correlates with patients¡¯ poor prognosis and liver metastasis. In vitro and in vivo experiments suggest that CD34+CD90+ ECs promote the GBC cell migration and metastasis, showing EndoMT properties. Moreover, CD34+CD90+ ECs display enhanced activation of TGF-¦Â signaling, and TGF-¦Â inhibition abolishes the CD34+CD90+ ECs¡¯ promotion effect on GBC cell migration. Collectively, our study provides a detailed profiling of endothelial cells in GBC and identifies an essential endothelial population that regulates GBC metastasis, laying new theoretical insight and offering a potential therapeutic target for GBC metastasis.

Background: Recurrent/metastatic head and neck squamous cell carcinoma ((R/M) HNSCC) represents one of the most aggressive and immunosuppressive cancers. Despite the introduction of immune checkpoint inhibitors (ICIs), only a limited number of patients obtain long-term benefits. In (R/M) HNSCC patients, the antitumor immune response is defective, conferring resistance and promoting tumor progression. Therefore, the identification of novel biomarkers for superior clinical outcomes and easily accessible in standard clinical settings is still an unmet clinical need. Methods: Blood liquid biopsies obtained from (R/M) HNSCC patients undergoing pembrolizumab therapy (monotherapy or in combination with chemotherapy) were analyzed by flow cytometry to evaluate the levels of circulating immunosuppressive regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs), at baseline and during therapy. Correlations between these immunosuppressive immune cell subsets and clinical parameters (clinical response rate, progression-free survival (PFS), overall survival (OS) and performance status (PS)) were performed. Results: Univariate analysis showed that before therapy, higher circulating levels of both CD137?Tregs and LOX-1?PMN-MDSCs, identified patients with significantly worse survival. Furthermore, CD137?Tregs resulted also positively correlated with worse PS, while high levels of LOX-1?PMN-MDSCs negatively affected response to pembrolizumab, with a significant increase in non-responsive patients during therapy. Interestingly, both CD137?Tregs as well as LOX-1?PMN-MDSCs exerted a higher immunosuppression on T cell proliferation than CD137?Tregs and LOX-1?PMN-MDSCs, respectively. Multivariate analysis revealed that the circulating LOX-1?PMN-MDSC subset resulted as an independent prognostic factor for survival by multivariate analysis, as confirmed in an independent validation cohort. Conclusions: The levels of blood circulating LOX-1?PMN-MDSCs may be proposed as non-invasive biomarkers to predict clinical outcomes of (R/M) HNSCC patients developing resistance to immunotherapy, improving patient selection and suggesting novel personalized therapies.

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2¨C6, suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states. The transcription factor FOXO1 has a key role in human T cell memory, and manipulating FOXO1 expression could provide a way to enhance CAR T cell therapies by increasing CAR T cell persistence and antitumour activity.