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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.

AbstractT cells in the human female genital tract (FGT) are key mediators of susceptibility to and protection from infection, including HIV and other sexually transmitted infections. There is a critical need for increased understanding of the distribution and activation of T cell populations in the FGT, but current sampling methods require a healthcare provider and are expensive, limiting the ability to study these populations longitudinally. To address these challenges, we have developed a method to sample immune cells from the FGT utilizing disposable menstrual discs which are noninvasive, self-applied, and low in cost. To demonstrate reproducibility, we sampled the cervicovaginal fluid of healthy, reproductive-aged individuals using menstrual discs across 3 sequential days. Cervicovaginal fluid was processed for cervicovaginal cells, and high-parameter flow cytometry was used to characterize immune populations. We identified large numbers of live, CD45+ leukocytes, as well as distinct populations of T cells and B cells. Within the T cell compartment, activation and suppression status of T cell subsets were consistent with previous studies of the FGT utilizing current approaches, including identification of both tissue-resident and migratory populations. In addition, the T cell population structure was highly conserved across days within individuals but divergent across individuals. Our approach to sample immune cells in the FGT with menstrual discs will decrease barriers to participation and empower longitudinal sampling in future research studies.