High content Image Analysis to study phenotypic heterogeneity in endothelial cell monolayers

Endothelial cells (EC) are heterogeneous across and within tissues, reflecting distinct, specialised functions. EC heterogeneity has been proposed to underpin EC plasticity independently from vessel microenvironments. However, heterogeneity driven by contact-dependent or short-range cell-cell crosstalk cannot be evaluated with single cell transcriptomic approaches as spatial and contextual information is lost. Nonetheless, quantification of EC heterogeneity and understanding of its molecular drivers is key to developing novel therapeutics for cancer, cardiovascular diseases and for revascularisation in regenerative medicine. Here, we developed an EC profiling tool (ECPT) to examine individual cells within intact monolayers. We used ECPT to characterise different phenotypes in arterial, venous and microvascular EC populations. In line with other studies, we measured heterogeneity in terms of cell cycle, proliferation, and junction organisation. ECPT uncovered a previously under-appreciated single-cell heterogeneity in NOTCH activation. We correlated cell proliferation with different NOTCH activation states at the single cell and population levels. The positional and relational information extracted with our novel approach is key to elucidating the molecular mechanisms underpinning EC heterogeneity.

Endothelial-T cell crosstalk contributes to vascular injury in fatty liver disease

Cardiovascular complications are often the fundamental causes of death in non-alcoholic fatty liver disease (NAFLD) patients. While there are known systemic mediators in NAFLD that may induce vascular inflammation, the mechanism of endothelial dysfunction remain understudied. In this work, we harnessed the replicative potential of blood outgrowth endothelial cells (BOECs) to develop personalized cell lines from NAFLD patients and healthy controls. Our transcriptomic analysis showed that the top interactome network enriched in NAFLD BOECs comprised of several C-C and C-X-C chemokine ligands involved in immune cell chemotaxis. We previously reported T cell infiltration in mouse model of non-alcoholic steatohepatitis, and here, we confirmed enhanced endothelial chemokine signatures in arterial histological sections. To elucidate endothelial-immune crosstalk, we performed single-cell analysis on human peripheral blood mononuclear cells and found T cell intensification in NAFLD patients compared to healthy controls. Our immunoprofiling by flow cytometry further revealed that NAFLD patients possessed higher levels CD8+ memory cells. Functionally, T cells, instead of monocytes, adhered more pronouncedly to NAFLD BOECs. In evaluating the CXCL12-CXCR4 axis in chemotaxis, CXCR4 antagonist (AMD3100) substantially modulated the migration of patient-derived CD8+ T cells towards NAFLD BOECs, which was not observed in healthy endothelial-T cell chemotaxis coculture. Finally, we validated NAFLD-associated endothelial dysfunction by enumerating two folds more circulating endothelial cells, a biomarker of vascular injury, in the blood samples of NAFLD patients than healthy controls. Our work provides insights for translation to restore blood vessel health and potentially mitigate adverse vascular events in NAFLD. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Industrial Alignment Fund Pre-Positioning grant from the Agency for Science, Technology and Research, Singapore Endothelial-T cell crosstalk in NAFLD

Cancer Stem Cell for Tumor Therapy

Tumors pose a significant threat to human health. Although many methods, such as operations, chemotherapy and radiotherapy, have been proposed to eliminate tumor cells, the results are unsatisfactory. Targeting therapy has shown potential due to its specificity and efficiency. Meanwhile, it has been revealed that cancer stem cells (CSCs) play a crucial role in the genesis, development, metastasis and recurrence of tumors. Thus, it is feasible to inhibit tumors and improve prognosis via targeting CSCs. In this review, we provide a comprehensive understanding of the biological characteristics of CSCs, including mitotic pattern, metabolic phenotype, therapeutic resistance and related mechanisms. Finally, we summarize CSCs targeted strategies, including targeting CSCs surface markers, targeting CSCs related signal pathways, targeting CSC niches, targeting CSC metabolic pathways, inducing differentiation therapy and immunotherapy (tumor vaccine, CAR-T, oncolytic virus, targeting CSCs–immune cell crosstalk and immunity checkpoint inhibitor). We highlight the potential of immunity therapy and its combinational anti-CSC therapies, which are composed of different drugs working in different mechanisms.

Two distinct Sertoli cell states are regulated via germ cell crosstalk†

Sertoli cells are a critical component of the testis environment for their role in maintaining seminiferous tubule structure, establishing the blood-testis barrier, and nourishing maturing germ cells in a specialized niche. This study sought to uncover how Sertoli cells are regulated in the testis environment via germ cell crosstalk in the mouse. We found two major clusters of Sertoli cells as defined by their transcriptomes in Stages VII–VIII of the seminiferous epithelium and a cluster for all other stages. Additionally, we examined transcriptomes of germ cell-deficient testes and found that these existed in a state independent of either of the germ cell-sufficient clusters. Altogether, we highlight two main transcriptional states of Sertoli cells in an unperturbed testis environment, and a germ cell-deficient environment does not allow normal Sertoli cell transcriptome cycling and results in a state unique from either of those seen in Sertoli cells from a germ cell-sufficient environment.