Nanopore Long-Read Transcriptomics Profiling Reveals Gene Expression Signatures of Mouse Tumor Endothelial Cells 2H-11

Background:Tumor endothelial cells (TECs) play an indispensable role in tumor growth and metastasis. Compared with normal endothelial cells (NECs), TECs exhibit unique phenotypic and functional heterogeneity in terms of metabolism, genetics, and transcriptomics. It is not only the key to coordinate tumor angiogenesis, but also an important factor of immune regulation in the tumor microenvironment. In recent years, the role of TECs in tumor metabolism and invasion has been continuously reported. However, the research on the mechanism behind the complex functions of TECs is still at the basic stage. We use Oxford Nanopore Technology (ONT) three-generation full-length transcriptome sequencing to detect all genetic structural changes in the transcriptome of mouse TECs 2H-11 and mouse NECs SVEC4-10.Results: In Tumor endothelial cells 2H-11,1847genes are up-regulated and 1202 genes are down-regulated. According to the Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs), we found that different functional trends related to metabolic processes, developmental processes, localization, immune system processes, and locomotion are the main reasons for the differences. DEGs are mainly enriched in signal pathways related to cancer, immunity and metabolism, involving Pathways in cancer,Antigen processing and presentation , Proteoglycans in cancer, Focal adhesion, MAPK signaling pathway ,Protein digestion and absorption，ECM-receptor interaction，PI3K-Akt signaling pathway and Glutathione metabolism. We also obtained the structural variation of transcripts such as alternative splicing, gene fusion, and alternative polyadenylation and accurately quantified the expression of the transcript. Some of our results have been confirmed in other documents. But other data have not been reported yet, which is the focus of our future exploration.Conclusion: We try to use transcriptomics and bioinformatics methods to characterize tumor endothelial cell-related genes and signaling pathways.It could help better understand the molecular mechanisms of tumor endothelial cells involved in tumorigenesis and development. DEGs in key pathways may be potential diagnostic markers or therapeutic targets of TECs. Our data also provide useful genetic resources for improving the genome and transcriptome annotations of TECs and NECs.

Optimal Strategy for Colorectal Cancer Patients’ Diagnosis Based on Circulating Tumor Cells and Circulating Tumor Endothelial Cells by Subtraction Enrichment and Immunostaining-Fluorescence In Situ Hybridization Combining with CEA and CA19-9

Background. Cancerous embryo antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) are commonly used in clinical practice to assist in diagnosing CRC. However, their sensitivity is very low. This study aims to investigate the clinical significance of circulating tumor cells (CTCs) and circulating tumor endothelial cells (CTECs) compared with CEA and CA19-9 in the auxiliary diagnosis of colorectal cancer (CRC) patients. Methods. 115 pathologically confirmed CRC patients and 20 healthy controls were enrolled in this study. CTCs and CTECs were enriched and identified by subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH). A logistic regression was used to establish a model for the receiver-operating characteristic (ROC) curve analysis, and the diagnostic efficacy of CTCs, CTECs, CEA, CA19-9, and their combinations was analyzed. Results. The CTC ( P < 0.0001 ) and CTEC ( P = 0.0009 ) level was significantly higher in CRC patients than that in healthy controls. For CRC patients, CTC and CTEC level was significantly correlated with tumor stage and lymph node metastasis status, but not with sex, age, tumor location, and degree of differentiation. The positive rate of CTCs, CTECs, CEA, and CA19-9 in CRC patients was 87.8%, 39.1%, 28.7%, and 26.1%, respectively. To distinguish CRC patients from controls, the area under the curve (AUC) of CTC was 0.889, which was much higher than 0.695 of CTEC, 0.696 of CEA, and 0.695 of CA19-9. Establishing ROC curve by logistic regression algorithm, the highest AUC was 0.935, which combined CTCs with CTEC, CEA, and CA19-9. Conclusions. CTCs combined with CTEC, CEA, and CA19-9 are useful to improve the diagnostic efficiency, which has high clinical significance in the diagnosis of colorectal cancer.

Vascular Microenvironment, Tumor Immunity and Immunotherapy

Immunotherapy holds great promise for treating cancer. Nonetheless, T cell-based immunotherapy of solid tumors has remained challenging, largely due to the lack of universal tumor-specific antigens and an immunosuppressive tumor microenvironment (TME) that inhibits lymphocyte infiltration and activation. Aberrant vascularity characterizes malignant solid tumors, which fuels the formation of an immune-hostile microenvironment and induces tumor resistance to immunotherapy, emerging as a crucial target for adjuvant treatment in cancer immunotherapy. In this review, we discuss the molecular and cellular basis of vascular microenvironment-mediated tumor evasion of immune responses and resistance to immunotherapy, with a focus on vessel abnormality, dysfunctional adhesion, immunosuppressive niche, and microenvironmental stress in tumor vasculature. We provide an overview of opportunities and challenges related to these mechanisms. We also propose genetic programming of tumor endothelial cells as an alternative approach to recondition the vascular microenvironment and to overcome tumor resistance to immunotherapy.

miRNA-1246 in extracellular vesicles secreted from metastatic tumor induces drug resistance in tumor endothelial cells

Tumor endothelial cells (TECs) reportedly exhibit altered phenotypes. We have demonstrated that TECs acquire drug resistance with the upregulation of P-glycoprotein (P-gp, ABCB1), contrary to traditional assumptions. Furthermore, P-gp expression was higher in TECs of highly metastatic tumors than in those of low metastatic tumors. However, the detailed mechanism of differential P-gp expression in TECs remains unclear. miRNA was identified in highly metastatic tumor extracellular vesicles (EVs) and the roles of miRNA in endothelial cell resistance were analyzed in vitro and in vivo. In the present study, we found that treatment of highly metastatic tumor-conditioned medium induced resistance to 5-fluorouracil (5-FU) with interleukin-6 (IL-6) upregulation in endothelial cells (ECs). Among the soluble factors secreted from highly metastatic tumors, we focused on EVs and determined that miR-1246 was contained at a higher level in highly metastatic tumor EVs than in low metastatic tumor EVs. Furthermore, miR-1246 was transported via the EVs into ECs and induced IL-6 expression. Upregulated IL-6 induced resistance to 5-FU with STAT3 and Akt activation in ECs in an autocrine manner. These results suggested that highly metastatic tumors induce drug resistance in ECs by transporting miR-1246 through EVs.

Bcl9 Depletion Modulates Endothelial Cell in Tumor Immune Microenvironment in Colorectal Cancer Tumor

Tumor endothelial cells are an important part of the tumor microenvironment, and angiogenesis inhibitory therapy has shown potential in tumor treatment. However, which subtypes of tumor endothelial cells are distributed in tumors, what are the differences between tumor endothelial cells and normal endothelial cells, and what is the mechanism of angiogenesis inhibitory therapy at the histological level, are all need to be resolved urgently. Using single-cell mRNA sequencing, we analyzed 12 CT26 colon cancer samples from mice, and found that knockdown of the downstream factor BCL9 in the Wnt signaling pathway or inhibitor-mediated functional inhibition can modulate tumor endothelial cells at a relatively primitive stage, inhibiting their differentiation into further extracellular matrix construction and angiogenesis functions. Furthermore, we propose a BCL9-endo-Score based on the differential expression of cells related to different states of BCL9 functions. Using published data sets with normal endothelial cells, we found that this score can characterize endothelial cells at different stages of differentiation. Finally, in the The Cancer Genome Atlas (TCGA) pan-cancer database, we found that BCL9-endo-Score can well predict the prognosis of diseases including colon cancer, kidney cancer and breast cancer, and identified the markers of these tumor subtypes, provide a basis for the prognosis prediction of patients with such types of tumor. Our data also contributed knowledge for tumor precision treatment with angiogenesis inhibitory therapy by targeting the Wnt signaling pathway.