Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Abstract

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Angiocrine Regulation of Epithelial Barrier Integrity in Inflammatory Bowel Disease

“…Next, candidates were selected based on data mining (inflammatory, angiocrine, epithel, extracellular, endothelial, barrier, cytokine, bowel, secreted). Subsequently, the resulting 257 genes were mapped to profiles from human endothelial cells of different origin, including human umbilical vein endothelial cells (HUVEC) exposed to shear stress ( 126 ), under LPS-stimulation ( 127 ), overexpressing γ-interferon-inducible protein (IFI) 16 ( 128 ) and unstimulated ( 129 ), as well as endothelial cells from brain, lung, heart ( 130 ) and colorectal carcinoma ( 119 , 131 ). This analysis identified in total 28 genes ( Table 1 ).…”

Cryopreservation of Whole Tumor Biopsies from Rectal Cancer Patients Enable Phenotypic and In Vitro Functional Evaluation of Tumor-Infiltrating T Cells

“…The surgically resected tumors were immediately forwarded to research nurses in the surgical department for cleaning of tumor tissue by removal of macroscopically visible adipose, muscle, and connective tissues as previously described [ 34 ]. After cleaning, the tumors were cut into 3 × 3 mm pieces with a scalpel and randomly divided into two portions (5 pieces per portion).…”

Analysis of the interferon-γ-induced secretome of intestinal endothelial cells: putative impact on epithelial barrier dysfunction in IBD

“…To this goal, HIEC were isolated from healthy colon areas of five patients who underwent surgical therapy for colorectal cancer (CRC) (see Supplementary Methods ). Endothelial cells were isolated by FACS-based cell sorting following previously established protocols ( Naschberger et al, 2016 ; Naschberger et al, 2018 ). A purity above 98% of all five cultures was determined with reverse transcription quantitative polymerase chain reaction (RT-qPCR) and cytochemistry as described previously ( Naschberger et al, 2016 ; Naschberger et al, 2018 ) and is exemplarily shown here by the uniform expression of the endothelial marker CD31 in all HIEC of the different patients but not in the CRC tumor cell line DLD1 ( Figure 1A , Supplementary Figures S1A ).…”