Use of multiple biomarkers for the localization and characterization of colon cancer stem cells by indirect immunocytochemistry

Abstract

Abstract. In this study, we used LGR5, γ-synuclein, p53, KRAS and epiregulin antibodies to localize stem cells by indirect immunocytochemistry in paraffin sections of normal and cancerous colon tissues. In the normal colon tissue, no staining of cells with LGR5, γ-synuclein, p53 and KRAS antibodies was observed, apart from a few scattered cells in between the colon villi that were faintly stained with antibodies to LGR5. Staining of highly differentiated cancer tissue with LGR5 antibodies revealed single cells or clusters of up to 4 cells in the interior space of the carcinoma cell layers. Staining of poorly differentiated cancer tissues (stage I-IV) revealed 9-81 clustered stem cells. The number of clustered stem cells increased significantly with the tumor stage, when comparing stage II to stage IV (p<00048). Occasionally, the clustered stem cells appeared in the interphase between the colon stroma and the tumor tissue. Surprisingly, antibodies to p53 clearly stained the clusters of stem cells both in the nuclei and the cytoplasm. The staining of the nuclei of other cells in the undifferentiated tumors was in general weaker, and no staining was found in the cytoplasm. Antibodies to γ-synuclein heavily stained the endothelial cells of the blood vessels and some other scattered cells in the highly differentiated tumors. Antibodies to γ-synuclein heavily stained the stem cells in both the cytoplasm and the nuclei of poorly differentiated tumors. Antibodies to KRAS stained the cytoplasm and the nuclei of stem cells in poorly differentiated tumors and also stained the cytoplasm of some scattered cells. Antibodies to epiregulin stained the cytoplasm of normal colon tissue cells in the crypt-villus axis. The antibodies weakly stained the highly differentiated tumor cells and moderately stained the moderately differentiated tumor cells.Of note, the antibodies intensively stained the clustered stem cells of the poorly differentiated tumor cells. These antibodies also clearly stained the clustered stem cells of poorly differentiated tumors but were not specific as they clearly stained cells in the crypt-villus axis of the normal colon wall. Our results show that LGR5 antibodies can serve as a reliable marker for colon cancer stem cells. Once the colon stem cells are identified, the targeting of specific drugs to kill these cells should be attempted in the future in order to cure this disease. Moreover, the fact that we did not find any stained cells with antibodies to LGR5 in normal tissues apart from a few scattered cells, suggests that the normal colon stem cells differ from the tumor stem cells at least as regards the expression of this protein.In addition, antibodies to γ-synuclein, p53 and KRAS only stained the tumor stem cells and not the normal tissue. Thus, they can serve as multiple biomarkers for the localization of colon cancer stem cells by indirect immunofluorescence.

The fermentation characteristics of Sparassis crispa polysaccharides and their effects on the intestinal microbes in mice

“…Moreover, the growth state of the colon can determine the efficiency of polysaccharide hydrolysis [61]. The intake of carbohydrates increases the wet weight of feces resulting in a more relaxed colonic state, which is considered to have a positive impact on colon health [62]. In this study, the protective effect of SCPs on colonic tissue was observed, which was characterized by significantly increase of the colonic index, the length of colon and villi, and the fecal water content in SCP-treated mice compared with those of the control group, suggesting that the intake of SCPs might improve the physiological state of the colon.…”

Current Technologies in Plant Molecular Breeding

“…In contrast, immunohistochemical samples are sections of tissue in which each cell is surrounded by tissue architecture and adjacent cells (Javois 1999 ;Amsterdam et al 2012 ). Immunocytochemistry is performed on intact cells with their surrounding extracellular matrix removed.…”

Targeting Human Cancer Stem Cells with Monoclonal Antibodies

“…Lgr5 is expressed on Wnt/β-catenin-dependent adult stem cell populations of the intestine, colon and hair-follicle [212]. Expression of Lgr5 has been demonstrated on intestinal crypt stem cells, which are the cells-of-origin of intestinal cancer [213], indicating that Lgr5 is a functional marker of intestinal CSCc involved in intestinal and colorectal carcinogenesis [214,215]. In patients with colorectal cancer, Lgr5 expression positively correlates with histological grade, invasion and metastasis, and inversely correlates with survival of patients, revealing an important role of Lgr5 in the progression and prognosis of colorectal cancer [217].…”