Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.
Int J Oncol. 2012 Jul;41(1):285-91. doi: 10.3892/ijo.2012.1430. Epub 2012 Apr 10.
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.
在这项研究中，我们使用了 LGR5、γ-突触核蛋白、p53、KRAS 和表皮调节素抗体，通过间接免疫细胞化学法对正常和癌结肠组织的石蜡切片中的干细胞进行定位。在正常结肠组织中，除了在结肠绒毛之间有少数散在的细胞用 LGR5 抗体呈弱阳性染色外，没有观察到细胞用 LGR5、γ-突触核蛋白、p53 和 KRAS 抗体染色。用 LGR5 抗体对高分化癌组织进行染色，显示在癌细胞层的内部空间中有单个细胞或多达 4 个细胞的细胞簇。对低分化癌组织（I-IV 期）进行染色，发现有 9-81 个簇集的干细胞。随着肿瘤分期的增加，簇集的干细胞数量显著增加，II 期与 IV 期相比（p<00048）。偶尔，簇集的干细胞出现在结肠基质与肿瘤组织之间的间隙中。令人惊讶的是，p53 抗体不仅在细胞核中，而且在细胞质中都能清晰地染色干细胞簇。未分化肿瘤中其他细胞的核染色通常较弱，细胞质中无染色。γ-突触核蛋白抗体强烈染色高分化肿瘤中的血管内皮细胞和一些其他散在细胞。γ-突触核蛋白抗体强烈染色低分化肿瘤的干细胞的细胞质和细胞核。KRAS 抗体染色低分化肿瘤干细胞的细胞质和细胞核，并染色一些散在细胞的细胞质。表皮调节素抗体染色隐窝-绒毛轴上的正常结肠组织细胞的细胞质。该抗体弱阳性染色高分化肿瘤细胞，中度染色中度分化肿瘤细胞。值得注意的是，这些抗体强烈染色低分化肿瘤细胞的簇集干细胞。这些抗体也能清晰地染色低分化肿瘤的簇集干细胞，但不具有特异性，因为它们能清晰地染色正常结肠壁隐窝-绒毛轴上的细胞。我们的结果表明，LGR5 抗体可作为结肠癌干细胞的可靠标志物。一旦确定了结肠干细胞，未来就应该尝试针对这些细胞的特定药物，以试图治愈这种疾病。此外，我们在正常组织中除了少数散在的细胞外，没有发现任何用 LGR5 抗体染色的细胞，这表明正常结肠干细胞至少在这种蛋白的表达上与肿瘤干细胞不同。此外，γ-突触核蛋白、p53 和 KRAS 抗体仅染色肿瘤干细胞，而不染色正常组织。因此，它们可以作为间接免疫荧光法定位结肠癌干细胞的多种生物标志物。
