The CSC theory has become an intensely investigated topic, even though the origin of CSCs remains elusive and the isolation and identification of CSCs has not been achieved for many types of human tumor. CSCs are defined as a small side population of tumor cells with the ability to self-renew and potentially promote the formation of tumors[
28‐
30]. Conventional cancer treatments indiscriminately kill proliferating cells and are always unsuccessful due to the survival of quiescent CSCs. Therefore, therapies could be designed to target cancer stem cells by inducing their differentiation or to eliminate them by inhibiting the maintenance of the stem-cell state[
6,
31]. CSCs can survive radiation therapy and chemotherapy and then return to a proliferative growth state, making them good targets for biomarker identification[
15]. The cellular origin of cancer stem cells has not been clearly determined. GPCRs have been hypothesized to be closely associated with CSCs during tumorigenesis[
13,
32]. Lgr5, a member of the GPCR superfamily, is known as a stem cell marker in the small intestine and colon, as well as in hair follicles[
11,
33]. Lgr5 over-expression has been reported in a few cancers[
10,
12‐
15], including hepatocellular carcinoma, colorectal cancer, ovarian cancer, basal cell carcinoma, and esophageal adenocarcinoma. Recent studies suggested that Lgr5 may be involved in colorectal carcinogenesis as a target of Wnt signaling[
8,
9] and may be an ideal marker of colorectal CSCs[
18,
19]. In this study, Lgr5 was significantly overexpressed in the majority of CRCs (56.3%) compared with distal normal mucosa (25%). Increased expression of Lgr5 was significantly correlated with depth of invasion, lymph node metastasis, and distant metastasis. These results suggest that high expression levels of Lgr5 receptors are usually correlated with more malignant and metastatic tumors. Furthermore, Lgr5 was seen more frequently in advanced colorectal cancer. Positive expression of Lgr5 was found in 83.1% (54/65) of stage III cancer tissues and 100% (10/10) of stage IV cancer tissues. These results suggest that Lgr5 may play an important role in the development and progression of the tumor. It was also found that Lgr5 is closed related to the worst prognosis of colorectal carcinoma. In the present study, Lgr5 expression correlated with poor survival of colorectal cancer, indicating that Lgr5-positive cells may contain more CSCs. Therefore, in agreement with CSC theories, the residual CSCs may lead to metastasis and recurrence of colorectal cancer through multilineage differentiation and self-renewal after routine therapy[
17,
34]. Our study also showed that Lgr5 expression was positively correlated with Ki-67, a recognized nuclear antigen-specific marker of cellular proliferation, suggesting that Lgr5-positive cancer cells have higher proliferative activity.
Furthermore, we attempted to identify the Hoechst 33342 low-staining (SP) cancer stem-like cells in colorectal cancers using immunocytochemistry after selecting the cells that extruded Hoechst 33342. In our experiments, the SP cells were observed at a frequency of 7% in the Colo205 cell line, which was higher than reported by Kondo[
2] and Patrawala
et al.[
35], and also proved the conclusion in the report by Telford
et al.[
36]. In order to better recognize the SP cells directly by microscopy, we first used immunofluorescence to observe and analyze the colon cancer cells expressing Lgr5 after Hoechst33342 staining. The results demonstrated that the expression of Lgr5 was obviously much stronger in the Hoechst 33342 low-staining SP cells than in the other cells. Therefore, it is suggested that the increased expression of Lgr5 may be a positive marker of SP cells in the Colo205 colon cancer cell line. Recently, many studies have focused on the identification of colon CSCs because of their potential for colorectal cancer treatment[
37‐
39]. CD133 has recently been identified as a potential cancer stem cell marker[
37,
38]. However, controversy still remained because it had been reported that CD133 expression is not restricted to intestinal stem or cancer-initiating cells, and both CD133+ and CD133- metastatic colon cancer cells could initiate tumors[
40]. It has been shown that CD133 expression is broadly distributed in colorectal cancer cells[
41,
42]. Thus, CD133 appears to be an inappropriate marker for the characterization of single colon CSCs[
41]. Our results suggested that the expression of Lgr5 may correspond to the formation and function of Hoechst 33342 low-staining SP cells in colon cancer, and may differentiate these cells from other non-SP cells. Our results should be considered preliminary and require further confirmation by more in-depth functional studies both in cell lines and solid tumor samples.