Recently, considerable efforts have been made in the discovery and characterization of CSLC markers. Most markers for sorting CSLC are empirical and derived from normal stem cells, which have been questioned for their specificity and reliability as CSLC markers. Up to now, several molecules have been proposed to be used as markers to characterize the ECSLCs. Neurotrophin receptor p75 (p75NTR), also known as CD271, is a stem cell marker for normal oesophageal epithelial cells [
23]. In ESCC cells, Huang et al. [
6] demonstrated that p75NTR-positive cells possess some characteristics of CSCs, namely, self-renewal and chemotherapy resistance. Recently, Yamaguchi et al. [
24] further demonstrated that p75NTR-positive cells express higher stem cell-related genes (Nanog, p63 and Bmi-1) and EMT-related genes (N-cadherin and fibronectin), exhibite higher abilities of the colony formation in vitro and the tumor formation in vivo than that of CD44 or CD90-positive cells. Also, they found that p75NTR-positive/CD44-negative and p75NTR-positive/CD90-negative cell fractions contain significantly higher proportions of mitotically quiescent cells. Hence, p75NTR expression may serve as a characteristic of the mitotically quiescent cancer stem cell population present in ESCC. With two primary ESCC cells, Zhao et al. [
5] selected ECSLC markers from the candidates that had been used in other solid carcinomas, including CD44, CD90, CD133, CD271 and CD326. They found that only CD44 expression is correlated with tumorigenicity. Aldehyde dehydrogenase 1A1 (ALDH1A1), an intracellular enzyme responsible for detoxifying aldehydes, is a cancer stem-like cell-associated protein in various malignant [
25]. Almanaa et al. [
26] reported that ALDH1-positive esophageal cancer cells have high cell proliferation rates and the ability to regenerate tumor bulk. Our previous work [
4] demonstrated that ALDH1A1-positive ESCC cells possess properties of cancer stem-like cells and highly invasive potential. CD90, also called Thy-1 (thymocyte differentiation factor 1), has been identified in various stem cells and CSLCs [
27]. Tang et al. [
8] reported that CD90-positive ESCC cells exhibit stem cell-like features and have high tumourigenic and metastatic capacities. Thus it can be seen that there are many inconsistencies and contradictions among these markers for characterization of ECSCs. To circumvent these challenging issues, more functional ECSLC markers should be exploited. Accordingly, CR-1 showed promising potential as a functional marker for the identification and isolation of CSLC from ESCC. Here, we demonstrated that CR-1
high ESCC cells express high level of stemness-related genes, and possess high potential of self-renewal and high ability of tumorigenesis. Silencing CR-1 expression significantly reduced the expression of stemness-related genes and abilities of self-renewal and tumorigenesis. We also evaluated the consistency between CR-1 expression with ALDH1 expression, and found that these two markers are highly overlapped in ESCC cell lines and 64% (85/132) cases are consistent in ESCC specimens. But the possible overlapping of CR-1 expression with that of CD90, CD44, p75
NTR in ECSLCs requires further investigation. In addition, antisense nucleic acid technique on CR-1 [
28], and antagonist of the CR-1 CFC domain have been reported to inhibit human breast cancer, colon cancer and testicular cancer cells growth [
29], suggesting that CR-1 is a valuable therapeutic target for targeting CSLCs in ESCC.
EMT describes a mechanism by which cells lose their epithelial characteristics and acquire more migratory mesenchymal properties. In the context of cancer, EMT facilitates the dissemination of cancer cells and endows them with properties essential for metastasis including stemness, invasiveness, and the ability to survive in the circulation and seed at a secondary site [
22,
30‐
32]. Indeed, a number of epigenetic regulators are known to functionally regulate genes important for EMT [
33]. Despite a few reported EMT-related molecules in ESCC [
34‐
39], the molecular mechanisms regulating EMT in ESCC remain elusive. Results from the current study suggest that CR-1 regulates EMT in ESCC as knockdown of CR-1 in EC109 and TE-1 cells reversed EMT as well as the invasive and metastatic properties of the cells. EMT has been shown to be associated with early steps of invasion and metastasis of epithelial origin cancer cells [
40]. These results are consistent with the previous reports that CR-1 is crucial for the activation of EMT in some cancers, including ESCC [
41‐
45]. Increasing evidence has suggested that EMT generates cells with properties of CSLC cells [
32], and our results demonstrate that EMT promoted by CR-1 is involved in generation of ECSLCs.
To emphasize the role of CR-1 in ESCC, we analyzed CR-1 levels in clinical ESCC specimens. We demonstrated that the most ESCCs had positive CR-1 expression, which is closely related to the depth of tumor invasion, lymph node metastasis and poor prognosis in the ESCC patients. Metastasis and invasion are the major causes of poor prognosis for ESCC patients. At present, the Tumor, Node, Metastasis (TNM) staging system is the primary tool to determine the extent of cancer and the prognosis of patients, and functions as a surrogate for survival. However, due to the existence of undetectable micrometastasis and low sensitivity of clinical imaging, this system does not always predict prognosis accurately. Discovering new molecular markers, related to the metastasis and invasion, is a promising strategy to achieve more accurate clinical outcome predictions and treatment options for ESCC. Our results suggest that CR-1 can serve as such a marker for ESCC, which is consistent with the previous reports in cervical carcinoma [
46], gastric cancer [
47], and non-small cell lung cancer [
48].