**4.2. Phenotypic characterization of gastric cancer stem cells**

Several phenotypic characteristics have been proposed to isolate GCSCs using fluorescenceactivated cell sorting (FACS), including (1) the expression of cell membrane markers (or combinations of markers), and among them CD44; (2) the exclusion of Hoechst 33342 dye by the "side population" of cells (SP cells); and (3) the enzymatic activity of aldehyde dehydrogenases (ALDH).

**CD44** was among the first markers of CSCs described in solid tumors and initially in breast carcinoma [61]. CD44 is a type I transmembrane glycoprotein expressed in many normal and tumoral cells. It plays a role in adhesion/homing, supporting cell migration and transmitting survival signals, thereby being pro-oncogenic by nature. The principal ligand of the CD44 receptor coordinating signalization is hyaluronic acid, but it can also interact with additional molecules of the extracellular matrix, such as collagen, fibronectin, fibrinogen, laminin, or osteopontin [62]. Cytoplasmic partner molecules of CD44 are the cytoskeletal proteins Ezrin, Radixin, Moesin, and Ankyrin, which influence the signaling pathway. Loss of CD44 in mice models results in a decrease in gastric mucosa proliferation in the isthmus region. The critical role of CD44 in proliferation involves its interaction with hyaluronic acid and the downstream activation of the STAT3 signaling pathway [17], RhoGTPases, the PI3K/AKT pathway, and the MAPK signaling pathway [63]. CD44 is encoded by the 20-exon *CD44* gene, in which exons 1–5 and 16–20 are spliced together and translated into CD44s, the standard or small isoform. In addition, the variant exons 6–15 can be alternatively spliced and assembled in different combinations with the standard exons to generate other variant (CD44v) protein isoforms [62, 64]. We and others reported that CD44 is expressed following *H. pylori* infection in patients and in mouse models in the case of regenerative hyperplasia, intestinal metaplasia, dysplasia, and gastric carcinoma (**Figure 2**) [35, 36, 50, 51, 65]. Histological and molecular analyses of tumor collections have shown that CD44 is positively and significantly associated with tumor recurrence and mortality in gastric cancer, and the expression of CD44 and CD44v has also been associated with metastasis formation [57, 65–70].

Takaishi et al. were the first to propose CD44 as a marker of GCSCs in a study performed on several gastric cancer cell lines [50]. Their CD44<sup>+</sup> cells were able to form tumorspheres and initiate tumors after subcutaneous and orthotopic engraftment in mice, and they were resistant to anticancer drugs, whereas CD44− sorted cells were not. Moreover, it seems that CD44 is not only a GCSC marker, but it also plays an oncogenic role, assessed by a decrease in tumor growth using siRNA targeting CD44. More recently, further relevant results from patient-derived xenograft models (PDXs) of gastric carcinoma have confirmed that CD44 is also a marker of GCSCs in primary gastric carcinoma. The FACS-sorted CD44<sup>+</sup> cells, but not their CD44− counterpart, displayed CSC properties such as growing as tumorspheres in vitro and lead to tumor formation in vivo that reconstitute the heterogeneity of the primary tumor of the patients and are more chemoresistant [51, 71, 72]. ESA, CD24, CD133, and CD166 are also expressed by CD44<sup>+</sup> cells, but they do not allow a better enrichment of GCSCs in combination with CD44 compared to CD44 alone [51, 73]. Although CD44 marks GCSCs, not all CD44<sup>+</sup> cells are tumorigenic [51]. CD44v8-10, also named CD44E, has been identified as the predominant CD44 variant expressed in gastric cancer cells, and its expression is low in normal tissues [57]. It plays a functional role in tumor initiation, most likely by increasing CSC resilience to adverse conditions such as hypoxia or oxidative stress. Indeed, there is evidence that CD44v8-10 stabilizes the cystine-glutamate transporter subunit xCT and promotes the synthesis of glutathione, thereby protecting cancer cells from reactive oxygen species [70]. Depletion of the expression of CD44 leads to a decrease in the tumorigenicity of cancer cell lines [50], and Yoon et al. demonstrated implication of the Hedgehog signaling in the maintenance, chemoresistance, and migration capacity of the GCSC CD44<sup>+</sup> cells [74].

**ALDH activity** has also been described as a GCSC marker [51, 75]. In an extensive screening of the expression of 10 putative CSC surface markers, as well as in eight PDX models, we found that CSCs expressed both CD44 and ALDH activity and that ALDH activity revealed a subpopulation within the CD44<sup>+</sup> cells that possessed CSC properties, i.e., the ability to generate a new heterogeneous tumor in vivo and a tumorsphere in vitro. Xenograft experiments using the ELDA mathematical model showed that the frequency of GCSCs expressing CD44 and ALDH was 0.1–3.5% of the cancer cells [51]. These CD44<sup>+</sup> /ALDH<sup>+</sup> cells did not incorporate the vital DNA dye Hoechst 33342, whereas the ALDH− cells incorporated it, suggesting that CD44<sup>+</sup> /ALDH<sup>+</sup> cells may correspond to **SP cells** with CSC properties as previously described in gastric carcinoma cell lines by others [76–78]. The ability of CD44<sup>+</sup> /ALDH<sup>+</sup> cells to efflux the Hoechst 33342 dye and to resist conventional chemotherapy was reversed by treatment with efflux pump inhibitors [51]. Nevertheless, Takaishi et al. found that both gastric SP and non-SP cells possess a tumorigenic ability in vitro and in vivo [50]. Therefore, the detection of the SP cells does not seem to be a good marker for GCSCs; rather the best markers to detect them are CD44 and ALDH.
