**5. Conclusion**

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

Takaishi et al. were the first to propose CD44 as a marker of GCSCs in a study performed

and initiate tumors after subcutaneous and orthotopic engraftment in mice, and they were

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

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,

of GCSCs in combination with CD44 compared to CD44 alone [51, 73]. Although CD44

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 migra-

**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

ate 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

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

cells may correspond to **SP cells** with CSC properties as previously described

counterpart, displayed CSC properties such as growing as tumorspheres

is also a marker of GCSCs in primary gastric carcinoma. The FACS-sorted CD44<sup>+</sup>

cells [74].

and ALDH was 0.1–3.5% of the cancer cells [51]. These CD44<sup>+</sup>

in gastric carcinoma cell lines by others [76–78]. The ability of CD44<sup>+</sup>

the vital DNA dye Hoechst 33342, whereas the ALDH−

cells were able to form tumorspheres

cells, but

sorted cells were not. Moreover, it seems that

cells, but they do not allow a better enrichment

cells are tumorigenic [51]. CD44v8-10, also named CD44E,

cells that possessed CSC properties, i.e., the ability to gener-

/ALDH<sup>+</sup>

cells did not incorporate

cells to efflux

cells incorporated it, suggesting that

/ALDH<sup>+</sup>

been associated with metastasis formation [57, 65–70].

on several gastric cancer cell lines [50]. Their CD44<sup>+</sup>

resistant to anticancer drugs, whereas CD44−

and CD166 are also expressed by CD44<sup>+</sup>

marks GCSCs, not all CD44<sup>+</sup>

tion capacity of the GCSC CD44<sup>+</sup>

subpopulation within the CD44<sup>+</sup>

CD44<sup>+</sup>

/ALDH<sup>+</sup>

not their CD44−

74 Gastric Cancer

Since 2007, researchers have increased efforts to identify real gastric stem cells, the cell population capable of replenishing an entire gastric gland containing of all cell lineages. Many of the markers involved have been reviewed here, and their stemness properties have been clearly demonstrated in mouse models. It will be of interest to understand why there are different localizations of stem cells, one in the isthmus and one at the bottom of the gland. These two stem/progenitor cell niches could play different roles, one being more proliferative than the other one which seems to behave like a reservoir, but they could also play distinct roles in response to different stimuli and damage to the gastric mucosa.

Regarding to the gastric tumor, an in-depth analysis of putative CSC markers identified CD44 as well as ALDH activity as the "gold" gastric CSC markers in cancer cell lines and in PDX models [51]. Determination of the signaling pathways controlling their properties is now instrumental to find new targeted therapies for gastric cancer, for which there is a crucial unmet need to find new efficient therapy. In this aim, we have shown by different strategies that the targeting of gastric CSCs expressing CD44 by blocking specific microRNAs or by inducing their differentiation by all-trans retinoic acid allows inhibition of tumor growth in vivo [58, 83].

Nevertheless, the characterization of gastric CSCs was limited in some publications by the cellular model used. To date, the best models to study the efficiency of new therapeutic strategies on primary gastric CSCs remain PDX models, with the restriction that almost all of those described are subcutaneous engraftments which never give rise to metastasis. Moreover, mouse models of gastric carcinogenesis induced by *Helicobacter* infection and/or carcinogens do not reproduce invasion of the deeper layers of the stomach, peritoneal carcinomatosis, and distant metastases as in humans. Consequently, there is an urgent need to develop mouse models of metastatic gastric carcinoma, in order to study the efficiency of new therapeutic strategies targeting CSCs not only on tumor initiation but also on metastasis formation.
