**3.1. Tumors can originate from epithelial stem cell transformation**

cells recruited to the isthmus and stimulated by Cxcl12 endothelial cell production. The effi-

gastric innate lymphoid cells [13], suggesting that cells in the niche regulate stem/progenitor

The enteric nervous system also has the ability to regulate gastric homeostasis via direct innervation of the glands. In three independent mouse models of gastric cancer, Zhao et al. elegantly demonstrated that surgical or pharmacological denervation suppresses gastric tumorigenesis, even if performed at an early preneoplastic step [21]. Further analyses revealed that cholinergic nerves surround the base of glands and modulate epithelial stem cells through activation

 cells. In stomach organoid models, coculture with neurons or treatment with pilocarpine, a cholinomimetic drug, increased organoid formation and the expression of *Lgr5* and *Cd44* stem cell markers, whereas the effects were reversed by botox treatment [21]. Another

gastric mucosa, induced nerve growth factor (NGF) secretion from epithelial cells that expand enteric nerves and promote carcinogenesis [22]. Remarkably, *Tff2-*Cre;*R29-*NGF mice devel-

The Notch signaling pathway is also inhibited in vagotomized mice [21]. The Notch inhibitor dibenzazepine (DBZ) reduced the proliferation in the isthmus region, decreased the Mist1 lineage tracing, and blocked the growth of corpus organoids in vitro, suggesting that Notch

Gastric carcinoma is a multifactorial disease, involving a chronic *Helicobacter pylori* infection as the main cause as well as the Epstein-Barr Virus to a small extent, diet (low vitamins, nitrosamines, chemicals, etc.), smoking, and genetic susceptibility of the host [23, 24]. At the histological level, the WHO described more than five different histological subtypes, divided into two main groups in the Lauren classification of gastric tumors, i.e., the intestinal type and the diffuse type [25]. At the molecular level, gastric carcinomas are classified in four main groups based on their mutational profile [26, 27]. These classifications are not currently used

More than 93% of distal gastric carcinoma cases are associated with a chronic *H. pylori* infection of the gastric mucosa [28]. Most of these cases represent the intestinal histological subtype. *H. pylori* infection induces a chronic inflammation of the gastric mucosa, i.e., gastritis. In 5–10% of cases, gastritis evolves into gastric or duodenal ulcer, and in 1% of cases, gastritis leads to stomach cancers. In this last case, the loss of specialized epithelial cells results in a chronic atrophic gastritis and to the compensatory cellular hyperproliferation and aberrant differentiation at the origin of the intestinal metaplasia (firstly appearing in the pylorus)

tuft cells and nerves, the main sources of acetylcholine in the

intraepithelial

R) expressed by

dysplastic cell expansion and

cells in this context led to

R-dependent manner [22].

ciency of organoid formation is enhanced by Wnt5a or coculture with Cxcr4<sup>+</sup>

of the Wnt signaling pathway via the muscarinic acetylcholine receptor 3 (M<sup>3</sup>

oped metaplasia and dysplasia by 8 months of age with CD44<sup>+</sup>

**3. Is gastric cancer a stem cell disease?**

intramucosal adenocarcinomas by 18 months. Ablation of Dclk1<sup>+</sup>

the inhibition of epithelial proliferation and tumorigenesis in a M<sup>3</sup>

in clinical practice to orientate toward a specific targeted therapy.

activity is important for corpus gastric stem cell maintenance and activity [13].

proliferation.

68 Gastric Cancer

Lgr5<sup>+</sup>

publication reported that Dclk1<sup>+</sup>

Interestingly, the parietal cell atrophy induced after *H. pylori* infection causes an increase in the proliferation of stem/progenitor cells in the isthmus [37] that is associated with an induction of CD44 expression in this region, which then expands toward the bottom of the gastric unit [17, 35, 36] (**Figure 2**). Sigal et al. also reported an increase of the number of antral Lgr5<sup>+</sup> cells in *H. pylori-*associated gastritis and carcinoma in humans. As these cells are susceptible to DNA damage, it suggests that Lgr5<sup>+</sup> stem cells could be at the origin of cancer [38, 39]. An Lgr5<sup>+</sup> gene signature in pyloric gastric units identified Wnt target genes, including *Sox9* and *Cd44*, suggesting canonical Wnt signaling activity at the base of the pyloric glands. Gastric cancer patients exhibit a dysregulation of Wnt signaling [21]. Spontaneous Wnt activation in the mouse model *APCmin* leads to the development of gastric adenomas in the pyloric region; *Apc* depletion specifically in Lgr5<sup>+</sup> via a single tamoxifen injection in *Lgr5-EGFP-CreERT2;APCflox/flox* mice leads to adenoma formation in the gastric antrum, but not in the corpus [7].

KRAS is one of the most commonly mutated oncogenes in gastric cancer. The *Kras* mutation in the Mist1<sup>+</sup> isthmus cells, and not in the Mist1<sup>+</sup> chief cells, results in the formation of metaplastic/dysplastic foci from the isthmus to the bottom of glands. Mist1<sup>+</sup> stem cells give rise to intramucosal intestinal-type gastric cancer induced by *Apc* loss of function but only in the context of KRAS-induced metaplasia [13].

E-cadherin expression is lost in most diffuse-type gastric carcinomas, but E-cadherin loss alone is not sufficient to initiate diffuse-type gastric cancer in mice [40]. Loss of the tight junction protein IQGAP1 is also insufficient to induce diffuse-type gastric carcinoma in transgenic mice after challenge with *Helicobacter* infection, as it indeed promotes intestinal-type carcinogenesis [41]. To try to reproduce the diffuse-type gastric cancer, some authors infected *Mist1*;CreERT2;*Cdh1*flox/flox mice with *H. felis* to induce chronic inflammation. In this context, E-cadherin loss in Mist1<sup>+</sup> cells resulted in the development of diffuse-type gastric carcinomas [13]. The double inactivation of E-cadherin and p53 in a conditional mouse model also successfully led to metastatic diffuse-type gastric cancer [42].

#### **3.2. Tumors originating from bone marrow-derived cells**

Some data have shown that bone marrow-derived cells (BMDCs) can migrate to peripheral tissues in case of injury or inflammation where they are engrafted and participate in tissue repair, giving rise to all cell lineages. Houghton et al. showed that BMDCs are recruited into the gastric mucosa of C57BL/6 mice chronically infected by *H. felis* and contribute overtime to metaplasia, dysplasia, and finally cancer [34]. In fact, to the contrary, they found that it was a rare event in the context of a normal homeostasis, without injury. Results from this study did not exclude the possibility that BMDCs participate in the development of lesions via fusion with epithelial cells. Our group confirmed these observations in the same mouse genetic background but with different strains of the human pathogen *H. pylori* and found that nearly a quarter of high-grade dysplastic lesions are composed of BMDCs [35]. BMDC epithelial gland repopulation was significantly associated with pseudointestinal metaplasia, suggesting that BMDC recruitment may play a role in preneoplastic lesion progression. These BMDCs are recruited only in response to chronic *H. felis* and *H. pylori* infection but not in response to acute injury [34]. BMDC recruitment occurs in response to the secretion of several chemokines such as SDF1 and TNFα by infected epithelial cells in a NF-κB-dependent manner [34, 43]. Once recruited, the BMDCs can differentiate into local gastric epithelial cells via transdifferentiation or cell/cell fusion with local gastric epithelial cells [35, 44]. Interestingly, BMDC recruitment into the gastric mucosa was a late event in the cascade of gastric carcinogenesis, occurring only in infected animals of more than 1 year of age. In these chimera mice, metaplastic lesions were comprised, inside the same gland, of a mosaic of tagged-BMDCs and native gastric epithelial cells, revealing a multiclonal composition [35]. These metaplastic lesions are now considered as a "point of no return," after which, most of the time, eradication of *H. pylori* cannot lead to a regression of the metaplastic and associated dysplastic lesions, because mutations deregulating stem cell properties and proliferation are already present. A monoclonal conversion will occur during the evolution of intestinal metaplasia toward dysplasia and finally carcinoma. As the mice never develop real metastatic gastric adenocarcinoma in contrast to the human situation, there is no evidence in the literature to date of the role of BMDCs composing metaplastic/dysplastic lesions as the tumor-initiating cells in invasive gastric adenocarcinoma. However, it is very interesting to note that BMDCs were also detected in gastric carcinoma of the esophagus in mice models and in humans, which also develop on a background of chronic inflammation and intestinal metaplasia cascade [45, 46]. Very few studies have been described in humans to strengthen the results obtained in animal models, because there is a limited possibility to trace BMDCs in an individual developing a carcinoma of the GI tract. The only technical approach tested was to detect the Y chromosome of BM cells of a male donor in female transplanted patients by fluorescence in situ hybridization (FISH). In such transplanted cases having developed carcinoma of the GI tract, BMDCs were detected in some rare cases of carcinoma and dysplasia of the esophagus [45, 46]. Concerning the stomach, the study of Whortley et al., performed on only four cases of sex-mismatch transplanted cases having developed gastric carcinoma, failed to report a carcinoma composed of cells of BM origin. However, one of the cases showed aneuploidy, so a contribution of the BMDCs cannot be totally excluded [47].

Unfortunately, in those models, full proof of the concept that gastric stem cells or other populations of differentiated cells or BMDCs are the cells of origin of cancer has not been found, because the tumorigenic effect mediated, for instance, by *Apc* and *Cdh1* inactivation or by *Kras* oncogenic activation in non-stem cell populations, i.e., in progenitor or differentiated cells, has not been followed. Moreover, in contrast to squamous skin tumors [48] or intestinal adenomas [49], the in vivo contribution of GCSCs to tumorigenesis has not yet been fully elucidated. Nevertheless, regardless of their origin, dysplastic lesions and gastric adenocarcinomas are composed of CD44<sup>+</sup> cells (**Figure 2**) [17, 36] that have been recently described to possess cancer stem cell properties [50, 51].
