Published OnlineFirst September 12, 2016; DOI: 10.1158/1078-0432.CCR-15-2157 Biology of Human Tumors Clinical Cancer Research Characterization of Biomarkers of Tumorigenic and Chemoresistant Cancer Stem Cells in Human Gastric Carcinoma Phu Hung Nguyen1,2, Julie Giraud1,2, Lucie Chambonnier1,2, Pierre Dubus2,3,4, Linda Wittkop2,5,6, Genevieve Belleannee 4, Denis Collet4, Isabelle Soubeyran7,8, Serge Evrard2,7,8, Benoit Rousseau2,9, Nathalie Senant-Dugot2,10, Francis Megraud 1,2,4, Fred eric Mazurier11, and Christine Varon1,2 Abstract Purpose: Gastric carcinomas are heterogeneous, and the current activity presented the properties to generate new heterogeneous therapy remains essentially based on surgery with conventional tumorspheres in vitro and tumors in vivo. CD44 and CD166 were chemotherapy and radiotherapy. This study aimed to characterize coexpressed, representing 6.1% to 37.5% of the cells; ALDH biomarkers allowing the detection of cancer stem cells (CSC) activity was detected in 1.6% to 15.4% of the cells; and the þ þ þ in human gastric carcinoma of different histologic types. ALDH cells represented a core within the CD44 /CD166 Experimental Design: The primary tumors from 37 patients subpopulation that contained the highest frequency of tumori- þ with intestinal- or diffuse-type noncardia gastric carcinoma were genic CSCs in vivo. The ALDH cells possessed drug efflux prop- studied, and patient-derived tumor xenograft (PDX) models in erties and were more resistant to standard chemotherapy than the – immunodeficient mice were developed. The expressions of 10 ALDH cells, a process that was partially reversed by verapamil putative cell surface markers of CSCs, as well as aldehyde dehy- treatment. drogenase (ALDH) activity, were studied, and the tumorigenic Conclusions: CD44 and ALDH are the most specific properties of cells were evaluated by in vitro tumorsphere assays biomarkers to detect and isolate tumorigenic and chemore- and in vivo xenografts by limiting dilution assays. sistant gastric CSCs in noncardia gastric carcinomas inde- Results: We found that a subpopulation of gastric carcinoma pendently of the histologic classification of the tumor. Clin cells expressing EPCAM, CD133, CD166, CD44, and a high ALDH Cancer Res; 1–12. Ó2016 AACR. Introduction pylori (H. pylori). Infection with H. pylori,classified as a class 1 carcinogen by the World Health Organization, induces a chron- Gastric cancer is the fourth most common cancer in frequen- ic inflammation evolving over decades from a chronic atrophic cy and the third leading cause of cancer mortality in the world. gastritis to intestinal metaplasia, dysplasia, and finally adeno- Ninety-five percent of gastric cancers are gastric carcinomas, carcinoma (1, 2). Some cases also include Lynch syndromes which are divided into two types depending on their localiza- (microsatellite instability, MSI) and Epstein–Barr Virus (EBV) tion in the stomach: adenocarcinomas of the cardia whose infection. The classification of gastric carcinomas is based etiology remains unclear, and noncardia gastric carcinomas for essentially on histologic criteria. The Lauren classification dis- which the main factor is a chronic infection by Helicobacter tinguishes two main subtypes, the intestinal type, which repre- sents the majority of the cases, and the diffuse type (3). The 1INSERM, U853 Helicobacter Infection, Inflammation and Cancer, Bordeaux, intestinal type is composed of glands having more or less 2 3 France. University of Bordeaux, Bordeaux, France. EA 2406, University of preserved their organization and differentiation state, or having Bordeaux, Bordeaux, France. 4University Hospital Center of Bordeaux, Bor- 5 acquired intestinal characteristics; it is subclassified into tubu- deaux, France. INSERM, ISPED, Centre INSERM U1219 Bordeaux Population fi Health, Bordeaux, France. 6Pole^ de Sante Publique, Service d'information lar, mucinous, or papillary carcinoma in the WHO classi ca- medicale, University Hospital Center of Bordeaux, Bordeaux, France. 7Institut tion of gastric carcinoma (4). The diffuse type is poorly cohe- Bergonie, Bordeaux, France. 8INSERM, U1012 Actions for onCogenesis under- sive, composed of isolated cells (often signet ring cells) pro- standing and Target Identification in Oncology (ACTION), Bordeaux, France. ducing mucins. These classification systems have little clinical 9 10 Service Commun des Animaleries, Animalerie A2, Bordeaux, France. SFR utility, as they cannot orientate patient therapy. With the 11 TransBioMed, Bordeaux, France. CNRS UMR 7292, GICC LNOx, Tours, France. exception of Her2 positivity which orientates toward a specific Note: Supplementary data for this article are available at Clinical Cancer treatment, treatment is still based on surgery combined with Research Online (http://clincancerres.aacrjournals.org/). conventional chemotherapy and/or radiotherapy, and the Corresponding Author: Christine Varon, University of Bordeaux, INSERM, U853 5-year survival rates remain under 30% in most countries (5). Helicobacter Infection, Inflammation and Cancer, 146 rue Leo Saignat, Bordeaux Recently, the Cancer Genome Atlas Research Network and F33076, France. Phone: 33557579575; Fax: 33556796018; E-mail: Wang and colleagues published a molecular profiling of gastric [email protected] carcinomas based on two studies with 295 cases and 100 cases, doi: 10.1158/1078-0432.CCR-15-2157 respectively. Both studies led to a classification of gastric carci- Ó2016 American Association for Cancer Research. nomas into four main subtypes according to their molecular www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst September 12, 2016; DOI: 10.1158/1078-0432.CCR-15-2157 Nguyen et al. gastric carcinoma cell lines proposed CD44 as a gastric CSC Translational Relevance marker, but this marker was expressed in three out of six cell We report the screening of the expression of 10 cell surface lines studied, and confirmation in primary tumors was lacking markers and aldehyde dehydrogenase (ALDH) activity on cells (21). Then, the study performed by Rocco and colleagues on from primary gastric carcinoma. We found that a subpopula- 12 human primary gastric carcinomas failed to demonstrate þ þ tion of tumor cells expressing CD133, CD166, CD44, and tumor-initiating properties of CD133 -andCD44 -sorted cells ALDH presented cancer stem cell (CSC) tumorigenic proper- after xenograft in both NOD/SCID and nude immunodeficient þ ties in vitro and in vivo. Among them, ALDH cells represented mice (22). 1.6% to 15.4% of the tumor cells and contained the highest Another important point concerns the origin of the CSCs. As þ frequency of tumorigenic CSCs before CD44 cells. In addi- Houghton and colleagues and our group reported in mouse þ þ tion, the tumorigenic CD44 ALDH cells possessed drug models of Helicobacter-induced gastric carcinogenesis, gastric efflux and chemoresistance properties, constituting the cells dysplasia and carcinoma may originate from the transformation to target in the development of new therapy. Results also of a local epithelial stem cell or of a bone marrow (BM)–derived showed that CD44, which is poorly expressed or absent in stem cell (23–25). In this model, dysplastic lesions were com- þ healthy gastric epithelium, is overexpressed in gastric carcino- posed of CD44 cells, regardless of their BM or local origin (24). ma and may constitute a good biomarker for the detection of In addition, the heterogeneity of gastric carcinomas suggests that CSCs by standard immunohistochemistry on patient tissue gastric CSC markers, if they indeed exist, may be different accord- samples, whereas detection of CSCs possessing a high ALDH ing to the origin and/or the histologic type of gastric carcinoma. activity is not yet possible by standard immunohistochemistry In this study, we performed an extensive screening of the and involves many ALDH isozymes. expression of putative cell surface markers of CSCs as well as ALDH activity in order to identify biomarkers allowing the detection and isolation of tumorigenic and chemoresistant CSCs in human primary intestinal- and diffuse-type noncardia gastric carcinoma. þ profiles: (1) EBV tumors (frequent PIK3CA mutations, extreme DNA hypermethylation), (2) MSI tumors (elevated mutation rates, hypermethylation), (3) genomically stable tumors Materials and Methods (enriched for the diffuse type; driver mutations include CDH1, Human samples and mouse xenografts RHOA, cytoskeleton, and cell junction regulators), and (4) chro- Fresh tumors samples were collected from gastric surgical mosomal instability tumors (marked aneuploidy, focal amplifi- wastes from patients who underwent gastrectomy for noncardia cation of tyrosine kinase receptors; refs. 6, 7). These studies were gastric carcinoma and for whose informed consent was performed without distinguishing between cardia and noncardia obtained. Fresh samples of tumor and paired nontumor tissues gastric carcinomas, whose etiology is different. were transported in DMEM medium with 20% FCS, 50 IU/mL Tumors are heterogeneous, composed of cells which are more penicillin, 50 mg/mL streptomycin, 50 mg/mL vancomycin, and or less differentiated, and not all proliferative. Over the last 15 mg/mL amphotericin-B. Samples were minced in small decade, extensive research has focused on the discovery and the pieces of 2 mm  2 mm size and were subcutaneously trans- characterization of cancer stem cells (CSC) at