characterization of biomarkers of tumorigenic and ......2016/09/10 · 9 - linda wittkop,...

Nguyen et al. Page 1

Characterization of biomarkers of tumorigenic and chemoresistant 1

cancer stem cells in human gastric carcinoma 2

Short title: biomarkers of tumorigenic and chemoresistant gastric CSCs 3

4

Phu Hung Nguyen1,2, Julie Giraud1,2, Lucie Chambonnier1,2, Pierre Dubus2,3,4, Linda 5

Wittkop2,5,6, Geneviève Belleannée4, Denis Collet4, Isabelle Soubeyran7, Serge Evrard7, Benoit 6

Rousseau2,8, Nathalie Senant-Dugot2,9, Francis Mégraud1,2,4, Frédéric Mazurier10, Christine 7

Varon1,2. 8

9

1 INSERM, U853, Bordeaux F-33000, France. 10

2 University of Bordeaux, Bordeaux F-33000, France. 11

3 EA 2406, Bordeaux F-33000, France. 12

4 University Hospital Center of Bordeaux, Bordeaux F-33000, France. 13

5 INSERM, ISPED, Centre INSERM U897-Epidemiologie- Biostatistique, F-33000 Bordeaux, 14

France. 15

6 Pôle de Santé Publique, Service d’information médicale, University Hospital Center of 16

Bordeaux, Bordeaux F-33000, France. 17

7 Institut Bergonié, Bordeaux F-33000, France. 18

8 Service Commun des Animaleries, Animalerie A2, Bordeaux F-33000, France. 19

9 SFR TransBioMed, Bordeaux F-33000, France. 20

10 CNRS UMR 7292, GICC LNOx, Tours F-37032, France. 21

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 12, 2016; DOI: 10.1158/1078-0432.CCR-15-2157

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Correspondence. Dr. Christine Varon, INSERM U853, Université de Bordeaux, 146 rue Leo 1

Saignat, Bordeaux F-33000, France. Email: [email protected]. Tel: 2

+33(0)557579575, Fax: +33(0)556514182. 3

4

Grant information 5

We thank the French ‘Association pour la Recherche contre le Cancer’ (grant number 8412), the 6

‘Institut National du Cancer’ (grant 07/3D1616/IABC-23-12/NC-NG and grant 2014-152), the 7

‘Conseil Regional d’Aquitaine’ (grant number 20071301017 and 20081302203), the French 8

National Society for Gastroenterology, and the Canceropole Grand Sud-Ouest (grant 2010-08-9

canceropole GSO-Université Bordeaux 2) for financial support. This project was supported by 10

SIRIC BRIO (Site de Recherche Intégrée sur le Cancer – Bordeaux Recherche Intégrée 11

Oncologie) (grant INCa-DGOS-Inserm 6046). 12

13

Acknowledgments 14

We thank Marie-Edith Lafon (CNRS UMR 5234, University of Bordeaux) and technicians from 15

the Department of Tumor Pathology (Haut-Leveque Hospital, University Hospital Center of 16

Bordeaux) for molecular analyses on tumor tissues, Pierre Costet (animal facilities, University of 17

Bordeaux), Vincent Pitard and Santiago Gonzalez (Flow Cytometry and FACS Platform, 18

University of Bordeaux), Philippe Brunet de la Grange (CNRS UMR5164 CIRID, University of 19

Bordeaux) for assistance on SP cells analyses, Alban Giese (Experimental Pathology Platform of 20

the Canceropole GSO and SIRIC BRIO, University of Bordeaux), Elodie Siffre and Lucie 21

Benejat (INSERM U853) for technical assistance. 22

23





Authors' contributions 1

- Phu Hung Nguyen, animal experiments; tumorsphere assays; data acquisition and analysis; 2

drafting of the manuscript; 3

- Julie Giraud, acquisition and analysis of tumorsphere assays and flow cytometry data; drafting 4

of the manuscript; 5

- Lucie Chambonnier, technical assistance for animal care and experiments, flow cytometry and 6

immunohistochemistry analyses; 7

- Pierre Dubus, study concept and design; histopathological analyses; 8

- Linda Wittkop, mathematical and statistical analyses; drafting of the manuscript; 9

- Geneviève Belleannée, Denis Collet, Isabelle Soubeyran and Serge Evrard, collection of 10

patient’s informed consent; collection of human tissue samples from consenting patients who 11

underwent gastrectomy; histopathological analyses; 12

- Benoit Rousseau, technical assistance for animal care and animal experiments; 13

- Nathalie Senant-Dugot, technical assistance for tissue processing and histology; 14

- Francis Mégraud, study concept and design: study supervision; drafting of the manuscript; 15

- Frédéric Mazurier, study concept and design; acquisition and analysis of data; 16

- Christine Varon, study concept and design; animal experiments; acquisition and analysis of 17

data; drafting of the manuscript; study supervision. 18

19

Disclosures 20

All authors declare no financial, professional, or personal conflict of interest. 21

22

23

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Abbreviations: 1

ABC, ATP-binding cassette. 2

ALDH, aldehyde dehydrogenase. 3

AML, acute myeloid leukemia. 4

BM, bone marrow. 5

CSC, cancer stem cell. 6

EBV, Epstein Barr Virus. 7

ELDA, extreme limiting dilution assay. 8

FCS, Fetal calf serum. 9

GC, gastric carcinoma. 10

MSI, microsatellite instability. 11

NSG, non obese diabetic/severe combined immunodeficiency/interleukin-2Rγ null. 12

P, passage by serial transplantation in mice. 13

PDX, patient derived tumor xenograft. 14

WHO, World Health Organization. 15

16





Statement of translational relevance (120-150 words) 1

We report the screening of the expression of ten cell surface markers and ALDH activity on cells 2

from primary gastric carcinoma. We found that a subpopulation of tumor cells expressing 3

CD133, CD166, CD44 and ALDH presented CSC tumorigenic properties in vitro and in vivo. 4

Among them, ALDH+ cells represented 1.6-15.4% of the tumor cells and contained the highest 5

frequency of tumorigenic CSCs before CD44+ cells. In addition, the tumorigenic CD44+ALDH+ 6

cells possessed drug efflux and chemoresistance properties, constituting the cells to target in the 7

development of new therapy. Results also showed that CD44, which is poorly expressed or 8

absent in healthy gastric epithelium, is overexpressed in gastric carcinoma and may constitute a 9

good biomarker for the detection of CSCs by standard immunohistochemistry on patients' tissue 10

samples, whereas detection of CSCs possessing a high ALDH activity is not yet possible by 11

standard immunohistochemistry and involves many ALDH isozymes. 12

13

14

Abstract. 15

Purpose: Gastric carcinomas (GC) are heterogeneous, and the current therapy remains 16

essentially based on surgery with conventional chemo- and radiotherapy. This study aimed to 17

characterize biomarkers allowing the detection of cancer stem cells (CSC) in human GC of 18

different histological types. 19

Experimental Design: The primary tumors from thirty-seven patients with intestinal or diffuse 20

type non-cardia GC were studied, and patient’s derived tumor xenograft (PDX) models in 21

immunodeficient mice were developed. The expression of ten putative cell surface markers of 22





CSCs, as well as ALDH activity, were studied and the tumorigenic properties of cells were 1

evaluated by in vitro tumorsphere assays and in vivo xenografts by limiting dilution assays. 2

Results: We found that a subpopulation of GC cells expressing EPCAM, CD133, CD166, CD44 3

and a high ALDH activity presented the properties to generate new heterogeneous tumorspheres 4

in vitro and tumors in vivo. CD44 and CD166 were co-expressed, representing 6.1 to 37.5% of 5

the cells; ALDH activity was detected in 1.6 to 15.4% of the cells and the ALDH+ cells 6

represented a core within the CD44+/CD166+ subpopulation that contained the highest 7

frequency of tumorigenic CSCs in vivo. The ALDH+ cells possessed drug efflux properties and 8

were more resistant to standard chemotherapy than the ALDH- cells, a process which was 9

partially reversed by verapamil treatment. 10

Conclusions: 11

CD44 and ALDH activity are the most specific biomarkers to detect and isolate tumorigenic and 12

chemoresistant gastric CSCs in non-cardia GCs independently of the histological classification 13

of the tumor. 14

15

Keywords: Gastric cancer, CSC, gastric adenocarcinoma, ALDH, CD44, CD133, CD166, 16

intestinal, diffuse, verapamil, 5-fluorouracil, doxorubicin, cisplatin. 17

18

Word count: 4999 words. 19





Introduction. 1

Gastric cancer is the fourth most common cancer in frequency and the third leading cause of 2

cancer mortality in the world. Ninety-five percent of gastric cancers are GCs, which are divided 3

into 2 types depending on their localization in the stomach: adenocarcinomas of the cardia whose 4

etiology remains unclear, and non-cardia GCs for which the main factor is a chronic infection by 5

Helicobacter pylori. Infection with H. pylori, classified as a class 1 carcinogen by the WHO, 6

induces a chronic inflammation evolving over decades from a chronic atrophic gastritis to 7

intestinal metaplasia, dysplasia and finally adenocarcinoma (1)(2). Some cases also include 8

Lynch syndromes (microsatellite instability, MSI) and Epstein Barr Virus (EBV) infection. The 9

classification of GCs is based essentially on histological criteria. The Lauren classification 10

distinguishes 2 main subtypes, the intestinal type which represents the majority of the cases, and 11

the diffuse type (3). The intestinal type is composed of glands having more or less preserved 12

their organization and differentiation state, or having acquired intestinal characteristics; it is sub-13

classified into tubular, mucinous or papillary carcinoma in the WHO classification of GC (4). 14

The diffuse type is poorly cohesive, composed of isolated cells (often signet ring cells) 15

producing mucins. These classification systems have little clinical utility, as they cannot 16

orientate patient therapy. With the exception of Her2 positivity which orientates toward a 17

specific treatment, treatment is still based on surgery combined with conventional chemotherapy 18

and/or radiotherapy, and the five-year survival rates remain under 30% in most countries (5). 19

Recently, the Cancer Genome Atlas Research Network and Wang et al. published a molecular 20

profiling of GCs based on two studies with 295 cases and 100 cases, respectively. Both studies 21

led to a classification of GCs into four main subtypes according to their molecular profiles: 1) 22





EBV+ tumors (frequent PIK3CA mutations, extreme DNA hypermethylation), 2) MSI tumors 1

(elevated mutation rates, hypermethylation), 3) genomically stable tumors (enriched for the 2

diffuse type; driver mutations include CDH1, RHOA, cytoskeleton and cell junction regulators), 3

and 4) chromosomal instability tumors (marked aneuploidy, focal amplification of tyrosine 4

kinase receptors) (6)(7). These studies were performed without distinguishing between cardia 5

and non-cardia GCs, whose etiology is different. 6

7

Tumors are heterogeneous, composed of cells which are more or less differentiated and not all 8

proliferative. Over the last decade, extensive research has focused on the discovery and the 9

characterization of CSCs at the origin of cancers in numerous organs. Tumors are hierarchically 10

organized with CSCs at the top of this pyramid and at the origin of tumor initiation, 11

heterogeneity and propagation (8-10). The CSCs correspond to a subpopulation of cells within 12

the tumor defined by self-renewal, asymmetrical division and differentiation properties, giving 13

rise to the more or less differentiated cells composing the tumor mass. CSCs can stay in 14

quiescence under some conditions, resist conventional therapies, and be at the origin of tumor 15

relapse and metastasis. The definition of CSCs remains largely operational and based on 16

functional assays that register their self-renewal and tumorigenic properties, assessed by the 17

formation of new heterogeneous tumors after xenograft in vivo, and of tumorspheres in particular 18

culture conditions in vitro (8-10). 19

Indeed CSC may display both genetic and phenotypic heterogeneity, markers allowing their 20

identification have been characterized in tumors of different organs, including CD133, CD44 and 21

CD24 among those studied (8)(10)(11-16). More recently, the activity of aldehyde 22





dehydrogenases (ALDH), intracellular enzymes involved in oxidation of aldehydes and retinoic 1

acid signaling, also led to the identification of CSCs in tumors of the breast (17), lung (18), colon 2

(19) and other organs (20). 3

In the stomach, the existence of CSCs has been subject to debate. The first study performed by 4

Takaishi et al. on GC cell lines proposed CD44 as a gastric CSC marker, but this marker was 5

expressed in three out of six cell lines studied, and confirmation in primary tumors was lacking 6

(21). Then, the study performed by Rocco et al. on twelve human primary GC failed to 7

demonstrate tumor-initiating properties of CD133+ and CD44+ sorted cells after xenograft in 8

both NOD/SCID and nude immunodeficient mice (22). 9

Another important point concerns the origin of the CSCs. As Houghton et al and our group 10

reported in mouse models of Helicobacter-induced gastric carcinogenesis, gastric dysplasia and 11

carcinoma may originate from the transformation of a local epithelial stem cell or of a bone-12

marrow (BM)-derived stem cell (23)(24)(25). In this model, dysplastic lesions were composed of 13

CD44+ cells, regardless of their BM or local origin (24). In addition, the heterogeneity of GCs 14

suggests that gastric CSC markers, if they indeed exist, may be different according to the origin 15

and/or the histological type of GC. 16

17

In this study, we performed an extensive screening of the expression of putative cell surface 18

markers of CSCs as well as ALDH activity in order to identify biomarkers allowing the detection 19

and isolation of tumorigenic and chemoresistant CSCs in human primary intestinal and diffuse 20

types non-cardia GC. 21

22





Materials and methods 1

Human samples and mouse xenografts 2

Fresh tumors samples were collected from gastric surgical wastes from patients who underwent 3

gastrectomy for non-cardia GC and for who informed consent was obtained. Fresh samples of 4

tumor and paired non-tumor tissues were transported in DMEM medium with 20% fetal calf 5

serum (FCS), 50 IU/ml penicillin, 50 µg/ml streptomycin, 50 µg/ml vancomycin and 15 µg/ml 6

amphotericin-B. Samples were minced in small pieces of 2 mm x 2 mm size, and were 7

subcutaneously transplanted into the right dorsal flank of 7-week-old male NSG mice under 8

2.5% isoflurane anesthesia (Belamont, Cournon, France). Alternatively, after mechanical 9

mincing, cells were dissociated by incubation in a solution of 1 mg/ml collagenase IV and 0.2 10

mg/ml hyaluronidase in DMEM (Sigma, Saint-Quentin Fallavier, France) for 1 h at 37°C with 11

shaking (15), then suspended in 100 μL of 7 mg/mL ice-cold Matrigel (BD Biosciences, Le Pont 12

de Claix, France) for subcutaneous injection. Xenografts were carried out within 3-5 hours 13

following gastrectomy. The tumor size was monitored with callipers once a week, and tumor 14

volume was estimated as (D2xd)/2 (D, large diameter; d, small diameter) (26). At the end of the 15

experiments (until 10 months post-engraftment for the primary xenograft) and when tumor 16

reached ~500 mm3, mice were sacrificed by cervical dislocation and tumors were immediately 17

harvested and processed for analyses. Secondary tumors were amplified subcutaneously in mice 18

by serial transplantation of pieces of tumor bulk, or by injection of tumor cells in Matrigel. For 19

xenograft experiments in extreme limiting dilution assay (ELDA), 10,000 to 30 FACS-sorted 20

cells were subcutaneously injected with Matrigel; tumor size was recorded twice a week. 21

22





GC cell lines 1

GC cell lines were cultured in DMEM/F12 media for AGS (ATCC CRL1739) and in RPMI1640 2

media for NCI87 (ATCC CRL-5822), MKN45, MKN74, MKN7 and MKN28 (all from RIKEN, 3

Japan) cells, supplemented with 10% heat-inactivated FCS, 50 IU/ml penicillin and 50 µg/ml 4

streptomycin (all from Invitrogen, Cergy-Pontoise, France) at 37°C in a 5% CO2 atmosphere 5

(26)(27). All cell lines were routinely verified mycoplasma free (by PCR) and were tested and 6

authenticated by STR profiling within 6 months preceding the experiments (last STR profiling 7

report, October 2015; LGC Standards, Teddington, UK). Cell viability was assessed by trypan 8

blue exclusion method. 9

10

Flow cytometry analysis 11

Cells were dissociated by collagenase/hyaluronidase procedure from fresh PDXs, passed through 12

a 70 µm mesh filter (BD), and red blood cells were removed by incubating in a solution 13

containing 2 mM KHCO3, 0.1 mM EDTA and 170 mM NH4Cl for 8 min at 4°C. Then, 100,000 14

cells in 100 µL PBS-0.5% bovine serum albumin (BSA) and 2 mM EDTA (Sigma) were stained 15

with 3-5 µL of fluorescent-labelled primary antibodies including EPCAM-FITC (Stem Cell 16

Technologies, Grenoble, France) or EPCAM-VioBlue (MACS-Miltenyi Biotec, Paris, France), 17

CD10-PE, CD24-PE, CD73-PE, CD49f-PE, CD105-PE, CD166-PE, CD90-PECy5, CD44-PE, 18

CD44-APC, CD338-APC (all from BD) and CD133-PE (MACS-Miltenyi Biotec, Paris, France) 19

for 20 min at 4°C. Cells were rinsed twice with PBS-0.5% BSA-2 mM EDTA containing 50 20

µg/ml 7-aminoactinomycin-D (7-AAD, BD) before being analysed using a FACSCanto II 21

instrument and DIVA software (BD) (26)(27). The ALDEFLUOR Kit (Stem Cell Technologies) 22





was used to detect ALDH activity according to the manufacturer's instructions. Dead cells were 1

excluded based on light scatter characteristics and 7-AAD positivity. For SP cells analysis, cells 2

were incubated with 10 µg/mL Hoechst-33342 in HBSS-2% FCS for 60 min at 37°C or, when 3

indicated, in ALDEFLUOR buffer for 30 min at room temperature, with or without 100 µM 4

verapamil or 50 µM reserpine (Sigma), then washed with ice cold HBSS-2% FCS. The Hoechst-5

33342 dye was excited at 375 nm, and its fluorescence was dual wavelength analyzed (blue, 402-6

446 nm; red, 650-670 nm) (28). Cell sorting was performed on 5-10 million cells stained with 7

primary fluorescent-labelled antibodies or ALDEFLUOR reagent and 7-AAD, on 7-AAD 8

negative cells using a FACSAria (BD). 9

10

Tumorsphere assay 11

1,000 FACS-sorted cells were plated in non-adherent 24 well plates (or alternatively 200 cells in 12

96 well plates) previously coated with a 10% poly(2-hydroxyethyl methacrylate) solution in 95% 13

ethanol (v/v) (Sigma), in DMEM-F12 media supplemented with 20 ng/ml human-epidermal 14

growth factor, 20 ng/ml basic-fibroblast growth factor, 5 µg/ml insulin, 0.3% glucose, 50 IU/ml 15

penicillin and 50 µg/ml streptomycin (Sigma) (26). For PDXs cells, the media was supplemented 16

with 5% FCS for the first 2 days of culture, and was then replaced by serum-free media. After 7 17

days, the number of spheroids/well was counted under light microscopy using a ×20 objective. 18

For drug treatment experiments, 5 days tumorspheres grown in non-adherent 96 well plates 19

(8


trypsin/EDTA procedure and seeded in new non-adherent 96 well plates (8


tumors xenografted, 8 led to the growth of secondary tumors serially transplantable in mice; 7 1

were intestinal type (GC04, GC07, GC10, GC35, GC40, GC44) and 1 was diffuse type (GC06) 2

(Supplementary Table 1). There was no significant association between PDX growth and the 3

following characteristics: patient's gender, age, preoperative treatment, WHO and Lauren 4

classifications of gastric tumor histological type, grade, TNM classification and stage 5

(Supplementary Table 1). PDXs reached a 500 mm3 size between 2 to 6 months (mean 16.7±3.4 6

weeks) after the first passage (P) (P1) in mice, and earlier following successive passages (after 7

10.9±5.9 weeks at P2 and 7.2±0.8 weeks at P5) (Figure 1B). Case GC42, a mucinous type 8

according to the WHO classification, was excluded because tumors developed slowly and were 9

mostly composed of mucus, rendering its study impossible. Histopathological analyses 10

confirmed that the PDXs obtained between P1 and P5 remained similar to the respective 11

patients’ primary tumor for all cases (Figure 1C) except GC07, which appeared to dedifferentiate 12

after P2 and therefore was excluded (data not shown). 13

14

Evaluation CD24, CD133 and CD44 cell surface markers expression on the patients' gastric 15

tissues and PDXs 16

The expression of CD24, CD133 and CD44 was evaluated by flow cytometry in live (7-AAD 17

negative) EPCAM+ epithelial cells dissociated from freshly collected paired non-tumor and 18

tumor gastric tissue samples from 7 cases. In non-tumor gastric tissues, CD24 and CD133 were 19

expressed in about half of the cells whereas CD44 was expressed only in 10±9% of cells. In 20

paired tumors, CD24, CD133 and CD44 expression was significantly higher compared to non-21

tumor cells, CD24 being expressed in most of the tumor cells (90±8%), CD133 in 71±17% and 22





CD44 in 27±17% of the tumor cells (Figure 2A). The percentage of tumor cells expressing these 1

markers were then evaluated on cells dissociated from freshly collected serial xenografts from P1 2

to P4 of the 6 PDXs cases (Figure 2B). The percentage of cells expressing the different markers 3

remained stable in serial PDXs from P1 to P4, EPCAM being expressed in most of the cells 4

(>80%) followed by CD24 and CD133 expressed at a relatively high proportion of cells, except 5

in GC35. In all cases, CD44 was expressed in less than a third of total cells. These results were 6

confirmed by immunohistochemistry analyses of the expression of CD44 on patient’s non-tumor 7

and tumor tissues and on the corresponding P2-3 PDXs for the 6 cases studied (Figure 2C). In 8

the non-tumor area, CD44 was expressed at a low level, preferentially in cells in the isthmus 9

region of gastric glands in the area of gastritis, as previously reported (26). In patients' tumors, 10

CD44 was expressed in some tumor cells but not all, mainly at the periphery of the tumor islets 11

for the intestinal type tumors. A similar pattern of CD44 expression was observed in PDXs, 12

confirming that the cellular heterogeneity of the primary tumor was reproduced in serial PDXs. 13

14

GC cells expressing CD133, CD166, CD44 and an ALDH activity have tumorigenic CSCs 15

properties 16

We then analyzed the expression of 7 additional cell surface markers as putative markers of 17

gastric CSCs, i.e. CD10, CD49f and CD166 described in CSCs of other organs (29)(30)(31), 18

CD73, CD90 and CD105 as the main markers of mesenchymal stem cells (32)(27), as well as 19

ALDH activity in cells from 5 PDXs (freshly collected at P2-P4) and 5 GC cell lines (7 intestinal 20

type: GC04, GC10, GC35, GC44, MKN74, MKN7, NCI87 ; and 3 diffuse type: GC06, AGS, 21

MKN45) (Figure 3A). CD10 was negative except for 2 of the 10 cases studied. CD49f was 22





expressed in more than 80% of the cells in all cases, similar to EPCAM. For the other markers, 1

the expression pattern was more homogeneous in PDXs than in cell lines which exhibited more 2

heterogeneity, being either highly positive or negative. CD133 was expressed only in PDXs cells 3

and not in GC cell lines. In PDXs, CD49f and CD24 were highly expressed, followed by CD133 4

and CD90 expressed in nearly half of the cells, then by CD73 expressed in more than a third of 5

the cells. At a level similar to CD44, CD166 was expressed in 21±13% of the cells. CD105 6

expression and ALDH activity were detected in only 9±6% and 8±5% of the cells, respectively 7

(Figure 3A). Results from flow cytometry co-staining analyses revealed that CD166 and CD44 8

were co-expressed and detected the same cells subpopulation (Figure 3B). The majority of 9

CD44+ cells were positive for CD24, CD133 and CD73. Less than 50% of CD44+ cells were 10

positive for ALDH, CD105 and CD90. Interestingly, ALDH activity but not CD90 and CD105 11

expression was recorded mainly in CD44+ cells, showing that ALDH+ cells representing a core 12

within the CD44+ subpopulation of cells (Figure 3B). 13

In order to evaluate the tumorigenic properties of the cells expressing or not CD44, CD133, 14

CD73, CD166, CD90, and CD105, P2-P3 tumors of 3 PDX cases, GC04, GC06 and GC10, were 15

freshly dissociated and 7-AAD-EPCAM+ cells either positive or negative for these markers were 16

sorted by FACS and submitted to the tumorsphere assay. Similar experiments were performed on 17

7-AAD- FACS-sorted cells based on ALDH activity. In all of the cases, EPCAM+CD133+, 18

EPCAM+CD44+, EPCAM+CD73+, EPCAM+CD166+, and ALDH+ cells formed significantly 19

more tumorspheres after 10 days of in vitro culture than their respective negative counterparts 20

(Figure 3C). CSCs forming tumorspheres were essentially present in CD44+, CD166+ and 21

ALDH+ subpopulations, and to a lesser extent in CD133+ and CD73+ subpopulations; they were 22





essentially CD90- and CD105-. The high tumorsphere capacity of ALDH+ cells was confirmed 1

on both MKN45 and MKN74 cell lines (Figure 3C). 2

To confirm these results in vivo, xenografts were performed in mice with 7-AAD-EPCAM+ 3

FACS-sorted cells based on the expression of CD133 and CD44 on GC04, GC06 and GC10 4

PDXs. The same experiments were performed based on ALDH activity on GC06, GC10, 5

MKN45 and MKN74 cells (Table 1). In all cases, tumors developed at a significant higher 6

frequency in EPCAM+CD44+ cells (1/29 to 1/1,020) than in their respective EPCAM+CD44- 7

cells (1/568 to 1/28,963), and in EPCAM+CD133+ cells (1/105 to 1/1,911) than in their 8

respective EPCAM+CD133- cells (1/781 to 1/66,876) (Table 1). CSC frequency was higher in 9

EPCAM+CD44+ cells than in EPCAM+CD133+ cells, confirming that CD44 is a better marker 10

of gastric CSCs than CD133, as also determined by the in vitro tumorsphere assays. ALDH+ 11

cells led to the development of tumors at a significant higher frequency than the respective 12

ALDH- cells (1/38 to 1/746 for ALDH+ cells versus 1/372 to 1/8,024 for ALDH- cells) for all 13

cases studied, with the exception of MKN45 which was highly tumorigenic at the doses studied 14

(Table1). For both PDXs cases GC10 (intestinal type) and GC06 (diffuse type), the CSC 15

frequencies were high in ALDH+ cells (1/81 and 1/38, respectively) and in EPCAM+CD44+ 16

cells (1/29 and 1/352, respectively) and both were higher than in EPCAM+CD133+ cells (1/105 17

and 1/1,658, respectively), suggesting that ALDH and CD44 are more specific markers of gastric 18

CSCs than CD133. Finally, we showed that the CSCs contained in EPCAM+CD44+ and 19

ALDH+ FACS-sorted cells generated tumors that recapitulated the phenotypic heterogeneity of 20

the initial tumors, giving rise both to CD44+ cells containing CSCs, and to more differentiated 21

CD44- cells (Figure 4A). The asymmetric division and differentiation properties of these CSCs 22





was confirmed in vitro with MKN45 cells; ALDH+ FACS-sorted cells were able to reproduce 1

heterogeneous tumorspheres in vitro, composed of a similar proportion of cells with ALDH+ 2

activity compared to the initial situation and expressing CD44, and of CD44+ALDH- and CD44-3

ALDH- cells incorporating the Hoechst-33342 stain (Figure 4B). In addition, the CD44+ALDH+ 4

FACS-sorted cells generated more tumorspheres than the CD44+ALDH- cells which generated 5

less but still a significant number of tumorspheres and which therefore could correspond to 6

progenitor/transit amplifying cells. The CD44-ALDH- cells may correspond to more 7

differentiated cells with very limited proliferation capacities as they formed significantly less or 8

no tumorspheres compared to the CD44+ALDH- and CD44+ALDH+ cells (Figure 4C). 9

10

ALDH+ CSCs are more resistant to conventional chemotherapy than ALDH- cells in GC 11

The combined analysis of CD44 expression, ALDH activity, and Hoechst-33342 incorporation 12

was assessed on live MKN45 tumorsphere (as in Figure 4B) during their development. In young, 13

small tumorspheres (after 5 days), most cells were positive for both CD44 and ALDH activity 14

and were negative for Hoechst-33342 (Supplementary Figure 1). When tumorspheres became 15

bigger (after 10-15 days), only a fraction of cells remained CD44+ALDH+ and Hoechst 33342-16

which may correspond to CSCs, with the appearance of CD44+ALDH- Hoechst 33342+ cells 17

which may correspond to progenitor/transit amplifying cells (Supplementary Figure 1). 18

An important property of CSCs is to be resistant to conventional therapies, leading to tumor 19

recurrence and metastasis after treatment (8)(9). Verapamil treatment, known to inhibit drug 20

efflux systems, restored Hoechst-33342 incorporation in ALDH+ cells in MKN45 and GC10 21

tumorspheres in vitro (Figure 5A). This effect was confirmed by flow cytometry analyses on 22





MKN45 cells, and confirmed at a lesser extent with reserpine, another inhibitor of drug efflux 1

systems (Figure 5B). The drug efflux properties of CSCs is usually assessed by the functional 2

analysis of the Side Population (SP), a minor subpopulation of cells defined by Hoechst-33342 3

stain efflux properties in specific experimental conditions. In these particular experimental 4

conditions, we found a consistent proportion of Hoechst- SP cells in MKN45 cell line (3.9±0.8 % 5

of total cells, Figure 5C) but not in MKN74 cell line (


conditions and that only ALDH+ cells and not ALDH- cells can form tumorspheres in vitro 1

(Figure 3C). In both cell lines, ALDH+ cells were more resistant than ALDH- cells to both 5-2

fluorouracil and doxorubicin treatments but not to cisplatin at the dose studied (Figure 5E). 3

Verapamil treatment sensitized ALDH+ cells to these chemotherapies (Figure 5E). This effect 4

was confirmed on the formation of tumorsphere by both cell lines, in which verapamil treatment 5

potentiated significantly the reduction of tumorsphere number in response to 5-fluorouracil, 6

doxorubicin and cisplatin treatments (Figure 5F). The same result was obtained on tumorspheres 7

of GC10 PDX (Supplementary Figure 4). Self-renewal assays with residual cells from MKN45 8

and MKN74 treated tumorspheres confirmed that the combination of verapamil with these 9

conventional chemotherapeutic drugs significantly reduced the number of tumorigenic CSCs 10

(Figure 5G). Altogether, these results indicate that, within the CD44+ subpopulation of tumor 11

cells, the determination of ALDH activity allows the detection and isolation of CSCs with 12

tumorigenic and chemoresistant properties in GC. 13

14

Discussion. 15

In this study, we characterized the expression of cell surface biomarkers and ALDH activity of 16

gastric CSCs in intestinal and diffuse type non-cardia GCs. 17

Contrary to the situation in colon cancer in which CD133+ cells containing colon CSCs 18

represent a small and rare subpopulation within tumors (13)(14), we demonstrated that CD133+ 19

cells (detected by similar experimental procedures) were frequent in GC. We also showed that 20

CD133 was a less specific marker for the enrichment of gastric CSCs than CD44 and ALDH, as 21

demonstrated by their lower capacity to form tumorspheres in vitro and a new tumor after 22





xenograft in vivo. CD44 was expressed in all patient derived primary GCs but not in the healthy 1

gastric mucosa or at a very low level in the isthmus of the corpus gastric glands where stem cells 2

reside. We and others previously reported that these CD44+ stem/progenitor cells expand from 3

the isthmus towards the base of the unit in metaplastic and dysplastic areas induced in response 4

to chronic H. pylori infection (24)(26)(33). This occurs via an epithelial-mesenchymal-like 5

transition, conferring CSC-like properties to CD44+ cells (26)(25). CD44 expression has been 6

reported in GC (34)(35)(36)(37). A recent study demonstrated that CD44 inhibition by peptide 7

inhibitors prevented the development of cellular hyperproliferation and chronic atrophic gastritis 8

in animal models of H. pylori-induced gastric carcinogenesis (38)(33). 9

Interestingly, we showed that CD166 was coexpressed with CD44 and, as a consequence, 10

CD166+ cells presented the same tumorigenic properties as CD44+ cells in vitro. Similar 11

analyses of in vitro co-expression with CD44 and tumorigenic properties led to the conclusion 12

that the gastric CSC phenotype corresponds to EPCAM+, CD24+, CD133+, CD73+, CD90-, 13

CD105-, CD166+, CD44+, associated with ALDH activity. Finally, in all PDXs studied, cells 14

with ALDH activity represented the smallest subpopulation of cells compared to all other 15

markers studied, with high tumorigenic properties both in vitro and in vivo, and with asymmetric 16

division and differentiation properties reproducing the heterogeneity of the initial tumors. As for 17

other cancers, we must consider that the gastric CSC phenotype may be plastic, subjected to 18

regulation by the surrounding tumor microenvironment in vivo. Recent work has demonstrated 19

that breast CSCs co-exist between two different phenotypic states: a more quiescent and 20

invasive, mesenchymal-like state characterized by a CD24-CD44+ phenotype and located mainly 21

at the tumor periphery and invasive front, and a more proliferative epithelial-like state, 22





characterized by ALDH activity and located more centrally (39)(9). However, unlike to the 1

situation in breast cancer, we have shown that gastric CSC express both CD44 and ALDH 2

activity, and that ALDH activity reveals a subpopulation within the CD44+ cells (see Figure 3B) 3

that possess the CSC properties, ie to generate a new heterogeneous tumor in vivo and 4

tumorsphere in vitro (Figure 4 and Figure 3C). 5

6

The ALDEFLUOR assay used to isolate CSCs in liquid and solid tumors detects the activity of 7

several isoforms of ALDH (40). Among them, the main isoforms expressed in tumors are 8

retinaldehyde dehydrogenases, ALDH1A1 and ALDH1A3, responsible for the oxidation of 9

retinal to retinoic acid and, to a lesser extent, ALDH3A1 (40)(20). They can metabolize and 10

detoxify chemotherapeutic agents such as cyclophosphamide in hematopoietic stem cells, and 11

their level of expression was shown to be predictive of response to treatment in breast cancer 12

(41)(42). In this study, we show that ALDH+ cells were more resistant to treatment with 13

conventional chemotherapeutic drugs than ALDH- cells. We also adapted the detection of 14

ALDH activity with the ALDEFLUOR reagent design for flow cytometry, to the detection by 15

fluorescent microscopy on live cells. Using both methods, we showed that these ALDH+ cells 16

did not incorporate the vital DNA dye Hoechst-33342 instead the ALDH- cells incorporated it, 17

confirming that ALDH+ cells may correspond to the side population of cells with CSCs 18

properties as previously described by Fukuda et al. in GC cell lines (28). The ability of ALDH+ 19

cells to efflux Hoechst-33342 and to resist conventional chemotherapy was reversed by 20

verapamil or reserpine treatment, two inhibitors of efflux pumps such as the ATP-binding 21

cassette (ABC) transporters family members, confirming that these cells are associated with 22





chemotherapy resistance as proposed in other cancers (43). In this study, we did not find a 1

noticeable coexpression of BCRP (ABCG2) and MDR-1 (not expressed), the two leaders of the 2

ABC transporters family, in ALDH+ gastric CSCs (flow cytometry and RT-qPCR analyses, data 3

not shown), suggesting that the Hoechst-33342 and drug efflux may result from the activity of 4

other members of the ABC transporters family. This family includes at least 49 genes grouped 5

into seven families, and at least 16 of these proteins have been implicated in cancer drug 6

resistance (44). 7

A limit to the use of ALDH as a biomarker of chemoresistant gastric CSC, is that ALDH activity 8

can be detected only by the ALDEFLUOR assay on live cells by flow cytometry or fluorescent 9

microscopy analyses. So, its detection as a biomarker of CSCs in current practice on patients' 10

specimens may be possible for circulating cancer cells and liquid cancers such as leukemia but 11

remain elusive for the analysis of solid tumors such as GC. These findings also imply that ALDH 12

isozymes can be considered not only as biomarkers of CSCs but also as putative targets to inhibit 13

tumor growth and to overcome resistance to cancer therapy. 14

It is of importance to note that GC PDXs always remained heterogeneous and composed of 15

tumor cell subpopulations expressing EPCAM, CD24, CD133 and CD44, similar to the patients’ 16

situation, while GC cell lines were found to be negative for CD133 and either positive or 17

negative for CD44 and others markers including ALDH. These results strengthen the importance 18

and the necessity to study CSC on models as close as possible to the patients’ situation, as is the 19

case in this study, and not only on cancer cell lines. 20

Currently, PDXs represent the most pertinent pre-clinical model to study the capacity of CSCs to 21

give rise to tumor growth, heterogeneity, and sensitivity/resistance to new treatment strategies. 22





However, PDXs models present some limitations, particularly because the contribution of the 1

patient’s tumor microenvironment _ including inflammation, CSCs niche within the organ of 2

origin, cross-talk with immune and stromal cells _ cannot be taken into account on CSCs 3

plasticity, tumor progression and metastasis. These limits are partly illustrated here by the low 4

tumor engraftment success of patient’s GC samples, being only ~20% of engraftment success, as 5

described by others for other type of cancers, unveiling the contribution of uncontrolled 6

microenvironment parameters for cancer propagation in the patient. Nevertheless, there is an 7

urgent unmet need of new, more efficient and better tolerated therapeutic strategies for GCs, that 8

could focus on gastric CSCs. 9

In this study, the development of original PDXs models allowed us to demonstrate that 10

tumorigenic and chemoresistant gastric CSCs co-express EPCAM, CD133, CD166, CD44 and 11

ALDH, ALDH activity being the most specific biomarker of CSC enrichment before CD44 in 12

both diffuse and intestinal type non-cardia GCs. This finding led to the hypothesis that treatment 13

strategy for non-cardia GCs can focus on CD44+ALDH+ CSCs, independently of the 14

histological classification of the tumor. 15

16

17

References 18

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38. Bertaux-Skeirik N, Feng R, Schumacher MA, Li J, Mahe MM, Engevik AC, et al. CD44 1 Plays a Functional Role in Helicobacter pylori-induced Epithelial Cell Proliferation. PLoS 2 Pathog 2015;11: e1004663. 3 39. Liu S, Cong Y, Wang D, Sun Y, Deng L, Liu Y, et al. Breast cancer stem cells transition 4 between epithelial and mesenchymal states reflective of their normal counterparts. Stem Cell 5 Reports 2014;2: 78-91. 6 40. Marcato P, Dean CA, Giacomantonio CA, Lee PW. Aldehyde dehydrogenase: its role as 7 a cancer stem cell marker comes down to the specific isoform. Cell Cycle 2011;10: 1378-84. 8 41. Magni M, Shammah S, Schiro R, Mellado W, Dalla-Favera R, Gianni AM. Induction of 9 cyclophosphamide-resistance by aldehyde-dehydrogenase gene transfer. Blood 1996;87: 1097-10 103. 11 42. Sladek NE, Kollander R, Sreerama L, Kiang DT. Cellular levels of aldehyde 12 dehydrogenases (ALDH1A1 and ALDH3A1) as predictors of therapeutic responses to 13 cyclophosphamide-based chemotherapy of breast cancer: a retrospective study. Rational 14 individualization of oxazaphosphorine-based cancer chemotherapeutic regimens. Cancer 15 Chemother Pharmacol 2002;49: 309-21. 16 43. Raha D, Wilson TR, Peng J, Peterson D, Yue P, Evangelista M, et al. The cancer stem 17 cell marker aldehyde dehydrogenase is required to maintain a drug-tolerant tumor cell 18 subpopulation. Cancer Res 2014;74: 3579-90. 19 44. Di C, Zhao Y. Multiple drug resistance due to resistance to stem cells and stem cell 20 treatment progress in cancer (Review). Exp Ther Med 2015;9: 289-93. 21 22 23 24 25 Figure Legends. 26

Figure 1. Establishment of mouse xenograft models using primary tumors from patients 27

with non-cardia GC. 28

A. Schematic representation of the strategy used to collect, analyze and perform serial tumor 29

xenografts in NSG immunodeficient mice from primary non-cardia GC freshly collected from 30

patients who underwent gastrectomy. At each passage (P) in mice, histology and flow cytometry 31

analyses were performed respectively on tissue samples and on cells freshly dissociated by 32

enzymatic procedures. In vitro tumorsphere assays and in vivo CSC frequency determination 33

were monitored after cell sorting by FACS of cell subpopulations based on the expression of 34

EPCAM, CD133, CD44 and ALDH activity following the second passage in mice (P2). B. 35





Number of weeks when a tumor size reached 500 mm3 after serial transplantation in mice (from 1

P1 to P5). Histograms represent the mean ± SD for each case, and numbers represent the global 2

mean ± SD of all cases (1


cell lines (CLs: AGS, MKN28, MKN45, MKN74 and NCI87) of diffuse and intestinal types. 1

Bars, median. B. Representative dot-plot analyses of CD24, CD133, CD73, CD166, CD90 and 2

CD105 stained with PE and PECy5-labeled antibodies and ALDH activity determined by 3

ALDEFLUOR assay in combination with anti-CD44/APC antibodies on GC10 cells (top panel). 4

Quantification of the percentage of positive cells for the different markers in the PDXs (5


nuclei staining with Hoechst-33342 (in blue), and of flow cytometry analysis (right panel) of 1

CD44 stained with anti-CD44/APC antibodies and ALDH activity detected by ALDEFLUOR 2

reagent. Scale bar, 25 µm. C. Cells from MKN45, NCI-87 and GC07 PDX were stained as in B 3

and with 7-AAD (to exclude 7AAD+ dead cells) and anti-EPCAM/VioBlue antibodies for GC07 4

cells dissociated from a fresh PDX (to select EPCAM+ carcinoma cells). The 7AAD-(EPCAM+) 5

cells were sorted by FACS on the expression of CD44 and ALDH activity and submitted to the 6

tumorsphere assay. Plots (min to max) represent the number of tumorspheres formed per 200 7

cells seeded per well after 5 to 8 days of culture (n=10 per condition). *, p


and Hoechst+ (MP-like) cells, detected after 30 min incubation in ALDEFLUOR buffer at 37°C, 1

then 30 min incubation with Hoechst-33342 at RT (n=3). E. Percentage of viable ALDH+ (black 2

bars) and ALDH- (white bars) FACS-sorted MKN45 and MKN74 cells after 48 h of adherent 3

culture and treatment without (control, CT) or with 10 µM verapamil with or without 50 µM 5-4

fluorouracil (5-FU), 1 µM doxorubicin (DOXO) or 50 µM cisplatin. B-E, Results represent the 5

mean ± SD. F-G. Plots (min to max) represent the number of MKN45 and MKN74 tumorspheres 6

formed: F, after a 48 h treatment of 5 days tumorspheres without (control, CT) or with verapamil 7

(dotted bars), 5-FU, DOXO and cisplatin as in E; G, after 5 days by cells dissociated from 8

residual treated tumorspheres (from experiment described in F). E-G, 8


Table 1: Gastric cancer-initiating cell frequencies for the markers CD133, CD44 and ALDH determined after tumor 1

xenografts in limiting dilutions in NSG immunodeficient mice. 2

Number of tumours/number of transplanted mice Number of transplanted cells

CASE Marker 10,000 3,000 1,000 300 100 30 Gastric cancer-initiating cell frequencies (95% Confidence interval)

Test for difference in stem cell frequencies between positive and negative cells

1 EPCAM+CD133+ 5/5 4/5 1/5 2/5 0/5 1/1,911 (1/4,019-1/908) p

0

5

10

15

20

25

30

35

P1 P2 P3 P4 P5

GC04

GC06

GC07

GC10

GC35

GC40

GC42

GC44

2-6 months

Cell sorting by FACS

Xenografts / ELDA

First

xenograft

EPCAM+/CD133+ EPCAMA+/CD133

- EPCAM+/CD44+ EPCAM+/CD44-

Primary

tumor from

patient (n=37)

Histological analyses

Flow cytometry analyses

Secondary tumors

(P1, n=8)

Second xenograft

Serial tumors (P2 to P5)

A B

Figure 1

Tumorsphere assay

5-15 weeks

16.7±3.4

10.9±5.9

10.6±6.9

7.9±1.0 7.2±0.8

GC04 GC06 GC10 GC35 GC40 GC44

Patient

tumor

Tumor

xenograft

C

EPCAM+/ALDH+ EPCAM+/ALDH-

N

um

ber

of

weeks f

or

tum

or

develo

pm

en

t aft

er

xen

og

raft




Figure 2

A

CD24 CD133 CD44

Healthy Tumor Healthy Tumor Healthy Tumor

0

50

100

150

* *

*

Pe

rcenta

ge o

f p

ositiv

e c

ells

GC44 P1 P2 P3 P4 P1 P2 P3 P4 P1 P2 P3 P4 P1 P2 P1 P2 P3 P1 P2 P3

GC40 GC35 GC10 GC06 GC04

Cu

mu

late

d p

erc

enta

ges

of p

ositiv

e c

ells

B

C GC04 GC06 GC10 GC35 GC40 GC44

Patient’s

distant

mucosa

Patient’s

Tumor

Tumor

xenograft

EPCAM CD24 CD133 CD44




78.2

2.0

0.0

86.7 EPCAM-FITC

CD

44-A

PC

+ DEAB - DEAB

ALDH activity

0.4 10.1

C

Figure 3

Tu

mors

phere

num

ber

(per

1000 c

ells

)

CD

44-

CD

44+

CD

73+

CD

73-

CD

166+

CD

166-

CD

90+

CD

90-

CD

105+

CD

105-

ALD

H+

ALD

H- 0

50

100

150

GC04 GC06 GC10

* * *

# #

CD

133+

CD

133-

*

A CLs (Cancer cell Lines), intestinal

PTs (Primary Tumors), intestinal

EPCAM CD24 CD133 CD44 CD10 CD49f CD73 CD166 CD90 CD105 ALDH

CL

s

PT

s

CL

s

PT

s

CL

s

PT

s

CL

s

PT

s

CL

s

PT

s

CL

s

PT

s

CL

s

PT

s

CL

s

PT

s

CL

s

PT

s

CL

s

PT

s

CL

s

PT

s 0

20

40

60

80

100

120 P

erc

enta

ge

of

positiv

e c

ells

CLs – diffuse

PTs – diffuse

CD

44-A

PC

EPCAM-FITC

ALD

H+

ALD

H- 0

20

40

60

80

100 MKN45 MKN74

Tum

ors

phere

num

ber

(per

500 c

ells

) *

B 2.2 17.5

77.3 3.0

34.4 8.1

42.7 14.8

6.0 1.7

67.6 24.7

2.3 10.8

73.3 13.6 CD

16

6-P

E

CD

90

-PE

Cy5

CD

10

5-P

E

AL

DH

13.3 15.7

57.9 9.1

CD

73

-PE

39.5 25.7

33.5 1.3

CD

24

44.7 23.0

29.9 2.4

CD

24

-PE

CD

13

3-P

E

CD44-APC

PTs - intestinal

PTs - diffuse

Perc

enta

ge o

f cells

20

40

60

80

100

0




B

C

A GC10 GC04 GC06 MKN45 MKN74

Tumors obtained with

ALDH+ sorted cells

Tumors

obtained with

CD44+/high

sorted cells

Figure 4

13.8 84.0

ALDH activity

CD

44

-AP

C

0.1 2.1

11.4%

+ DEAB - DEAB

SS

C-A

ALDH activity

0.0% ALDH+

sorted cells

ALDH activity - + - - + - - +

CD44 + + - + + - + +

MKN45 NCI-N87 GC07

0.8 0.0

80.0 19.2

CD

44

-AP

C

ALDH activity

sorted cells

ALDH activity (green) CD44 (red) merge

*

* * *

*

* *




B

-

Ver

apam

il

Res

erpi

ne

0

10

20

30

40

*

Ho

ech

st

neg

ati

ve s

ub

po

pu

lati

on

C

3.9±0.8 0.6±0.1 1.2±0.2

+Verapamil +Reserpine

E

G Self-renewal of treated cells

Figure 5

E Viability 48h post-treatment MKN45 MKN74

F Tumorsphere formation 48h post-treatment MKN45 MKN74

MKN45 MKN74

D

+ D

EA

B

1.7±0.5

24.6±3.5

SS

C-A

ALDH activity

0.1

- DE

AB




Published OnlineFirst September 12, 2016.Clin Cancer Res Phu Hung Nguyen, Julie Giraud, Lucie Chambonnier, et al. chemoresistant cancer stem cells in human gastric carcinomaCharacterization of biomarkers of tumorigenic and

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