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. 2011 Nov;68(21):3589-605.
doi: 10.1007/s00018-011-0672-z. Epub 2011 Mar 30.

Cancer spheres from gastric cancer patients provide an ideal model system for cancer stem cell research

Myoung-Eun Han  1 , Tae-Yong Jeon, Sun-Hwi Hwang, Young-Suk Lee, Hyun-Jung Kim, Hye-Eun Shim, Sik Yoon, Sun-Yong Baek, Bong-Seon Kim, Chi-Dug Kang, Sae-Ock Oh
Affiliations

Cancer spheres from gastric cancer patients provide an ideal model system for cancer stem cell research

Myoung-Eun Han et al. Cell Mol Life Sci. 2011 Nov.

Abstract

Cancer stem cells have been hypothesized to drive the growth and metastasis of tumors. Because they need to be targeted for cancer treatment, they have been isolated from many solid cancers. However, cancer stem cells from primary human gastric cancer tissues have not been isolated as yet. For the isolation, we used two cell surface markers: the epithelial cell adhesion molecule (EpCAM) and CD44. When analyzed by flow cytometry, the EpCAM(+)/CD44(+) population accounts for 4.5% of tumor cells. EpCAM(+)/CD44(+) gastric cancer cells formed tumors in immunocompromised mice; however, EpCAM(-)/CD44(-), EpCAM(+)/CD44(-) and EpCAM(-)/CD44(+) cells failed to do so. Xenografts of EpCAM(+)/CD44(+) gastric cancer cells maintained a differentiated phenotype and reproduced the morphological and phenotypical heterogeneity of the original gastric tumor tissues. The tumorigenic subpopulation was serially passaged for several generations without significant phenotypic alterations. Moreover, EpCAM(+)/CD44(+), but not EpCAM(-)/CD44(-), EpCAM(+)/CD44(-) or EpCAM(-)/CD44(+) cells grew exponentially in vitro as cancer spheres in serum-free medium, maintaining the tumorigenicity. Interestingly, a single cancer stem cell generated a cancer sphere that contained various differentiated cells, supporting multi-potency and self-renewal of a cancer stem cell. EpCAM(+)/CD44(+) cells had greater resistance to anti-cancer drugs than other subpopulation cells. The above in vivo and in vitro results suggest that cancer stem cells, which are enriched in the EpCAM(+)/CD44(+) subpopulation of gastric cancer cells, provide an ideal model system for cancer stem cell research.

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Conflict of interest statement

There are no conflicts of interest.

Figures

Fig. 1
Fig. 1
EpCAM/CD44 expression profiles in primary gastric cancers and normal gastric mucosa. a The pattern of EpCAM/CD44 expression in primary gastric cancers and normal gastric mucosa was compared. To minimize experimental variability and contributions of genetic background, primary cancers (right panels) were compared with autologous normal mucosa (left panels) for patients 15 and 3 (Table 1), and analyzed on the same day. The upper two panels were derived from the intestinal type of gastric cancer (patient 15) and lower two panels from the diffuse type of gastric cancer (patient 3). The percentages reported in flow plots indicate the percentage of cells contained within gate P2. b Comparison of EpCAM/CD44 expression between the intestinal and the diffuse types of gastric cancers. Data are the mean ± SE of 19 patients (*P < 0.05, Student's t test)
Fig. 2
Fig. 2
Xenografts generated from EpCAM+/CD44+ gastric cancer cells resemble the original patient tumor (patient 6). The histology of the two tumors, as examined by H&E staining, shows similar structures. The immunohistochemical staining using various markers, including MUC5AC, MUC6, Pan-cytokeratin (Pan-CK) and CD44, showed comparable staining patterns in both the xenograft and parental tumors. In the invasive front of gastric cancer, CD44 staining was similarly observed in both tumors. Production of two gastric mucins (MUC5AC and MUC6) was also similar in both tumors. Scale bar = 200 µm
Fig. 3
Fig. 3
The tumorigenic potential was restricted to the EpCAM+/CD44+ subpopulation. The tumorigenic potential of freshly isolated EpCAM+/CD44+, EpCAM−/CD44− and unseparated (total) gastric cancer cells was evaluated after subcutaneous injection of 104 cells in matrigel into immunocompromised mice. No tumor growth was observed on injection of EpCAM−/CD44− cells (a, circled area, patient 6). Data concerning tumor volumes are the mean ± SE of five independent experiments in duplicate (b)
Fig. 4
Fig. 4
Long-term tumorigenic potential of gastric cancer EpCAM+/CD44+ cells. a Analysis of tumors grown from injection of sorted EpCAM+/CD44+ cells showed reconstitution of parental expression profiles (patient 2). Percentage reported in flow plots indicated the percentage of cells contained within gate P2. b Tumorigenic potential of gastric cancer cells derived by tumors induced by injection of 106 freshly isolated EpCAM+/CD44+ cells (secondary xenografts), gastric cancer cells derived by such secondary xenografts (tertiary xenografts) and gastric cancer cells derived by tertiary xenografts (quaternary xenografts). Data are the mean ± SE of three independent experiments in duplicate
Fig. 5
Fig. 5
The tumorigenic potential of EpCAM+/CD44+ gastric cancer cells is lost upon differentiation. a Microscopic analysis of gastric cancer spheres (patient 16) cultivated in differentiation condition for 7 days. CD44 expression in undifferentiated and differentiated gastric cancer cells was analyzed by immunocytochemistry. Before immunocytochemistry, undifferentiated cancer spheres were disaggregated into single cells by trypsin treatment. b Tumorigenic potential of cancer spheres and differentiated EpCAM+/CD44+ gastric cancer cells. Differentiated cells are not CD44-postive. Tumor volumes of mice injected with 104 cells are shown. Data are the mean ± SE of three independent experiments in duplicate
Fig. 6
Fig. 6
Representative example of a sphere formation originating from a single cancer stem cell. After FACS using CD44 and EpCAM antibody, a single cancer stem cell was cultured in a well of 96-well plate, and images were taken at the indicated times (patient 16). Original magnifications: days 1–3, 40× magnification; days 4–5, 20× magnification; days 6–7, 10× magnification; and day 8, 4× magnification
Fig. 7
Fig. 7
Differentiation status of cancer spheres. a Cancer spheres (patient 16) were dehydrated and processed for paraffin block. The sections were stained with the indicated markers and analyzed. Images are representative of spheres in three independent experiments. Scale bar = 20 µm. b TEM images of cancer spheres. Microvilli, interceullar junctions (arrow) and various kinds of vesicles were observed. Scale bar = 1 µm
Fig. 8
Fig. 8
Gastric cancer stem cells resist chemotherapy. Each subpopulation EpCAM+/CD44+, EpCAM+/CD44−, EpCAM−/CD44− and EpCAM−/CD44+ was isolated from cancer spheres, and the chemo-sensitivity was assessed in each subpopulation. Cells were treated with various concentrations of anti-cancer drugs including 5-fluorouracil (5-FU, a), doxorubicin (b), vinblastine (c) and paclitaxel (d) for 48 h. Data are the mean ± SE of three independent experiments in quintuplicate [*P < 0.01, one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison]
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References

    1. Han ME, Lee YS, Baek SY, Kim BS, Kim JB, Oh SO. Hedgehog signaling regulates the survival of gastric cancer cells by regulating the expression of bcl-2. Int J Mol Sci. 2009;10(7):3033–3043. doi: 10.3390/ijms10073033. - DOI - PMC - PubMed
    1. Yuasa Y. Control of gut differentiation and intestinal-type gastric carcinogenesis. Nat Rev Cancer. 2003;3(8):592–600. doi: 10.1038/nrc1141. - DOI - PubMed
    1. Correia M, Machado JC, Ristimaki A. Basic aspects of gastric cancer. Helicobacter. 2009;14(Suppl 1):36–40. doi: 10.1111/j.1523-5378.2009.00696.x. - DOI - PubMed
    1. Vogiatzi P, Vindigni C, Roviello F, Renieri A, Giordano A. Deciphering the underlying genetic and epigenetic events leading to gastric carcinogenesis. J Cell Physiol. 2007;211(2):287–295. doi: 10.1002/jcp.20982. - DOI - PubMed
    1. Wu CW, Hsieh MC, Lo SS, Lui WY, P’Eng FK. Results of curative gastrectomy for carcinoma of the distal third of the stomach. J Am Coll Surg. 1996;183(3):201–207. - PubMed

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