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. 2013 Aug 28;8(8):e72438.
doi: 10.1371/journal.pone.0072438. eCollection 2013.

CD49f(high) cells retain sphere-forming and tumor-initiating activities in human gastric tumors

Hiroshi Fukamachi  1 , Hyang Sook Seol, Shu Shimada, Chikako Funasaka, Kanako Baba, Ji Hun Kim, Young Soo Park, Mi Jeung Kim, Keiji Kato, Mikito Inokuchi, Hiroshi Kawachi, Jeong Hwan Yook, Yoshinobu Eishi, Kazuyuki Kojima, Woo Ho Kim, Se Jin Jang, Yasuhito Yuasa
Affiliations

CD49f(high) cells retain sphere-forming and tumor-initiating activities in human gastric tumors

Hiroshi Fukamachi et al. PLoS One. .

Abstract

Identification of gastric tumor-initiating cells (TICs) is essential to explore new therapies for gastric cancer patients. There are reports that gastric TICs can be identified using the cell surface marker CD44 and that they form floating spheres in culture, but we could not obtain consistent results with our patient-derived tumor xenograft (PDTX) cells. We thus searched for another marker for gastric TICs, and found that CD49f(high) cells from newly-dissected gastric cancers formed tumors with histological features of parental ones while CD49f(low) cells did not when subcutaneously injected into immunodeficient mice. These results indicate that CD49f, a subunit of laminin receptors, is a promising marker for human gastric TICs. We established a primary culture system for PDTX cells where only CD49f(high) cells could grow on extracellular matrix (ECM) to form ECM-attaching spheres. When injected into immunodeficient mice, these CD49f(high) sphere cells formed tumors with histological features of parental ones, indicating that only TICs could grow in the culture system. Using this system, we found that some sphere-forming TICs were more resistant than gastric tumor cell lines to chemotherapeutic agents, including doxorubicin, 5-fluorouracil and doxifluridine. There was a patient-dependent difference in the tumorigenicity of sphere-forming TICs and their response to anti-tumor drugs. These results suggest that ECM plays an essential role for the growth of TICs, and that this culture system will be useful to find new drugs targeting gastric TICs.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CD49fhigh cells form tumors while CD49flow cells do not.
(A) A moderately-differentiated adenocarcinoma from patient #8 and (B) a poorly-differentiated one from patient #9 in Table 1 were dissociated, and FACS-sorted cells were subcutaneously injected into NOD-SCID mice to examine their tumorigenicity. Histological features of parental tumors (left) correspond well with those of tumors (right) formed by injection of sorted cells (shown by red rectangles in FACS analysis). Tissue specimens are stained with hematoxylin and eosin. Scale bars represent 100 µm.
Figure 2
Figure 2. Localization of CD49f in gastric tissues.
(A–C) CD49f is localized at the epithelial-stromal interface in (A) normal mucosa, (B) chronic gastritis and (C) intestinal metaplasia. (D–G) In (D) gastric dysplasia adjacent to carcinoma and (E–G) gastric tumor tissues, expression of CD49f is diversified: It is localized at the epithelial-stromal interface in some cells (G), but on apical, lateral and basal surfaces in some cells (E, arrows), and expression can hardly be detected in some cells (E, arrowheads). Scale bars represent 50 µm.
Figure 3
Figure 3. CD49fhigh cells form spheres in primary culture.
(A) Phase contrast micrographs showing the growth of spheres in culture of CD49fhigh HGC-1 tumor cells on collagen gel. The same area on days 8, 10 and 14 are shown. It is clear that spheres in the white circles grow rapidly in culture. Scale bar represents 200 µm. (B) The increase in diameters of spheres in culture of CD49fhigh HGC-1 tumor cells, shown in (A). (C) Light micrographs of spheres formed by culture of CD49fhigh HGC-1 tumor cells for 2 weeks. Tissue specimens are stained with PAS-hematoxylin. Scale bar represents 100 µm. (D) Phase contrast micrographs showing the growth of spheres in culture of unsorted HGC-4 tumor cells on collagen gel. The same area on days 2, 6, and 12 are shown. Scale bar represents 200 µm. (E) Phase contrast micrographs showing the growth of spheres in culture of unsorted HGC-2 tumor cells on collagen gel. Many flat cells closely attach to ECM to form a thin layer. Sphere-forming cells (arrows) grow slowly on the flat cells to form large spheres at 6 weeks. Scale bar represents 500 µm. (F) Changes in the ratio of CD49fhigh cells in total cells in culture of sorted CD49fhigh HGC-1 tumor cells (for 2 weeks), unsorted HGC-1 tumor cells (for 2 weeks), unsorted HGC-4 tumor cells (for 2 weeks) and unsorted HGC-2 tumor cells (for 8 weeks). Significant increases in the ratio of CD49fhigh cells are always found in culture of unsorted tumor cells. ***, P<0.001; *, P<0.05 by Student’s t-test.
Figure 4
Figure 4. Sphere cells form tumors with histological features of parental ones.
Light micrographs of tumors formed by subcutaneous injection of sphere cells into NOD-SCID mice (right panels), which are obtained by cultures of (A) unsorted HGC-1, (B) unsorted HGC-2 and (C) unsorted HGC-4 tumor cells. Histological features of tumors formed in mice correspond well with those of parental ones (left panels). Tissue specimens are stained with hematoxylin and eosin. Scale bars represent 50 µm.
Figure 5
Figure 5. Some TICs are more resistant to anti-tumor drugs than gastric tumor cell lines and other TICs.
HGC-1, HGC-2 and HGC-4 tumor cells, and MKN45 and MKN74 tumor cell lines are seeded on day 0, treated with various concentrations of (A) doxorubicin, (B) 5-fluorouracil, and (C) doxifluridine from days 1 to 14, and cell numbers on day 14 are determined by MTT assay. Black and blue asterisks show that HGC-1 and/or HGC-4 cells are significantly (**, P<0.01; *, P<0.05 by Student’s t-test) more resistant to the drugs than MKN45 and MKN74 cell lines, respectively, while pink and brown asterisks indicate that HGC-4 cells are significantly (**, P<0.01; *, P<0.05 by Student’s t-test) more resistant to the drugs than HGC-1 and HGC-2 cells, respectively.
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