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

Fukamachi H, Seol HS, Shimada S, Funasaka C, Baba K, Kim JH, Park YS, Kim MJ, Kato K, Inokuchi M, Kawachi H, Yook JH, Eishi Y, Kojima K, Kim WH, Jang SJ, Yuasa Y - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

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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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.
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pone-0072438-g003: 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.

Mentions: When HGC-1 gastric tumor cells were cultured after FACS-sorting, the cloning efficiency was usually less than 10%, but CD49fhigh cells attached to collagen gel to form spheres (Figure 3A), whereas cell growth was not observed in culture of CD49flow cells (Figure 3B). These spheres were consisted of epithelial cells closely attached to each other with occasional PAS-positive droplets (Figure 3C). It was difficult to cultivate FACS-sorted HGC-2 and HGC-4 cells, and we were not certain whether only CD49fhigh cells could form spheres in their culture. We thus concluded that some gastric TICs proliferated to form spheres in primary culture as has been reported in other TICs.


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

Fukamachi H, Seol HS, Shimada S, Funasaka C, Baba K, Kim JH, Park YS, Kim MJ, Kato K, Inokuchi M, Kawachi H, Yook JH, Eishi Y, Kojima K, Kim WH, Jang SJ, Yuasa Y - PLoS ONE (2013)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756075&req=5

pone-0072438-g003: 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.
Mentions: When HGC-1 gastric tumor cells were cultured after FACS-sorting, the cloning efficiency was usually less than 10%, but CD49fhigh cells attached to collagen gel to form spheres (Figure 3A), whereas cell growth was not observed in culture of CD49flow cells (Figure 3B). These spheres were consisted of epithelial cells closely attached to each other with occasional PAS-positive droplets (Figure 3C). It was difficult to cultivate FACS-sorted HGC-2 and HGC-4 cells, and we were not certain whether only CD49fhigh cells could form spheres in their culture. We thus concluded that some gastric TICs proliferated to form spheres in primary culture as has been reported in other TICs.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

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.

Show MeSH
Related in: MedlinePlus