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Identification and Characterization of CXCR4-Positive Gastric Cancer Stem Cells.

Fujita T, Chiwaki F, Takahashi RU, Aoyagi K, Yanagihara K, Nishimura T, Tamaoki M, Komatsu M, Komatsuzaki R, Matsusaki K, Ichikawa H, Sakamoto H, Yamada Y, Fukagawa T, Katai H, Konno H, Ochiya T, Yoshida T, Sasaki H - PLoS ONE (2015)

Bottom Line: In clinical samples, these CXCR4-positive cells were found from not only late metastasis stage (accumulated ascites) but also earlier stage (peritoneal washings).Moreover, treatment with transforming growth factor-β enhanced the anti-cancer effect of docetaxel via induction of cell differentiation/asymmetric cell division of the CXCR4-positive gastric CSCs even in a dormant state.Therefore, differentiation inducers hold promise for obtaining the maximum therapeutic outcome from currently available anti-cancer drugs through re-cycling of CSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Oncology, National Cancer Center Research Institute, Tokyo, Japan; Second Department of Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan.

ABSTRACT
Diffuse-type solid tumors are often composed of a high proportion of rarely proliferating (i.e., dormant) cancer cells, strongly indicating the involvement of cancer stem cells (CSCs) Although diffuse-type gastric cancer (GC) patients have a poor prognosis due to high-frequent development of peritoneal dissemination (PD), it is limited knowledge that the PD-associated CSCs and efficacy of CSC-targeting therapy in diffuse-type GC. In this study, we established highly metastatic GC cell lines by in vivo selection designed for the enrichment of PD-associated GC cells. By microarray analysis, we found C-X-C chemokine receptor type 4 (CXCR4) can be a novel marker for highly metastatic CSCs, since CXCR4-positive cells can grow anchorage-independently, initiate tumors in mice, be resistant to cytotoxic drug, and produce differentiated daughter cells. In clinical samples, these CXCR4-positive cells were found from not only late metastasis stage (accumulated ascites) but also earlier stage (peritoneal washings). Moreover, treatment with transforming growth factor-β enhanced the anti-cancer effect of docetaxel via induction of cell differentiation/asymmetric cell division of the CXCR4-positive gastric CSCs even in a dormant state. Therefore, differentiation inducers hold promise for obtaining the maximum therapeutic outcome from currently available anti-cancer drugs through re-cycling of CSCs.

No MeSH data available.


Related in: MedlinePlus

Tumorigenicity of CXCR4+ small cells sorted from 60As6 and primary cultured GC cells.(A) Representative images of anchorage independent growth of CXCR4+ small cells and CXCR4- small cells in colony formation assay (left). Quantitative analysis of cell growth using CyQuant GR dye (right) (n = 3, mean + SE; ***p<0.001). Scale bars represent 200μm. (B) Tumor formation frequency of CXCR4+ small cells and CXCR4- large cells in xenograft mice (Bi). The lower panels show representative macroscopic findings of multiple tumor nodules (Bii, arrowhead) along with the intestine and bloody ascites (Biii) in a SCID/SCID mouse. (C) RT-PCR (left panel) and immunocytochemical staining (right panels) of CXCR4 (green) and CXCR7 (green) in primary cultured GC cells (NSC-20C). Scale bar represents 200μm. (D) Flow cytometric analysis in primary cultured GC cells (NSC-20C) (Di) and sorted CXCR4+ cells after cultivating for 14 days (Dii). Colony formation assay of CXCR4+ and CXCR4- small cells derived from NSC-20C cells (n = 3, mean + SE; **p<0.005) (Diii).
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pone.0130808.g003: Tumorigenicity of CXCR4+ small cells sorted from 60As6 and primary cultured GC cells.(A) Representative images of anchorage independent growth of CXCR4+ small cells and CXCR4- small cells in colony formation assay (left). Quantitative analysis of cell growth using CyQuant GR dye (right) (n = 3, mean + SE; ***p<0.001). Scale bars represent 200μm. (B) Tumor formation frequency of CXCR4+ small cells and CXCR4- large cells in xenograft mice (Bi). The lower panels show representative macroscopic findings of multiple tumor nodules (Bii, arrowhead) along with the intestine and bloody ascites (Biii) in a SCID/SCID mouse. (C) RT-PCR (left panel) and immunocytochemical staining (right panels) of CXCR4 (green) and CXCR7 (green) in primary cultured GC cells (NSC-20C). Scale bar represents 200μm. (D) Flow cytometric analysis in primary cultured GC cells (NSC-20C) (Di) and sorted CXCR4+ cells after cultivating for 14 days (Dii). Colony formation assay of CXCR4+ and CXCR4- small cells derived from NSC-20C cells (n = 3, mean + SE; **p<0.005) (Diii).

Mentions: Next, we validated the anchorage independency and tumor-initiating ability of the two small cell subpopulation (I and II). In the colony formation assay, CXCR4+ small cells formed many more colonies than did the CXCR4- small cells (Fig 3A). Serial dilution experiments of intraperitoneal inoculation in immunodeficient mice revealed that CXCR4+ small cells also possessed a higher tumorigenic ability (tumor development in 4/5 and 1/5 mice upon inoculation of 105 and 104 cells, respectively) compared with CXCR4- small cells (1/10 and 0/10 mice following 105 and 104 cells, respectively) (Fig 3Bi). Notably, all five tumor-bearing mice after inoculation of CXCR4+ small cells had multiple tumor nodules (Fig 3Bii), and began to develop bloody ascites within 3 weeks (Fig 3Biii).


Identification and Characterization of CXCR4-Positive Gastric Cancer Stem Cells.

Fujita T, Chiwaki F, Takahashi RU, Aoyagi K, Yanagihara K, Nishimura T, Tamaoki M, Komatsu M, Komatsuzaki R, Matsusaki K, Ichikawa H, Sakamoto H, Yamada Y, Fukagawa T, Katai H, Konno H, Ochiya T, Yoshida T, Sasaki H - PLoS ONE (2015)

Tumorigenicity of CXCR4+ small cells sorted from 60As6 and primary cultured GC cells.(A) Representative images of anchorage independent growth of CXCR4+ small cells and CXCR4- small cells in colony formation assay (left). Quantitative analysis of cell growth using CyQuant GR dye (right) (n = 3, mean + SE; ***p<0.001). Scale bars represent 200μm. (B) Tumor formation frequency of CXCR4+ small cells and CXCR4- large cells in xenograft mice (Bi). The lower panels show representative macroscopic findings of multiple tumor nodules (Bii, arrowhead) along with the intestine and bloody ascites (Biii) in a SCID/SCID mouse. (C) RT-PCR (left panel) and immunocytochemical staining (right panels) of CXCR4 (green) and CXCR7 (green) in primary cultured GC cells (NSC-20C). Scale bar represents 200μm. (D) Flow cytometric analysis in primary cultured GC cells (NSC-20C) (Di) and sorted CXCR4+ cells after cultivating for 14 days (Dii). Colony formation assay of CXCR4+ and CXCR4- small cells derived from NSC-20C cells (n = 3, mean + SE; **p<0.005) (Diii).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4481351&req=5

pone.0130808.g003: Tumorigenicity of CXCR4+ small cells sorted from 60As6 and primary cultured GC cells.(A) Representative images of anchorage independent growth of CXCR4+ small cells and CXCR4- small cells in colony formation assay (left). Quantitative analysis of cell growth using CyQuant GR dye (right) (n = 3, mean + SE; ***p<0.001). Scale bars represent 200μm. (B) Tumor formation frequency of CXCR4+ small cells and CXCR4- large cells in xenograft mice (Bi). The lower panels show representative macroscopic findings of multiple tumor nodules (Bii, arrowhead) along with the intestine and bloody ascites (Biii) in a SCID/SCID mouse. (C) RT-PCR (left panel) and immunocytochemical staining (right panels) of CXCR4 (green) and CXCR7 (green) in primary cultured GC cells (NSC-20C). Scale bar represents 200μm. (D) Flow cytometric analysis in primary cultured GC cells (NSC-20C) (Di) and sorted CXCR4+ cells after cultivating for 14 days (Dii). Colony formation assay of CXCR4+ and CXCR4- small cells derived from NSC-20C cells (n = 3, mean + SE; **p<0.005) (Diii).
Mentions: Next, we validated the anchorage independency and tumor-initiating ability of the two small cell subpopulation (I and II). In the colony formation assay, CXCR4+ small cells formed many more colonies than did the CXCR4- small cells (Fig 3A). Serial dilution experiments of intraperitoneal inoculation in immunodeficient mice revealed that CXCR4+ small cells also possessed a higher tumorigenic ability (tumor development in 4/5 and 1/5 mice upon inoculation of 105 and 104 cells, respectively) compared with CXCR4- small cells (1/10 and 0/10 mice following 105 and 104 cells, respectively) (Fig 3Bi). Notably, all five tumor-bearing mice after inoculation of CXCR4+ small cells had multiple tumor nodules (Fig 3Bii), and began to develop bloody ascites within 3 weeks (Fig 3Biii).

Bottom Line: In clinical samples, these CXCR4-positive cells were found from not only late metastasis stage (accumulated ascites) but also earlier stage (peritoneal washings).Moreover, treatment with transforming growth factor-β enhanced the anti-cancer effect of docetaxel via induction of cell differentiation/asymmetric cell division of the CXCR4-positive gastric CSCs even in a dormant state.Therefore, differentiation inducers hold promise for obtaining the maximum therapeutic outcome from currently available anti-cancer drugs through re-cycling of CSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Oncology, National Cancer Center Research Institute, Tokyo, Japan; Second Department of Surgery, Hamamatsu University School of Medicine, Shizuoka, Japan.

ABSTRACT
Diffuse-type solid tumors are often composed of a high proportion of rarely proliferating (i.e., dormant) cancer cells, strongly indicating the involvement of cancer stem cells (CSCs) Although diffuse-type gastric cancer (GC) patients have a poor prognosis due to high-frequent development of peritoneal dissemination (PD), it is limited knowledge that the PD-associated CSCs and efficacy of CSC-targeting therapy in diffuse-type GC. In this study, we established highly metastatic GC cell lines by in vivo selection designed for the enrichment of PD-associated GC cells. By microarray analysis, we found C-X-C chemokine receptor type 4 (CXCR4) can be a novel marker for highly metastatic CSCs, since CXCR4-positive cells can grow anchorage-independently, initiate tumors in mice, be resistant to cytotoxic drug, and produce differentiated daughter cells. In clinical samples, these CXCR4-positive cells were found from not only late metastasis stage (accumulated ascites) but also earlier stage (peritoneal washings). Moreover, treatment with transforming growth factor-β enhanced the anti-cancer effect of docetaxel via induction of cell differentiation/asymmetric cell division of the CXCR4-positive gastric CSCs even in a dormant state. Therefore, differentiation inducers hold promise for obtaining the maximum therapeutic outcome from currently available anti-cancer drugs through re-cycling of CSCs.

No MeSH data available.


Related in: MedlinePlus