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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

TGF-β promotes differentiation of CXCR4+ small cells and enhances the effect of anti-tumor drugs.(A) Smad2/3 nuclear localization (Ai, green) and CXCR4 expression (Aii) in TGF-β-treated 60As6 cells analyzed by immunohistochemistry and RT-PCR. Nuclei were counterstained with DAPI (blue). Scale bars represent 50μm. (B) Flow cytometry analysis of 60As6 cells with TGF-β treatment under a normal nutrient condition (10% FBS). (C) Caspase 3/7 assay in 60As6 cells with or without TGF-β treatment (n = 3, mean + SE; *p<0.01). (D) Flow cytometry analyses of 60As6 cells with TGF-β treatment under a serum starvation (0% FBS) for 5 days. Dot plotting of the cells (Di). Propidium iodide-based cell cycle analysis (Dii). (e) Flow cytometry analyses of 60As6 cells with TGF-β treatment under a serum starvation (0% FBS) for 3 days. Dot plotting of the cells (Ei). Propidium iodide-based cell cycle analysis (Eii). (F) The viability of 60As6 cells after Doc treatment with or without TGF-β (n = 3, mean + SE; ***p<0.001).
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pone.0130808.g006: TGF-β promotes differentiation of CXCR4+ small cells and enhances the effect of anti-tumor drugs.(A) Smad2/3 nuclear localization (Ai, green) and CXCR4 expression (Aii) in TGF-β-treated 60As6 cells analyzed by immunohistochemistry and RT-PCR. Nuclei were counterstained with DAPI (blue). Scale bars represent 50μm. (B) Flow cytometry analysis of 60As6 cells with TGF-β treatment under a normal nutrient condition (10% FBS). (C) Caspase 3/7 assay in 60As6 cells with or without TGF-β treatment (n = 3, mean + SE; *p<0.01). (D) Flow cytometry analyses of 60As6 cells with TGF-β treatment under a serum starvation (0% FBS) for 5 days. Dot plotting of the cells (Di). Propidium iodide-based cell cycle analysis (Dii). (e) Flow cytometry analyses of 60As6 cells with TGF-β treatment under a serum starvation (0% FBS) for 3 days. Dot plotting of the cells (Ei). Propidium iodide-based cell cycle analysis (Eii). (F) The viability of 60As6 cells after Doc treatment with or without TGF-β (n = 3, mean + SE; ***p<0.001).

Mentions: TGF-β has been reported to diminish the side population of some diffuse-type GC cell lines [34]. Therefore, we investigated whether TGF-β has a potential to change the characteristics of CXCR4+ diffuse-type GC cells. We first examined the expression levels of TGF-β receptors in 60As6 cells and GC primary cultures derived from two independent patients (NSC-16C and -22C). RT-PCR experiments showed that both of two TGF-β receptor genes (TGFBR1 and TGFBR2) expressed in all of these cells (S6 Fig). We also examined the localization of SMAD2/3, since these transcriptional factors are widely known to accumulate in the nuclei in response to the activation of TGF-β signaling. As shown in Fig 6Ai, the nuclear accumulation of SMAD2/3 was detected in 60As6 cells after a 30 min treatment with 2 ng/ml TGF-β.


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)

TGF-β promotes differentiation of CXCR4+ small cells and enhances the effect of anti-tumor drugs.(A) Smad2/3 nuclear localization (Ai, green) and CXCR4 expression (Aii) in TGF-β-treated 60As6 cells analyzed by immunohistochemistry and RT-PCR. Nuclei were counterstained with DAPI (blue). Scale bars represent 50μm. (B) Flow cytometry analysis of 60As6 cells with TGF-β treatment under a normal nutrient condition (10% FBS). (C) Caspase 3/7 assay in 60As6 cells with or without TGF-β treatment (n = 3, mean + SE; *p<0.01). (D) Flow cytometry analyses of 60As6 cells with TGF-β treatment under a serum starvation (0% FBS) for 5 days. Dot plotting of the cells (Di). Propidium iodide-based cell cycle analysis (Dii). (e) Flow cytometry analyses of 60As6 cells with TGF-β treatment under a serum starvation (0% FBS) for 3 days. Dot plotting of the cells (Ei). Propidium iodide-based cell cycle analysis (Eii). (F) The viability of 60As6 cells after Doc treatment with or without TGF-β (n = 3, mean + SE; ***p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4481351&req=5

pone.0130808.g006: TGF-β promotes differentiation of CXCR4+ small cells and enhances the effect of anti-tumor drugs.(A) Smad2/3 nuclear localization (Ai, green) and CXCR4 expression (Aii) in TGF-β-treated 60As6 cells analyzed by immunohistochemistry and RT-PCR. Nuclei were counterstained with DAPI (blue). Scale bars represent 50μm. (B) Flow cytometry analysis of 60As6 cells with TGF-β treatment under a normal nutrient condition (10% FBS). (C) Caspase 3/7 assay in 60As6 cells with or without TGF-β treatment (n = 3, mean + SE; *p<0.01). (D) Flow cytometry analyses of 60As6 cells with TGF-β treatment under a serum starvation (0% FBS) for 5 days. Dot plotting of the cells (Di). Propidium iodide-based cell cycle analysis (Dii). (e) Flow cytometry analyses of 60As6 cells with TGF-β treatment under a serum starvation (0% FBS) for 3 days. Dot plotting of the cells (Ei). Propidium iodide-based cell cycle analysis (Eii). (F) The viability of 60As6 cells after Doc treatment with or without TGF-β (n = 3, mean + SE; ***p<0.001).
Mentions: TGF-β has been reported to diminish the side population of some diffuse-type GC cell lines [34]. Therefore, we investigated whether TGF-β has a potential to change the characteristics of CXCR4+ diffuse-type GC cells. We first examined the expression levels of TGF-β receptors in 60As6 cells and GC primary cultures derived from two independent patients (NSC-16C and -22C). RT-PCR experiments showed that both of two TGF-β receptor genes (TGFBR1 and TGFBR2) expressed in all of these cells (S6 Fig). We also examined the localization of SMAD2/3, since these transcriptional factors are widely known to accumulate in the nuclei in response to the activation of TGF-β signaling. As shown in Fig 6Ai, the nuclear accumulation of SMAD2/3 was detected in 60As6 cells after a 30 min treatment with 2 ng/ml TGF-β.

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