FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2116661033> ?p ?o ?g. }

• W2116661033 endingPage "1799" @default.
• W2116661033 startingPage "1790" @default.
• W2116661033 abstract "Cancer recurrence has been suggested to be induced by residual cancer-initiating cells (CICs) or cancer stem cells (CSCs) after chemotherapy. Moreover, it is possible that CICs/CSCs acquire more aggressive behavior after therapy as shown by invasion and metastasis. In the cancer microenvironment, CICs/CSCs may localize in a specific area, the so-called stem cell niche, and isolation of this niche is important to elucidate the molecular mechanism of how CICs/CSCs acquire malignancy. We analyzed whether CICs acquire drug resistance after cancer drug treatment in a tumor cell allograft model in which we could identify and isolate living CICs by detecting a higher level of transcriptional activity of the PSF1 gene promoter. In our models using Lewis lung carcinoma (LLC) mouse lung cancer and colon26 mouse colon cancer cell lines, we found that CICs in both tumors acquired drug resistance after cancer drug treatment. Interestingly, response to the anticancer drug was quite different between LLC and colon26 original tumors (ie, the proportion of CICs in LLC tumors increased but in colon26 tumors the proportion decreased). We found that CICs frequently localized near mature blood vessels in which endothelial cells were covered with mural cells and that the incidence of mature blood vessels in LLC tumors was four times higher than in colon26 tumors. These results suggest a relationship between mature blood vessels and CIC drug resistance. Cancer recurrence has been suggested to be induced by residual cancer-initiating cells (CICs) or cancer stem cells (CSCs) after chemotherapy. Moreover, it is possible that CICs/CSCs acquire more aggressive behavior after therapy as shown by invasion and metastasis. In the cancer microenvironment, CICs/CSCs may localize in a specific area, the so-called stem cell niche, and isolation of this niche is important to elucidate the molecular mechanism of how CICs/CSCs acquire malignancy. We analyzed whether CICs acquire drug resistance after cancer drug treatment in a tumor cell allograft model in which we could identify and isolate living CICs by detecting a higher level of transcriptional activity of the PSF1 gene promoter. In our models using Lewis lung carcinoma (LLC) mouse lung cancer and colon26 mouse colon cancer cell lines, we found that CICs in both tumors acquired drug resistance after cancer drug treatment. Interestingly, response to the anticancer drug was quite different between LLC and colon26 original tumors (ie, the proportion of CICs in LLC tumors increased but in colon26 tumors the proportion decreased). We found that CICs frequently localized near mature blood vessels in which endothelial cells were covered with mural cells and that the incidence of mature blood vessels in LLC tumors was four times higher than in colon26 tumors. These results suggest a relationship between mature blood vessels and CIC drug resistance. A recent concept has suggested that the recurrence of cancer is induced by cancer-initiating cells (CICs)/cancer stem cells (CSCs) that are resistant to conventional chemoradiotherapy.1Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Abstract Full Text Full Text PDF PubMed Scopus (41049) Google Scholar, 2Gupta P.B. Chaffer C.L. Weinberg R.A. Cancer stem cells: mirage or reality?.Nat Med. 2009; 15: 1010-1012Crossref PubMed Scopus (796) Google Scholar They are defined as malignant cancer cells. Moreover, cells in this population have a greater ability to spread to other organs to form metastatic lesions and to digest matrices for invasion even during chemotherapy. In addition, a poor prognosis in a diverse set of human and mouse malignancies was associated with the expression of an embryonic stem cell–like genetic program,3Ben-Porath I. Thomson M.W. Carey V.J. Ge R. Bell G.W. Regev A. Weinberg R.A. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors.Nat Genet. 2008; 40: 499-507Crossref PubMed Scopus (1927) Google Scholar, 4Wong D.J. Liu H. Ridky T.W. Cassarino D. Segal E. Chang H.Y. Module map of stem cell genes guides creation of epithelial cancer stem cells.Cell Stem Cell. 2008; 2: 333-344Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar, 5Somervaille T.C. Matheny C.J. Spencer G.J. Iwasaki M. Rinn J.L. Witten D.M. Chang H.Y. Shurtleff S.A. Downing J.R. Cleary M.L. Hierarchical maintenance of MLL myeloid leukemia stem cells employs a transcriptional program shared with embryonic rather than adult stem cells.Cell Stem Cell. 2009; 4: 129-140Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar suggesting that CSCs express an embryonic stem cell–specific genetic code. There is no doubt that CSC death is crucial for a permanent cancer cure.It is widely accepted that CSCs/CICs have multidrug resistance and radiotherapy resistance mediated by activation of DNA damage responses.6Bao S. Wu Q. McLendon R.E. Hao Y. Shi Q. Hjelmeland A.B. Dewhirst M.W. Bigner D.D. Rich J.N. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.Nature. 2006; 444: 756-760Crossref PubMed Scopus (4762) Google Scholar In addition, the association of CSCs/CICs with the expression of multidrug-resistant genes was reported.7Frank N.Y. Schatton T. Frank M.H. The therapeutic promise of the cancer stem cell concept.J Clin Invest. 2010; 120: 41-50Crossref PubMed Scopus (509) Google Scholar For example, ABCB5 is well known to play a role in drug resistance, and is one of a number of molecules proposed as a marker of CSC melanoma.7Frank N.Y. Schatton T. Frank M.H. The therapeutic promise of the cancer stem cell concept.J Clin Invest. 2010; 120: 41-50Crossref PubMed Scopus (509) Google Scholar, 8Loebinger M.R. Giangreco A. Groot K.R. Prichard L. Allen K. Simpson C. Bazley L. Navani N. Tibrewal S. Davies D. Janes S.M. Squamous cell cancers contain a side population of stem-like cells that are made chemosensitive by ABC transporter blockade.Br J Cancer. 2008; 98: 380-387Crossref PubMed Scopus (107) Google Scholar In a human breast cancer cell line, ABCB1 is co-regulated with CD44 expression (one of the CSC markers).9Miletti-González K.E. Chen S. Muthukumaran N. Saglimbeni G.N. Wu X. Yang J. Apolito K. Shih W.J. Hait W.N. Rodríguez-Rodríguez L. The CD44 receptor interacts with P-glycoprotein to promote cell migration and invasion in cancer.Cancer Res. 2005; 65: 6660-6667Crossref PubMed Scopus (183) Google Scholar Moreover, it was reported that CD133, which is used as a stem cell marker, promotes up-regulation of ABCB1 and higher ABC transporter activity in rat glioma cells.10Angelastro J.M. Lamé M.W. Overexpression of CD133 promotes drug resistance in C6 glioma cells.Mol Cancer Res. 2010; 8: 1105-1115Crossref PubMed Scopus (71) Google Scholar Another possibility is that cancer stromal cells in the tumor microenvironment prevent CSC cell death by several cues through the interaction between CSCs and stromal cells. In organs and tissues, the stem cell population localizes in a specific area, the so-called niche, and the interaction between stem cells and cells composing the niche is important to maintain the stemness of stem cells, such as self-renewal, immature status, and dormancy in such a niche.11Cabarcas S.M. Mathews L.A. Farrar W.L. The cancer stem cell niche–there goes the neighborhood?.Int J Cancer. 2011; 129: 2315-2327Crossref PubMed Scopus (195) Google ScholarIn the field of hematopoiesis, the niche has been analyzed extensively. The vascular niche was suggested to be important for self-renewal of hematopoietic stem cells (HSCs) because proliferation of HSCs was observed in vascular areas in embryos and in adult bone marrow.12Kiel M.J. Morrison S.J. Maintaining hematopoietic stem cells in the vascular niche.Immunity. 2006; 25: 862-864Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 13Mikkola H.K. Orkin S.H. The journey of developing hematopoietic stem cells.Development. 2006; 133: 3733-3744Crossref PubMed Scopus (378) Google Scholar On the other hand, HSC quiescence is induced in the osteoblastic niche where osteoblasts adhere to endosteum in the bone marrow.14Adams G.B. Scadden D.T. The hematopoietic stem cell in its place.Nat Immunol. 2006; 7: 333-337Crossref PubMed Scopus (359) Google Scholar Several tumor models have proposed that CSCs also may localize in vascular areas.15Calabrese C. Poppleton H. Kocak M. Hogg T.L. Fuller C. Hamner B. Oh E.Y. Gaber M.W. Finklestein D. Allen M. Frank A. Bayazitov I.T. Zakharenko S.S. Gajjar A. Davidoff A. Gilbertson R.J. A perivascular niche for brain tumor stem cells.Cancer Cell. 2007; 11: 69-82Abstract Full Text Full Text PDF PubMed Scopus (1728) Google Scholar, 16Nagahama Y. Ueno M. Miyamoto S. Morii E. Minami T. Mochizuki N. Saya H. Takakura N. PSF1, a DNA replication factor expressed widely in stem and progenitor cells, drives tumorigenic and metastatic properties.Cancer Res. 2010; 70: 1215-1224Crossref PubMed Scopus (44) Google Scholar In a skin cancer model, vascular endothelial growth factor derived from CSCs expanded the vascular area, including the number of blood vessels, and resulted in the proliferation of CSCs.17Beck B. Driessens G. Goossens S. Youssef K.K. Kuchnio A. Caauwe A. Sotiropoulou P.A. Loges S. Lapouge G. Candi A. Mascre G. Drogat B. Dekoninck S. Haigh J.J. Carmeliet P. Blanpain C. A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumours.Nature. 2011; 478: 399-403Crossref PubMed Scopus (340) Google Scholar On the other hand, although not connected with the localization of CSCs, it has been suggested that hepatocyte growth factor derived from myofibroblasts maintains the stemness of CSCs in colon cancer indirectly via the frizzled/Wnt pathway and that myofibroblast-secreted factors play pivotal roles in the induction of cancer cells into malignant CSCs.18Vermeulen L. De Sousa E. Melo F. van der Heijden M. Cameron K. de Jong J.H. Borovski T. Tuynman J.B. Todaro M. Merz C. Rodermond H. Sprick M.R. Kemper K. Richel D.J. Stassi G. Medema J.P. Wnt activity defines colon cancer stem cells and is regulated by the microenvironment.Nat Cell Biol. 2010; 12: 468-476Crossref PubMed Scopus (1377) Google Scholar Myofibroblasts are α-smooth muscle actin (α-SMA)–positive cells that usually localize near blood vessels and may work as a mural cell population. Therefore, based on previous reports, the vascular niche in which endothelial cells are covered with α-SMA–positive cells may maintain the stemness of CSCs; however, this has not been verified yet. Therefore, it is important to evaluate whether mural cell–covered blood vessels work as a niche for CSC/CICs.For the evaluation of drug resistance, CSCs or CICs should be isolated as living cells from the tumor that has remained after therapy and it should be observed whether those cell types indeed do form secondary tumors. Several methods have been suggested for the detection of CSCs/CICs (ie, surface expression of CD133, surface-high expression of CD44 and negative-low expression of CD24, and high aldehyde dehydrogenase activity). However, data obtained by these methods have not always resulted in the same conclusion in mice. Therefore, we have generated a new technique to identify CICs by using promoter activity of the partner of sld five 1 (Psf1) gene (PSF1-promoter), a member of the GINS complex (from the Japanese go-ichi-ni-san meaning 5-1-2-3, derived from four related subunits of the complex Sld5, Psf1, Psf2 and Psf3), which is required for DNA replication by associating with CDC45 in yeast.19Takayama Y. Kamimura Y. Okawa M. Muramatsu S. Sugino A. Araki H. GINS, a novel multiprotein complex required for chromosomal DNA replication in budding yeast.Genes Dev. 2003; 17: 1153-1165Crossref PubMed Scopus (278) Google Scholar PSF1 protein is composed of coiled-coil, arginine-rich, and PEST-like domains from the N to the C terminus, and the C-terminal domain of PSF1 is crucial for chromatin binding and replication activity.20Kamada K. Kubota Y. Arata T. Shindo Y. Hanaoka F. Structure of the human GINS complex and its assembly and functional interface in replication initiation.Nat Struct Mol Biol. 2007; 14: 388-396Crossref PubMed Scopus (71) Google Scholar PSF1 expression was observed abundantly in bone marrow, thymus, and testis in mice.21Ueno M. Itoh M. Kong L. Sugihara K. Asano M. Takakura N. PSF1 is essential for early embryogenesis in mice.Mol Cell Biol. 2005; 25: 10528-10532Crossref PubMed Scopus (66) Google Scholar Immature cell populations and stem cell populations express PSF1 [ie, epiblasts during embryogenesis, bone marrow hematopoietic stem cell populations, sperm stem cells (spermatogonia), and others] in mice.21Ueno M. Itoh M. Kong L. Sugihara K. Asano M. Takakura N. PSF1 is essential for early embryogenesis in mice.Mol Cell Biol. 2005; 25: 10528-10532Crossref PubMed Scopus (66) Google Scholar, 22Han Y. Ueno M. Nagahama Y. Takakura N. Identification and characterization of stem cell-specific transcription of PSF1 in spermatogenesis.Biochem Biophys Res Commun. 2009; 380: 609-613Crossref PubMed Scopus (14) Google Scholar, 23Ueno M. Itoh M. Sugihara K. Asano M. Takakura N. Both alleles of PSF1 are required for maintenance of pool size of immature hematopoietic cells and acute bone marrow regeneration.Blood. 2009; 113: 555-562Crossref PubMed Scopus (32) Google Scholar Based on the phenotypes of mutant mice, PSF1 expression was required for acute proliferation of immature cell types such as epiblasts in embryos21Ueno M. Itoh M. Kong L. Sugihara K. Asano M. Takakura N. PSF1 is essential for early embryogenesis in mice.Mol Cell Biol. 2005; 25: 10528-10532Crossref PubMed Scopus (66) Google Scholar and HSCs after bone marrow ablation.23Ueno M. Itoh M. Sugihara K. Asano M. Takakura N. Both alleles of PSF1 are required for maintenance of pool size of immature hematopoietic cells and acute bone marrow regeneration.Blood. 2009; 113: 555-562Crossref PubMed Scopus (32) Google Scholar The PSF1 in mice comprises the GINS complex, as observed in yeast. Therefore, it is possible that PSF1 is involved in the formation of DNA replication in mammals; however, the function of PSF1 has not been clarified in mice yet.We found that promoter activity of the Psf1 gene correlated with cancer cell malignancy.16Nagahama Y. Ueno M. Miyamoto S. Morii E. Minami T. Mochizuki N. Saya H. Takakura N. PSF1, a DNA replication factor expressed widely in stem and progenitor cells, drives tumorigenic and metastatic properties.Cancer Res. 2010; 70: 1215-1224Crossref PubMed Scopus (44) Google Scholar When cells containing high or low levels of PSF1-promoter activity were compared, PSF1-promoterhigh cancer cells had high proliferative activity, serial transplantation potential, and metastatic ability. Moreover, PSF1-promoterhigh cancer cells displayed embryonic stem cell–like gene expression signatures. These results suggest that PSF1-promoterhigh cancer cells are CICs. In the present work, we analyzed the in vitro and in vivo drug resistance of PSF1-promoterhigh cancer cells and analyzed the microenvironment that supports the drug resistance of PSF1-promoterhigh cancer cells.Materials and MethodsCell CultureLewis lung carcinoma (LLC) cells or colon26 (mouse colon cancer) cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO) or RPMI 1640 (Sigma-Aldrich), respectively, with 10% fetal bovine serum (Equitech-Bio, Kerville, TX) and penicillin/streptomycin. Colon26 cells or LLC cells expressing enhanced green fluorescent protein (EGFP) under the transcriptional control of the PSF1-promoter (colon26-PSF1p-EGFP or LLC-PSF1p-EGFP) were generated as previously reported.16Nagahama Y. Ueno M. Miyamoto S. Morii E. Minami T. Mochizuki N. Saya H. Takakura N. PSF1, a DNA replication factor expressed widely in stem and progenitor cells, drives tumorigenic and metastatic properties.Cancer Res. 2010; 70: 1215-1224Crossref PubMed Scopus (44) Google ScholarMiceSeven- to 8-week-old C57BL/6 female mice (for LLC experiments), BALB/c female mice of the same age (for colon26 experiments), and 6- to 7-week-old KSN female nude mice were purchased from Japan SLC (Shizuoka, Japan). All animal studies were approved by the Osaka University Animal Care and Use Committee. Subcutaneous allografts were established by injecting 106 cells into the backs of mice. Tumor volume was measured with a caliper and calculated according to the following formula: V = width × width × length × 0.5.Anticancer Agent AdministrationOn day 7 after subcutaneous inoculation of cancer cells as described earlier, mice were treated with intraperitoneal injections of saline, 60 mg/kg body weight 5-fluorouracil (5-FU; Kyowa Hakko Kirin, Tokyo, Japan), or 5 mg/kg body weight cisplatin (Bristol-Myers, Tokyo, Japan) every other day. LLC-PSF1p-EGFP–bearing mice were administered five treatments with 5-FU and the other mice received four treatments with either saline or cisplatin. Flow cytometric analysis or fluorescence-activated cell sorting was performed 3 days after the last administration of an anticancer agent.Flow Cytometric Analysis and Cell SortingMice were euthanized and tumor tissues were excised, minced, and digested with Dispase II (Godo Shusei, Corp., Chiba, Japan) and collagenase (Wako, Osaka, Japan) with continuous shaking at 37°C. Single-cell suspensions of tumor cells were prepared using a standard protocol.16Nagahama Y. Ueno M. Miyamoto S. Morii E. Minami T. Mochizuki N. Saya H. Takakura N. PSF1, a DNA replication factor expressed widely in stem and progenitor cells, drives tumorigenic and metastatic properties.Cancer Res. 2010; 70: 1215-1224Crossref PubMed Scopus (44) Google Scholar Flow cytometric analysis was performed using a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ), and cell sorting was performed using a FACSAria (Becton Dickinson). For the EGFP-high cell population, 5% of the most brightly fluorescing cells were sorted, and for the EGFP-low cell population, 5% of the least fluorescent cells were sorted. We used parental LLC or colon26 as negative controls.In Vitro Colony Formation Analysis and in Vivo Tumor Initiation AnalysisIsolated cells were plated at a concentration of 500 cells per dish on 10-cm culture dishes and cultured in culture media with or without various doses of anticancer agents. For in vivo experiments, sorted cells were suspended in 100 μL of PBS with growth factor–reduced Matrigel (BD Biosciences, San Jose, CA) and injected subcutaneously into the mice. Tumorigenic ability was judged for 8 weeks after inoculation of tumor cells.ImmunohistochemistryDissected tissues were fixed in 4% paraformaldehyde and dehydrated in methanol. Fixed specimens were embedded in polyester wax (VWR International, Ltd., Lutterworth, UK) and cut into 7-μm sections as described.24Takakura N. Watanabe T. Suenobu S. Yamada Y. Noda T. Ito Y. Satake M. Suda T. A role for hematopoietic stem cells in promoting angiogenesis.Cell. 2000; 102: 199-209Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar The staining procedure of tissue sections was almost the same as previously reported.25Takakura N. Huang X.L. Naruse T. Hamaguchi I. Dumont D.J. Yancopoulos G.D. Suda T. Critical role of the TIE2 endothelial cell receptor in the development of definitive hematopoiesis.Immunity. 1998; 9: 677-686Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar Rabbit anti-GFP antibody (Invitrogen, Eugene, OR), rat anti-CD31 antibody (BD Biosciences), and mouse anti–α-SMA antibody (Sigma-Aldrich) were used for primary antibodies.For the immunohistochemical analyses, biotin-conjugated goat anti-rabbit Ig (Dako, Carpinteria, CA) or biotin-conjugated goat anti-rat Ig (Invitrogen) was used as a secondary antibody. The Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) was used to amplify the target antigen signal before the visualization of horseradish peroxidase by diaminobenzidine (Dojindo, Kumamoto, Japan). The 5-bromo-4-chloro-3-indoxyl phosphate and nitro blue tetrazolium chloride substrate system (Dako) was used for the visualization of alkaline phosphatase.For the immunofluorescence analyses, Alexa Fluor 488–conjugated IgGs (Invitrogen) or streptavidin-allophycocyanin (APC) conjugate (BD Pharmingen, Franklin Lakes, NJ) was used as a second-step reagent. Images were acquired using a DFC 500 digital camera (Leica Microsystems, Nussloch, Germany) and processed with the Leica application suite and Adobe Photoshop CS2 software (Tokyo, Japan). All images shown are representative of three to six independent experiments.Statistical AnalysisResults are expressed as the means ± SEM. The Student’s t-test was used for statistical analysis except for the cumulative survival rate data, for which the log-rank test was used. Differences were considered statistically significant when the P value was less than 0.05.ResultsPSF1 Promoter Activity Does Not Affect Drug Sensitivity Directly in Colon26 and LLC CellsWe previously designated PSF1-promoterhigh cancer cells as a population of CICs/CSCs in a mouse tumor cell allograft model because they had higher tumorigenic, invasive, and metastatic abilities compared with PSF1-promoterlow cancer cells and had an embryonic stem cell–specific immature gene signature.16Nagahama Y. Ueno M. Miyamoto S. Morii E. Minami T. Mochizuki N. Saya H. Takakura N. PSF1, a DNA replication factor expressed widely in stem and progenitor cells, drives tumorigenic and metastatic properties.Cancer Res. 2010; 70: 1215-1224Crossref PubMed Scopus (44) Google Scholar First, we observed whether PSF1-promoterhigh cancer cells showed drug resistance. We used colon cancer (colon26) cell lines and lung cancer (LLC) cell lines that stably expressed EGFP under the transcriptional control of the PSF1-promoter (colon26- and LLC-PSF1p-EGFP, respectively). In these cells, the intensity of EGFP is correlated with endogenous PSF1 mRNA expression.16Nagahama Y. Ueno M. Miyamoto S. Morii E. Minami T. Mochizuki N. Saya H. Takakura N. PSF1, a DNA replication factor expressed widely in stem and progenitor cells, drives tumorigenic and metastatic properties.Cancer Res. 2010; 70: 1215-1224Crossref PubMed Scopus (44) Google Scholar After inoculation of colon26- or LLC-PSF1p-EGFP, tumors were dissected and EGFP-high (5% most bright) or EGFP-low (5% least bright) cancer cells were sorted by a fluorescence-activated cell sorter (Figure 1, A and B). Sorted cells were cultured for 10 days in culture media containing various doses of 5-FU. As previously reported, EGFP-high cells formed significantly larger colonies, as well as a higher number of colonies, compared with EGFP-low cells for both colon26 and LLC cells (Figure 1, C and D). The number of colonies formed by both EGFP-high and EGFP-low cells decreased in a dose-dependent manner in the presence of 5-FU. At a glance, EGFP-low cells seemed to be more sensitive to 5-FU because colonies were not formed at higher doses of 5-FU. However, when the sensitivity to 5-FU was evaluated by the relative ratio of the number of colonies in the presence of 5-FU to that in the absence of 5-FU, no significant differences in sensitivity to 5-FU were observed between EGFP-high and EGFP-low cells (Figure 1, E and F). Therefore, we concluded that PSF1-promoter activity does not alter the drug sensitivity of the original cancers before 5-FU treatment, at least in colon26 and LLC cancer cells.Expression Pattern of PSF1 Promoter Activity in Remaining Cancer Cells after Treatment with Cancer Drug Is Different Depending on Cancer Cell TypeWe next observed the level of PSF1-promoter activity in cancer cells after administration of a cancer drug in an allograft model using colon26- and LLC-PSF1p-EGFP cells. The administration of 5-FU in vivo had a marked cytoreductive effect in both tumors (Figure 2, A and B). However, there were obvious differences in EGFP fluorescence intensity of the residual cancer cells between colon26- and LLC-PSF1p-EGFP cells. In the case of colon26-PSF1p-EGFP cells, with 5-FU administration in vivo there was a marked reduction in the proportion of EGFP-high cells compared with the control group (Figure 2C). On the other hand, with LLC-PSF1p-EGFP cells, the proportion of EGFP-high cells increased after 5-FU administration in comparison with the control group (Figure 2D). Similar results were obtained with the administration of cisplatin (Supplemental Figure S1, A and B). These results showed a difference in the expression pattern of PSF1-promoter activity in residual cancer cells after cancer drug administration, depending on the cancer cell type.Figure 2Expression pattern of PSF1-promoter activity in cancer cells that remain after treatment with cancer drug. A: Time course of tumor volume after 5-FU administration in vivo. 5-FU was administered to tumor-bearing mice inoculated by colon26-PSF1p-EGFP cells on days 7, 9, 11, and 13 as indicated by red arrows. Data show the means ± SEM (n = 6). B: Flow cytometric analysis of cells from tumor tissues on day 16 (as in A). Data obtained by flow cytometry in the saline (control) group (purple area) and the 5-FU–injected group (green line) represent the results of multiple experiments. C: Time course of tumor volume after 5-FU administration in vivo. 5-FU was administered to tumor-bearing mice inoculated by LLC-PSF1p-EGFP cells on days 7, 9, 11, 13, and 15 as indicated by red arrows. Data show the means ± SEM (n = 7). D: Flow cytometric analysis of cells from tumor tissues on day 18 (as in C). Data obtained by flow cytometry in the saline (control) group (purple area) and the 5-FU–injected group (green line) represent the results of multiple experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PSF1-Promoterhigh Cancer Cells Acquire Drug Resistance after Cancer Drug AdministrationWe previously reported that PSF1-promoterhigh cancer cells had significantly higher tumorigenic activity than PSF1-promoterlow cancer cells.16Nagahama Y. Ueno M. Miyamoto S. Morii E. Minami T. Mochizuki N. Saya H. Takakura N. PSF1, a DNA replication factor expressed widely in stem and progenitor cells, drives tumorigenic and metastatic properties.Cancer Res. 2010; 70: 1215-1224Crossref PubMed Scopus (44) Google Scholar We next assessed whether residual EGFP-high cancer cells after treatment with an anticancer drug (5-FU) in a tumor allograft model also would show tumor initiation activity and drug resistance in a second in vitro treatment with an anticancer drug. As shown in Figure 3A, LLC-PSF1p-EGFP cells were inoculated into mice and tumor-bearing mice were treated with 5-FU using the same schedule as described in Figure 2C. On day 18, tumors were dissected and two fractions of cancer cells (EGFP-low and EGFP-high cells) were sorted by a fluorescence-activated cell sorter as indicated (Figure 3A). Cancer cells (50, 100, or 1000) secondarily were inoculated into mice for observation of tumor initiation capacity. Moreover, cells were cultured in the presence or absence of 5-FU for 14 days and drug resistance was evaluated by calculating the number of colonies.Figure 3Acquisition of drug resistance after cancer drug administration. A: Analysis of the drug resistance capacity and tumor initiation capacity. Tumor-bearing mice by inoculation with LLC-PSF1p-EGFP cells were administered 5-FU as in Figure 2C. Tumor cells from the remaining tumor tissue were analyzed by flow cytometry and EGFP-high or EGFP-low cells were obtained. The black line shows LLC cells (no EGFP expression), the red line shows LLC-PSF1p-EGFP cells without 5-FU treatment, and the green line indicates LLC-PSF1p-EGFP cells treated with 5-FU in vivo. Cells from these two fractions were analyzed for in vivo tumorigenic activity and in vitro colony formation. B: Drug resistance analysis of residual cancer cells after 5-FU administration in vivo. Five hundred sorted cells as in A were seeded onto 10-cm culture dishes and cultured in various doses of 5-FU for 14 days. Colonies generated from different fractions were stained with Giemsa solution and assessed quantitatively. Data show the means ± SEM. ∗P < 0.05, ∗∗P < 0.01 (n = 3). C: Sensitivity to 5-FU. Relative ratio of the number of colonies in the presence of 5-FU to those in the absence of 5-FU obtained in experiment B was evaluated quantitatively. Data show the means ± SEM. ∗P < 0.05 (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)A smaller number of EGFP-high cells generated a secondary tumor more effectively than similar numbers of EGFP-low cells (Table 1). The difference in tumor initiation capacity between EGFP-low cells and EGFP-high cells was significant. When cultured in the presence or absence of 5-FU, EGFP-high cells formed significantly larger as well as a higher number of colonies than EGFP-low cells under all conditions (Figure 3B). In an evaluation of the sensitivity to 5-FU by the relative ratio of the number of colonies in the presence of 5-FU to those in the absence of 5-FU, EGFP-high cells showed significant resistance to 5-FU compared with EGFP-low cells (Figure 3C).Table 1Tumor Initiation Rate in Serial TransplantationLLC-PSF1p-EGFP cellsEGFP lowEGFP high1000 cells100%100%(n = 5)(n = 5)100 cells70%100%(n = 10)(n = 10)50 cells80% ± 6.7%∗P < 0.05 (n = 10).100% ± 0%(n = 10 × 3)(n = 10 × 3)A small number of sorted cells as indicated were inoculated subcutaneously into mice and tumor formation was observed until 8 weeks after tumor cell inoculation. Quantitat" @default.
• W2116661033 created "2016-06-24" @default.
• W2116661033 creator A5048494715 @default.
• W2116661033 creator A5055453369 @default.
• W2116661033 creator A5068249524 @default.
• W2116661033 creator A5068623243 @default.
• W2116661033 creator A5077760366 @default.
• W2116661033 date "2013-05-01" @default.
• W2116661033 modified "2023-09-26" @default.
• W2116661033 title "Possible Role of Mural Cell–Covered Mature Blood Vessels in Inducing Drug Resistance in Cancer-Initiating Cells" @default.
• W2116661033 cites W1967600348 @default.
• W2116661033 cites W1968762000 @default.
• W2116661033 cites W1970309962 @default.
• W2116661033 cites W1976741685 @default.
• W2116661033 cites W1987966760 @default.
• W2116661033 cites W2006202974 @default.
• W2116661033 cites W2011745548 @default.
• W2116661033 cites W2015831734 @default.
• W2116661033 cites W2017908190 @default.
• W2116661033 cites W2026379260 @default.
• W2116661033 cites W2029470215 @default.
• W2116661033 cites W2032580087 @default.
• W2116661033 cites W2035754643 @default.
• W2116661033 cites W2040006430 @default.
• W2116661033 cites W2040129329 @default.
• W2116661033 cites W2041357218 @default.
• W2116661033 cites W2044634784 @default.
• W2116661033 cites W2044905529 @default.
• W2116661033 cites W2045000546 @default.
• W2116661033 cites W2060551076 @default.
• W2116661033 cites W2062945727 @default.
• W2116661033 cites W2067755505 @default.
• W2116661033 cites W2072702826 @default.
• W2116661033 cites W2073759231 @default.
• W2116661033 cites W2081026902 @default.
• W2116661033 cites W2084156847 @default.
• W2116661033 cites W2103628876 @default.
• W2116661033 cites W2114834794 @default.
• W2116661033 cites W2117692326 @default.
• W2116661033 cites W2121417385 @default.
• W2116661033 cites W2124977778 @default.
• W2116661033 cites W2134156532 @default.
• W2116661033 cites W2140088548 @default.
• W2116661033 cites W2147650118 @default.
• W2116661033 cites W2151627674 @default.
• W2116661033 cites W2152387543 @default.
• W2116661033 cites W2165435676 @default.
• W2116661033 doi "https://doi.org/10.1016/j.ajpath.2013.01.019" @default.
• W2116661033 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23473746" @default.
• W2116661033 hasPublicationYear "2013" @default.
• W2116661033 type Work @default.
• W2116661033 sameAs 2116661033 @default.
• W2116661033 citedByCount "9" @default.
• W2116661033 countsByYear W21166610332014 @default.
• W2116661033 countsByYear W21166610332016 @default.
• W2116661033 countsByYear W21166610332019 @default.
• W2116661033 countsByYear W21166610332021 @default.
• W2116661033 countsByYear W21166610332023 @default.
• W2116661033 crossrefType "journal-article" @default.
• W2116661033 hasAuthorship W2116661033A5048494715 @default.
• W2116661033 hasAuthorship W2116661033A5055453369 @default.
• W2116661033 hasAuthorship W2116661033A5068249524 @default.
• W2116661033 hasAuthorship W2116661033A5068623243 @default.
• W2116661033 hasAuthorship W2116661033A5077760366 @default.
• W2116661033 hasConcept C111758988 @default.
• W2116661033 hasConcept C114851261 @default.
• W2116661033 hasConcept C121608353 @default.
• W2116661033 hasConcept C126322002 @default.
• W2116661033 hasConcept C142362112 @default.
• W2116661033 hasConcept C142724271 @default.
• W2116661033 hasConcept C1491633281 @default.
• W2116661033 hasConcept C153349607 @default.
• W2116661033 hasConcept C205783811 @default.
• W2116661033 hasConcept C2777420470 @default.
• W2116661033 hasConcept C2779395532 @default.
• W2116661033 hasConcept C2780035454 @default.
• W2116661033 hasConcept C2992686903 @default.
• W2116661033 hasConcept C3020340455 @default.
• W2116661033 hasConcept C502942594 @default.
• W2116661033 hasConcept C54355233 @default.
• W2116661033 hasConcept C71924100 @default.
• W2116661033 hasConcept C86803240 @default.
• W2116661033 hasConcept C96232424 @default.
• W2116661033 hasConcept C98274493 @default.
• W2116661033 hasConceptScore W2116661033C111758988 @default.
• W2116661033 hasConceptScore W2116661033C114851261 @default.
• W2116661033 hasConceptScore W2116661033C121608353 @default.
• W2116661033 hasConceptScore W2116661033C126322002 @default.
• W2116661033 hasConceptScore W2116661033C142362112 @default.
• W2116661033 hasConceptScore W2116661033C142724271 @default.
• W2116661033 hasConceptScore W2116661033C1491633281 @default.
• W2116661033 hasConceptScore W2116661033C153349607 @default.
• W2116661033 hasConceptScore W2116661033C205783811 @default.
• W2116661033 hasConceptScore W2116661033C2777420470 @default.
• W2116661033 hasConceptScore W2116661033C2779395532 @default.
• W2116661033 hasConceptScore W2116661033C2780035454 @default.
• W2116661033 hasConceptScore W2116661033C2992686903 @default.
• W2116661033 hasConceptScore W2116661033C3020340455 @default.

• next