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Diphenyleneiodonium (DPI) has long been evaluated as an anticancer drug inhibiting NADPH oxidase, the IC50 in several cancer cell lines was reported 10 µM, which is too high for efficacy. In this study, we employed miPS-Huh7cmP cells, which we previously established as a cancer stem cell (CSC) model from induced pluripotent stem cells, to reevaluate the efficacy of DPI because CSCs are currently one of the main foci of therapeutic strategy to treat cancer, but generally considered resistant to chemotherapy. As a result, the conventional assay for the cell growth inhibition by DPI accounted for an IC50 at 712 nM that was not enough to define the effectiveness as an anticancer drug. Simultaneously, the wound-healing assay revealed an IC50 of approximately 500 nM. Comparatively, the IC50 values shown on sphere formation, colony formation, and tube formation assays were 5.52, 12, and 8.7 nM, respectively. However, these inhibitory effects were not observed by VAS2780, also a reputed NADPH oxidase inhibitor. It is noteworthy that these three assays are evaluating the characteristic of CSCs and are designed in the three-dimensional (3D) culture methods. We concluded that DPI could be a suitable candidate to target mitochondrial respiration in CSCs. We propose that the 3D culture assays are more efficient to screen anti-CSC drug candidates and better mimic tumor microenvironment when compared to the adherent monolayer of 2D culture system used for a conventional assay, such as cell growth inhibition and wound-healing assays.
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Antineoplásicos , Células Madre Pluripotentes Inducidas , Neoplasias , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular , NADPH Oxidasas/metabolismo , Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Compuestos OnioRESUMEN
Many tumors are resistant to conventional cancer therapies because a tumor is composed of heterogeneous cell population. Especially, subpopulation of cancer stem cells, which have self-renewal and differentiation properties and responsible for the tumor initiation, is generally considered resistant to chemo-, radio-, and immune therapy. Understanding the mechanism of drug resistance in cancer stem cells should lead to establish more effective therapeutic strategies. Actually, different molecular mechanisms are conceivable for cancer stem cells acquiring drug resistance. These mechanisms include not only cytoplasmic signaling pathways but also the intercellular communications in the tumor microenvironment. Recently, a great deal of successful reports challenged to elucidate the mechanisms of drug resistance and to develop novel treatments targeting cancer stem cells.
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Resistencia a Antineoplásicos , Neoplasias , Humanos , Neoplasias/patología , Transformación Celular Neoplásica/patología , Diferenciación Celular , Células Madre Neoplásicas/patología , Microambiente TumoralRESUMEN
Cancer stem cells (CSCs) are small subpopulation sharing similar properties like normal stem such as self-renewal and differentiation potential to direct tumor growth. Last few years, scientists considered CSCs as the cause of phenotypic heterogeneity in diverse cancer types. Also, CSCs contribute to cancer metastasis and recurrence. The cellular and molecular regulators influence on the CSCs' phenotype changing their behaviors in different stages of cancer progression. CSC markers play significance roles in cancer diagnosis and characterization. We delineate the cross-talks between CSCs and the tumor microenvironment that supports their intrinsic properties including survival, stemness, quiescence and their cellular and molecular adaptation. An insight into the markers of CSCs specific to organs is described.
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Neoplasias , Humanos , Neoplasias/genética , Neoplasias/patología , Células Madre Neoplásicas/patología , Diferenciación Celular , Fenotipo , Microambiente Tumoral/genéticaRESUMEN
Cancer stem cells (CSCs) are responsible for cancer initiation, drug resistance, and aggressive tumor phenotypes. Our lab has established a novel method to induce CSCs from induced pluripotent stem (iPS) cells in a microenvironment mimicking chronic inflammation. The converted cells acquired CSC characteristics and developed malignant tumors. Recently, we demonstrated that nonmutagenic chemical inhibitors accelerated the conversion of mouse iPS (miPS) cells into CSCs. Here, we investigated the effects of AZD-6244, a MEK1/2-specific inhibitor, on the conversion of iPS cells into CSCs. The miPS cells were cultured for one week in the presence of the conditioned medium (CM) of Lewis lung carcinoma (LLC) cells and AZD-6244, PD0325901, a pan-MEK inhibitor, or GDC-0879, a B-Raf inhibitor. As a result, AZD-6244 enhanced the conversion of iPS cells into CSCs and upregulated AKT phosphorylation as same as GDC-0879 and PD0325901. The converted cells maintained their self-renewal ability and stemness gene expression. The expression of the CSC markers CD24, CD44 and CD133 was higher in the cells cultured with MAPK inhibitors than in those cultured without MAPK inhibitors. Moreover, converted cells gained migration and invasion abilities assessed by in vitro assays. Therefore, the inhibition of MEK1/2 was found to be critical for the conversion of normal stem cells into CSCs in the tumor-inducing microenvironment.
RESUMEN
Angiogenesis is generally involved in tumor growth and metastasis. Cancer stem cells (CSCs) are considered to facilitate the angiogenesis. Therefore, CSCs could be the effective targets to stop angiogenesis. Recently, our group successfully generated CSC models from induced pluripotent stem cells (iPSCs) in the presence of conditioned medium derived from cancer derived cells. These novel model CSCs has been characterized by highly tumorigenic, angiogenic and metastatic potentials in vivo. The angiogenic potential of CSCs has been explained by the expression of both angiogenic factors and their receptors implying the angiogenesis in autocrine manner. In this protocol we optimized the method to evaluate tumor angiogenesis with the CSC model, which was described effective to assess sorafenib as an antiangiogenic drug, on chick chorioallantoic membrane (CAM) assay. Our results demonstrate that CSCs developed from iPSCs and CAM assay are a robust and cost-effective tool to evaluate tumor angiogenesis with CSCs. Collectively, CSCs in CAM assay could serve as a very useful model for the screening of potential therapeutic agents targeting tumor angiogenesis.
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Neoplasias , Neovascularización Patológica , Sorafenib/farmacología , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Embrión de Pollo , Membrana Corioalantoides/metabolismo , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Células Madre NeoplásicasRESUMEN
Metformin exhibits anti-cancer activities in various types of tumours while it is prescribed as the first-line drug for type 2 diabetes. Since new evidence has recently suggested that metformin could target cancer stem cells (CSCs) and prevent their recurrence, repositioning of metformin could be considered as a candidate for anti-CSC agent. In this study, we assessed the effect of metformin on the cancer stem cells developed from induced pluripotent stem cells. As the result, metformin significantly suppressed the self-renewal ability of CSCs when assessed by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cell counting methods exhibiting the IC50 as approximately 20 mM, which suppressed tube formation by CSCs on Matrigel reducing the angiogenic potential of CSCs. Cell cycle analysis showed that metformin reduced the percentage of cells in the S phase increasing the percentage of cells in G0/G1 phase. Moreover, the tumorigenicity of CSCs was found to be attenuated when the cells were injected with metformin. From these results, we concluded that metformin could be promising for targeted therapy by repositioning the widely available drugs with safety. SIGNIFICANCE OF THE STUDY: Metformin could target CSCs and prevent their recurrence, repositioning of metformin could be considered as a candidate for the anti-CSC agent. In this paper, we assessed the effect of metformin on the CSCs developed from induced pluripotent stem cells. Here, we show that metformin suppresses the self-renewal and tube formation abilities of CSCs. We also show that metformin reduces the percentage of cells in the S phase increasing the percentage of cells in G0/G1 phase. Moreover, the tumorigenicity of CSCs was found to be attenuated when grafted in vivo after treatment with metformin.
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Antineoplásicos/farmacología , Autorrenovación de las Células/efectos de los fármacos , Metformina/farmacología , Modelos Biológicos , Células Madre Pluripotentes/citología , Animales , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales CultivadasRESUMEN
Cripto-1 is a member of the EGF-CFC/FRL1/Cryptic family and is involved in embryonic development and carcinogenesis. We designed a novel anti-Cripto-1 artificial antibody and assessed the recognition to the antigen and the potential to suppress the growth of cancer stem cells. First, single chain antibody clones were isolated by bio-panning with the affinity to recombinant Cripto-1 protein from our original phage-display library. Then, the variable regions of heavy chain VH and light chain VL in each clone were fused to constant regions of heavy chain CH and light chain CL regions respectively. These fused genes were expressed in ExpiCHO-S cells to produce artificial humanized antibodies against Cripto-1. After evaluation of the expression levels, one clone was selected and the anti-Cripto-1 antibody was produced and purified. The purified antibody showed affinity to recombinant Cripto-1 at 1.1 pmol and immunoreactivity to cancer tissues and cell lines. The antibody was available to detect the immunoreactivity in tissue microarrays of malignant tumors as well as in Cripto-1 overexpressing cells. Simultaneously, the antibody exhibited the potential to suppress the growth of human colon cancer derived GEO cells overexpressing Cripto-1 with IC50 at approximately 110 nM. The artificially humanized antibody is proposed to be a good candidate to target cancer cells overexpressing Cripto-1.
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Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas Ligadas a GPI/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Proteínas de Neoplasias/inmunología , Teratocarcinoma/tratamiento farmacológico , Neoplasias Testiculares/tratamiento farmacológico , Secuencia de Aminoácidos , Anticuerpos Monoclonales Humanizados/inmunología , Antineoplásicos/inmunología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Femenino , Humanos , Masculino , Homología de Secuencia , Teratocarcinoma/inmunología , Teratocarcinoma/metabolismo , Teratocarcinoma/patología , Neoplasias Testiculares/inmunología , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Liver cancer is the second most common cause of cancer-related death. Every type of tumours including liver cancer contains cancer stem cells (CSCs). To date, the molecular mechanism regulating the development of liver CSCs remains unknown. METHODS: In this study, we tried to generate a new model of liver CSCs by converting mouse induced pluripotent stem cells (miPSCs) with hepatocellular carcinoma (HCC) cell line Huh7 cells conditioned medium (CM). miPSCs treated with CM were injected into the liver of BALB/c nude mice. The developed tumours were then excised and analysed. RESULTS: The primary cultured cells from the malignant tumour possessed self-renewal capacity, differentiation potential and tumorigenicity in vivo, which were found rich in liver cancer-associated markers as well as CSC markers. CONCLUSIONS: We established a model of liver CSCs converting from miPS and showed different stages of stemness during conversion process. Our CSC model will be important to assess the molecular mechanisms necessary to develop liver CSCs and could help in defeating liver cancer.
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Carcinogénesis/efectos de los fármacos , Carcinoma Hepatocelular/genética , Medios de Cultivo Condicionados/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Neoplasias Hepáticas/genética , Animales , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Medios de Cultivo Condicionados/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/patología , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , Células Madre Neoplásicas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Paclitaxel (PTX) is one of the front-line drugs approved for the treatment of ovarian cancer. However, the application of PTX is limited due to the significant hydrophobicity and poor pharmacokinetics. We previously reported target-directed liposomes carrying tumor-selective conjugated antibody and encapsulated glycosylated PTX (gPTX-L) which successfully overcome the PTX limitation. The tubulin stabilizing activity of gPTX was equivalent to that of PTX while the cytotoxic activity of gPTX was reduced. In human ovarian cancer cell lines, SK-OV-3 and OVK18, the concentration at which cell growth was inhibited by 50% (IC50) for gPTX range from 15â»20 nM, which was sensitive enough to address gPTX-L with tumor-selective antibody coupling for ovarian cancer therapy. The cell membrane receptor CD44 is associated with cancer progression and has been recognized as a cancer stem cell marker including ovarian cancer, becoming a suitable candidate to be targeted by gPTX-L therapy. In this study, gPTX-loading liposomes conjugated with anti-CD44 antibody (gPTX-IL) were assessed for the efficacy of targeting CD44-positive ovarian cancer cells. We successfully encapsulated gPTX into liposomes with the loading efficiency (LE) more than 80% in both of gPTX-L and gPTX-IL with a diameter of approximately 100 nm with efficacy of enhanced cytotoxicity in vitro and of convenient treatment in vivo. As the result, gPTX-IL efficiently suppressed tumor growth in vivo. Therefore gPTX-IL could be a promising formulation for effective ovarian cancer therapies.
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Receptores de Hialuranos/metabolismo , Terapia Molecular Dirigida , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Paclitaxel/uso terapéutico , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Glicosilación , Humanos , Liposomas/ultraestructura , Neoplasias Ováricas/patología , Paclitaxel/farmacologíaRESUMEN
Cripto-1 is a glycophosphatidylinositol (GPI) anchored signaling protein of epidermal growth factor (EGF)-Cripto-1-FRL1-Cryptic (CFC) family and plays a significant role in the early developmental stages and in the different types of cancer cells, epithelial to mesenchymal transition and tumor angiogenesis. Previously, we have developed cancer stem cells (miPS-LLCcm) from mouse iPSCs by culturing them in the presence of conditioned medium of Lewis Lung Carcinoma (LLC) cells for four weeks. Nodal and Cripto-1 were confirmed to be expressed in miPS-LLCcm cells by quantitative reverse transcription PCR (rt-qPCR) implying that Cr-1 was required in maintaining stemness. To investigate the biological effect of adding exogenous soluble CR-1 to the cancer stem cells, we have prepared a C-terminally truncated soluble form of recombinant human CR-1 protein (rhsfCR-1), in which the GPI anchored moiety was removed by substitution of a stop codon through site-directed mutagenesis. rhsfCR-1 effectively suppressed the proliferation and sphere forming ability of miPS-LLCcm cells in a dose-dependent manner in the range of 0 to 5 µg/mL, due to the suppression of Nodal-Cripto-1/ALK4/Smad2 signaling pathway. Frequency of sphere-forming cells was dropped from 1/40 to 1/69 by rhsfCR-1 at 1 µg/mL. Moreover, rhsfCR-1 in the range of 0 to 1 µg/mL also limited the differentiation of miPS-LLCcm cells into vascular endothelial cells probably due to the suppression of self-renewal, which should reduce the number of cells with stemness property. As demonstrated by a soluble form of exogenous Cripto-1 in this study, the efficient blockade would be an attractive way to study Cripto-1 dependent cancer stem cell properties for therapeutic application.
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Autorrenovación de las Células , Proteínas Ligadas a GPI/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/citología , Animales , Diferenciación Celular , Línea Celular , Humanos , Ratones , Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Proteína Smad2/metabolismoRESUMEN
Induced pluripotent stem cells (iPSCs) are useful tools for modeling diseases and developing personalized medicine. We have been developing cancer stem cells (CSCs) from iPSCs with conditioned medium (CM) of cancer-derived cells as the mimicry of the microenvironment of tumor initiation. However, the conversion of human iPSCs has not always been efficient with only CM. In this study, human iPSCs reprogrammed from monocytes of healthy volunteers were cultured in a media containing 50% of the CM from human pancreatic cancer derived BxPC3 cells supplemented with a MEK inhibitor (AZD6244) and a GSK-3α/ß inhibitor (CHIR99021). The survived cells were assessed for the characteristics of CSCs in vitro and in vivo. As a result, they exhibited CSC phenotypes of self-renewal, differentiation, and malignant tumorigenicity. Primary culture of the malignant tumors of the converted cells exhibited the elevated expression of CSC related genes CD44, CD24 and EPCAM maintaining the expression of stemness genes. In conclusion, the inhibition of GSK-3α/ß and MEK and the microenvironment of tumor initiation mimicked by the CM can convert human normal stem cells into CSCs. This study could provide insights into establishing potentially novel personalized cancer models which could help investigate the tumor initiation and screening of personalized therapies on CSCs. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00575-1.
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The heterogeneous cell population in the stromal microenvironment is considered to be attributed to the multiple sources from which the cells originate. Tumor associated myoepithelial cells (TAMEs) are one of the most important populations in the tumor microenvironment (TME) especially in breast cancer. On the other hand, cancer stem cells (CSCs) have previously been described to be the origin of tumor-associated cellular components in the TME. We prepared a cancer stem cell model converting mouse-induced pluripotent stem cells (miPSCs) in the presence of conditioned medium of breast cancer cell line MDA-MB-231 cells. The converted cells developed tumors progressing into invasive carcinoma with ductal carcinoma in situ (DCIS) like structure when transplanted into mouse mammary fat pads. The primary cultured cells from the tumor further exhibited markers of CSC such as Sox2, Oct3/4, - CD133 and EpCAM, and mammary gland-related TAME markers such as α-smooth muscle actin, cytokeratin 8, whey acidic protein, prolactin receptor and progesterone receptor as well. These results indicated that the CSCs could be an origin of TAMEs contributing to mammary gland epithelial cell differentiation and the progression to invasive carcinoma during tumor development. The gene expression profiles confirmed the enhanced signaling pathways of PI3K/AKT and MAPK, which have been demonstrated to be enriched in the CSC models, together with the estrogen receptor signaling which was peculiar to mammary gland-derived character.
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Carcinoma Intraductal no Infiltrante , Ratones , Animales , Carcinoma Intraductal no Infiltrante/patología , Microambiente Tumoral , Fosfatidilinositol 3-Quinasas , Biomarcadores de Tumor , Células Madre Neoplásicas/patologíaRESUMEN
Cancer stem cells (CSCs) are suggested to be responsible for drug resistance and aggressive phenotypes of tumors. Mechanisms of CSC induction are still under investigation. Our lab has established a novel method to generate CSCs from iPSCs under a cancerous microenvironment mimicked by the conditioned medium (CM) of cancer-derived cells. Here, we analyzed the transcriptome of CSCs, which were converted from iPSCs with CM from pancreatic ductal adenocarcinoma cells. The differentially expressed genes were identified and used to explore pathway enrichment. From the comparison of the CSCs with iPSCs, genes with elevated expression were related to the ErbB2/3 signaling pathway. Inhibition of either ErbB2 with lapatinib as a tyrosine kinase inhibitor or ErbB3 with TX1-85-1 or siRNAs arrested cell proliferation, inhibited the in vitro tumorigenicity, and lead to loss of stemness in the converting cells. The self-renewal and tube formation abilities of cells were also abolished while CD24 and Oct3/4 levels were reduced, and the MAPK pathway was overactivated. This study shows a potential involvement of the ErbB2/ErbB3 pathway in CSC generation and could lead to new insight into the mechanism of tumorigenesis and the way of cancer prevention.
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Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Neoplásicas/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Acrilamidas/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Autorrenovación de las Células/efectos de los fármacos , Medios de Cultivo Condicionados , Regulación Neoplásica de la Expresión Génica , Humanos , Lapatinib/farmacología , Sistema de Señalización de MAP Quinasas , Neoplasias Pancreáticas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-3/antagonistas & inhibidores , Receptor ErbB-3/genética , Transducción de Señal , Neoplasias PancreáticasRESUMEN
Previously, our group has demonstrated establishment of Cancer Stem Cell (CSC) models from stem cells in the presence of conditioned medium of cancer cell lines. In this study, we tried to identify the factors responsible for the induction of CSCs. Since we found the lipid composition could be traced to arachidonic acid cascade in the CSC model, we assessed prostaglandin E2 (PGE2) as a candidate for the ability to induce CSCs from induced pluripotent stem cells (iPSCs). Mouse iPSCs acquired the characteristics of CSCs in the presence of 10 ng/mL of PGE2 after 4 weeks. Since constitutive Akt activation and pik3cg overexpression were found in the resultant CSCs, of which growth was found independent of PGE2, chronic stimulation of the receptors EP-2/4 by PGE2 was supposed to induce CSCs from iPSCs through epigenetic effect. The bioinformatics analysis of the next generation sequence data of the obtained CSCs proposed not only receptor tyrosine kinase activation by growth factors but also extracellular matrix and focal adhesion enhanced PI3K pathway. Collectively, chronic stimulation of stem cells with PGE2 was implied responsible for cancer initiation enhancing PI3K/Akt axis.
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Dinoprostona , Neoplasias , Animales , Ácido Araquidónico/metabolismo , Medios de Cultivo Condicionados/farmacología , Dinoprostona/metabolismo , Dinoprostona/farmacología , Ratones , Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Cancer stem cells (CSCs) are generated under irregular microenvironment in vivo, of which mimic is quite difficult due to the lack of enough information of the factors responsible for cancer initiation. Here, we demonstrated that mouse induced pluripotent cells (miPSCs) reprogrammed from normal embryonic fibroblasts were susceptible to the microenvironment affected by cancer cells to convert into CSCs in vivo. METHODS: Three different pancreatic cancer line cells, BxPC3, PANC1, and PK8 cells were mixed with miPSCs and subcutaneously injected into immunodeficient mice. Tumors were evaluated by histological analysis and cells derived from iPSCs were isolated and selected from tumors. The isolated cells were characterized for cancer stem cell characters in vitro and in vivo as well as their responses to anticancer drugs. The impact of co-injection of iPSCs with cancer cells on transcriptome and signaling pathways of iPSCs was investigated. RESULTS: The injection of miPSCs mixed with human pancreatic cancer cells into immunodeficient mice maintained the stemness of miPSCs and changed their phenotype. The miPSCs acquired CSC characteristics of tumorigenicity and self-renewal. The drug responses and the metastatic ability of CSCs converted from miPSCs varied depending on the microenvironment of cancer cells. Interestingly, transcriptome profiles of these cells indicated that the pathways related with aggressiveness and energy production were upregulated from the levels of miPSCs. CONCLUSIONS: Our result suggests that cancer-inducing microenvironment in vivo could rewire the cell signaling and metabolic pathways to convert normal stem cells into CSCs.
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Expresión Génica/genética , Células Madre Pluripotentes Inducidas/metabolismo , Redes y Vías Metabólicas/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Microambiente TumoralRESUMEN
Paclitaxel (PTX) is a chemotherapeutical agent commonly used to treat several kinds of cancer. PTX is known as a microtubule-targeting agent with a primary molecular mechanism that disrupts the dynamics of microtubules and induces mitotic arrest and cell death. Simultaneously, other mechanisms have been evaluated in many studies. Since the anticancer activity of PTX was discovered, it has been used to treat many cancer patients and has become one of the most extensively used anticancer drugs. Regrettably, the resistance of cancer to PTX is considered an extensive obstacle in clinical applications and is one of the major causes of death correlated with treatment failure. Therefore, the combination of PTX with other drugs could lead to efficient therapeutic strategies. Here, we summarize the mechanisms of PTX, and the current studies focusing on PTX and review promising combinations.
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Cancer stem cell (CSC) is considered as a cause of cancer recurrence and metastasis. Simultaneously CSCs are responsible for the heterogeneous population in tumor tissues due to their differentiation potential. However, the characterizations of CSCs are still not enough and cancer stem cell lines widely available is desired to be established for the advancement of cancer research. In this study, we tried to isolate and characterize stem like cells from human glioblastoma cell line U-251MG cells. U-251MG P1 cells, which was previously condensed in the presence of hyaluronic acid as CD44 positive population were subjected to single cell isolation procedure. Although 5 clones were isolated, only one clone exhibited high expression of CD44, Nanog, OCT3/4 and SOX2, and named U-251MGSC1. The sphere forming ability of U-251MGSC1 cell was significantly higher than the parental U-251MG cells. Tumorigenicity of U-251MG-SC1 cells were higher than that of U-251MG cells. U-251MGSC1 cells exhibited higher expression of CD44, SOX2, Nestin and A2B5 than U-251MG cells in vitro and in vivo. The expression of GFAP and NF-M was enhanced when the cells were treated with the conditioned medium of U-251MG cells indicating the potential of differentiation. Sphere forming ability was more efficient than that of U-251MG cells and was enhanced in the presence of hyaluronic acid, which enhanced the cell growth as well. U-251MGSC1 cells exhibited rapid growth tumor in nude mice and efficient metastatic ability in transmembrane assay when compared with U-251MG cells. As the result, we concluded U-251MGSC1 cell was a glioblastoma CSC line derived from the parental U-251MG cells. U-251MGSC1 cells will be a good tool to develop effective therapeutic agents against CSCs and to elucidate the properties of glioma derived CSCs and the mechanism of tumor development in brain.
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We previously demonstrated the conversion of normal stem cells, including induced pluripotent stem cells (iPSCs), into cancer stem cells (CSCs) without genetic manipulation. Herein, we designed a meta-analysis to assess gene expression profiles in different breast cancer cell lines focusing on the secretory factors responsible for conversion. As a result, fibroblast growth factor 2 (FGF2) was found to be the best candidate in T47D and BT549 cells, of which conditioned medium was previously successful in inducing CSCs. When treated with 3.1 µg/ml FGF2, mouse iPSCs not only maintained survival without LIF for three weeks but also acquired growth ability independent of FGF2. The resultant cells exhibited expression of stemness and cancer stem cell markers, sphere-forming ability, differentiation, and tumorigenicity with malignancy. The primary cultures of the tumor confirmed the signatures of CSCs with two different phenotypes with or without GFP expression under control of the Nanog promoter. Bioinformatic analysis of gene expression profiles suggested constitutive autocrine activation of the FGF receptor, integrins, focal adhesions, and PI3K/AKT pathways. FGF2 could potently initiate cancer as a component of the inflammatory microenvironment.
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Breast cancer is the first common cause of cancer-related death in women worldwide. Since the malignancy and aggressiveness of breast cancer have been correlated with the presence of breast cancer stem cells, the establishment of a disease model with cancer stem cells is required for the development of a novel therapeutic strategy. Here, we aimed to evaluate the availability of cancer stem cell models developed from mouse induced pluripotent stem cells with the conditioned medium of different subtypes of breast cancer cell lines, the hormonal-responsive T47D cell line and the triple-negative breast cancer BT549 cell line, to generate in vivo tumor models. When transplanted into the mammary fat pads of BALB/c nude mice, these two model cells formed malignant tumors exhibiting pronounced histopathological characteristics similar to breast cancers. Serial transplantation of the primary cultured cells into mammary fat pads evoked the same features of breast cancer, while this result was perturbed following subcutaneous transplantation. The tumors formed in the mammary fat pads exhibited immune reactivities to prolactin receptor, progesterone receptor, green florescent protein, Ki67, CD44, estrogen receptor α/ß and cytokeratin 8, while all of the tumors and their derived primary cells exhibited immunoreactivity to estrogen receptor α/ß and cytokeratin 8. Cancer stem cells can be developed from pluripotent stem cells via the secretory factors of cancer-derived cells with the capacity to inherit tissue specificity. However, cancer stem cells should be plastic enough to be affected by the microenvironment of specific tissues. In summary, we successfully established a breast cancer tumor model using mouse induced pluripotent stem cells developed from normal fibroblasts without genetic manipulation.
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Cancer stem cells (CSCs) are subpopulations in the malignant tumors that show self-renewal and multilineage differentiation into tumor microenvironment components that drive tumor growth and heterogeneity. In previous studies, our group succeeded in producing a CSC model by treating mouse induced pluripotent stem cells. In the current study, we investigated the potential of CSC differentiation into blood cells under chemical hypoxic conditions using CoCl2. CSCs and miPS-LLCcm cells were cultured for 1 to 7 days in the presence of CoCl2, and the expression of VEGFR1/2, Runx1, c-kit, CD31, CD34, and TER-119 was assessed by RT-qPCR, Western blotting and flow cytometry together with Wright-Giemsa staining and immunocytochemistry. CoCl2 induced significant accumulation of HIF-1α changing the morphology of miPS-LLCcm cells while the morphological change was apparently not related to differentiation. The expression of VEGFR2 and CD31 was suppressed while Runx1 expression was upregulated. The population with hematopoietic markers CD34+ and c-kit+ was immunologically detected in the presence of CoCl2. Additionally, high expression of CD34 and, a marker for erythroblasts, TER-119, was observed. Therefore, CSCs were suggested to differentiate into erythroblasts and erythrocytes under hypoxia. This differentiation potential of CSCs could provide new insight into the tumor microenvironment elucidating tumor heterogenicity.