RESUMEN
INTRODUCTION: Tumor-initiating cells (TICs), aka "cancer stem cells", are believed to fuel tumors and to sustain therapy resistance and systemic metastasis. Breast cancer is the first human carcinoma in which a subpopulation of cells displaying a specific CD44+/CD24-/low/ESA+ antigenic phenotype was found to have TIC properties. However, CD44+/CD24-/low/ESA+ is not a universal marker phenotype of TICs in all breast cancer subtypes. The aim of this study was to identify novel antigens with which to isolate the TIC population of the basal-A/basal-like breast cancer cell lines. METHODS: We used polychromatic flow-cytometry to characterize the cell surface of several breast cancer cell lines that may represent different tumor molecular subtypes. We next used fluorescence-activated cell sorting to isolate the cell subpopulations of interest from the cell lines. Finally, we explored the stem-like and tumorigenic properties of the sorted cell subpopulations using complementary in vitro and in vivo approaches: mammosphere formation assays, soft-agar colony assays, and tumorigenic assays in NOD/SCID mice. RESULTS: The CD44+/CD24+ subpopulation of the BRCA1-mutated basal-A/basal-like cell line HCC1937 is enriched in several stemness markers, including the ABCG2 transporter (i.e., the CD338 antigen). Consistently, CD338-expressing cells were also enriched in CD24 expression, suggesting that coexpression of these two antigenic markers may segregate TICs in this cell line. In support of ABCG2 expression in TICs, culturing of HCC1937 cells in ultra-low adherent conditions to enrich them in precursor/stem-cells resulted in an increase in CD338-expressing cells. Furthermore, CD338-expressing cells, unlike their CD338-negative counterparts, displayed stemness and transformation potential, as assessed in mammosphere and colony formation assays. Lastly, CD338-expressing cells cultured in ultra-low adherent conditions maintained the expression of CD326/EpCAM and CD49f/α6-integrin, which is a combination of antigens previously assigned to luminal progenitors. CONCLUSION: Collectively, our data suggest that CD338 expression is specific to the tumor-initiating luminal progenitor subpopulation of BRCA1-mutated cells and is a novel antigen with which to sort this subpopulation.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Proteína BRCA1/metabolismo , Neoplasias de la Mama/patología , Citometría de Flujo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/patología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Animales , Antígeno CD24/metabolismo , Adhesión Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Ratones Endogámicos NOD , Ratones SCID , Esferoides Celulares/patologíaRESUMEN
Maternal obesity is associated to increased fetal risk of obesity and other metabolic diseases. Human amniotic mesenchymal stem cells (hA-MSCs) have not been characterized in obese women. The aim of this study was to isolate and compare hA-MSC immunophenotypes from obese (Ob-) and normal weight control (Co-) women, to identify alterations possibly predisposing the fetus to obesity. We enrolled 16 Ob- and 7 Co-women at delivery (mean/SEM prepregnancy body mass index: 40.3/1.8 and 22.4/1.0 kg/m2, respectively), and 32 not pregnant women. hA-MSCs were phenotyped by flow cytometry; several maternal and newborn clinical and biochemical parameters were also measured. The expression of membrane antigen CD13 was higher on Ob-hA-MSCs than on Co-hA-MSCs (P = 0.005). Also, serum levels of CD13 at delivery were higher in Ob- versus Co-pregnant women and correlated with CD13 antigen expression on Ob-hA-MSCs (r2 = 0.84, P < 0.0001). Adipogenesis induction experiments revealed that Ob-hA-MSCs had a higher adipogenic potential than Co-hA-MSCs as witnessed by higher peroxisome proliferator-activated receptor gamma and aP2 mRNA levels (P = 0.05 and P = 0.05, respectively), at postinduction day 14 associated with increased CD13 mRNA levels from baseline to day 4 postinduction (P < 0.05). Adipogenesis was similar in the two sets of hA-MSCs after CD13 silencing, whereas it was increased in Co-hA-MSCs after CD13 overexpression. CD13 expression was high also in Ob-h-MSCs from umbilical cords or visceral adipose tissue of not pregnant women. In conclusion, antigen CD13, by influencing the adipogenic potential of hA-MSCs, could be an in utero risk factor for obesity. Our data strengthen the hypothesis that high levels of serum and MSC CD13 are obesity markers.
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Adipogénesis/genética , Amnios/enzimología , Antígenos CD13/metabolismo , Células Madre Mesenquimatosas/enzimología , Obesidad/genética , ARN Mensajero/metabolismo , Adulto , Amnios/crecimiento & desarrollo , Amnios/patología , Índice de Masa Corporal , Antígenos CD13/antagonistas & inhibidores , Antígenos CD13/genética , Estudios de Casos y Controles , Femenino , Expresión Génica , Humanos , Inmunofenotipificación , Recién Nacido , Células Madre Mesenquimatosas/citología , Obesidad/enzimología , Obesidad/patología , PPAR gamma/genética , PPAR gamma/metabolismo , Embarazo , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factores de RiesgoRESUMEN
Acute promyelocytic leukemia (APL) is a hematological emergency in which a rapid diagnosis is essential for early administration of appropriate therapy, including all-trans retinoic acid before the onset of fatal coagulopathy. Currently, the following methodologies are widely used for rapid initial diagnosis of APL: 1) identification of hypergranular leukemic promyelocytes by using classical morphology; 2) identification of cells with diffuse promyelocytic leukemia (PML) protein distribution by immunofluorescence microscopy; 3) evidence of aberrant promyelocyte surface immunophenotype by conventional flow cytometry (FCM). Here, we show a method for immunofluorescent detection of PML localization using ImageStream FCM. This technique provides objective per-cell quantitative image analysis for statistically large sample sizes, enabling precise and operator-independent PML pattern recognition even in electronic and real dilution experiments up to 10% of APL cellular presence. Therefore, we evidence that this method could be helpful for rapid and objective initial diagnosis and the prompt initiation of APL treatment.
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Citometría de Flujo/métodos , Células Precursoras de Granulocitos/fisiología , Citometría de Imagen/métodos , Leucemia Promielocítica Aguda/diagnóstico , Proteínas Nucleares/análisis , Factores de Transcripción/análisis , Proteínas Supresoras de Tumor/análisis , Línea Celular Tumoral , Humanos , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/química , Proteínas Nucleares/química , Proteína de la Leucemia Promielocítica , Coloración y Etiquetado/métodos , Fijación del Tejido/métodos , Factores de Transcripción/química , Proteínas Supresoras de Tumor/químicaRESUMEN
Mollicutes contaminations of cellular models can have marked effects on gene expression and cell behaviour in vitro leading to the production of unreliable data, unsafe biopharmaceutical drugs or to the loss of cell culture itself. Fortunately, irreplaceable cell culture can be cured by decontamination with the specific antibiotic regimen. Here, we describe the treatment of 35 mycoplasma-positive cell lines by the use of the novel antibiotic Mycozap(®) as well as evaluate its eradication performance versus the well-known routinely employed BM-Cyclins and fluoroquinolones molecules (175 treatments). Our data evidenced: i) the permanent elimination of mycoplasma infection by MycoZap(®), MRA, Enrofloxacin, Ciprofloxacin and BM-Cyclins in 46%, 29%, 40%, 43%, and 57% of the cultures, respectively; ii) a significant correlation between MRA and Ciprofloxacin eradication profile, as determined by the Spearman correlation coefficient (r = 0.3469, p < 0.05); iii) a mycoplasma eradication in 100% of cell lines by the exclusive adoption of MycoZap(®), Ciprofloxacin, Enrofloxacin, BM-Cyclin 1-2 antibiotic regimen, with the MRA exclusion; iv) the MycoZap(®) effectiveness even in case of a mycoplasmal load higher than 50 CFU/mL, as for SH-SY5Y and Neuro2A cells. In conclusion, we want to suggest an optimized antibiotic panel to get 100% mycoplasma-clearance especially in case of unique or treatment-resistant cellular models.
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Antiinfecciosos/farmacología , Viabilidad Microbiana/efectos de los fármacos , Infecciones por Mycoplasma/tratamiento farmacológico , Mycoplasma , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , HumanosRESUMEN
BACKGROUND: Mollicutes contamination is recognized to be a critical issue for the cultivation of continuous cell lines. In this work we characterized the effect of Mycoplasma hyorhinis contamination on CD133 expression in human colon cancer cell lines. METHODS: MycoAlert and mycoplasma agar culture were used to detect mycoplasma contamination on GEO, SW480 and HT-29 cell lines. Restriction fragment length polymorphism assay was used to determine mycoplasma species. All cellular models were decontaminated by the use of a specific antibiotic panel (Enrofloxacin, Ciprofloxacin, BM Cyclin 1 and 2, Mycoplasma Removal Agent and MycoZap). The percentage of CD133 positive cells was analyzed by flow cytometry on GEO, SW480 and HT-29 cell lines, before and after Mycoplasma hyorhinis eradication. RESULTS: Mycoplasma hyorhinis infected colon cancer cell lines showed an increased percentage of CD133+ cells as compared to the same cell lines rendered mycoplasma-free by effective exposure to antibiotic treatment. The percentage of CD133 positive cells increased again when mycoplasma negative cells were re-infected by Mycoplasma hyorhinis. CONCLUSIONS: Mycoplasma hyorhinis infection has an important role on the quality of cultured human colon cancer cell lines giving a false positive increase of cancer stem cells fraction characterized by CD133 expression. Possible explanations are (i) the direct involvement of Mycoplasma on CD133 expression or (ii) the selective pressure on a subpopulation of cells characterized by constitutive CD133 expression.In keeping with United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines, the present data indicate the mandatory prerequisite, for investigators involved in human colon cancer research area, of employing mycoplasma-free cell lines in order to avoid the production of non-reproducible or even false data.
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Antígenos CD/biosíntesis , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/microbiología , Glicoproteínas/biosíntesis , Infecciones por Mycoplasma/inmunología , Mycoplasma hyorhinis/inmunología , Antígeno AC133 , Línea Celular Tumoral , Células HT29 , Humanos , Péptidos , TenericutesRESUMEN
AIMS: The UbcH10 ubiquitin-conjugating enzyme plays a key role in regulating mitosis completion. We have previously reported that UbcH10 overexpression is associated with aggressive thyroid, ovarian and breast carcinomas. The aim of this study was to investigate UbcH10 expression in human lymphomas. METHODS AND RESULTS: Cell lines and tissue samples of Hodgkin's lymphoma (HL) and of non-Hodgkin's lymphoma (NHL) were screened for UbcH10 expression at transcriptional and translational levels. UbcH10 expression was related to the grade of malignancy. In fact, it was low in indolent tumours and high in a variety of HL and NHL cell lines and in aggressive lymphomas. It was highest in Burkitt's lymphoma, as shown by quantitative real-time polymerase chain reaction and by tissue microarray immunohistochemistry. Flow cytometry of cell lines confirmed that UbcH10 expression is cell-cycle dependent, steadily increasing in S phase, peaking in G(2)/M phase and dramatically decreasing in G(0)/G(1) phases. We also showed that UbcH10 plays a relevant role in lymphoid cell proliferation, since blocking of its synthesis by RNA interference inhibited cell growth. CONCLUSIONS: Taken together, these results indicate that UbcH10 is a novel lymphoid proliferation marker encompassing the cell cycle window associated with exit from mitosis. Its overexpression in aggressive lymphomas suggests that UbcH10 could be a therapeutic target in this setting.
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Enfermedad de Hodgkin/enzimología , Linfoma no Hodgkin/enzimología , Enzimas Ubiquitina-Conjugadoras/genética , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Enfermedad de Hodgkin/patología , Humanos , Linfoma no Hodgkin/patología , Enzimas Ubiquitina-Conjugadoras/metabolismoAsunto(s)
Células de la Médula Ósea/citología , Antígeno CD56 , Integrina alfa4 , Células Madre Mesenquimatosas/citología , Células Madre Multipotentes/citología , Neprilisina , Placenta/citología , Adolescente , Adulto , Células de la Médula Ósea/enzimología , Diferenciación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12/farmacología , Endotelio/efectos de los fármacos , Endotelio/metabolismo , Femenino , Citometría de Flujo , Fucosa/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glicosiltransferasas/metabolismo , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/enzimología , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/enzimología , Fenotipo , Placenta/enzimología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Selectinas/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
BACKGROUND: Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. RESULTS: In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA). CONCLUSION: Our study, comparing surface antigen expression of ALDH+/CD34+, ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow, clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.