Presentation on theme: "Autores / Authors: C. Soriano 3 ; C. Gil-Gas 1 ; P. Castro-García 1 ; P. Honrubia 1 ; C.B. Álvarez-Simón 1 ; J. Ferre 1 ; S. Sabater 1,3 ; G. Aparicio."— Presentation transcript:
Autores / Authors: C. Soriano 3 ; C. Gil-Gas 1 ; P. Castro-García 1 ; P. Honrubia 1 ; C.B. Álvarez-Simón 1 ; J. Ferre 1 ; S. Sabater 1,3 ; G. Aparicio 2 ; L. A. Aparicio; Misco Jones 1 y C. Ramírez-Castillejo 1. Filiación / Hospital: (1) Laboratorio de Biología Celular y Molecular de la Célula Madre. Centro Regional de Investigaciones Biomédicas / Facultad de Medicina. Universidad de Castilla La Mancha. (2) Oncology Research Unit. CHU A Coruña, Spain. (3) Complejo Hospitalario Universitario de Albacete. (CHUA). Introducción y objetivos: Objetives: The cancer stem cell hypothesis suggests that cancer contains cancer cells with stem characteristics. (Reya et al, 2001). Several publications have described the cancer stem cell population in cell lines, for instance in the C6 glioma cell line (Kondo et al, 2004). Our work consist of analyzing the characteristics of this small “cancer stem cell” population in the C6 glioma. We have found a small population defined by the expression of two biomarkers that have previously reported in both stem and cancer stem cells. We have found 0,6% BCRP1+ cells, which Kondo et al. reported as the principal protein responsible for the Side Population in the same cell line, counting for similar values as we described in this report. BCRP1+ protein is related with the bone marrow stem cell population and confers resistance against toxics. On the other hand, we have found a smaller CD133+ population than the previous one. CD133 is a self-renewal related protein. Moreover, the whole CD133+ population match with a part of the BCRP1+ cells (BCRP1+ AC133+), suggesting that CD133+ cells could be a subpopulation of the BCRP1+ cells, and therefore, a more specific cancer stem cell population than previously reported in the C6 glioma cell line. Furthermore, these CD133+BCRP1+ cells display an aggressive phenotype when injected in NOD/SCID mice. Eventually, we have also found the cancer stem cell population (BCRP1+ CD133+) in glioma patients with aggressive disease, but not in patients with good prognosis. A Cancer Stem Cell population in the C6 glioma cell line. (1) Reya et al, Nature 2001 (2) Kondo et al, PNAS, 2004 Referencias: References: Métodos: Methods: Resultados: Results: AC133 AND BCRP1 POSITIVES CELLS STUDY IN XENOGRAFT SCID MOUSE MODEL. (A) Sorted cells in the C6 glioma line (Left) AC133 + /BCRP1 -+ population (B) Positives and negatives cells were injectedintraperitoneal into nude mice (4000 cells per inject), four weeks after the injection, mice were photographed and killed. Tumor sizes were measured and tumor growth volume were obtained (Right) the bars indicate differences between AC133 + /BCRP1 -+ and AC133 - /BCRP1 - tumors (C) Tumors formed were fixed and photographed by confocal microscopy. Histology was verified by hematoxiline stain. The graphic shows the significative difference (p<0.001) in number of BCRP1+ cells between positive tumors and the negatives ones. Conclusiones: Conclusions: *The patient with high levels of AC133+ cell has a poor prognosis. *In SCID mice, BCRP1+ cells form more aggressive tumors than BCRP1- cells *BCRP1- derived tumors do not contain AC133+ cells neither BCRP1+ cells, conversely to BCRP1+ tumors, which contains high rates of AC133+ and BCRP1+ cells. *AC133+ cell could be a subpopulation of the BCRP1+ cell population Anti-neoplasic treatment Tumors could contain Cancer Stem CellsCancer Stem Cell resist treatmenttumour reappears OUR WORK CONSIST OF LOCALIZATE THE CSC POPULATION USING THE BIOMARKERS: BCRP1 PROTEIN CD133 PROTEIN LONG CYCLE CELLS CÉLULAS MADRE TUMORALES EN LA LÍNEA CELULAR C6 DE GLIOMA Tema: SNC PROTEIN EXPRESSION LEVELS OF AC133+ AND BCRP1+ PRESENTS IN BRAIN TUMOURS AND CELL LINE LONG CELL CYCLE CELLS EXPRESS CD133 SELF RENEWAL RELATED PROTEIN (A) C6 glioma cells were stained with the fluorescent dye DFFDA. The 99.71% populations (green) is represent at 0 days in vitro, and the red circle make a distinction of slow kinetic 4.35% after 5 days in vitro (B) C6 cells were stained with DFFDA and CD133 inmunomagnetic antibodies, then cells were separated using MACS technology depending on CD133 expression. Cells were seed and analized by flow cytometry before 4 days in vitro. Flow citometry and cell sorting analysis Inmunomagnetic cell sorting Long retaining labeling cells monitoring assay Cell injection in SCID mice Inmunofluorescence and histology CSC (A-B) Flow cytometric analysis of BCRP1+ and AC133+ surface expression in two brain tumours. The firsts panels are controls of the stain (staining controls), graphic Right division represent positive staining for BCRP1+ and AC133+ association to each patient respectively. (C) Immunofluorescent images of C6 glioma cells (objetive 63× magnification) show BCRP1 expression as a green signal at the cell surface and blue signal is a DAPI nuclear expression as well as the evaluation of the expression was then performed by flow cytometry. (D) The expression in AC133 was detected In the same way in C6 glioma cells.