Research article
Genomic profiling of tumor initiating prostatospheres
1 Cancer Stem Cell Section. Laboratory of Cancer Prevention. National Cancer Institute at Frederick, 1050 Boyles Street, Frederick, MD 21702, USA
2 Laboratorio de Interacciones Moleculares. Departamento de BiologÍa Molecular y Celular. Facultad de Ciencias. Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay
3 Departamento de Genética. Facultad de Medicina. Universidad de la República, General Flores 2125, Montevideo 11800, Uruguay
4 Laboratory of Molecular Technology. Advanced Technology Program, SAIC-Frederick, Inc. National Cancer Institute at Frederick, 915 Toll House Avenue, Frederick, MD 21702, USA
5 Facultad de Ciencias. Universidad de la República, Iguá 4225, Montevideo 11400, Uruguay
6 Departamento de NeurobiologÍa Molecular y Celular. Instituto de Investigaciones Biológicas Clemente Estable, Avenida Italia 3318, Montevideo 11600, Uruguay
BMC Genomics 2010, 11:324 doi:10.1186/1471-2164-11-324
Published: 25 May 2010Additional files
Additional file 1:
Characterization of PCSCs tumor cell lines. A. Expression of stem cell makers at the protein level. OCT3/4, NANOG, BMI1, SSE4, AR and KRT14 were assessed by western blot using total extracts and infrared conjugated antibodies in a LICOR's Odyssey Infrared Imaging System. Positive and negative signals are indicated by "+" and "-"respectively. Percentage of cells expressing CD44 and CD133 surface markers was determined by flow cytometry and is expressed as percentages of the total cell line. Aldehyde dehydrogenase (ALDH) activity was assessed with ALDEFLUOR® kit (StemCell Technologies Inc.) and is expressed as percentage of positive cells.
B. Anchorage independent growth of PCSCs. Soft agar assays were carried out in 96 well plates using Cell Biolabs CytoSelect™ 96-well Cell Transformation Assay. 500, 2500 and 10000 cells were plated in triplicates. Upper panel shows 2 × lens low magnification pictures of the entire well (of a 96 well plate) at 2500 cells density after 10 days in culture. Lower plot presents the averaged relative fluorescent units for the three cell densities assessed at 10 days in culture.
C. Kapplan-Meier survival curves of NOD/SCID mice xenograft experiments injecting parental PCSCs. 100, 1000, 10000 and 300000 cells of PCSC-1, PCSC-2 or PCSC-3 were used per injection as described in Materials and Methods.
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Additional file 2:
qRT-PCR validation of selected genes. Stem cell markers in LNCaP (A) and PCSCs (B) and the 66 gene signature genes (C). The data for each gene is the average of at least two biological replicates. Error bars represent averaged standard error in the measurements of a given gene.
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Additional file 3:
Most significant (p ≤ E-4) functional categories represented in the 66 genes PS gene set. The numbers indicate the number of genes in each category.
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Additional file 4:
List of TF binding sites enrichments retrieved from MSigDB. Upper chart presents the five PSs that are common to LNCaP and PCSCs (≥2-fold change and p ≤ 0.01). Lower chart describes those TF that are specific for each type of cell line.
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