Figure 1.
Expression profiling of SP/MP cells from ascites specimens.
(A) Heatmap showing the expression pattern of 438 probesets that discriminated SP cells from MP cells in ovarian cancer patients. Vertical columns represent individual samples; horizontal rows represent individual genes. The red and blue color indicates up and down-regulation respectively. (B) Validation of microarray data using qRT-PCR: 19 randomly selected genes were used to validate the microarray data. To calculate the relative expression for each gene, the 2−ΔΔCT method was used averaging the CT values for the three housekeeping genes (Cyclophilin A, GUSB, GAPDH) for a single reference gene value. (C) Representative plots for correlation analysis between the microarray and qRT-PCR data: The 2−ΔCT values (qRT-PCR) were plotted against signal intensity values (Microarray). The correlation analysis was performed for each gene by Pearson's and Spearman's method using GraphPad Prism version 4.00.
Table 1.
The percentage of the SP cells identified in ascites samples.
Figure 2.
Identification of putative signaling events contributing to ovarian cancer SP cell survival, self-renewal and tumor maintenance.
(A) Gene ontology analysis of microarray data (P value<0.01): The pie diagram shows the biological functions of the differentially expressed genes among SP and MP cells. The gene signature was enriched for genes in gene ontology biological processes of apoptosis, cell cycle, cell proliferation, transport, signal transduction, transcription, translation, protein modification, metabolism and proteolysis. Other represents genes involved in defense response, vasculogenesis, blood coagulation, visual perception, ontogenesis, cell matrix adhesion, and initiation of primordial ovarian follicle growth. (B) Graphical representation of the literature derived facts about the biological pathways involved in SP cells. Pathway Studio 6.0 software was used to identify the activated pathways in SP cells. Solid symbols representing the genes (EGFR, TNFRSF6, BCL2L11, FOXO3A, RUNX1) as down regulated in SP cells, the open symbols represents the upregulated genes (EPHB4, EPHB2, PAWR, AKT1) and gray symbols are the genes whose expression did not change significantly between SP and MP cells. (C) The Notch signaling pathway: The figure shows the schematic of Notch signal transduction elements. The overexpressed proteins in SP cells (FPTG, ST3GAL6 and ADAM19) are shown in blue color. Post-translational modification of precursor Notch-protein includes cleavage by proteases and glycosylations in the trans-Golgi. Adherence of Notch extracellular domain (ECN) with Notch intracellular domain (ICN) results in mature Notch heterodimers and they are transferred to the cell membrane. Receptor interaction with the ligands of the DSL family (Delta, Serrate, Lag3) on neighboring cells is modulated by glycosylations of ECN. Successful interaction of extracellular ligand regions with EGF-like repeats of ECN lead to the proteolytic cleavages of Notch transmembrane domain by two sequential steps by ADAM proteases and presenilins with γ–secretase activity. The released Notch intracellular domain is translocated to the nuclease where it interacts with CSL1, replacing CSL repressors and forming a transcription complex with Mastermind-like factors and transcriptional coactivators. This transcriptional complex activates downstream target genes and may account for cancer stem cell survival, self-renewal and tumor maintenance in ovary.
Figure 3.
Identification and validation of side population cells from ovarian cancer cell lines.
(A) Identification of SP cells in established human ovarian cancer cell lines. SKOV3 and A224 cell lines were labeled with Hoechst 33342 dye and analyzed by flow cytometry. The SP cells, which disappeared in the presence of Verapamil (a multidrug transporter inhibitor), are outlined and shown as the percentage of the total cell population. Similar results were obtained for three independent measurements. (B) Validation of randomly selected genes from the SP gene list on the SP and MP cells isolated from the SKOV3 cell lines. (C, D) Colony forming efficiency assay: Colony forming efficiency of sorted SP and MP cells from SKOV3 and A224 cell lines. For the analysis of colony forming efficiency (CFE), SP and MP cells were sorted and plated in equal numbers in tissue culture six well plates and grown for 14 days. The cells were then fixed with cold methanol and stained with 0.5% crystal violet solution to count the number of colonies by microscopy. The experiments were carried out in triplicates. (E, F) Passage Number: Colony forming efficiency of sorted SP and MP cells from SKOV3 and A224 cell lines were evaluated as a function of passage number. Cells were fixed with cold methanol and stained with 0.5% crystal violet.
Figure 4.
Biological validation of SP cells isolated from ovarian cancer cell lines.
(A, B) Anchorage independent growth of the sorted SP and MP cells from SKOV3 and A224 cell lines. (C, D) Single cell assay in 96 well plates: Single cells of A224 and SKOV3 SP and MP cells were cultured in individual wells. The single cells were allowed to grow for 14 days and the colony forming efficiency was estimated using crystal violet staining. (E) In vivo tumor growth of SKOV3 SP and MP cells in female athymic nude mice.
Figure 5.
Validation of SP cells from cell lines and the effect of GSI-IX inhibitor on SP cells colony forming efficiency.
(A) Repopulation Assay: The SKOV3 and A224 cells were stained with Hoechst 33342 dye and sorted for SP and MP populations. The cells were cultured for 8 days for repopulation, and then reanalyzed by flow cytometry. This demonstrated the enrichment of SP cells (22.5% and 10.4% for SKOV3 and A224 resp.) with a capacity to regenerate to MP cells. (B, C, D) qRT-PCR analysis for stem cell marker genes and transporter genes (p<0.01). To calculate the relative expression for each gene, the 2−ΔΔCT method was used averaging the CT values for the three housekeeping genes (Cyclophilin A, GUSB, GAPDH) for a single reference gene value. (E, F) Inhibitory effect of GSI-IX on Colony forming efficiency of sorted SP and MP cells from SKOV3. Cells were sorted to SP and MP populations and treated with 10 and 20 µg of GSI-IX. The inhibitor carrier DMSO is used as a control.
Figure 6.
Validation of SP cell signature on ovarian tumor recurrent expression database.
(A, B) qRT-PCR Validation of SP cell gene signature in recurrent ovarian cancer specimens. (A) Group 1 consists of tumor recurrence observed between 1 to 12 months and (B) group 2 in between 13 to 24 months. (C) Electronic validation of SP cell gene signature in recurrent ovarian cancer patients.