Figure 1.
Growth characteristics of human pancreatic cancer cell lines in culture.
(A) Representative photomicrographs of PANC-1 and BxPc-3 cells 72 h after seeding. (B) & (C) Growth curves of PANC-1 and BxPc-3 cells. Symbols are experimental data and curves represent the best fits to exponential growth equation: Y = Y0 × exp(k × X), where Y0 is the Y value when X (time) is zero and K is the rate constant. τ is the time constant (day) and DT (doubling-time) is in the time units of the X axis, computed as ln2/K.
Figure 2.
Analysis of the side population (SP) in three human pancreatic cancer cell lines PANC-1, SW1990, and BxPc-3, using flow cytometry and Hoechst33342 dye.
(A) Examples of floe cytometry analysis of SP cells in the presence or absence of 30 µM verapamil, cdODN-SOC (a complex decoy oligodeoxynucleotide carrying cis-elements for Sox2, Oct4 and c-Myc), or NC–ODN (negative control cdODN). (B) The SP proportions as percentage of total population. ***p<0.001 vs Control; n=4.
Figure 3.
Validation of the ability of cdODN-SOC to bind target transcription factors, assessed by electrophoretic mobility shift assay (EMSA).
(A) The sequences of the cdODN-SOC carrying cis-elements for Sox2, Oct4 and c-Myc and the negative control ODN with nucleotide replacements (NC–ODN). (B) Examples of EMSA images showing the ability of cdODN-SOC to bind human recombinant Sox2, Oct4 or c-Myc protein. The shift bands indicate the positions of the DNA-protein complexes and the supershift bands indicate the specific DNA-protein binding picked up by the respective antibody. Lane labels: 1, control with no proteins; 2: in the presence of cold or unlabeled cdODN-SOC; 3: in the presence of cdODN-SOC; 4: in the presence of NC–ODN; and 5: with antibody.
Figure 4.
Differential gene expression between PANC-1 SP and NSP cells.
(A) Expression of CD133, ALDH1, ABCG2, Sox2, Oct4, Nanog, c-Myc, and Notch1 at the mRNA level, determined by real-time RT-PCR. The values were obtained by first normalized to GAPDH for internal control and then presented as a ratio of SP over NSP. **p<0.01 SP vs NSP; ***p<0.001 SP vs NSP; n=4. (B) Expression of CD133, ALDH1, ABCG2, Sox2, Oct4, Nanog, c-Myc, and Notch1 at the protein level, determined by Western blot analysis. The values were obtained by first normalized to GAPDH for internal control and then presented as a ratio of SP over NSP. CD133: 120 kDa; ALDH1: 55 kDa; ABCG2: 72 kDa; Sox2: 40 kDa; Oct4: 45 kDa; Nanog: 35 kDa; c-Myc: 62 kDa; Notch1: 300 kDa. **p<0.01 SP vs NSP; ***p<0.001 SP vs NSP; n=4 (Similar results were observed in SW1990 and BxPc-3 cells; Figure S1 and Figure S2 online).
Figure 5.
Cell cycle characteristics and differentiation ability of PANC-1 SP and NSP cells.
(A) Percentage of PANC-1 SP and NSP cells in G0-G1 and S cell cycles. Note that cdODN-SOC minimized the difference between SP and NSP. *P<0.05 vs SP alone; n=4. (B) Freshly sorted PANC-1 SP and NSP cells were immediately reanalyzed to ensure the purity, and were cultured for 1 week before re-staining with Hoechst33342 dye and resorted. Note that the SP cells repopulated both SP and significant NSP proportions from the original sort, whereas the NSP generated only NSP cells, and cdODN-SOC eliminated the ability of SP cells to repopulate SP cells. ***P<0.001 vs SP alone; n=4.
Figure 6.
Suppression of the clonogenic ability of CSCs in h-PCCLs by cdODN-SOC.
(A) Upper left: representative examples of soft agar assays for clonogenic ability of PANC-1 CSCs under varying conditions and lower left: the amplified view of colonies. Right panel: mean data showing the effects of cdODN-SOC on the number of colonies. SP cells were more clonogenic than NSP cells and cdODN-SOC suppressed the clonogenic ability. ***p<0.001 vs SP alone. (B) Serial sphere formation assays for clonogenic ability of CSCs, showing the suppressive effects of cdODN-SOC on spherical formation. ***p<0.001 vs SP alone. Quantitatively the same results were obtained from SW1990 and BxPc-3 CSCs (data not shown).
Figure 7.
Inhibition of SP cell invasion and migration by cdODN-SOC.
(A) Invasion assay. PANC-1 CSCs were plated onto the Matrigel-coated membrane in the top chamber of the transwell and transfected or treated with gemcitabine for 48 h. All cells were mock-treated with lipofectamine 2000. Cells invaded to the lower chambered were fixed with methanol, stained with crystal violet and counted. ***p<0.001 vs SP alone; n=6. (B) Migration assay. PANC-1 CSCs were plated in the top chamber of the transwell and transfected or treated with gemcitabine for 36 h. All cells were mock-treated with lipofectamine 2000. Cells migrated to the lower chambered were fixed with methanol, stained with crystal violet and counted. ***p<0.001 vs SP alone; n=5. Quantitatively the same results were obtained from SW1990 and BxPc-3 CSCs (data not shown).
Figure 8.
Enhanced drug resistance of SP cells and suppression by cdODN-SOC.
(A) Percent cell survival measured by MTT assay in PANC-1 CSCs. All cells were all treated with varying concentrations of gemcitabine and with lipofectamine 2000. Symbols are experimental data and the lines represent best fits to Hill equation. ***p<0.001 vs SP alone for comparison of IC50 values; n=4. (B) Percent apoptotic cells measured by ELISA to quantify DNA fragmentation in PANC-1 CSCs. All cells were all treated with varying concentrations of gemcitabine and with lipofectamine 2000. Symbols are experimental data and the lines represent best fits to Hill equation. ***p<0.001 vs SP alone for comparison of IC50 values; n=4. Quantitatively the same results were obtained from SW1990 and BxPc-3 CSCs (data not shown).
Figure 9.
Enhanced in vivo tumorigenicity of SP cells and the anti-tumor effects of cdODN-SOC in BALB/C nude mice.
PANC-1 SP and NSP cells were orthotopically inoculated s.c. to the left dorsal flank of mice. On 50 days after inoculation, the mice were sacrificed to detect tumor formation. (A) The SP cells regenerated larger tumors than corresponding NSP cells at every cell dose. ***p<0.001 SP vs NSP; n=6. (B) Representative images of tumors in nude mice showing the difference in the tumorigenic potentials between SP and NSP and the anti-tumor effects of cdODN-SOC, as well as the drug resistance of SP-induced tumor to gemcitabine. For the PANC-1 cell line, as few as 1×104 SP cells formed tumors, while 1×106 NSP cells were required to initiate a tumor. (C) Mean data of tumor volume under different conditions. Note the ability of cdODN-SOC to reduce the tumor volume. ***p<0.001 vs SP; n=6.