Figure 1.
Photomicrographs (10X) of passage 2 day 7 sphere cultures of (A) TC-1 spheres, (B) HM-LLC spheres and (C) LM-LLC sphere media culture. Bar = 100 µm. (D) Total fold expansion and (E) symmetric division frequency over eight passages for TC-1 spheres (red) and HM-LLC spheres (black), and two passages for LM-LLC sphere media culture (grey) shows significant differences; *P<0.001. (F–G) Fold expansion over seven passages for TC-1 spheres (red) and HM-LLC spheres (black). (H) Passage 2 spheres were cultured and counted for total number of cells on the days as shown. Experiment repeated twice, representative results shown. On day 1 the total number of cells in culture is reduced. The surviving mitogen-responsive cancer initiating cells are responsible for cell proliferation and generating fresh multi-cellular spheres that can be harvested on day 7. Bars: ±Standard error of the mean (SEM).
Table 1.
Murine lung cancer cell lines sphere forming capacity.
Figure 2.
Oct-4 expression is increased in spheres compared to adherent cells.
Real time PCR quantitation was done for Oct-4, a gene associated with stem cell self-renewal, mRNA expression. TC-1 spheres (A) and HM-LLC spheres (B) demonstrate increased Oct-4 expression compared to matched adherent cells; *P≤0.001. HM-LLC cells expressed significantly more Oct-4 than LM-LLC adherent cells; **P = 0.005. Plots represent combined data from 2–4 biological replicates, each with three technical replicates. TC-1 cells (red); LM-LLC adherent (grey); HM-LLC cells (black). Bars: ±SEM.
Figure 3.
Aldefluor expression is enhanced in HM-LLC but not in TC-1 spheres compared to adherent cells.
(A–B) Representative plots showing Aldefluor staining (blue) and DEAB control staining (red) for HM-LLC, LM-LLC and TC-1 adherent cells compared to passage 2, day 1 and day 7 sphere-derived cells. Aldefluor high expressing cells (rectangular box) are significantly greater in (C) frequency and (D) mean fluorescent intensity (MFI) of Aldefluor within HM-LLC spheres compared to LM-LLC or HM-LLC adherent cells, and HM-LLC adherent cells express more Aldefluor than LM-LLC adherent cells; frequency (C) *P≤0.001; MFI (D) *P≤0.002. Compared to DEAB staining, Aldefluor positivity (non-rectangular box) was found to be significantly higher in TC-1 adherent cells compared to spheres (E); *P≤0.004. MFI of Aldefluor within the TC-1 groups (F) was not found to be significantly different. No Aldefluor high population was noted for TC-1 spheres or adherent cells. Plots represent combined data from two biological repeats with three technical replicates each. Bars: ±SEM.
Figure 4.
Hoechst 33342 side population (SP) expression is enhanced in HM-LLC but not in TC-1 spheres compared to adherent cells.
Representative SP plots for (A) LLC cells and (B) TC-1 cells comparing matched adherent cells to passage 2, day 1 and day 7 sphere-derived cells. Hoechst 33342 staining was confirmed with verapamil blockade. (C) SP frequency was significantly increased in HM-LLC spheres compared to adherent cells; *P<0.001; **P≤0.012. HM-LLC and LM-LLC adherent cells were not significantly different for SP frequency. (D) SP was not found to be upregulated in sphere-derived TC-1 cells compared to matched adherent cells. Day 1 sphere-derived cells had a significantly lower frequency of SP cells compared to adherent cells and day 7 sphere-derived cells; *P = 0.03. Plots represent combined data from three biological replicates with three technical replicates each. Bars: ±SEM.
Figure 5.
CD133/CD44 cell surface expression.
(A) Representative flow cytometry plots for CD133 (Y-axis) and CD44 (X-axis) co-staining. (B and D) The mean fluorescent intensity (MFI) of CD44 is shown corrected for intensity of isotype staining for LLC and TC-1 cells. (B) HM-LLC spheres and adherent cells as well as LLC (ATCC) cells express higher amounts of CD44 compared to LM-LLC cells. Sphere-derived cells were found to have a lower MFI for CD44 compared to HM-LLC adherent cells. D7 sphere-derived cells had a lower MFI for CD44 compared to D1 cells; *P<0.001. (D) Day 1 TC-1 sphere-derived cells express less CD44 than day 7 or adherent cells; *P≤0.002. (C) CD133 frequency of positive cells is higher in HM-LLC spheres compared to LM-LLC adherent cells; *P≤0.043. CD133 expression was not significantly different between HM-LLC adherent cells and spheres or amongst the three different LLC adherent cells. (E) No significant difference in CD133 expression amongst the TC-1 culture conditions. Experiment done in triplicate, representative results shown. Bars: ±SEM.
Figure 6.
In vivo tumorigenicity of TC-1 and LLC cells in immunocompetent and immunodeficient mice.
Confidence interval plot of limiting dilution analysis is shown with the y-axis displaying the estimated frequency of cancer initiating cells (CIC). (A) A limiting dilution of TC-1 cells (120,000, 80,000, 40,000, 10,000, 5,000, and 1,000) were injected subcutaneously into syngeneic C57BL/6 mice (n = 4 to 8). Sphere derived cells were found to be more tumorigenic than adherent cells; *P≤0.001; **P<0.02. Day 1 sphere-derived cells were more tumorigenic than day 7; ***P = 0.04. A limiting dilution (10,000, 1,000, and 500 cells) (n = 6 to 8) of the same three groups were injected into NSG mice and CIC frequency was found to be below 1/500 for all groups. (B) Analysis of LLC limiting dilution tumor experiment for syngeneic (80,000, 40,000, 20,000, and 10,000 cells) and immunocompromised (NSG) mice (80,000, 40,000, 10,000, and 1,000 cells) (n = 4 to 9). No significant differences noted; however, sphere-derived cells trended towards being more tumorigenic than HM or LM adherent cells in C57BL/6 mice. Lower numbers of implanted cells were needed to initiate tumors in immunocompromised animals than in syngeneic animals with the estimated CIC frequency below 1/1000 for all LLC groups.