Figure 1.
Analysis of Sp17 expression in ID8 cells.
Sp17 mRNA in the murine ID8 tumor cells and in mouse testis (positive control). The mRNA levels were analyzed by RT-PCR for Sp17 expression. A cellular housekeeping gene, B-actin, was included as a control. A PCR-only control (no RT step) failed to generate a product, indicating that there was no DNA contamination in the samples. In addition to the RT-PCR (see figure 1a), Western blot was performed and figure 1b shows the expression of Sp17 at the protein level. The ID8 cell line was further characterized by an immunocytochemistry (ICC) and immunofluorescence (IF). This figure shows a cytospin of ID8 cells permeabilized (P) and a positive staining for Sp17 at the cytoplasm level. In addition, Sp17 was confirmed by IF in the cytoplasm. Additionally, in figure 1c, the non permeabilized (NP) cells show clear expression via IF and less by ICC. Panel d shows ID8 characterization for surface expression of Sp17 and MHC-I; isot. ctrl, isotypic control antibody; percentage indicate positive-staining cells. MHC-I expression was evaluated with or without IFN-g stimulation.
Figure 2.
(a, b, c, d) Tumor growth in ID8-injected mice.
Shows the progressive formation of ascites/tumors when i.p. injected with ID8 cells. ID8 injected mice are shown as m1-m5. Control mice were injected with PBS alone. 1a shows mice injected with 105 ID8 cells. The tumors did not reach a significant size until after 150 days. 2b shows the progressive formation of ascites/tumors when i.p. injected with 5×105 ID8 cells. The tumors did not reach a significant size until after 120 days. 2c shows the progressive formation of ascites/tumors when i.p. injected with 106 ID8 cells. The tumors reached a significant size in 90–120 days. 2d shows the progressive formation of ascites/tumors when i.p. injected with 2×106 ID8 cells. The tumors reached a significant size in 35–60 days (p<0,0001); a visible enlargement of the mice was noticed at 30 days.
Figure 3.
(a, b, c, d) Analysis of ID8 cells growth and dissemination in vivo.
a shows on the left side a control mouse (width of mouse 20 mm) not injected with ID8 cells and on the right side a mouse injected with 2×106 ID8 cells after 40 days (width of mouse 44 mm). These mice represent experiments with similar results. b shows the full peritoneal cavity of ascites induced from an i.p injection of 2×106 ID8 in 40 days (left) and an open view of the cavity with several metastatic nodes and tumor masses (center and right, after aspirating 22 mL of ascites). c shows a peritoneum negative for Sp17 from a control mouse not injected with ID8 cells. f shows a positive expression of Sp17 on the peritoneum of a mouse injected with 2×106 ID8 cell after 40 days. Testis is the positive control for Sp17 by ICH (g). Figure d shows PCR for Sp17 DNA (and B-actin control): a tissue panel derived from (1) the organs of a 2×106 ID8 injected mouse was positive for Sp17 and a panel of tissues derived from organs of a healthy mouse revealed no expression of SP17. Positive controls (ID8 and testis) for Sp17 are also shown. Panel h shows in vivo fluorescence pictures of a control (left) and ID8-injected (right) mouse for the localization of GFP-positive ID8 cells.
Figure 4.
(a,b) Survival analysis with different vaccination schemes.
Overall survival of mice from the four prophylaxis (a) or therapeutic (b) groups, detailed in the text (tumor free, Sp17+CpG vaccinated, unvaccinated, Sp17 or CpG vaccinated). Tumor-bearing mice were i.p. injected with 106 ID8 cells for both the prophylactic and therapeutic regimens. Tumor cells were injected 30 days after the 3rd vaccination of the prophylactic regimen, or 21 days before the first therapeutic vaccination. In the therapeutic group, the mean difference in weight between tumor-challenged and tumor-free animals was 10 grams at the beginning of vaccination regimen. Horizontal axis displays time expressed as days from initiation of treatment. Log-rank test indicated statistically significant difference between unvaccinated and Sp17+CpG vaccinated versus unvaccinated or CpG treated group (p<0.0001) for both regimens.
Figure 5.
Measurement of circulating anti-Sp17 IgG following different vaccinations.
Serum from mice undergone different treatments was collected and analyzed by E.L.I.S.A. for the levels of circulating anti-Sp17 IgG. The X axis shows serial dilutions of serum. a, b and c show results obtained in the prophylactic vaccination schemes, while d displays the response of mice from the therapeutic regimen. On day 7 (a) the immune response was low for Sp17. On day 97 (b), the immune response was higher than on day 7. On day 270 (c), the response for Sp17 was little less than on day 97.
Figure 6.
(a,b) Measurement of cytokines in mice treated with different vaccines.
On day 300, serum was collected and analyzed by E.L.I.S.A. for the measurement of the indicated cytokine levels in prophylactically (a) or therapeutically (b) vaccinated mice. For both regimens, IFN-gamma, TNF-alpha and GM-CSF statistically significant increments were detected only in Sp17+CpG vaccinated mice compared with controls (PBS). No significant differences were evidenced for IL-2, IL-4, IL-5 or IL-10 levels.
Figure 7.
ELISPOT for the assessment of IFN-gamma and TNF-alpha serum levels.
On day 270 (9th vaccine) splenocytes from different formulations of vaccinated mice and controls were collected and analyzed by E.L.I.SPOT assay. a,b) frequency of IFN-gamma and TNF-alpha positive cells in prophylactic vaccinations; c,d) frequency of IFN-gamma and TNF-alpha positive cells in therapeutic vaccinations. These results are presented as spot-forming cells per 106 splenocytes. Spot numbers represent the mean of ten mice in each vaccinated group; bars, SE.
Figure 8.
Cytotoxicity assay in the prophylaxis regimen.
Splenocytes from the three different formulations of prophylactically vaccinated mice and controls were collected and analyzed by 51Chromium-release assay on days 7 (first vaccine), 97 (third vaccine) and 270 (ninth vaccine), using splenocytes as effector cells and ID8 as target cells. These results were obtained from three independent experiments. X axis indicate effector:target ratios.
Figure 9.
Cytotoxicity assay in the therapeutic regimen.
Splenocytes from the three different formulations of therapeutically vaccinated mice and controls were collected and analyzed by 51Chromium-release assay. These results show three independent experiments after third vaccine (day 97) and ninth vaccine (day 270), using splenocytes as effector cells and ID8 as target cells. X axis indicate effector:target ratios.
Figure 10.
Analysis of Th-17 and T-reg population frequencies.
Splenocytes from mice that received prophylactic or therapeutic vaccinations or controls (mice with no tumor or tumor-bearing mice without vaccinations) were collected at different time points (no tumor: day 0; ID8 only: day 45; prophylactic and therapeutic vaccines: day 270) analyzed by flow-cytometry for the measurement of a) Th-17 population (CD4/IL-17-double positive) or b)T-reg population (CD4/Foxp3-double positive) frequency.