Figure 6: Cryogel vaccines confer long-term prophylactic and therapeutic protection against melanoma.
(a) A comparison of the survival rate in challenged C57BL/6 mice previously prophylactically vaccinated with (A) bolus vaccine injection (4 × 105 irradiated B16-F10 cells+soluble 3 μg GM-CSF+soluble 100 μg CpG ODN); (B) Cryogel vaccines (2 × 2 × 105 irradiated B16-F10 cells+2 × 1.5 μg GM-CSF+2 × 50 μg CpG ODN); (C) Cryogel vaccines (2 × 2 × 105 irradiated B16-F10 cells+2 × 1.5 μg GM-CSF); (D) Cryogel vaccines (2 × 2 × 105 irradiated B16-F10 cells+2 × 50 μg CpG ODN); (E) Blank cryogel (control; negative control); (F) naive mice (no immunization). At day 6 following immunization, C57BL/6 J mice (10 mice/group) were challenged with 105 B16-F10 tumour cells and monitored for animal survival. (b) Tumour growth curve after the first tumour challenge (105 cells) on prophylactically vaccinated mice. (c) A comparison of the survival rate in re-challenged mice prophylactically treated with (A) bolus injection (4 × 105 irradiated B16-F10 cells+soluble 3 μg GM-CSF+soluble 100 μg CpG ODN); (B) Cryogel vaccine (2 × 2 × 105 irradiated B16-F10 cells+2 × 1.5 μg GM-CSF+2 × 50 μg CpG ODN); (D) Cryogel vaccine (2 × 2 × 105 irradiated B16-F10 cells+2 × 50 μg CpG ODN); and (F) naive mice (no immunization). At day 126 following immunization, C57BL/6 J mice (10 mice/group) from the first challenge study were challenged a second time with 105 B16-F10 tumour cells and monitored for survival. (d) Overall survival rate after two consecutive tumour challenges in prophylactically immunized mice to evaluate long-term immunological protection in the context of melanoma. Overall survival is defined as the fraction of mice that survive both challenges. (e) A comparison of the survival of mice bearing established melanoma tumours (inoculated with 5 × 105 B16-F10 cells and allowed to develop for 3 days, tumour area≥10 mm2) and therapeutically treated with cryogel vaccines (2 × 2 × 105 irradiated B16-F10 cells+2 × 1.5 μg GM-CSF+2 × 50 μg CpG ODN) either once at day 3 (Cryogel Vax – 1X) or at both days 3 and 10 (Cryogel Vax – 2X), or naive mice (Control, no immunization). (f) Individual tumour growth curves for each mouse surviving tumour challenge (5 × 105 cells) after a two-time treatment with cryogel vaccines at days 3 and 10. Values represent mean and s.d. (n=10 per condition) from at least two independent studies. For simplification, only Fig. 6b shows the legend but it applies also to Fig.6a, c and d. Data were analysed using χ2-test, log-rank test (survival curves comparison), or one-way analysis of variance (ANOVA) (**P<0.01, ***P<0.001).
