Immunotherapeutic Potential of Mollusk Hemocyanins in Combination with Human Vaccine Adjuvants in Murine Models of Oral Cancer

Figure 3

Antitumor effects of KLH and FLH in combination with QS-21 adjuvant in an orthotopic mouse model of oral cancer. (a) Experimental scheme. Five C3H/He mice were primed subcutaneously in the superior right flank (day −14) with the following treatments: 100 μg of hemocyanin alone in 100 μl of PBS (vehicle) or 50 μg of each hemocyanin in combination with 10 μg of QS-21 in a total volume of 100 μl of PBS. The controls were 10 μg of QS-21 alone in 100 μl of PBS and 100 μl of PBS. For the tumor cell challenge (0 days), the mice received AT-87 Luc cells in the mouth floor. After 24 days, the animals received a subcutaneous injection in the superior right flank identical to the priming dose, which was then repeated 4 times at intervals of 3 days. DTH determination was performed on day 15 of the bioassay. Tumor growth was monitored with the IVIS® Lumina imaging system and palpation. For acquisition, the mice were anesthetized and injected intraperitoneally with 150 mg/kg D-luciferin; 10 minutes later, the animals were exposed to the CCD camera for 5 minutes. (b) Tumor growth at day 16. The tumor areas of the mice receiving PBS treatment were compared to those of the mice treated with hemocyanins alone and in combination with QS-21 as well with the adjuvant alone. Data are shown as of one representative experiment with mice per group for FLH + QS-21 and QS-21, four mice per group for KLH and FLH, and three mice per group for KLH + QS-21. and , two-tailed Student’s -test. (c) Representative murine bioluminescence images. Representative in vivo images (IVIS) of luminescence shown in the mouth floor of live C3H/He mice on days 7, 11, and 16 after the start of the experiment. Images obtained from ventral views of the mice show relative levels of luminescence ranging from low (blue) to medium (green) to high (yellow/red); the scale on the right indicates the average radiance. The green crosses represent animals that were euthanized, and the red crosses represent animals that died after the application of anesthesia. (d) Survival analysis of mice. A Kaplan-Meier survival analysis was performed using the log-rank test. for one representative experiment with mice per group, with the exception of the FLH plus QS-21 group (four mice).

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