Figure 3. In vivo efficacy of AK750 in a syngeneic B-16/F10 melanoma C57BL/6 mice model.

(a) Representative image shows the temporal accumulation of a NIR dye-tagged AK750 in the tumor in a melanoma-bearing mouse model. (b) Graph shows the quantification of actual drug concentration reached in the tumor, in vivo, as measured using LC-MS. Tumor-bearing animals were injected with equimolar dose of the drugs. Data shows mean ± SEM (n = 3), *p < 0.05 (Student’s t-test, two-sided). (c) Tumor growth curves show effect of different treatments on tumor volume. Each animal was injected with three doses of either vehicle (for control group), 45 mg/kg of free BLZ945, and AK750 (at equimolar dose to BLZ945) on day 0, day 4 and day 8. First day of treatment was considered as Day 0. Treatment with AK750 was significantly more effective than BLZ945. One should note the different routes of administration, and i.p. administration can result in lower bioavailability than i.v. administration. Data shown are mean ± SEM (n = 5), ***p < 0.001 (One-way ANOVA). (d) Graph shows drug toxicity assessed by measurements in overall body weight. Data shown are mean ± SEM (n = 5). (e) Western blot shows expression of phosphor-CSF-1R and total CSF-1R in 3 representative tumors in each treatment group, in vivo. The cropped blots are used in the figure, and full-length blots are presented in Supplementary Fig. 14; (f) Graphs show the quantification of expression of different M2 markers (CD11b+CD206+), or M1 markers (CD11b+MHC-II+, CD11b+CD86+ and CD11b+CD80+) in single cell suspension of the harvested tumor post-treatment, as quantified using flow cytometry. Tumors were harvested on day 10 and single cell suspension was prepared. Data shown are mean ± SEM (n = 3), p values are shown in the graphs. Statistical analysis was performed with One-way ANOVA with Newman-Keuls post Test.