.. IGF-1R antagonists inhibit proliferation of ACC cell lines IGF signaling regulates several aspects of cellular function including the enhancement of proliferation. To test the functional consequence of IGF-1R inhibition, MTS proliferation assays were performed on H295 cells treated with ARQ197 IMC-A12 or NVP-AEW541 (Fig. 44).). We used the RL251 cell line as a negative control in these assays because it lacks expression of IGF-1R protein in comparison with other ACC lines. As predicted, increasing doses of IMC-A12 had no substantial effect on the RL251 cells, whereas high concentrations of the antibody exhibited antiproliferative effects on H295 cells (Fig. 44,, left panel). The IMC-A12 IC50 value for the H295 cells was 90 nm, but 50% inhibition was not achieved with RL251 cells, even at the highest IMC-A12 dose (P < 0.

001 comparing RL251 vs. H295 at 100 nm A12). The NVP-AEW541 IC50 value for H295 cells was 2.7 ��m, whereas for RL251 cells, the IC50 was 36 ��m (P < 0.001 comparing RL251 vs. H295 at 3 ��m NVP-AEW541), again demonstrating targeted suppression of the H295 cell line��s ability to proliferate due to the presence of IGF-1R. Figure 4 Antiproliferative effects of IGF-1R antagonist treatments in vitro. H295 and RL251 cells were incubated with increasing micromolar concentrations of NVP-AEW541 (right panel) or nanomolar concentrations of IMC-A12 (left panel), and proliferation was assessed ... Targeting of IGF-1R delays tumor xenograft growth Stemming from our data revealing IGF-1R inhibition results in a significant decrease of downstream signaling and proliferation in vitro, we assessed its in vivo activity using human ACC cell lines.

Using the rationale discussed above, we used the RL251 cell line as an additional control in these assays. H295 or RL251 cells were inoculated sc into athymic nude mice, and the mice were subsequently randomized into three treatment groups (n = 8 tumors per group for H295 and n = 10 for RL251): placebo, IMC-A12 treatment, and NVP-AEW541 treatment. Tumor volume measurements were taken three times a week for 21 d. The data were plotted as the log ratios of tumor size over initial tumor size to more accurately reflect the percent reduction in tumor size and, at the same time, account for the variable initial tumor sizes in each mouse (Fig. 5A5A).). RL251 xenografts had a higher tumor engraftment rate than H295 xenografts (94 vs.

75%, data not shown). IMC-A12 was well tolerated in treated mice with no substantial adverse Dacomitinib effects or weight changes observed between groups (data not shown). The treatment of H295 tumors with IMC-A12 over a 21-d span (Fig. 5A5A,, left panel) resulted in a significant reduction in tumor size over untreated controls (P < 0.001 from day 7 onwards). Treatment in RL251 xenografts (Fig. 5A5A,, right panel) resulted in a statistically insignificant reduction in tumor volume at each time point, underscoring the specificity of IMC-A12.