Abstract
Colorectal cancer (CRC) is a common cause of oncological deaths worldwide. In clinical practice, a main concerning for choosing immune checkpoints inhibitors (ICI) is the low response rate, despite multiple predictive biomarkers have been adopted for patient selection. Recent clinical trials showed that ICI could effectively induce tumor regression in combination therapy with anti-angiogenetic treatment because the latter damages blood vessel and reprograms the tumor immune microenvironment. Here we tested sunitinib in combination with anti-PD-1 (nivolumab and CD279) in both in vitro and in vivo CRC models in order to assess the antitumor potential and shid light on cellular mechanisms underpinning drugs combination effects. To this purpose we utilized CaCo-2, Colo205, LoVo and HT-29 CRC cell lines for in vitro testing and CT-26 cell line for generating the syngenic in vivo model. For in vitro study the cells were challenged with scalar concentrations of sunitinib and nivolumab alone and in combination, for cytotoxicity evaluation by MTT and the combination index (CI) was calculated to determine the pharmacological interaction. The effects on cell cycle progression and apoptosis induction were determined by propidium iodide and Annexin V staining, respectively, followed by FACS analysis. Cell targets modulation was evaluated by western blotting. The in vivo study was performer in CT-26 syngenic mouse model. To treat established tumours (∼100 mm3), from day 8 after cell inoculation sunitinib and the anti-PD-1 CD279 were administered intraperitoneally to mice as follows: group (1) vehicle alone (control group); group (2) Sunitinib 1 mg/mouse for 7 consecutive days; group (3) on day 8, CD279 250 µg/mouse, three times at 2-day intervals; group (4) combination treatment of Sunitinib 1 mg/mouse for 7 consecutive days, followed by CD279 250 µg/mouse, three times at 2-day intervals. After treatment, the mice were observed for 12 days and then killed by an anaesthetic overdose. Moreover, the tumor rate has been calculated. In in vitro characterization, the combination resulted in a synergistic inhibition of cells proliferation with a CI ranging between 0.6-0.8 and the study of cell cycle progression evidenced a significant arrest at G0/G1 phase of cell cycle. Sunitinib strongly induced apoptosis, however the combination didn't lead to a further increase compared to sunitinib alone. Of note both sunitinib and drugs combination led to the LC3 A/B authophagic marker activation. Furthermore sunitinib reduced the levels of VEGF released by cells to the same extent of drugs combination. The in vivo study showed a time dependent synergistic effect of drugs combination compared to each drug alone with an improved overall survival of mice. Our study provides a rationale to further develop combination strategies with anti-angiogenic drug and PD-1 inhibitors for the management of CRC patients.
Citation Format: Letizia Porcelli, Roberta Di Fonte, Domenica Rea, Claudio Arra, Amalia Azzariti, Angelo V. Paradiso. Synergistic effect of sunitinib and PD-1 inhibitor nivolumab on colorectal cancer in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2238.
RSS Feeds