The Tie2 antagonist rebastinib reduces ovarian cancer growth in a syngeneic murine model

Abstract

Background: The receptor tyrosine kinase TIE2 and its ligands, angiopoietins (ANGPTs), promote angiogenesis. In addition to expression on vascular endothelial cells, TIE2 is expressed on M2-like pro-tumorigenic macrophages. Thus, the TIE2 inhibitor rebastinib was developed as a potential therapy to address multiple cancers. The objective of this study was to determine the effects of rebastinib alone and combined with chemotherapy in a syngeneic murine model of ovarian cancer.

Methods: Female C57Bl6J mice were intraperitoneally injected with syngeneic ID8 ovarian cancer cells. Once tumors were established, mice were untreated (control) or treated with rebastinib, carboplatin plus paclitaxel (chemotherapy), or rebastinib plus chemotherapy. In one set of experiments, survival was followed for 140 days. In other experiments, ascites was harvested 24 h after the last treatment and analyzed by flow cytometry. In in vitro experiments, RNA sequencing was performed on ID8 cells and murine peritoneal macrophage cells (PMJ2R) after treatment with rebastinib, chemotherapy, or rebastinib plus chemotherapy.

Results: Tumor-bearing mice treated with rebastinib plus chemotherapy had longer median survival than mice treated with chemotherapy (132.5 vs. 127 days, P < 0.01). Ascites from mice treated with rebastinib had more CD45 + macrophages (P < 0.03) and cytotoxic T cells (P < 0.0001) than ascites from mice treated with chemotherapy. Rebastinib had no significant effect on the numbers of regulatory T cells, Tie2 + macrophages, or Tie2 + M2 macrophages. In ID8 cells, in vitro, rebastinib treatment upregulated 1528 genes and downregulated 3115 genes. In macrophages, in vitro, rebastinib treatment upregulated 2302 genes and downregulated 2970 genes. Rebastinib differentially regulated ANGPT-like proteins in both types of cells, including several ANGPT-like genes involved in tumorigenesis, angiogenesis, and proliferation. ANGPTL1, an anti-angiogenic and anti-apoptotic gene, was increased tenfold in ID8 cells treated with rebastinib (P < 0.001) but was not altered in macrophages.

Conclusions: Rebastinib plus chemotherapy extends survival in a syngeneic murine model of ovarian cancer. Rebastinib alters proportions of immune cell subsets, increases cytotoxic T cells in ascites, and alters gene expression in tumor cells and macrophages.

Keywords: Angiogenesis; Ovarian cancer; Rebastinib; Tie2.

Fig. 1

Rebastinib significantly improved survival in syngeneic ID8 murine model. A ID8-GFP mouse ovarian cancer cells were injected on day 0 (6 × 10^6 cells/ 300 ul/ ip). Rebastinib (Reb) treatment was initiated on day 25 after cell injection (Research Diet, Rebastinib 10 mg/kg Open Standard Diet #D16110801Ri lot19040309A7VP2.5i, irradiated 10–20 kGy with red dye; Control Open Standard Diet with 15 kcal%fat D11112201i, lot 19040309A4VP2.5i irradiated 10 to 20 kGy with green dye). Group 1 and 2 Mice remained on Control / Rebastinib chow for the study duration. Group 3 and 4 mice were treated with Carboplatin (Carbo, 20 mg/kg ip) and paclitaxel (Pac, 12 mg/kg ip) once on day 32, 39, and 46. All mice were observed daily for behavioral changes, disease progression and survival till the end of study period (140 days). B Rebastinib treated mice survived significantly longer than control (P < 0148). Further, rebastinib significantly increased the survival of paclitaxel + carboplatin treated mice (P < 0.001). P value was determined using Log-rank Mantel-Cox test

Fig. 2

Rebastinib significantly increased CD45+ macrophages and cytotoxic T cells in syngeneic ID8 mouse model. A ID8-GFP mouse ovarian cancer cells were injected on day 0 (6 × 10^6 cells/ 300 ul/ ip). Rebastinib treatment was initiated on day 25 after cell injection (Research Diet, Rebastinib 10 mg/kg Open Standard Diet #D16110801Ri lot19040309A7VP2.5i, irradiated 10-20 kGy with red dye; Control Open Standard Diet with 15 kcal%fat D11112201i, lot 19040309A4VP2.5i irradiated 10 to 20 kGy with green dye). Mice remain on Rebastinib / Control chow for the study duration. Mice were treated with Carboplatin (20 mg/kg ip) and paclitaxel (12 mg/kg ip) once on day 32, 39, and 46. One day after the last dose of carboplatin/paclitaxel and ascites / peritoneal immune cell status was assessed by flow cytometry. B Rebastinib treated mice ascites showed significantly higher CD45+ macrophages (P < 0.02 compared to control, P < 0.01 compared to chemotherapy and P < 0.02 compared to chemotherapy plus rebastinib treated mice), T helper leukocytes (P < 0.03) compared to chemotherapy and chemotherapy plus rebastinib) and cytotoxic T leukocytes (P<0.03 compared to control, P < 0.002 compared to chemotherapy and P < 0.007 compared to chemotherapy plus rebastinib). C There was no significant difference in expression of other immune cells. Statistical analysis was carried out using ordinary one-way ANOVA Sidak’s multiple comparisons test

Fig. 3

Rebastinib altered both anti-tumorigenic and pro-tumorigenic ANGPTL mRNA in ID8 cells and PMJ2R mouse derived peritoneal macrophages, potentially leading to low in vivo efficacy of rebastinib. Single cell RNA sequencing was used to determine differentially expressed genes between ID8 cells and C57BL6 peritoneal macrophages. A Heat map shows upregulated and downregulated gene clusters. B, C Volcano plots showing downregulated and upregulated genes between treated and control ID8 cells and macrophages

Fig. 4

Graphical abstract describing possible mechanism of action of Tie2 inhibitiors