Rebastinib improves survival in syngeneic ID8 murine model of ovarian cancer.
To assess the effects of rebastinib in immunocompetent mice, we intraperitoneally injected mouse ID8 ovarian cancer cells into immune-competent C57Bl6/J mice (Fig. 1A). After allowing tumors to establish for 25 days, mice were divided into four groups. Group 1 received no treatment, Group 2 received rebastinib, Group 3 received carboplatin and paclitaxel, and Group 4 received rebastinib, carboplatin, and paclitaxel (Fig. 1A). Mice treated with rebastinib had longer median survival than untreated mice (79 days vs. 74.5 days; P < 0.0148, Log-rank Mantel-Cox test), and mice treated with rebastinib plus chemotherapy had significantly longer mean survival than those treated with chemotherapy (132.5 days vs. 127 days; P < 0.001, Log-rank Mantel-Cox test) (Fig. 1B). There was no significant difference in body weight gain between vehicle-treated and rebastinib treated mice. Mice treated with chemotherapy or rebastinib plus chemotherapy had slower body weight gain than control mice or mice treated with rebastinib, but there was no significant difference between mice treated with chemotherapy and those treated with chemotherapy plus rebastinib (Fig. S1). We conclude that treatment with rebastinib plus chemotherapy led to modest but statistically significant increases in survival over chemotherapy alone in a syngeneic model of ovarian cancer.
Figure 1. Rebastinib significantly improved survival in syngeneic ID8 murine model. A) ID8-gfp mouse ovarian cancer cells were injected on day 0 (6x10^6 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). 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.
Rebastinib modulates the tumor immune environment in murine ascites.
Next, we wanted to determine whether and how rebastinib affects the tumor immune environment. We thus injected immunocompetent mice with ID8 cells, allowed tumors to develop, treated them as above (Fig. 2A), and harvested ascites one day after the last chemotherapy treatment. Ascites from mice treated with rebastinib contained significantly more CD45 + macrophages (P < 0.02 compared to control; P < 0.01 compared to chemotherapy and P < 0.02 compared to rebastinib plus chemotherapy), higher 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) (Fig. 2B). However, ascites from mice treated with rebastinib plus chemotherapy had similar numbers of all immune cell types analyzed as ascites from mice treated with chemotherapy (Fig. 2C). We conclude that rebastinib treatment leads to greater recruitment of macrophages and cytotoxic T cells than chemotherapy but does not increase recruitment of these cell types when provided in combination with chemotherapy. In addition, rebastinib helps maintain helper T leukocytes, which are otherwise depleted by chemotherapy.
Figure 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 (6x10^6 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. Statiscal analysis was carried out using ordinary one-way ANOVA Sidak’s multiple comparisons test.
Rebastinib modulates the expression of angiopoietin-like receptors in macrophages and ID8 cells.
Finally, we wondered about the effects of rebastinib on gene expression in tumor cells and macrophages. We treated ID8 cells and PMJ2R murine peritoneal macrophages (PMJ2R CRL-2458 commercially available from ATCC) with vehicle or with 5 nM rebastinib for 6 hours and performed RNA sequencing. In ID8 cells, 1,528 genes were upregulated, and 3,115 genes were downregulated following rebastinib treatment. In macrophages, 2302 genes were upregulated, and 2970 genes were downregulated following rebastinib (Fig. 3A). Several ANGPT-like genes (ANGPTL2, ANGPTL4, ANGPTL6) involved in tumorigenesis, angiogenesis, and proliferation were downregulated by two- to ten-fold in both ID8 cells and macrophages treated with rebastinib (Figs. 3B and 3C).
Figure 3. 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.
Table 1 summarizes some of the key genes that are upregulated or downregulated. To highlight a few, the expression of ANGPTL1, an antiangiogenic and anti-apoptotic gene, was increased ten-fold in ID8 cells treated with rebastinib but was not altered in macrophages. Expression of ANGPTL3 was increased by 50- to 80-fold in ID8 cells and macrophages treated with rebastinib. Interestingly certain non-angiogenic genes like EZH2, PAX8, CSF were also modulated in ID8 cells and PMJ2R cells. Finally, expression of ANGPT2, a context-dependent agonist/antagonist of the TIE2 pathway, was increased 1.4-fold in ID8 cells and 4.6-fold in macrophages treated with rebastinib. We conclude that rebastinib treatment alters the expression of both angiogenic and non-angiogenic genes in ID8 tumor cells and macrophages.