Dabigatran and Cisplatin Co-Treatment Enhances the Antitumor Efficacy of Immune Checkpoint Blockade in A Murine Model of Resistant Ovarian Cancer

and Cisplatin Co-Treatment Enhances the Antitumor Efficacy of Immune Checkpoint Blockade in A Model of Resistant Ovarian Cancer. Abstract The standard treatment for ovarian cancer is surgical debulking followed by platinum- taxane-based chemotherapy. Although most patients are initially responsive to this therapy, patients in advanced stages eventually relapse and die. New therapeutic approaches using immune checkpoint blockade (ICB) have been less promising in ovarian cancer compared to other tumor types, resulting in durable tumor regression in only a small subset of ovarian cancer patients. Because previous studies showed immunomodulatory effects following co-treatment with cisplatin and the thrombin inhibitor dabigatran etexilate (C/D) in a preclinical animal model of ovarian cancer, we explored to what extent this co-treatment may enhance the anti-tumor efficacy of ICB in the ID8 tumor model that is resistant to ICB. Whereas cisplatin or dabigatran treatment alone or co-treatment with cisplatin and anti-PD-1 monoclonal antibody (mAb) demonstrated little significant effect on tumor spread, co-treatment with C/D with or without anti-PD-1 mAb significantly reduced ID8 tumor burden and increased peritoneal INF-γ producing CD8+ T-cells after only 2 weeks of treatment. Moreover, C/D cotreatment with ICB conferred a durable survival advantage over C/D or ICB alone. The enhanced anti-tumor effect and survival with C/D co-treatment and ICB compared to that with C/D or ICB alone was accompanied by decreases in immunosuppressive M2- macrophages, decreases in pro-tumorigenic cytokines, and corresponding increases in tumor-infiltrating, IFN-γ-producing CD8+ T-cells. Our findings provide proof-of-concept evidence that the addition of ICB with thrombin inhibition in frontline platinum-based chemotherapy may be a potential new therapeutic treatment combination for advanced ovarian cancer.


Introduction
Although ovarian cancer accounts for only 3% of all cancer in women, it is the fifth leading cause of cancer-related deaths for women in the United States [1]. Patients typically present with advanced-stage disease, and they often initially respond well to standard primary treatment with surgery and firstline platinum and taxane-based chemotherapy. However, the majority of patients experience recurrence of their cancer within 12-18 months and die of the disease [2]. Clearly, there is an urgent need for new therapeutic strategies for treating ovarian cancer. An exciting new cancer immunotherapy approach is to block two key immune checkpoint pathways mediated by immunosuppressive co-signaling, the first via programmed cell death-1 (PD-1) and programmed death ligand-1 (PD-L1) and the second via CTL-associated antigen 4 (CTLA-4) and its ligands B7-1 or B7-2 [3,4]. The immune checkpoint proteins, CTLA-4 and PD-1, normally keep immune responses in check by preventing overly intense responses that might damage normal tissue. Tumors can hijack these immune checkpoint proteins and use them to suppress immune responses. Blocking the activity of immune checkpoint proteins releases the "brakes" on the immune system, thus increasing its ability to destroy tumor cells. These new ICI treatments have led to dramatic tumor regressions in patients with some solid malignancies, including ovarian cancer [5,6].
Unfortunately, clinical studies have shown that the administration of inhibitors of CTLA-4, PD-1, and PD-L1 alone leads to durable tumor regression in only a subset of cancer patients [7,8]. In ovarian cancer patients treated with immune checkpoint blockade, symptomatic disease progression is common and often leads to early discontinuation of treatment [9]. Because tumors employ multiple and non-overlapping immunosuppressive mechanisms that can mitigate the clinical benefit of immunotherapy such as immune checkpoint blockers, it is important to identify and block these resistance mechanisms.
The clinical association between cancer and thrombosis has been recognized for more than a century [10], and expression of coagulation factors and biomarkers of hemostatic system activation correlates strongly with poor prognosis for multiple cancer types [11][12][13]. Indeed, ovarian cancer is associated with a high risk of thrombotic events (20%) which sometimes can be exacerbated by treatment with standard chemotherapeutic agents [14][15][16]. The pro-thrombotic microenvironment in tumors also directly promotes tumor growth and metastasis [17].
Thrombin is the primary effector protease of the coagulation cascade generated by the action of tissue factor and other coagu-lation factors. The critical role of thrombin in promoting tumor progression reflects its many functions, including fibrin formation [18], platelet activation [19], activation of protease-activated receptor (PAR) signaling [20] and the proteolytic breakdown of the extracellular matrix. In addition to its role in generating fibrin to promote hemostasis, thrombin acts directly on multiple effector cells of the immune system affecting both acute and chronic inflammatory processes [21,22]. The ablation of PAR-1 from the tumor microenvironment, but not the tumor, has been shown to dramatically reduce tumor growth and metastasis in multiple tumor models [23,24], in part by reducing infiltration of M2-like macrophages into the tumor [23]. Thrombin-activated platelets release immunosuppressive cytokines including TGF-β that can inhibit natural killer cell activity, helping the tumor evade host immunosurveillance [25,26]. Taken together, there is strong evidence that thrombin influences cancer pathogenesis via multiple mechanisms, including the tumor immune response, with thrombin emerging as a target for novel therapies in cancer. Using the murine ID8 ovarian tumor model, we have shown that the thrombin inhibitor, dabigatran etexilate, significantly enhances the anti-tumor efficacy of cisplatin in an immunomodulatory way [27]. Dabigatran is an oral anticoagulant that is a direct thrombin inhibitor [28]. The anti-tumor effect of this co-treatment was significantly greater than the reduction in tumor load from either cisplatin or dabigatran alone. The present investigation was designed to explore to what extent cisplatin and dabigatran co-treatment, that decreases the tumor infiltration of myeloid immunosuppressive cells, may enhance the efficacy of immune checkpoint inhibitors in a murine model of resistant ovarian cancer. animal experiments. ID8-luc cells were cultured in DMEM supplemented with 4% fetal bovine serum, 1x insulin/transferrin/ sodium selenite media supplement (Corning) and 1x Penicillin/ Streptomycin (Cellgro). The cells were freshly thawed from early passage cells, cultured for no more than 2 months, and regularly checked by virtue of their morphologic features to avoid cross-contamination or misuse.

Murine ID8 Tumor Model
Female C57/Bl6 mice were intraperitoneally (i.p.) injected with 2.0 x 10 6 ID8-luc cells. To monitor tumor growth and spread throughout the peritoneal cavity, mice were imaged for bioluminescence using an IVIS bioluminescence imager. Three weeks after ID8-luc cells were injected, anti-CTLA-4 therapy was started with 100 μg of anti-CTLA-4 antibody (clone 9D9, BioX-Cell) injected i.p. every third day for a total of three injections.

Cytokine analyses
Ascites samples were spun at 300 g for 10 minutes to pellet cells. Ascites supernatants were collected and analyzed for tumor necrosis factor-alpha (TNF-α), monocyte chemoattrac-

Statistics
All in vivo experiments were carried out using multiple animals (n = 7-10 per experimental group) in 3 separate experiments. All in vitro experiments were performed in at least triplicate, and data compiled from 2-3 separate experiments. Analyses were done using a 1-way ANOVA with a Tukey test for multiple comparison correction. 4 Survival rates were analyzed using the Kaplan-Meier method and evaluated with the log-rank test with Bonferroni correction. For analyzing ID8 tumor spread over time, a linear mixed-effects model and a linear growth model were fit to assess the change in average radiant flux over time among the control and three treatment groups. The models contained the main effects of treatment over time and included the interaction between the main effects. Additionally, they were fit using a random-intercept, which allowed each mouse to have a different baseline radiant flux at day 29. Due to the significant interaction, post-hoc tests were performed to identify significant differences among treatments, and all p-values were adjusted using a Bonferroni correction. All tests were two-sided and the significance level was p ≤ 0.05.

T-cells during the early stages of tumor progression
We evaluated the effect of cisplatin or cisplatin/dabigatran etexilate (C/D) co-treatment with immune checkpoint blockade (ICB) on tumor progression and survival using the ID8 tumor model of ovarian cancer, given that the two therapies modulate the immune response to cancer cells by different, and potentially, complementary mechanisms. Studies conducted using the ID8 model, a highly clinically relevant murine model of ICB-resistant ovarian cancer, have shown that treatment with anti-PD-1 monoclonal antibody (mAb) alone is ineffective in preventing peritoneal tumor growth [29]. At four weeks, daily treatment with C/D or vehicle control was initiated along with anti-PD-1 mAb or control isotype mAb. To evaluate tumor burden and the immune response at an early time point during disease progression, mice were sacrificed after 2 weeks of treatment.
The tumor growth of the luciferase-expressing ID8 tumor cells was measured by bioluminescence. Whereas treatment with cisplatin, dabigatran, or anti-PD-1 mAb individually had no effect on overall tumor burden, co-treatment with cisplatin and dabigatran with or without anti-PD-1 mAb significantly reduced ID8 tumor burden even after only 2 weeks of treatment ( Figure 1A).
Interestingly, only mice treated with cisplatin plus dabigatran had significantly higher levels of peritoneal INF-γ producing CD8 + T-cells compared to that in mice treated with vehicle control, anti-PD-1 mAb, dabigatran, or cisplatin alone ( Figure 1B

Co-treatment with dabigatran, cisplatin, and immune checkpoint inhibitors inhibit ID8 tumor growth and ascites development in vivo
To examine what effect co-treatment with C/D and ICB had on ID8 tumor progression as tumor burden increased, mice were injected with ID8-luc tumor cells and treated with C/D or C/D plus either anti-CTLA-4 or anti-PD-1 mAb and then sacrificed 10 weeks following tumor injection to allow for direct comparisons between treatment groups. Mono-therapy or cisplatin/anti-PD-1 co-treatment was not included due to a lack of effectiveness (data not shown, Figure 1A

Co-treatment with cisplatin and dabigatran and anti-CTLA-4 or anti-PD-1 antibody decreases cytokines in the ascites
Tumors develop many mechanisms to evade immune responses including the secretion of inhibitory cytokines such as tumor growth factor β (TGF-β) and IL-10 in addition to inhibitor cell types such as M2 macrophages or Tregs [31]. Cell-free ascites from ID8 tumor-bearing mice was analyzed by cytokine

Discussion
Previously, we showed significantly greater anti-tumor efficacy with dabigatran etexilate and cisplatin co-treatment that was accompanied by a decrease in immunosuppressive myeloid cell populations and pro-tumorigenic cytokines as well as a concomitant increase in CD8 + effector T-cell activity in the tumor ascites [27]. Here we show that co-treatment with chemotherapeutic cisplatin and the thrombin inhibitor dabigatran significantly enhances the efficacy of ICB in a murine model of ovarian cancer that is resistant to ICB alone. Of particular significance was that approximately a third of the mice treated with C/D plus ther anti-CTLA-4 or anti-PD-1 mAb further lowered levels of TNF-α and MCP-1 compared to that in mice treated with C/D alone. However, only co-treatment with anti-PD-1 and C/D significantly reduced levels of IL-6 in the ascites compared to control mice.
anti-PD-1 mAb survived, without treatment, for an additional three months beyond the survival of mice treated with C/D alone.
Thrombin has the potential to directly modulate the immune response to the developing tumor. The chronic pro-inflammatory state in the tumor microenvironment has been shown to induce thrombin expression [32,33] mediated by the pro-tumorigenic, pro-inflammatory cytokine IL-6 [34]. Thrombin signals through the PAR-1 receptor which is abundantly expressed in the tumor microenvironment including infiltrating immune cells [35]. Ablation of PAR-1 from the tumor microenvironment,    and heparins in that it requires no monitoring has less bleeding risk, and a direct antidote is available [39]. Our findings provide proof-of-concept evidence that the addition C/D with ICB may be a potential new therapeutic treatment combination that further harnesses the immune system for the treatment of advanced ovarian cancer.