More recently, doxorubicin has been established as an inducer of immunogenic cell death and has been shown to increase IFN gamma production and induce dendritic and T-cell tumor infiltration in mouse models [14], [15], [16], [17], [18], [19], [20]
More recently, doxorubicin has been established as an inducer of immunogenic cell death and has been shown to increase IFN gamma production and induce dendritic and T-cell tumor infiltration in mouse models [14], [15], [16], [17], [18], [19], [20]. Based on these immunomodulatory effects, we hypothesized that doxorubicin, or liposomal doxorubicin (Doxil), could potentiate the antitumor activity of immunotherapeutic agents in syngeneic mouse models. [3], [4]. One challenge that remains is that not all patients respond despite the durable effect these therapies can have. This is likely due to an immunosuppressive tumor microenvironment and/or poor immunogenicity of patients tumors. To increase the response rate of tumors to immunotherapy, rational combination approaches of different cancer immunotherapies have been investigated, including combining Rabbit Polyclonal to Tyrosine Hydroxylase mediators of checkpoint blockade (i.e., antiCPD-1 and PD-L1) and TNFR-family agonists (i.e., OX40) with small-molecule drugs [5], [6], [7], [8], [9], [10], [11]. Although significant progress has been achieved in the evaluation of combination therapies preclinically, there remains a great need for rational testing of immunotherapies in combination settings, in particular with established cancer treatments, and translation of novel combinations with improved activity into the clinic. Doxorubicin is a widely used chemotherapeutic drug for patients with sarcoma, lung, breast, and other cancers. Previously, doxorubicin has been well characterized as a DNA intercalator and an inhibitor of topoisomerase II [12]. Other mechanisms of action of doxorubicin that are reported are DNA cross-linking, interference with DNA strand separation, free-radical formation, helicase activity, and Mollugin direct membrane effects [13]. Doxorubicin therefore has been viewed as a cytotoxic agent with direct cell-killing effects on tumor cells. More recently, doxorubicin has been founded as an inducer of immunogenic cell death and has been shown to increase IFN gamma production and induce dendritic and T-cell tumor infiltration in mouse models [14], [15], [16], [17], [18], [19], [20]. Based on these immunomodulatory effects, we hypothesized that doxorubicin, or liposomal doxorubicin (Doxil), could potentiate the antitumor activity of immunotherapeutic providers in syngeneic mouse models. Doxil is an authorized drug for paclitaxel- and platinum-resistant ovarian carcinoma and Kaposis sarcoma. In preclinical models, Doxil has been shown to have more antitumor activity; consequently, assessment of this drug to free doxorubicin was explored with this study [21], [22]. Here, we demonstrate that both doxorubicin and Doxil synergize with several T-cellCtargeted immunotherapies in two syngeneic mouse models. Importantly, Mollugin combination activity was durable, led to high rates of total response (CR), and generated immunological memory space in mouse models. Furthermore, the results reveal for Mollugin the first time that Doxil offers effects on dendritic and immature myeloid cells in tumors, as well as on CD8+ T cells and regulatory T cells (Tregs). Materials and Methods Antibodies, Reagents, and Cell Lines CT26 cells were from ATCC (Manassas, VA) and were cultivated with RPMI 1640 medium supplemented with 10% fetal bovine serum. MCA205 cells were from Agonox (Portland, OR) and cultivated in the same growth medium. Following receipt of cell lines, both cell lines were reauthenticated using STR-based DNA profiling and multiplex polymerase chain reaction (IDEXX Bioresearch, Columbia, MO). Antibodies from BioXCell (Western Lebanon, NH) included the following: antiCPD-1 (RMP1-14), antiCPD-L1 (10?F.9G2), antiCCTLA-4 (9D9), and mouse IgG2b control (MPC-11). Mouse OX40 ligand fusion protein (OX40L FP), mouse GITR ligand fusion protein (GITRL FP), and rat IgG2a isotype control antibodies were produced by MedImmune. Doxil, gemcitabine, and oxaliplatin were purchased from Bluedoor Pharma (Rockville, MD), and doxorubicin was from Henry Schein Inc. (Melville, NY). Animal Studies Cells were cultivated in monolayer tradition, harvested by trypsinization, and implanted subcutaneously into the right flank of 6- Mollugin to 8-week-old female Balb/C (CT26) or C57BL/6 (MCA205), or 4- to 6-week-old athymic female nude mice (Harlan, Indianapolis, IN). For the CT26 tumor model, 5??105 cells were implanted in the right flank using a 26-gauge needle. For the MCA205 tumor model, 2.5??105 cells were implanted. All antibodies, OX40L FP, GITRL FP, gemcitabine, and oxaliplatin were dosed via intraperitoneal injection. Doxil and doxorubicin were dosed via intravenous injection. In some studies, isotype settings were given to mice like a cocktail of rat IgG2a and mouse IgG2b. At the beginning of treatment, mice were randomized either by tumor volume (established-tumor studies) or by body weight (preventative studies). The number of animals per group ranged from 10 to 12 animals per group as identified based on sample size calculations using nQuery software. Both tumor and body weight measurements were collected twice weekly, and tumor volume was determined using the equation (and refer to the space and width sizes, respectively. Error bars were calculated as the standard error of the mean. The general health of mice was monitored daily, and all experiments were conducted in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care and MedImmune Institutional Animal Care and Use Committee recommendations for humane treatment and care of laboratory animals. Kaplan-Meier statistical analysis was performed.