Histamine receptor 2 (H2) antagonists are widely used clinically for the control of gastrointestinal symptoms, but also impact immune function. They have been reported to reduce tumor growth in established colon and lung cancer models. Histamine has also been reported to modify populations of myeloid-derived suppressor cells (MDSCs). We have examined the impact of the widely used H2 antagonist ranitidine, on both myeloid cell populations and tumor development and spread, in three distinct models of breast cancer that highlight different stages of cancer progression. Oral ranitidine treatment significantly decreased the monocytic MDSC population in the spleen and bone marrow both alone and in the context of an orthotopic breast tumor model. H2 antagonists ranitidine and famotidine, but not H1 or H4 antagonists, significantly inhibited lung metastasis in the 4T1 model. In the E0771 model, ranitidine decreased primary tumor growth while omeprazole treatment had no impact on tumor development. Gemcitabine treatment prevented the tumor growth inhibition associated with ranitidine treatment. In keeping with ranitidine-induced changes in myeloid cell populations in non-tumor-bearing mice, ranitidine also delayed the onset of spontaneous tumor development, and decreased the number of tumors that developed in LKB1−/−/NIC mice. These results indicate that ranitidine alters monocyte populations associated with MDSC activity, and subsequently impacts breast tumor development and outcome. Ranitidine has potential as an adjuvant therapy or preventative agent in breast cancer and provides a novel and safe approach to the long-term reduction of tumor-associated immune suppression.

Monocytes and myeloid derived suppressor cells (MDSC) have been implicated on the regulation of tumor growth. Histamine is also important for regulating MDSC responses. Oral administration of the H2 receptor antagonist ranitidine can inhibit breast tumor growth and metastasis. In the current study, we examined the impact of oral ranitidine treatment, at a clinically relevant dose, on multiple murine tumor models. The impact of ranitidine on monocyte responses and the role of CCR2 in ranitidine-induced tumor growth inhibition were also investigated. Oral ranitidine treatment did not reduce tumor growth in the B16-F10 melanoma, LLC1 lung cancer and EL4 thymoma models. However, it consistently reduced E0771 primary tumor growth and metastasis in the 4T1 model. Ranitidine had no impact on E0771 tumor growth in mice deficient in CCR2, where monocyte recruitment to tumors was limited. Analysis of splenic monocytes also revealed an elevated ratio of H2 versus H1 expression from tumor-bearing compared with naïve mice. More detailed examination of the role of ranitidine on monocyte development demonstrated a decrease in monocyte progenitor cells following ranitidine treatment. Taken together, these results reveal that H2 signaling may be a novel target to alter the monocyte population in breast tumor models, and that targeting H2 on monocytes via oral ranitidine treatment impacts effective tumor immunity. Ranitidine is widely used for control of gastrointestinal disorders. The potential role of ranitidine as an adjunct to immunotherapies for breast cancer and the potential impact of H2 antagonists on breast cancer outcomes should be considered.

BackgroundThe histamine receptor 2 antagonist ranitidine is a commonly used, non-prescription, medication. It limits the development, growth, and metastasis of breast cancers in mouse models of disease. In this study, we examined the role of B cells in this response, the impact of ranitidine on the development of antitumor antibodies and subpopulations of natural killer cells using murine breast cancer models.MethodsPeripheral blood granulocyte populations were assessed in both E0771-GFP and 4T1 orthotopic tumor-bearing mice by evaluation of stained blood smears. Antibody responses were assessed both in terms of the levels of anti-GFP antibodies detected by enzyme-linked immunosorbent assay and also by antibody binding to the surface of tumor cells evaluated by flow cytometry. B cell and NK cell populations were examined in the draining lymph nodes and spleens of tumor-bearing animals, by flow cytometry with and without ranitidine treatment.ResultsOral ranitidine treatment was not associated with changes in peripheral blood granulocyte populations in tumor-bearing mice. However, ranitidine treatment was associated with the development of enhanced antitumor antibody responses. This was not limited to the tumor setting since ranitidine-treated mice immunized with ovalbumin also demonstrated increased IgG antibody responses. Analysis of B cell populations indicated that while B1 cell populations remained unchanged there was a significant decrease in B2 cells in the tumor-draining inguinal lymph nodes. Notably, ranitidine did not significantly inhibit primary tumor growth in B cell-deficient animals. Examination of NK cell populations revealed a significant decrease in the proportion of intermediately functionally mature NK cells populations (CD27+CD11b−) in ranitidine-treated tumor-bearing mice compared with untreated tumor-bearing controls.ConclusionThese data demonstrate an important role for B cells in the enhanced antitumor immune response that occurs in response to ranitidine treatment. Our findings are consistent with a model, whereby ranitidine reduces tumor-associated immune suppression allowing for the development of more effective antitumor responses mediated by B cells which may include the participation of NK cells. These data underline the importance of considering widely used histamine receptor antagonists as modulators of antitumor immunity to breast cancer.

The induction of tumor-targeted, cytotoxic T lymphocytes has been recognized as a key component to successful immunotherapy. DPX-based treatment was previously shown to effectively recruit activated CD8 + T cells to the tumor. Herein, we analyze the unique phenotype of the CD8 + T cells recruited into the tumor in response to DPX-based therapy, and how combination with checkpoint inhibitors impacts T cell response. C3-tumor-bearing mice were treated with cyclophosphamide (CPA) for seven continuous days every other week, followed by DPX treatment along with anti-CTLA-4 and/or anti-PD-1. Efficacy, immunogenicity, and CD8 + T cells tumor infiltration were assessed. The expression of various markers, including checkpoint markers, peptide specificity, and proliferation and activation markers, was determined by flow cytometry. tSNE analysis of the flow data revealed a resident phenotype of CD8 + T cells (PD-1 + TIM-3 + CTLA-4 +) within untreated tumors, whereas DPX/CPA treatment induced recruitment of a novel population of CD8 + T cells (PD-1 + TIM-3 + CTLA-4 −) within tumors. Combination of anti-CTLA-4 (ipilimumab) with DPX/CPA versus DPX/CPA alone significantly increased survival and inhibition of tumor growth, without changing overall systemic immunogenicity. Addition of checkpoint inhibitors did not significantly change the phenotype of the newly recruited cells induced by DPX/CPA. Yet, anti-CTLA-4 treatment in combination with DPX/CPA enhanced a non-antigen specific response within the tumor. Finally, the tumorrecruited CD8 + T cells induced by DPX/CPA were highly activated, antigen-specific, and proliferative, while resident phenotype CD8 + T cells, seemingly initially exhausted, were reactivated with combination treatment. This study supports the potential of combining DPX/CPA with ipilimumab to further enhance survival clinically.

Neoantigens are emerging targets for personalized cancer vaccines that provide patient specific cancer immunotherapies. Many algorithms have been developed to select the most immunogenic neoantigens to include, however not all neoantigens chosen will generate equivalent immune responses, nor may they induce effective anti-tumour activity. To maximize immunological activity, selected peptides should be delivered simultaneously in a formulation that can stimulate potent, sustained immune responses to many different peptides. The DepoVaxTM platform is an oil-based system that uses lipids to incorporate many different types of antigens and adjuvants into a single formulation. Using a set of neoantigens identified from murine B16-F10 melanoma, we optimized a DepoVax formulation method that allows us to incorporate up to 14 neoantigens with a polynucleotide based adjuvant in a single formulation. These selected neoantigens irrespective of their solubility and hydrophobicity were formulated in DepoVax with the contents completely soluble in oil. C57BL/6 mice were vaccinated with 14 synthetic long peptide neoantigens (each 27 amino acids in length) prepared in DepoVax or in an aqueous formulation containing poly ICLC adjuvant. The immune responses were assessed eight days later by IFN-γ ELISPOT using splenocytes. Several of the peptides generated strong immune responses that were significantly higher in mice vaccinated with the DepoVax formulation compared to the aqueous formulation. To assess the contribution of CD8+ and CD4+ T cell responses, splenocytes from vaccinated mice were stimulated with an immunogenic peptide and intracellular IFN-γ/TNF-α producing CD8+ or CD4+ T cells were detected by flow cytometry. The highest production of TNF-α was detected by CD8+ T cells. Biological activity of the vaccines was assessed after one month storage at -20, 5 and 25 °C by IFN-γ ELISPOT assay; no significant difference was detected compared to the initial results. Analytical characterization of 14 peptides in DepoVax carried out using high-performance liquid chromatography (RP-HPLC), detected no significant chemical modifications or degradation of peptides after storage at -20 °C for up to 3 months compared to the initial results. These results demonstrate that the DepoVax platform can incorporate at least 14 neoantigens in a single formulation. Neoantigens formulated in DepoVax are stable for at least 3 months and our manufacturing method can incorporate peptides with a wide range of physical and chemical characteristics. This formulation generates strong CD8+ T cell responses, in excess of those induced by an aqueous formulation. The DepoVax platform is a promising solution to inducing robust immune responses to multiple neoantigens in a single formulation. Citation Format: Valarmathy Kaliaperumal, Genevieve Weir, Rajkannan Rajagopalan, Arthvan Sharma, Heather Torrey, Alecia MacKay, Ava Vila-Leahey, Cynthia Tram, Andrea Penwell, Leeladhar Sammatur, Marianne Stanford. A novel delivery platform containing up to 14 neoantigens can induce robust immune responses in a single formulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1726.