Search

Breast cancer is the second leading cause of cancer-related deaths in women in the United States. The triple-negative breast cancer (TNBC) subtype associates with higher rates of relapse, shorter overall survival, and aggressive metastatic disease. Hormone therapy is ineffective against TNBC, leaving patients with limited therapeutic options. Mammalian orthoreovirus (reovirus) preferentially infects and kills transformed cells, and a genetically engineered reassortant reovirus infects and kills TNBC cells more efficiently than prototypical strains. Reovirus oncolytic efficacy is further augmented by combination with topoisomerase inhibitors, including the frontline chemotherapeutic doxorubicin. However, long-term doxorubicin use correlates with toxicity to healthy tissues. Here, we conjugated doxorubicin to reovirus (reo-dox) to control drug delivery and enhance reovirus-mediated oncolysis. Our data indicate that conjugation does not impair viral biology and enhances reovirus oncolytic capacity in TNBC cells. Reo-dox infection promotes innate immune activation, and crosslinked doxorubicin retains DNA-damaging properties within infected cells. Importantly, reovirus and reo-dox significantly reduce primary TNBC tumor burden in vivo, with greater reduction in metastatic burden after reo-dox inoculation. Together, these data demonstrate that crosslinking chemotherapeutic agents to oncolytic viruses facilitates functional drug delivery to cells targeted by the virus, making it a viable approach for combination therapy against TNBC., Graphical Abstract, The chemotherapeutic drug doxorubicin was conjugated to oncolytic reovirus (reo-dox) to control drug delivery and enhance viral-mediated oncolysis of cancer cells. Conjugation of the drug to the virus does not impair viral biology, enhances reovirus oncolytic capacity, and retains the damaging properties of doxorubicin.

Imaging techniques based on fluorescence and bioluminescence have been important tools in visualizing tumor progression and studying the effect of drugs and immunotherapies on tumor immune microenvironment in animal models of cancer. However, transgenic expression of foreign proteins may induce immune responses in immunocompetent syngeneic tumor transplant models and augment the efficacy of experimental drugs. In this study, we show that the growth rate of Lewis lung carcinoma (LL/2) tumors was reduced after transduction of tdTomato and luciferase (tdTomato/Luc) compared to the parental cell line. tdTomato/Luc expression by LL/2 cells altered the tumor microenvironment by increasing tumor-infiltrating lymphocytes (TILs) while inhibiting tumor-induced myeloid-derived suppressor cells (MDSCs). Interestingly, tdTomato/Luc expression did not alter the response of LL/2 tumors to anti-PD-1 and anti-CTLA-4 antibodies. These results suggest that the use of tdTomato/Luc-transduced cancer cells to conduct studies in immune competent mice may lead to cell-extrinsic tdTomato/Luc-induced alterations in tumor growth and tumor immune microenvironment that need to be taken into consideration when evaluating the efficacy of anti-cancer drugs and vaccines in immunocompetent animal models.

Immune checkpoint inhibitor (ICI) immunotherapy improved the survival of head and neck squamous cell carcinoma (HNSCC) patients. However, more than 80% of the patients are still resistant to this therapy. To test whether the efficacy of ICI therapy can be improved by vaccine-induced immunity, we investigated the efficacy of a tumor membrane-based vaccine immunotherapy in murine models of HNSCC. The tumors, grown subcutaneously, are used to prepare tumor membrane vesicles (TMVs). TMVs are then incorporated with glycolipid-anchored immunostimulatory molecules GPI-B7-1 and GPI-IL-12 by protein transfer to generate the TMV vaccine. This TMV vaccine inhibited tumor growth and improved the survival of mice challenged with SCCVII tumor cells. The tumor-free mice survived for several months, remained tumor-free, and were protected following a secondary tumor cell challenge, suggesting that the TMV vaccine induced an anti-tumor immune memory response. However, no synergy with anti-PD1 mAb was observed in this model. In contrast, the TMV vaccine was effective in inhibiting MOC1 and MOC2 murine oral cancer models and synergized with anti-PD1 mAb in extending the survival of tumor-bearing mice. These observations suggest that tumor tissue based TMV vaccines can be harnessed to develop an effective personalized immunotherapy for HNSCC that can enhance the efficacy of immune checkpoint inhibitors.

Dendritic cells (DCs) are the most effective antigen presenting cells for the development of T cell responses. The only FDA approved DC-based immunotherapy to date is Sipuleucel-T, which utilizes a fusion protein to stimulate DCs ex vivo with GM-CSF and simultaneously deliver the antigen PAP for prostate cancer. This approach is restricted by the breadth of immunity elicited to a single antigen, and to cancers that have a defined tumor associated antigen. Other multi-antigen approaches have been restricted by poor efficacy of vaccine adjuvants. We have developed a vaccine platform that consists of autologous DCs pulsed with cytokine-adjuvanted tumor membrane vesicles (TMVs) made from tumor tissue, that encapsulate the antigenic landscape of individual tumors. Here we test the efficacy of DCs pulsed with TMVs incorporated with glycolipid-anchored immunostimulatory molecules (GPI-ISMs) in HER2-positive and triple negative breast cancer murine models. Pulsing of DCs with TMVs containing GPI-ISMs results in superior uptake of vesicles, DC activation and cytokine production. Adaptive transfer of TMV-pulsed DCs to tumor bearing mice results in the inhibition of tumor growth, reduction in lung metastasis, and an increase in immune cell infiltration into the tumors. These observations suggest that DCs pulsed with TMVs containing GPI-GM-CSF and GPI-IL-12 can be further developed to be used as a personalized immunotherapy platform for cancer treatment.

Immunotherapy has emerged as a promising treatment of breast cancer; however, response rates to therapy remain far from ideal. We have developed a new platform for personalized vaccine immunotherapy that utilizes tumor tissue to generate tumor membrane vesicles (TMVs). These TMVs can be incorporated with immunostimulatory molecules such as IL-12 and B7-1 and used for immunization. In this study, we investigated the combinatorial effect of TMV-based vaccine immunotherapy, to induce an antitumor immune response, with the type 2 diabetes drug metformin, which has been reported to exert antitumor effects by promoting CD8 T-cell activity in the tumor microenvironment. Our results show that TMV vaccination inhibits primary tumor growth in preclinical tumor models, while the combination with metformin resulted in greater inhibition of primary growth. TMV vaccination reduced the metastatic burden in the lungs while the combination with metformin further abrogated metastatic spread. Interestingly, TMV vaccination alone induced a significant increase in T-cell infiltration to the tumor, and combination with metformin caused a decrease in the accumulation of T cells, despite having better control of tumor growth. Further, we show that metformin can reduce PD-L1 expression within the tumor microenvironment and result in more IFN-γ-producing CD8 T cells within the tumor, providing a mechanistic insight for the improvement of TMV-based vaccine immunotherapy. Citation Format: Luis E. Munoz, Ramireddy Bommireddy, Haley L. Huang, Christopher D. Pack, Sampath Ramachandiran, Periasamy Selvaraj. Metformin reduces PD-L1 expression in the tumor and enhances the efficacy of vaccine-generated CD8 T cells in a murine model of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B32.

Lung cancer is the second most common cancer among men and women and the leading cause of cancer deaths in the world. Despite the advancements in diagnostic technologies and the development of novel treatments in the recent years such as immune checkpoint inhibitor (ICI) therapy, the prognosis of lung cancer patients is still poor, and the survival rate is low. The patient-to-patient heterogeneity is the major problem in treating lung cancer, which results in differential responses to ICI therapy. In this study, we investigated the efficacy of a vaccine immunotherapy prepared by modification of tumor membrane vesicles (TMVs) prepared from the tumors to express immunostimulatory molecules in combination with ICI therapy in a partially ICI resistant murine lung cancer model. We demonstrate that prophylactic vaccination of TMV adjuvanted with immunostimulatory molecules prevents the CMT-167 tumor growth in mice and helps the mice develop immunological memory to the tumor cell challenge. In a therapeutic setting, the TMV vaccine inhibited tumor growth and induced primary tumor regression in 40% of mice and significantly reduced the metastatic tumor burden. However, the combination of TMV vaccination with anti-PD-1 mAb doesn’t have a synergistic or additive effect on the primary or metastatic tumor growth. Overall, these studies provide insight into using the TMV vaccine immunotherapy for treating lung cancer. Funding: Supported by the Lung Cancer seed grant from the Winship Cancer Institute of Emory University Citation Format: Lei Huang, Dr. Selvaraj's lab. Immunotherapy with TMV vaccine inhibits metastatic tumor growth in an immune checkpoint resistant murine lung cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-202.

Breast cancer remains the most commonly diagnosed cancers among women, with over 250,000 new cases per year in the United States. Immunotherapy has emerged as a promising treatment of breast cancer, however response rates to therapy remain far from ideal. In this study, we investigated the combinatorial effect of tumor membrane vesicle (TMV)-based vaccine immunotherapy, to induce an anti-tumor immune response, with the type 2 diabetes drug metformin, which has been reported to exert anti-tumor effects by maintaining CD8 T cell activity in the tumor microenvironment. Our results show that TMV vaccination inhibits primary tumor growth in preclinical tumor models, while combination with metformin resulted in greater inhibition of primary growth. Importantly, TMV vaccination reduced the metastatic burden in the lungs while combination with metformin further abrogated metastatic spread. Interestingly, TMV vaccination alone induced a significant increase in T cell infiltration to the tumor, while combination with metformin caused a decrease in the accumulation of T cells, despite having better outcomes. Further, we show that metformin is capable of reducing PD-L1 expression within the tumor microenvironment in both tumor cells as well as myeloid-derived suppressor cells, providing insight for the improvement of TMV-based immunotherapy. Citation Format: Luis E. Munoz, Ramireddy Bommireddy, Haley Huang, Janet Kim, Periasamy Selvaraj. Metformin enhances the efficacy of tumor membrane vesicle (TMV)-based vaccine immunotherapy in a murine model of breast [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-203.

Introduction: Head and neck cancer is a leading cause of cancer related deaths accounting for approximately 3% of all cancer related mortalities in the US. Currently, there is no cure for the advanced squamous cell carcinoma of the head and neck (SCCHN) thus development of efficacious therapies is urgently needed. To test whether vaccine-induced immunity inhibits tumor growth, we investigated efficacy of a tumor membrane-based vaccine immunotherapy in a murine SCCHN (SCC VII) model. Materials and Methods: The SCC VII tumors grown subcutaneously in C3H/HeJ mice were harvested to generate tumor membrane vesicles (TMVs). TMVs were then protein transferred with glycolipid-anchored immunostimulatory molecules mouse GPI-B7.1 and mouse GPI-IL-12 to generate the TMV vaccine. Mice were vaccinated with TMV vaccine either before (prophylactic) or after SCC VII tumor cell challenge (therapeutic) and tumor growth was monitored every 3 days. Survival was then assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Results: SCCVII cells express MHC I, CD44, CD47 and respond to IFN-γ in vitro. The therapeutic TMV vaccine inhibited tumor growth and improved the survival of mice challenged with SCCVII tumor cells. Tumor-free mice remained protective from rechallenge with SCCVII cells. Conclusions: These observations suggest that tumor tissue-based vaccines can be harnessed to develop an effective immunotherapy for SCCHN. Funding: Supported by Head and Neck SPORE pilot funding from Emory Winship Cancer Institute. Citation Format: Ramireddy Bommireddy, Luis Munoz, Chrstopher D. Pack, Sampath Ramachandiran, Shaker JC Reddy, Janet Kim, Georgia Chen, Nabil F. Saba, Dong M. Shin, Periasamy Selvaraj. TMV vaccine inhibits growth of squamous cell carcinoma of head and neck in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4097.

Neurocysticercosis (NCC) is one of the most common helminth parasitic diseases of the central nervous system (CNS) and the leading cause of acquired epilepsy worldwide. NCC is caused by the presence of the metacestode larvae of the tapeworm Taenia solium within brain tissues. NCC patients exhibit a long asymptomatic phase followed by a phase of symptoms including increased intra-cranial pressure and seizures. While the asymptomatic phase is attributed to the immunosuppressive capabilities of viable T. solium parasites, release of antigens by dying organisms induce strong immune responses and associated symptoms. Previous studies in T. solium-infected pigs have shown that the inflammatory response consists of various leukocyte populations including eosinophils, macrophages, and T cells among others. Because the role of eosinophils within the brain has not been investigated during NCC, we examined parasite burden, disease susceptibility and the composition of the inflammatory reaction in the brains of infected wild type (WT) and eosinophil-deficient mice (ΔdblGATA) using a murine model of NCC in which mice were infected intracranially with Mesocestoides corti, a cestode parasite related to T. solium. In WT mice, we observed a time-dependent induction of eosinophil recruitment in infected mice, contrasting with an overall reduced leukocyte infiltration in ΔdblGATA brains. Although, ΔdblGATA mice exhibited an increased parasite burden, reduced tissue damage and less disease susceptibility was observed when compared to infected WT mice. Cellular infiltrates in infected ΔdblGATA mice were comprised of more mast cells, and αβ T cells, which correlated with an abundant CD8+ T cell response and reduced CD4+ Th1 and Th2 responses. Thus, our data suggest that enhanced inflammatory response in WT mice appears detrimental and associates with increased disease susceptibility, despite the reduced parasite burden in the CNS. Overall reduced leukocyte infiltration due to absence of eosinophils correlates with attenuated tissue damage and longer survival of ΔdblGATA mice. Therefore, our study suggests that approaches to clear NCC will require strategies to tightly control the host immune response while eradicating the parasite with minimal damage to brain tissue., Author Summary Eosinophils are known to mediate a protective response against several parasitic infections. This is largely accomplished by eosinophil degranulation (direct killing) and modulating effective adaptive immune responses. Consequently, eosinophils can also contribute to host pathology via a bystander effect. However, the outcome of infection varies depending upon the parasite species. In the case of neurocysticercosis (NCC), the role of eosinophils in disease progression has not been investigated despite the known eosinophilic response in patients. NCC is one of the most common parasitic diseases of the brain which is caused by the metacestode (larva) of the tapeworm Taenia solium. To determine the role of eosinophils in NCC disease outcome, we used a murine model of NCC in which wildtype (WT) or eosinophil deficient mice (ΔdblGATA) were infected intracranially with Mesocestoides corti, a cestode parasite related to T. solium. Our data show that murine NCC is characterized by a robust eosinophil response that correlates with lower parasite burden in the brain. Comparison of T cell response reveals a mixed Th1/Th2 in the WT brain, and ΔdblGATA mice showed a significant decrease in both population but in particular in the Th2 response. In addition, the strong eosinophil reaction observed in WT brains correlates with exacerbated pathology and increased morbidity. Thus, our study suggest that eosinophils act as a double-edged sword playing a role in controlling the infection but worsening the disease outcome by contributing to host pathology.

The production of reactive oxygen species (ROS) via the oxidative burst has been connected with promotion of inflammation and tissue damage, but in recent years has been implicated in regulation of inflammation. We investigated the effects of ROS on the murine model of lupus and in patients (SLE). Aims The aim of this project was to elucidate the impact of the oxidative burst on the development of lupus-like autoimmunity. Lupus was induced by i.p. injection of pristane oil. Ex vivo phagocytosis assays were deployed to assess the uptake of cell debris in ROS deficient mice. The formation of neutrophil extracellular traps (NETs) was analysed by immune fluorescence microscopy. ROS activators were injected into mice to investigate the possible beneficial clinical effects on lupus pathology. Results The absence of ROS gives rise to dramatically exacerbated lupus. Aberrant phagocytosis in ROS-deficient animals leading to production of inflammatory mediators, accompanied by diminished pristane-induced but higher spontaneous formation of NETs could be responsible for this phenotype. Treatment with NOX2 agonists ameliorated the clinical course in mice, while application of a ROS scavenger worsened disease outcome. A fine-tuned balance of ROS-production is necessary to prevent autoimmunity and to avert the development of lupus in mice and men.

Apoptosis is important for the regulation of many physiological and pathological processes. Th e molecular machinery of apoptosis is composed of the death receptor pathway (extrinsic), the mitochondrial pathway (intrinsic), and other complicated signaling networks. In the death receptor pathway, following ligands binding to the receptors, intracellular adaptor molecules are recruited to the receptor and subsequently the apoptotic signal is transmitted. In the mitochondrial pathway, apoptotic signals trigger the release of mitochondrial apoptogenic factors and mitochondrial dysfunction. Apoptosis signaling pathway can be abnormally activated in various pathological processes in the liver, including acute and chronic hepatitis, fi brogenesis, liver cirrhosis and liver carcinogenesis. Th us delineation of apoptosis mechanisms has brought out new paradigms of understanding in the pathogenesis of liver diseases. Inhibition of apoptosis could reverse liver injury by blocking the death process. Hence, dissection of the mechanisms in apoptosis underlying liver diseases would provide the basis of novel therapeutic strategies. Corresponding addresses: Dr. Ying-Hong Shi, Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, 200032, China, E-mail: shi.yinghong@zs-hospital.sh.cn Dr. Xiao-Ming Yin, Department of Pathology, University of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh, PA 15261, E-mail: xmyin@pitt.edu Dr. Jia Fan, Dr. Zhen-Bin Ding, Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, 200032, China, E-mai: jiafan@zs-hospital.sh.cn Dr. Zhen-Bine Ding, E-mail: dzb2007fudan@gmail.com