Murine Tumor Microenvironment Research Articles

ADGRE5-centered Tsurv model in T cells recognizes responders to neoadjuvant cancer immunotherapy.

Neoadjuvant immunotherapy with anti-programmed death-1 (neo-antiPD1) has revolutionized perioperative methods for improvement of overall survival (OS), while approaches for major pathologic response patients' (MPR) recognition along with methods for overcoming non-MPR resistance are still in urgent need. We utilized and integrated publicly-available immune checkpoint inhibitors regimens (ICIs) single-cell (sc) data as the discovery datasets, and innovatively developed a cell-communication analysis pipeline, along with a VIPER-based-SCENIC process, to thoroughly dissect MPR-responding subsets. Besides, we further employed our own non-small cell lung cancer (NSCLC) ICIs cohort's sc data for validation in-silico. Afterward, we resorted to ICIs-resistant murine models developed by us with multimodal investigation, including bulk-RNA-sequencing, Chip-sequencing and high-dimensional cytometry by time of flight (CYTOF) to consolidate our findings in-vivo. To comprehensively explore mechanisms, we adopted 3D ex-vivo hydrogel models for analysis. Furthermore, we constructed an ADGRE5-centered Tsurv model from our discovery dataset by machine learning (ML) algorithms for a wide range of tumor types (NSCLC, melanoma, urothelial cancer, etc.) and verified it in peripheral blood mononuclear cells (PBMCs) sc datasets. Through a meta-analysis of multimodal sequential sc sequencing data from pre-ICIs and post-ICIs, we identified an MPR-expanding T cells meta-cluster (MPR-E) in the tumor microenvironment (TME), characterized by a stem-like CD8+ T cluster (survT) with STAT5-ADGRE5 axis enhancement compared to non-MPR or pre-ICIs TME. Through multi-omics analysis of murine TME, we further confirmed the existence of survT with silenced function and immune checkpoints (ICs) in MPR-E. After verification of the STAT5-ADGRE5 axis of survT in independent ICIs cohorts, an ADGRE5-centered Tsurv model was then developed through ML for identification of MPR patients pre-ICIs and post-ICIs, both in TME and PBMCs, which was further verified in pan-cancer immunotherapy cohorts. Mechanistically, we unveiled ICIs stimulated ADGRE5 upregulation in a STAT5-IL32 dependent manner in a 3D ex-vivo system (3D-HYGTIC) developed by us previously, which marked Tsurv with better survival flexibility, enhanced stemness and potential cytotoxicity within TME. Our research provides insights into mechanisms underlying MPR in neo-antiPD1 and a well-performed model for the identification of non-MPR.

203P Early remodeling of systemic antitumor T cell immunity in head and neck cancer patients treated by chemoradiation

A combination of radiotherapy and chemotherapy (CRT: chemoradiation) is particularly used as a cytotoxic therapy in locally advanced cancers. CRT also gained great interest as a combination approach with immune checkpoint inhibitors (ICI). In this regard, our recent report showed in the murine tumor microenvironment that CRT synergistically improves the antitumor T cell immunity suitable for ICI action (Lauret et al, JITC, 2021). Here, we performed a high throughput blood-based immune analysis in head and neck squamous cell carcinoma (HNSCC) treated by CRT.

Assessment of the Murine Tumor Microenvironment by Multiplex Immunofluorescence.

We describe the staining methods used for simultaneous detection of tumor microenvironment components as well as the automated quantification methodologies. This method uses mouse formalin-fixed paraffin-embedded tissues and multiplex immunofluorescence (Multiplex IF) followed by multispectral imaging. Currently, this methodology has shown to have a valuable role in murine immunoprofiling, and can be useful when evaluating the changes incurred on the tumor microenvironment upon various immunopreventive strategies.

An R0-resectable, Genetically Engineered Mouse Model of Pancreatic Cancer Mimics the Response to Neoadjuvant Chemotherapy in Human PDAC

Purpose: The understanding of the complex tumor-stromal interactions in PDAC microenvironment has been dramatically enhanced by the development of mouse models. However, recent studies demonstrated that the immune response in existing murine models does not correspond to that of the human disease. In this study, we analyzed the effects of neoadjuvant chemotherapy (neoCTx) on the histopathological features of a novel R0-resectable, genetically induced mouse model (termed Pfl mice) and compared it to human PDAC. Methods: Using double immunofluorescence stainings with multiple markers and software-based analysis, we quantified the angiogenesis, tumor desmoplasia (activated stroma index/ASI), and the tumor-associated infiltration with cytotoxic T-lymphocytes, myeloid-derived-suppressor cells/MDSC and natural killer cells (NK) in tissue specimens of 36 neoadjuvantly treated PCa patients and 6 Pfl mice and compared the results with matched cohorts of primarily resected specimens. Results: In contrast to human PDAC, the stromal activity in the murine tumor microenvironment remained constant after neoCTx. No significant change was observed in the microvessel density in both human and mice (p=0,179; p=0,560). NeoCTx led to a reduction of the CD45+ leucocytes (human: 266,7±22,75 vs 186,6±25,33; p=0,021; mice: 6,3x10-4±1,0 x10-4 vs 1,3x10-4±5,0 x10-5; p=0,005) as well as to an increase of the CD8+ T-lymphocytes/CD45+ leucocyte ratio in both populations (human: 0,22±0,0,01 vs 0,37±0,01; p<0,001; mice: 0,01267±0,03972; p=0,035). Simultaneously, our findings showed a marked decrease of MDSCs (human:16,95±2,36 vs 5,58±0,88; p<0,0001; mice: 6,4x10-5±1,0x10-5 vs 2,6x10-5±1,8x10-5; p=0,074) and NCAM+ NK cells (human: 1,33x10-4±2,13x10-4 vs 2,13x10-5±3,03x10-6; p<0,0001; mice: 12,2x10-5±3,2x10-5 vs 2,5x10-5±1,7x10-5; p=0,069) after neoCTx in human and murine tissue specimens. Conclusions: Our data suggest that the reactivation of anti-tumoral immune responses in the tumor microenvironment of the Pfl PDAC mouse model after neoCTx strongly mimics that of the human disease and therefore proved its suitability for its use in future preclinical trials for molecular immunotherapy.

Obesity Shapes Metabolism in the Tumor Microenvironment to Suppress Anti-Tumor Immunity

Obesity is a major cancer risk factor, but how differences in systemic metabolism change the tumor microenvironment (TME) and impact anti-tumor immunity is not understood. Here, we demonstrate that high-fat diet (HFD)-induced obesity impairs CD8+ Tcell function in the murine TME, accelerating tumor growth. We generate a single-cell resolution atlas of cellular metabolism in the TME, detailing how it changes with diet-induced obesity. We find that tumor and CD8+ Tcells display distinct metabolic adaptations to obesity. Tumor cells increase fat uptake with HFD, whereas tumor-infiltrating CD8+ Tcells do not. These differential adaptations lead to altered fatty acid partitioning in HFD tumors, impairing CD8+ Tcell infiltration and function. Blocking metabolic reprogramming by tumor cells in obese mice improves anti-tumor immunity. Analysis of human cancers reveals similar transcriptional changes in CD8+ Tcell markers, suggesting interventions that exploit metabolism to improve cancer immunotherapy.

Isolation and Characterization of Innate Lymphoid Cells within the Murine Tumor Microenvironment.

Innate lymphoid cells (ILCs) are important for both tissue immunity and tissue homeostasis. They are classified into three groups: Group 1 ILCs include NK cells, which are important in eliciting immunity against intracellular pathogens; group 2 ILCs protect against parasitic helminths; and group 3 ILCs protect against extracellular pathogens. The role of ILCs in cancer immunity remains unclear. In this chapter, we discuss methods for isolating and characterizing tumor-infiltrating ILC subsets within the tumor microenvironment in an experimental murine model of B16 melanoma. The chapter also highlights the expression of PD-1 on the various ILC subsets within the tumor microenvironment.

Mouse Ovarian Cancer Models Recapitulate the Human Tumor Microenvironment and Patient Response to Treatment

SummaryAlthough there are many prospective targets in the tumor microenvironment (TME) of high-grade serous ovarian cancer (HGSOC), pre-clinical testing is challenging, especially as there is limited information on the murine TME. Here, we characterize the TME of six orthotopic, transplantable syngeneic murine HGSOC lines established from genetic models and compare these to patient biopsies. We identify significant correlations between the transcriptome, host cell infiltrates, matrisome, vasculature, and tissue modulus of mouse and human TMEs, with several stromal and malignant targets in common. However, each model shows distinct differences and potential vulnerabilities that enabled us to test predictions about response to chemotherapy and an anti-IL-6 antibody. Using machine learning, the transcriptional profiles of the mouse tumors that differed in chemotherapy response are able to classify chemotherapy-sensitive and -refractory patient tumors. These models provide useful pre-clinical tools and may help identify subgroups of HGSOC patients who are most likely to respond to specific therapies.

A Mixture of Polyunsaturated Fatty Acids ω-3 and ω-6 Reduces Melanoma Growth by Inhibiting Inflammatory Mediators in the Murine Tumor Microenvironment.

Background: Although it has been previously demonstrated that acute inflammation can promote the tumor growth of a sub-tumorigenic dose of melanoma cells through of 5-lipoxygenase inflammatory pathway and its product leukotriene B4, and also that the peritumoral treatment with eicosapentaenoic acid and its product, leukotriene B5, reduces the tumor development, the effect of the treatment by gavage with omega-3 and omega-6 in the tumor microenvironment favorable to melanoma growth associated with acute inflammation has never been studied. Methods: C57BL/6 mice were coinjected with 1 × 106 apoptotic cells plus 1 × 103 viable melanoma cells into the subcutaneous tissue and treated by gavage with omega-3-rich fish oil or omega-6-rich soybean oil or a mixture of these oils (1:1 ratio) during five consecutive days. Results: The treatment by gavage with a mixture of fish and soybean oils (1:1 ratio) both reduced the melanoma growth and the levels of leukotriene B4 (LTB4), prostaglandin E2 (PGE2), PGE2/prostaglandin E3 (PGE3) ratio, and CXC ligand 1 (CXCL1) and increased the levels of interleukin 10 (IL-10) to IL-10/CXCL1 ratio in the melanoma microenvironment. Conclusion: The oral administration of a 1:1 mixture of fish oil and soybean oil was able to alter the release of inflammatory mediators that are essential for a microenvironment favorable to the melanoma growth in mice, whereas fish oil or soybean oil alone was ineffective.

Radiation-Induced CXCL12 Upregulation via Histone Modification at the Promoter in the Tumor Microenvironment of Hepatocellular Carcinoma.

Tumor cells can vary epigenetically during ionizing irradiation (IR) treatment. These epigenetic variegations can influence IR response and shape tumor aggressiveness. However, epigenetic disturbance of histones after IR, implicating in IR responsiveness, has been elusive. Here, we investigate whether altered histone modification after IR can influence radiation responsiveness. The oncogenic CXCL12 mRNA and protein were more highly expressed in residual cancer cells from a hepatoma heterotopic murine tumor microenvironment and coculture of human hepatoma Huh7 and normal IMR90 cells after radiation. H3K4 methylation was also enriched and H3K9 methylation was decreased at its promoter region. Accordingly, invasiveness and the subpopulation of aggressive CD133+/CD24- cells increased after IR. Histone demethylase inhibitor IOX1 attenuated CXCL12 expression and the malignant subpopulation, suggesting that responses to IR can be partially mediated via histone modifications. Taken together, radiation-induced histone alterations at the CXCL12 promoter in hepatoma cells are linked to CXCL12 upregulation and increased aggressiveness in the tumor microenvironment.

Radiation-Induced CXCL12 Upregulation via Histone Modification at the Promoter in the Tumor Microenvironment of Hepatocellular Carcinoma

Tumor cells can vary epigenetically during ionizing irradiation (IR) treatment. These epigenetic variegations can influence IR response and shape tumor aggressiveness. However, epigenetic disturbance of histones after IR, implicating in IR responsiveness, has been elusive. Here, we investigate whether altered histone modification after IR can influence radiation responsiveness. The oncogenic CXCL12 mRNA and protein were more highly expressed in residual cancer cells from a hepatoma heterotopic murine tumor microenvironment and coculture of human hepatoma Huh7 and normal IMR90 cells after radiation. H3K4 methylation was also enriched and H3K9 methylation was decreased at its promoter region. Accordingly, invasiveness and the subpopulation of aggressive CD133+/CD24− cells increased after IR. Histone demethylase inhibitor IOX1 attenuated CXCL12 expression and the malignant subpopulation, suggesting that responses to IR can be partially mediated via histone modifications. Taken together, radiation-induced histone alterations at the CXCL12 promoter in hepatoma cells are linked to CXCL12 upregulation and increased aggressiveness in the tumor microenvironment.

451 - Inhibitory functions of PD-L1 and PD-L2 in the regulation of anti-tumor immunity in murine tumor microenvironment

451 - Inhibitory functions of PD-L1 and PD-L2 in the regulation of anti-tumor immunity in murine tumor microenvironment

Mouse Ovarian Cancer Models Recapitulate the Human Tumor Microenvironment and Patient Response to Treatment

Although there are many prospective targets in the tumor microenvironment (TME) of high-grade serous ovarian cancer (HGSOC), pre-clinical testing is challenging especially as there is limited information on the murine TME. Here, we have characterized the TME of six orthotopic, transplantable syngeneic murine HGSOC lines established from genetic models and compared these to patient biopsies. This analysis identified significant correlations between the transcriptome, host cell infiltrates, matrisome, vasculature and tissue modulus of mouse and human TMEs, with several stromal and malignant targets in common. However, each model showed distinct differences and potential vulnerabilities that enabled us to predict response to chemotherapy and an anti-IL-6 antibody. Using machine learning, the transcriptional profiles of the mouse tumors that differed in chemotherapy response were able to classify chemotherapy-sensitive and -refractory patient tumors. These models provide useful pre-clinical tools and may help identify subgroups of HGSOC patients most likely to respond to specific therapies.

Inhibitory functions of PD-L1 and PD-L2 in the regulation of anti-tumor immunity in murine tumor microenvironment.

Although a role of PD-L1 in the suppression of anti-tumor immunity and its value as a predictive biomarker has been suggested by various preclinical and clinical studies, theprecise mechanisms how PD-L1 and PD-L2, another ligand of PD-1, regulate anti-tumor immunity in the tumor microenvironment are yet to be fully explored. Here, we address this issue using PD-L1-deficient tumor cells, PD-L1-knockout (KO) mice, anti-PD-L1 monoclonal antibody (mAb), and anti-PD-L2 mAb. Firstly, PD-L1-deficient or competent tumor cells were inoculated into wild-type or PD-L1-KO mice. Results of tumor growth and mouse survival indicated that both tumor- and host-derived PD-L1 are functional to suppress anti-tumor immunity, while the former contributes predominantly than the latter. Experiments using bone marrow (BM) chimeric mice, generated by transferring PD-L1-KO BM cells into wild-type mice or vice versa, further suggested that PD-L1 expressed on BM-derived hematopoietic cells mediates the suppressive effects on anti-tumor immunity. Secondly, anti-PD-L2 mAb treatment demonstrated a profound synergy with anti-PD-L1 mAb therapy, whereas anti-PD-L2 mAb alone hardly induced any anti-tumor effects, suggesting that PD-L2's function becomes evident when the effects of PD-L1 are abrogated by anti-PD-L1 mAb. Consistent with this notion, PD-L2 expression was upregulated on tumor-associated macrophages (TAM) when mice were treated with anti-PD-L1 mAb. Taken together, our study elucidated the importance of PD-L1 associated with tumor cells and non-tumor host cells, particularly BM-derived hematopoietic cells, as well as PD-L2 inducibly expressed on TAM in the suppression of anti-tumor immunity in the tumor microenvironment.

Abstract A57: Adoptive T cell therapy for ovarian cancer: Application of a surgically relevant model

Abstract Ovarian cancer is the most lethal gynecologic cancer, with more than 20,000 new diagnoses and 15,000 cancer-related deaths annually. Current standard therapy for advanced ovarian cancer incorporates upfront surgical cytoreduction followed by cytotoxic chemotherapy. But even with advances in surgical technique, chemotherapeutics and targeted delivery methods, the overall five-year survival rate remains ~50%, highlighting the need for novel treatment strategies. We have focused our efforts on immunotherapy by adoptive transfer of engineered T cells that can target proteins uniquely overexpressed in ovarian cancer. Recent work has identified mesothelin (MSLN) as a potential target, as this protein is overexpressed in malignant ovarian tissue, promotes the oncogenic phenotype, and has limited expression in healthy cells. Preclinical studies have demonstrated that mouse and human T cells engineered to express T cell receptors (TCR) with high-affinity for MSLN kill respectively murine and human ovarian tumor cells in vitro. Furthermore, in vivo studies in mice treating metastatic disease developing after intraperitoneal introduction of mouse ID8 ovarian cancer cells have demonstrated that engineered, adoptively transferred T cells preferentially accumulate in ID8 ovarian cancers, reduce tumor burden and prolong survival. However, our results also revealed that the tumor microenvironment (TME) can limit the persistence and killing capacity of engineered T cells. Immunosuppressive cells, inhibitory ligands that reduce T cell function, and cell death-inducing ligands are abundant within ID8 tumors, and cellular and molecular analyses of human ovarian cancer specimens showed that human T cell therapy will face similar TME-mediated obstacles. The ovarian cancer TME is also a nutrient- and oxygen-deprived milieu, and the adaptive metabolic responses required for survival by infiltrating T cells have protean effects on T cell function. Thus, strategies that modulate T cell responses to inhibitory signals, including metabolic pathways, have the potential to influence activity in the TME, and to enhance T cell function and improve anti-tumor efficacy. Ongoing studies exploring strategies to overcome elements common to the human and murine TME, including both direct modulation of the microenvironment and engineering of T cells to overcome critical components of immune evasion by solid tumors and to promote T cell survival and function, will be discussed. Building upon this work, we have been developing a novel model that more directly reflects the clinical setting requiring intervention. Following orthotopic injection of ID8 ovarian cancer cells directly beneath the ovarian bursa, tumorigenesis occurs initially within the ovary/fallopian tube microenvironment, and is followed by development of small volume metastatic disease. This orthotopic model, which results in formation of intraperitoneal metastatic implants originating from the ovary, replicates the development and patterns of metastatic spread of human ovarian cancer. Following intervention with primary surgical cytoreduction that includes hysterectomy and bilateral salpingo-oophorectomy, mice are being treated by adoptive transfer of antigen-specific T cells to target minimal residual disease in the peritoneal cavity, which models the clinical situation of patients requiring treatment after optimal surgical cytoreduction. Ongoing studies will be discussed that have been designed to modulate the function and persistence of transferred, MSLN-specific T cells and assess the impact on in vivo tumor development and survival in this clinically relevant model of ovarian cancer treatment. Citation Format: Christopher B. Morse, Kristin G. Anderson, Breanna M. Bates, Edison Y. Chiu, Nicolas M. Garcia, Philip D. Greenberg. Adoptive T cell therapy for ovarian cancer: Application of a surgically relevant model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A57.

Abstract B17: A dual in vivo and in silico system to model ectopic lymph node structure formation and antitumor immune response in the murine tumor microenvironment

Abstract The tumor microenvironment is a dynamic and complex system consisting of the tumor bed, surrounding stroma, vasculature, and immune infiltrate that self-organizes in response to contextual queues and polarizing factors. The nature and extent of this organization can determine if an immune response is beneficial, suppressed, or tumor promoting. Previous efforts have identified a gene expression signature (GES) that is composed of twelve chemokines and that is associated with the presence of ectopic lymph node structures (ELNS) within the tumor microenvironment. These ELNS are predictive of increased survival across a spectrum of solid tumors, and are thought to be foci of active antigen presentation, lymphocyte recruitment, and immune activation. Understanding how and why these structures form is paramount for improving existing immunotherapies and identifying novel treatment modalities. Most in vivo models utilize subcutaneous orthotopic inoculations that, while producing a tumorous mass, lack the surrounding context of a physiologically relevant microenvironment and completely lack ELNS. Alternatively, naturally occurring or chemically induced models offer a more physiologically relevant setting, but are often highly variable systems in which controlling experimental parameters becomes problematic. Here, we build upon insights gained from the GES using an implantable three-dimensional bioscaffold system to study the interaction of a developing tumor with endogenous immune infiltrates and implanted stroma. This allows for the introduction of carefully controlled microenvironmental elements in a more consistent mouse model. In this system, we can selectively interrogate individual chemokines using slow-release microparticles, and visualize immune infiltrates in real time using intravital microscopy. Concurrently, we use this system to parameterize an integrated mathematical model of the tumor microenvironment, which can then reinform our three-dimensional bioscaffold model. This dual-model system has identified an important role for three components of the GES--CCL19, CCL21, and CXCL13--in the induction of ELNS, and suggests that the type and extent of stromal activation can greatly enhance or prevent effective antitumor immunity. In particular, stromal activation using the lymphotoxin beta receptor ligand, LTα1β2, can induce organized aggregates of endogenous lymphocytes; significantly increase infiltration of T cells, B cells, and dendritic cells (all p&amp;lt;0.05); and prevent the growth of MC-38 colon carcinoma cells (p&amp;lt;0.001 by implant weight). In silico model runs parameterized by ex vivo microtaxis assays and in vivo ELNS formation assays predict that polarized activation of stromal cells is sufficient to compartmentalize lymphoid aggregates into discrete B cell and T cell zones and promote antitumor activity. Conversely, stromal activation in the presence of certain type-II promoting factors, such as all-trans retinoic acid, can result in the infiltration of suppressive populations that promote tumorigenesis. Taken together, this novel dual-model system of the tumor microenvironment suggests that the context of stromal activation in the tumor microenvironment can either promote effective antitumor immunity by inducing ELNS, or suppress antitumor immune response by recruiting suppressive populations. Citation Format: Adam W. Mailloux, Falahat Rana, Yohsuke Yagawa, Mark Robertson-Tessi, Zhou L. Susan, Alexander R. A. Anderson, James J. Mulé. A dual in vivo and in silico system to model ectopic lymph node structure formation and antitumor immune response in the murine tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B17.

Abstract 4980: Engineering adoptive T cell therapy for efficacy in ovarian cancer

Abstract Over 20,000 women are diagnosed with ovarian cancer annually - more than half will die within 5 years and this rate has changed very little in the last 20 years, highlighting the need for innovative therapies. One promising new treatment strategy has the potential to control tumor growth without toxicity to healthy tissues, by employing immune T cells engineered to target proteins uniquely overexpressed in tumors. Recent technological advances have helped identify and validate Wilms’ Tumor Antigen 1 (WT1) and mesothelin (MSLN) as valid antigen targets for ovarian cancer, as these proteins contribute to malignant and invasive phenotypes and have limited expression in healthy cells. In preclinical studies using either patient-derived cell lines or the mouse ID8 ovarian tumor model, we found that T cells engineered to express either a WT1- or MSLN- specific high-affinity T cell receptor (TCR) can kill human and murine ovarian tumor cells in vitro. Moreover, in a disseminated in vivo murine model, adoptively transferred TCR-engineered T cells preferentially accumulated within established ID8 tumors, delayed ovarian tumor growth and prolonged mouse survival. However, our data also revealed that the tumor microenvironment (TME) can limit engineered T cell persistence and killing capacity. Cellular and molecular analyses showed human therapy will face similar TME-mediated obstacles. The ovarian cancer TME is a nutrient- and oxygen-deprived milieu, and adaptive metabolic responses by infiltrating T cells have protean effects on T cell function. Thus, strategies that modulate T cell metabolic pathways, and thereby influence activity in the TME, might enhance T cell function and improve anti-tumor efficacy by overcoming a critical component of immune evasion by solid tumors. Ongoing studies will be discussed that are exploring strategies to overcome elements common to the human and murine TME, including direct modulation of the environment and T cell engineering to promote T cell survival and function. Citation Format: Kristin G. Anderson, Breanna M. Bates, Edison Y. Chiu, Philip D. Greenberg. Engineering adoptive T cell therapy for efficacy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4980. doi:10.1158/1538-7445.AM2017-4980

Abstract NTOC-078: ENGINEERING ADOPTIVE T CELL THERAPY FOR EFFICACY IN OVARIAN CANCER

Abstract Over 20,000 women are diagnosed with ovarian cancer annually—more than half will die within 5 years and this rate has changed very little in the last 20 years, highlighting the need for innovative therapies. Reprogramming immune system cells to target and kill cancer cells represents a promising new treatment strategy. Immune T cells have the potential to control tumor growth without toxicity to healthy tissues when engineered to target proteins uniquely overexpressed in tumors. Recent technological advances have helped identify and validate Wilms' Tumor Antigen 1 (WT1) and mesothelin (MSLN) as valid antigen targets for ovarian cancer; these proteins contribute to malignant and invasive phenotypes and have limited expression in healthy cells. In preclinical studies using either patient-derived cell lines or the mouse ID8 ovarian tumor model, we show that T cells engineered to express either a WT1- or MSLN- specific high-affinity T cell receptor (TCR) can kill human and murine ovarian tumor cells in vitro. Moreover, in a disseminated in vivo murine model, adoptively transferred TCRengineered T cells preferentially accumulated within established ID8 tumors, delayed ovarian tumor growth, and prolonged mouse survival. However, our data also reveal that the tumor microenvironment (TME) limits the persistence and killing capacity of the engineered T cells. Cellular and molecular analyses of human tumor specimens show human therapy will face similar obstacles posed by the TME. Ongoing studies will be discussed that are exploring strategies to overcome elements common to the human and murine TME, including direct modulation of the environment and T cell engineering to promote T cell survival and function. Citation Format: Kristin G. Anderson, Breanna M. Bates, Edison Y. Chiu, Philip D. Greenberg. ENGINEERING ADOPTIVE T CELL THERAPY FOR EFFICACY IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-078.

Abstract TMEM-019: NEW OVARIAN CANCER METASTASIS MODELS DEMONSTRATE PREFERENTIAL HEMATOGENOUS SPREAD OF OVARIAN CANCER CELLS TO THE OVARY AND A REQUIREMENT FOR THE OVARY FOR ABDOMINAL DISSEMINATION

Abstract BACKGROUND: Mounting evidence suggests that many high grade serous ‘ovarian’ cancers (HGSOC) start in the fallopian tube. Cancer cells are then recruited to the ovary and subsequently spread diffusely through the abdomen. The mechanism of ovarian cancer spread has long been thought to be largely due to direct shedding of tumor cells into the peritoneal cavity with vascular spread being of limited importance. Recent work challenges this dogma, suggesting hematogenous spread of ovarian cancer may play a larger role in ovarian cancer cell metastasis than previously thought. One reason the role of vascular spread of ovarian cancer has not been fully elucidated is the lack of easily accessible models of vascular ovarian cancer metastasis. METHODS: We developed three metastatic models of ovarian cancer which confirm the ability of ovarian cancer to hematogenously spread. A murine tail vein injection model using human ovarian cancer cell lines was used to observe the general pattern of hematogenous spread when cells are directly introduced into the vasculature. A subcutaneous murine ovarian tumor model was developed to investigate patterns of metastasis in the setting of a fully murine tumor microenvironment. Finally, a human subcutaneous xenograft model using primary patient derived ovarian cancer cells, human carcinoma-associated mesenchymal stem cells and human endothelial cells was developed to investigate patterns of metastasis in the setting of a humanized microenvironment. RESULTS: In all three models, we demonstrate the formation of distant metastasis via vascular spread. Strikingly, we observe a high rate of metastasis to the ovary (40-100%) in all three models representing a disproportionately large fraction of total metastatic burden. Mice which developed ovarian metastatic disease also developed further intra-abdominal metastatic disease and ascites. Interestingly, in the tail vein injection model, oophorectomy prior to ovarian cancer cell injection resulted in a complete loss of peritoneal metastases, ascites and decreased burden of liver metastasis. This ovary tropism appears to be unique to ovarian cancer cells as intravenous tail vein injection of breast cancer and lung cancer cell lines, while resulting in typical sites of disease in the lung and bone, did not result in ovary involvement. CONCLUSIONS: Taken together our data indicates that hematogenously disseminated high grade serous ovarian cancer cells have a unique tropism for the ovary and that hematogenous spread in ovarian cancer may be more common than previously appreciated. Furthermore our studies support a critical role for the ovary in promoting high grade serous ovarian cancer cell metastasis to the abdomen. The models developed here represent important new tools to evaluate both the mechanism of cancer cell recruitment to the ovary and to understand and target key steps in ovarian cancer metastasis. Citation Format: Lan G Coffman, Daniela Burgos-Ojeda, Rong Wu, Kathleen Cho, Shoumei Bai, and Ronald J Buckanovich. NEW OVARIAN CANCER METASTASIS MODELS DEMONSTRATE PREFERENTIAL HEMATOGENOUS SPREAD OF OVARIAN CANCER CELLS TO THE OVARY AND A REQUIREMENT FOR THE OVARY FOR ABDOMINAL DISSEMINATION [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-019.

IDO, PTEN-expressing Tregs and control of antigen-presentation in the murine tumor microenvironment.

The tumor microenvironment is profoundly immunosuppressive. This creates a major barrier for attempts to combine immunotherapy with conventional chemotherapy or radiation, because the tumor antigens released by these cytotoxic agents are not cross-presented in an immunogenic fashion. In this Focused Research Review, we focus on mouse preclinical studies exploring the role of immunosuppressive Tregs expressing the PTEN lipid phosphatase, and the links between PTEN+ Tregs and the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO). IDO has received attention because it can be expressed by a variety of human tumor types in vivo, but IDO can also be induced in host immune cells of both humans and mice in response to inflammation, infection or dying (apoptotic) cells. Mechanistically, IDO and PTEN+ Tregs are closely connected, with IDO causing activation of the PTEN pathway in Tregs. Genetic ablation or pharmacologic inhibition of PTEN in mouse Tregs destabilizes their suppressive phenotype, and this prevents transplantable and autochthonous tumors from creating their normal immunosuppressive microenvironment. Genetic ablation of either IDO or PTEN+ Tregs in mice results in a fundamental defect in the ability to maintain tolerance to antigens associated with apoptotic cells, including dying tumor cells. Consistent with this, pharmacologic inhibitors of either pathway show synergy when combined with cytotoxic agents such as chemotherapy or radiation. Thus, we propose that IDO and PTEN+ Tregs represent closely linked checkpoints that can influence the choice between immune activation versus tolerance to dying tumor cells.

Engineering adoptive T cell therapy for efficacy in ovarian cancer

Abstract Over 20,000 women are diagnosed with ovarian cancer annually; more than half will die within 5 years, which has changed little over the last 20 years. One promising new treatment strategy employs immune T cells engineered to target proteins uniquely overexpressed in tumors, with the potential to control tumor growth without toxicity to healthy tissues. Recently, mesothelin (MSLN) has been identified as a valid antigen target in ovarian cancer; minimally expressed in healthy cells, MSLN contributes to malignant and invasive phenotypes. In preclinical studies using patient-derived cell lines or the mouse ID8 ovarian tumor model, we found that T cells engineered to express a high-affinity MSLN-specific T cell receptor (TCR) can kill ovarian tumor cells in vitro. In vivo, adoptively transferred TCR-engineered T cells preferentially accumulate within disseminated ID8 tumors, delay tumor growth and prolong mouse survival, but our data also show the tumor microenvironment (TME) can limit engineered T cell persistence and function. Other analyses show human therapy will face similar TME-mediated obstacles. The ovarian cancer TME is a nutrient- and oxygen-deprived milieu, and adaptive metabolic responses by infiltrating T cells have protean effects on T cell function. Thus, strategies that modulate T cell metabolic pathways, and thereby influence activity in the TME, might enhance T cell function and improve anti-tumor efficacy by overcoming a critical component of immune evasion by solid tumors. Ongoing studies will be discussed that are exploring strategies to overcome elements common to the human and murine TME, including direct modulation of the environment and T cell engineering to promote T cell survival and function.