Project Details


Background: Natural Killer (NK) cells, lymphocytes of the innate immune system, are essential for rapid and efficient tumor surveillance. NK cells control tumor growth through production of IFN-gamma and TNF-alpha, direct cytolysis of tumor cells, and recruitment of dendritic cells. During cancer, diverse immunosuppressive axes between NK cells and non-hematopoietic cells adversely impact NK cell function. However, these axes have largely been demonstrated using reductionist murine models, and in-depth evaluation of similar mechanisms of dysfunction in human cancers is lacking. Our preliminary data suggest that NK cells in human bladder tumors (BlCa) are severely compromised functionally, and progressively acquire high levels of inhibitory checkpoint molecules including Tim-3, TIGIT, and NKG2A, as early non-invasive tumors transition into a more advanced muscle invasive stage. Furthermore, this phenotype parallels a partial dysfunction of NK cells in the blood, indicating a simultaneous, systemic imprinting process by the tumor microenvironment (TME) on distal tissues. Importantly however, our data suggest that the NK cells are not terminally 'exhausted,' and that their effector functions are enhanced or restored after ex vivo blockade of the inhibitory receptor Tim-3. These findings suggest that: (a) an adverse TME-NK cell axis compromises effective innate immune responses both locally and systemically, and (b) that NK cell-centric checkpoint blockade has the potential to ameliorate these defects. The proposed research addresses the Department of Defense Peer Reviewed Cancer Research Program (PRCRP) Fiscal Year 2018 (FY18) Topic Areas of Bladder Cancer and Immunotherapy, as well as the FY18 Military Relevance Focus Area of “militarily relevant risk factors for cancer.”Hypothesis: We hypothesize that NK cell tumor surveillance is progressively subverted as bladder tumors become more invasive, through activation of inhibitory modules unique to disease stage. Additionally, we hypothesize that targeting combinations of these modules can restore NK function and enhance tumor control.Specific Aims:Aim 1: Identify differences in disease stage-, and tissue-specific landscapes that preclude optimal NK cell effector function in bladder cancer patients. Aim 2: Interrogate NK and T cell specific transcriptional programs to define the mechanistic axes underlying dysfunction in tumor and peripheral blood. Aim 3: Externally validate gene signatures of NK cell dysfunction, and test rational, pre-clinical interventions to reverse it.Study Design: We will assemble high-resolution phenotypes of NK cell dysfunction and its evolution during NMI and MI BlCa by using single-cell approaches such as mass cytometry, scRNASeq, and highly-multiplexed IHC to query fresh surgical specimens (peripheral blood mononuclear cells [PBMC], tumor tissue), as well as an existing repository of the same tissues from ~100 patients. Healthy donor PBMC and non-involved bladder tissue will serve as controls. Similar approaches will be used to characterize the reversal of NK cell dysfunction after Tim-3 blockade and tumor 'rest' to empirically identify new therapeutic targets. Importantly, ex vivo combination blockade of Tim-3, TIGIT, NKG2A, Siglec-7, and other novel checkpoints will guide translational efforts to enhance bladder immunosurveillance with NK cell-centric approaches.Collaboration: Our team brings extensive expertise in NK cell immunology (Bhardwaj, Horowitz), immuno-oncology (Bhardwaj, Galsky) medical oncology, (Galsky), urology (Sfakianos) and statistical genomics (Zhu), and has had an existing collaboration in GU malignancies for 4 years. This proposal relies extensively on, and benefits from: (1) Access to, and clinical classification of fresh and banked patient tissues (Sfakianos, Galsky), (2) Overall experimental design and data interpretation (Bhardwaj), (3) Implementation and analysis of mass cytometry (Horowitz), and (4) Implementation and analysis of scRNASeq (Zhu).Military Relevance and Impact: BlCa disproportionately affects United States (US) Veterans, and is the fourth most common cancer diagnosed in the Department of Veterans Affairs Health System. Additionally, US Veterans exposed to Agent Orange during their Service in Vietnam may face an increased risk for BlCa. This proposal addresses the FY18 PRCRP Military Relevance Focus Area prioritizing studies examining relevant cancer risk factors, and will accelerate advances in the detection, diagnosis, and treatment of BlCa and other solid tumors for the benefit of US Service members.

Effective start/end date15/07/1914/07/22


  • Congressionally Directed Medical Research Programs: $1,584,753.00


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