BackgroundPatient-derived tumor models are the new standard for pre-clinical drug testing and biomarker discovery. However, the emerging technology of primary pancreatic cancer organoids has not yet been broadly implemented in research, and complex organotypic models using organoids in co-culture with stromal and immune cellular components of the tumor have yet to be established. In this study, our objective was to develop and characterize pancreatic cancer organoids and multi-cell type organotypic co-culture models to demonstrate their applicability to the study of pancreatic cancer.MethodsWe employed organoid culture methods and flow cytometric, cytologic, immunofluorescent and immunohistochemical methods to develop and characterize patient-derived pancreatic cancer organoids and multi-cell type organotypic co-culture models of the tumor microenvironment.ResultsWe describe the culture and characterization of human pancreatic cancer organoids from resection, ascites and rapid autopsy sources and the derivation of adherent tumor cell monocultures and tumor-associated fibroblasts from these sources. Primary human organoids displayed tumor-like cellular morphology, tissue architecture and polarity in contrast to cell line spheroids, which formed homogenous, non-lumen forming spheres. Importantly, we demonstrate the construction of complex organotypic models of tumor, stromal and immune components of the tumor microenvironment. Activation of myofibroblast-like cancer associated fibroblasts and tumor-dependent lymphocyte infiltration were observed in these models.ConclusionsThese studies provide the first report of novel and disease-relevant 3D in-vitro models representing pancreatic tumor, stromal and immune components using primary organoid co-cultures representative of the tumor-microenvironment. These models promise to facilitate the study of tumor-stroma and tumor-immune interaction and may be valuable for the assessment of immunotherapeutics such as checkpoint inhibitors in the context of T-cell infiltration.

BackgroundMultiple myeloma is characterized by the presence of transformed neoplastic plasma cells in the bone marrow and is generally considered to be an incurable disease. Successful treatments will likely require multi-faceted approaches incorporating conventional drug therapies, immunotherapy and other novel treatments. Our lab previously showed that a combination of transient lymphodepletion (sublethal whole body irradiation) and PD-1/PD-L1 blockade generated anti-myeloma T cell reactivity capable of eliminating established disease. We hypothesized that blocking a combination of checkpoint receptors in the context of low-dose, lymphodepleting whole body radiation would boost anti-tumor immunity.MethodsTo test our central hypothesis, we utilized a 5T33 murine multiple myeloma model. Myeloma-bearing mice were treated with a low dose of whole body irradiation and combinations of blocking antibodies to PD-L1, LAG-3, TIM-3, CD48 (the ligand for 2B4) and CTLA4.ResultsTemporal phenotypic analysis of bone marrow from myeloma-bearing mice demonstrated that elevated percentages of PD-1, 2B4, LAG-3 and TIM-3 proteins were expressed on T cells. When PD-L1 blockade was combined with blocking antibodies to LAG-3, TIM-3 or CTLA4, synergistic or additive increases in survival were observed (survival rates improved from ~30% to >80%). The increased survival rates correlated with increased frequencies of tumor-reactive CD8 and CD4 T cells. When stimulated in vitro with myeloma cells, CD8 T cells from treated mice produced elevated levels proinflammatory cytokines. Cytokines were spontaneously released from CD4 T cells isolated from mice treated with PD-L1 plus CTLA4 blocking antibodies.ConclusionsThese data indicate that blocking PD-1/PD-L1 interactions in conjunction with other immune checkpoint proteins provides synergistic anti-tumor efficacy following lymphodepletive doses of whole body irradiation. This strategy is a promising combination strategy for myeloma and other hematologic malignancies.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-014-0043-z) contains supplementary material, which is available to authorized users.

CD 200 is a widely expressed transmembrane glycoprotein that transmits an inhibitory signal after ligation of the structurally homologous CD 200-receptor-1 (CD 200 R1). Recently, we showed that CD 200 is expressed on keratinocytes and plays a role in protecting hair follicles from autoimmune attack. Here, we report the characterization of cell surface and mRNA expression of CD 200 R1 by cells of the murine epidermis. In addition, we report mRNA expression for other members of the CD 200 R-family (R2-R4) by quantitative real-time RT-PCR. Variable levels of CD 200 R1, R2, R3, and R4 mRNA were detected in bulk epidermal cell suspensions. Freshly isolated Langerhans cells (LC) preferentially expressed CD 200 R1. Consistent with an inhibitory role for CD 200:CD 200 R1 interaction, LC obtained from mice deficient in CD 200 (CD 200(-/-)) were in a heightened state of activation as compared with wild-type (CD 200(+/+)) cells. Freshly isolated dendritic epidermal T cells (DETC) expressed low levels of CD 200 R1, R2, and R3 mRNA, but they preferentially increased cell surface and mRNA expression of CD 200 R1 upon activation in vitro. In functional assays using sub-optimal CD3 signaling, immobilized CD 200 inhibited DETC proliferation and cytokine secretion. Collectively, these results suggest that CD 200:CD 200 R interactions may play a role in regulating both LC and DETC in cutaneous immune reactions.

Summary Metabolic heterogeneity within the tumor microenvironment promotes cancer cell growth and immune suppression. We determined the impact of mitochondria-targeted complex I inhibitors (Mito-CI) in melanoma. Mito-CI decreased mitochondria complex I oxygen consumption, Akt-FOXO signaling, blocked cell cycle progression, melanoma cell proliferation and tumor progression in an immune competent model system. Immune depletion revealed roles for T cells in the antitumor effects of Mito-CI. While Mito-CI preferentially accumulated within and halted tumor cell proliferation, it also elevated infiltration of activated effector T cells and decreased myeloid-derived suppressor cells (MDSC) as well as tumor-associated macrophages (TAM) in melanoma tumors in vivo . Anti-proliferative doses of Mito-CI inhibited differentiation, viability, and the suppressive function of bone marrow-derived MDSC and increased proliferation-independent activation of T cells. These data indicate that targeted inhibition of complex I has synchronous effects that cumulatively inhibits melanoma growth and promotes immune remodeling.

Our data demonstrate that pancreatic cancer cells actively signal broad antitumor immune responses through STING agonist induced type I IFN signaling and chemokine production that drive the infiltration and activation of cytolytic T cells in local and distant pancreatic tumors. BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDA) is a lethal chemoresistant cancer that exhibits early metastatic spread. The highly immunosuppressive PDA tumor microenvironment renders patients resistant to emerging immune-targeted therapies. Building from our prior work, we evaluated stimulator of interferon genes (STING) agonist activation of PDA cell interferon-a/b-receptor (IFNAR) signaling in systemic antitumor immune responses. METHODS: PDA cells were implanted subcutaneously to wildtype, IFNAR-, or CXCR3-knockout mice. Tumor growth was monitored, and immune responses were comprehensively profiled. RESULTS: Human and mouse STING agonist ADU-S100 reduced local and distal tumor burden and activated systemic tumor reactive antitumor immune responses in PDA-bearing mice. Effector T-cell infiltration and inflammatory cytokine and chemokine production, including IFN-dependent CXCR3agonist chemokines, were elevated, whereas suppressive immune populations were decreased in treated tumors. Intratumoral STING agonist treatment also generated inflammation in distal noninjected tumors and peripheral immune tissues. STING agonist treatment of type I IFN-responsive PDA tumors engrafted to IFNAR-/recipient mice was sufficient to contract tumors and stimulate local and systemic T-cell activation. Tumor regression and CD8 þ T-cell infiltration were abolished in PDA engrafted to CXCR3-/mice treated with STING agonist. CONCLUSIONS: These data indicate that STING agonists promote T-cell infiltration and counteract immune suppression in locally treated and distant tumors. Tumor-intrinsic type I IFN signaling initiated systemic STING-mediated antitumor inflammation and required CXCR3 expression. STING-mediated induction of systemic immune responses provides an approach to harness the immune system to treat primary and disseminated pancreatic cancers.

BackgroundAdoptive cellular therapy (ACT) with cancer antigen-reactive T cells following lymphodepletive pre-conditioning has emerged as a potentially curative therapy for patients with advanced cancers. However, identification and enrichment of appropriate T cell subsets for cancer eradication remains a major challenge for hematologic cancers.MethodsPD-1+ and PD-1− T cell subsets from myeloma-bearing mice were sorted and analyzed for myeloma reactivity in vitro. In addition, the T cells were activated and expanded in culture and given to syngeneic myeloma-bearing mice as ACT.ResultsMyeloma-reactive T cells were enriched in the PD-1+ cell subset. Similar results were also observed in a mouse AML model. PD-1+ T cells from myeloma-bearing mice were found to be functional, they could be activated and expanded ex vivo, and they maintained their anti-myeloma reactivity after expansion. Adoptive transfer of ex vivo-expanded PD-1+ T cells together with a PD-L1 blocking antibody eliminated established myeloma in Rag-deficient mice. Both CD8 and CD4 T cell subsets were important for eradicating myeloma. Adoptively transferred PD-1+ T cells persisted in recipient mice and were able to mount an adaptive memory immune response.ConclusionsThese results demonstrate that PD-1 is a biomarker for functional myeloma-specific T cells, and that activated and expanded PD-1+ T cells can be effective as ACT for myeloma. Furthermore, this strategy could be useful for treating other hematologic cancers.

Inhibitory cell surface proteins on T cells are often dynamically regulated, which contributes to their physiologic function. PECAM-1 (CD31) is an inhibitory receptor that facilitates TGF-β-mediated suppression of T cell activity. It is well established in CD4 T cells that PECAM-1 is expressed in naïve recent thymic emigrants, but is down-regulated after acute T cell activation and absent from memory cells. The extent to which PECAM-1 expression is similarly regulated in CD8 T cells is much less well characterized. We evaluated T cells recovered from mice after infection with a model intracellular pathogen and determined that, in CD8 T cells, PECAM-1 expression was strongly down-regulated during acute infection but re-expressed to intermediate levels in memory cells. Down-regulation of PECAM-1 expression in CD8 T cells was transcriptionally regulated and affected by the strength and nature of TCR signaling. PECAM-1 was also detected on the surface of human activated/memory CD8 , but not CD4 T cells. These data demonstrate that PECAM-1 expression is dynamically regulated, albeit differently, in both CD4 and CD8 T cells. Furthermore, unlike memory CD4 T cells, memory CD8 T cells retain PECAM-1 expression and have the potential to be modulated by this inhibitory receptor.

Data from animal models have shown that hepatic graft-versus-host disease (GVHD) may be mediated by donor T cells interacting with liver adhesion molecules, other minor histocompatibility antigens, or both. We hypothesized that T-cell infiltrates within a liver biopsy during clinical GVHD would show a restricted T-cell response because the T cells would be responding to a limited number of antigens. We studied the peripheral T-cell repertoire and the liver-infiltrating T-cell repertoire of a patient who developed skin GVHD and subsequent liver GVHD after a matched sibling bone marrow transplantation for acute myeloid leukemia. Spectratype analysis of peripheral blood at the time of liver GVHD revealed that the patient had reconstituted a complex peripheral T-cell repertoire as evidenced by the presence of complementarity-determining region 3 (CDR3) length heterogeneity in most of the T-cell families. The repertoire complexity was skewed in variable gene beta (VB) 5.3, VB4, VB7, VB8, and VB15. Spectratype analysis on the liver biopsy sample revealed a limited infiltrate with an oligoclonal expansion in VBs 4, 7, and 8. We evaluated the T-cell infiltrate in more detail by sequencing the relevant expansions noted by spectratype and developing probes for the predominant CDR3 sequences. These clonotype probes were hybridized to peripheral blood and liver samples from the patient, a T-cell line developed from the patient's peripheral blood at the time of the initial skin GVHD, the donor's blood and marrow, and control samples. The results showed that the T-cell infiltrate during liver GVHD is mediated by a limited number of T cells, and that those cells are mostly different from the ones expanded from the peripheral blood during an acute skin GVHD reaction. These data support the concept that liver GVHD is a response to tissue-specific minor histocompatibility antigens.