Next Generation 3D Tissue Culture Systems with Tertiary Lymphoid Organs

Fast-Track proposals will be accepted. Direct-to-Phase II proposals will NOT be accepted Number of anticipated awards: 3-5 Budget (total costs, per award): Phase I: up to $400,000 for up to 9 months Phase II: up to $2,000,000 for up to 2 years PROPOSALS THAT EXCEED THE BUDGET OR PROJECT DURATION LISTED ABOVE MAY NOT BE FUNDED. Summary Tertiary lymphoid organs (TLOs) are lymph node-like structures that form in tissues in the presence of chronic inflammation in response to molecular and pathological damage from exposures (both internal and external). The purpose of their formation is an active area of study, but they likely form to fight damage at the tissue level while also collecting, processing, and delivering and presenting tissue- and tumor- antigens to primary and secondary lymphoid organs. TLOs are characterized by many of the same morphological features as lymph nodes. These morphological features include germinal center B-cell zones, T-cell follicles, and high endothelial venules (HEVs) through which immune cells traffic into/out of the TLO. TLOs are also populated with many of the same cell types as lymph nodes, including mesenchymal derived follicular dendritic cells that recruit lymphocytes and assist in the formation of the morphological features; and hematopoietic stem cell derived antigen presenting dendritic cells (DCs), macrophages, central and effector memory lymphocytes, etc. TLOs are found within almost all tumors and may play a critical gatekeeping role in the ability for T cells to access the tumor microenvironment and participate in immune surveillance. Recent studies show that intra-tumoral TLOs can sequester cytotoxic T cells, and ectopic TLO formation immediately adjacent to the tumor margin prevents the infiltration of T lymphocytes (TILs) into tumor. While TLOs are likely to play a critical role in the ability of the immune system to mount an immune response to tumor neoantigens, and also control effector cell access to tumor cells, TLO containing in vitro models do not exist and in vivo approaches to induce TLOs in animal models are extremely limited. Therefore, there is a need for more efforts to develop both intra and ectopic tumor associated TLO models to understand their role in the development of cancer, immune tolerance, tumor immune evasion, and antitumor immunity. Generating these 3D systems with TLOs that are viable for longer periods is challenging but once established using right elements that best represent the biology and pathology, they can be valuable tools for obtaining insights in basic and translational research in cancer, autoimmune diseases and chronic inflammatory and infectious diseases. Establishing TLO containing 3D systems that survive for a longer duration is not only important for understanding the immune system’s role in keeping cancers in check but also for unraveling how immune system may contribute ques for cell transformation, cancer development and progression, and all these processes require a longer interaction of the immune system with the tissue cells. Project Goals The goal is to advance the development of next generation 3D tissue/tumor cell culture systems that develop and maintain self-assembled TLOs for months. There are several potential research uses for 3D culture models that incorporate TLOs, including: 1) studying immune system’s role in clearing autophagy, apoptosis and/or necrosis mediated tissue damage; 2) studying immune system’s role in prevention, initiation and development of cancer as well as metastasis, 3) studying interactions among immune cells and tumor cells in the tumor microenvironment, 4) serving as an innovative and simple in vitro approach for identifying neoantigens that are collected, processed and displayed by the antigen presenting cells in the TLOs, 5) studying movements and interactions of TILs in tumors, and 6) facilitate development of personalized immunotherapy. The activities and deliverables in the solicitation will focus on the development of 3D tissue cultures that contain self- assembling TLOs with key morphological and functional characteristics. Critical morphological characteristics of the TLOs are B cell and T cell zones. Offerors will be required to interrogate the functional aspects of the TLOs including tumor antigen presentation by DCs within the TLO, and activation and expansion of T cells and B cells. This topic will require developers to establish a 3D culture system representing at least one tumor type and develop 3D cultures from multiple donors. Furthermore, the activities will require that the offerors evaluate the longevity of TLOs in vitro, and ideally demonstrating that a 3D culture systems containing TLOs can be maintained for a minimum of 2 months, with a preference for longer periods of 8-10 months, to allow testing the utility of the established systems, particularly for studying tumor microenvironment interactions. Responsive proposals must develop 3D culture systems containing TLOs using human tumor and immune tissue. Offerors must propose to use tumor tissue and immune cells from the same donor for each 3D system’s development. The immune cells must be differentiated and/or obtained using progenitors with tumor cells providing the cues for their differentiation and activation. The stromal-like cells, to improve structural organization and functionality of the TLOs, must be differentiated/obtained from mesenchymal stromal cells. Preference may be given to systems that use matrices from human sources. Systems that use matrices from non-human sources will be of low priority. Offerors will not be required to develop new 3D tissue culture systems to respond to the solicitation, and companies that have expertise in building 3D culture systems and previously built 3D culture systems through a previous NCI SBIR contract opportunity or other opportunities will be eligible to compete for this topic assuming their proposals meet all of the requirements laid out in this solicitation. Activities not responsive to announcement: 3D systems 1) without functional immune cells, 2) that are simply made by incubating the 3D systems with peripheral blood mononuclear cells (PBMCs) to allow infiltration of immune cells, 3) made using immune cells not properly allowed to mature, differentiate and activate using the cues from the tissue/tumor cells used for creating the 3D system, and 4) 3D systems with TLOs made using synthetic polymer-based dendritic cells Phase I Activities and Deliverables: • Project team: Establish a project team with proven expertise in development of 3D complex tissue models, immunology, clinicians with access to patient samples, including subject matter experts in the tumor(s) being studied • Identify appropriate cell types needed to create the 3D systems • Show resources and expertise needed to mature, differentiate and/or activate the cells needed for creating 3D systems containing TLOs • Create the 3D systems with TLOs representing at least one cancer type (such as pancreas, breast, prostate, lung, colon or liver) • Show that the 3D systems can be developed reproducibly, by showing that the developed systems maintain genotypic and phenotypic characteristics for at least a month. Determine growth and expansion of cells and continued maintenance of genotype and phenotype in different compartments of the 3D systems • Characterize the cells in the 3D system: Show that the mesenchymal derived follicular dendritic cells can recruit immune cells and form TLOs in the 3D systems. Determine antigen presenting capacity of dendritic cells, and central and effector memory cells divide, expand and maintain the TLOs. Analyze T cell receptor excision circles and kappa-deleting recombination excision circles to determine half-life of the T/B cell clonotypes and number of cell divisions, and T cell and B cell receptor sequencing to determine suitability of the T/B cells for forming TLOs that represent the TLOs present in tumor/tissue that is being created • Show access to samples needed to conduct comparative analysis in phase II • Establish workflows for creation and maintenance of 3D systems, and morphological and molecular characterization of the 3D systems and component cells • Submit a detailed statement of performance characteristics along with SOPs for establishing and characterizing the 3D systems to NCI. Phase II Activities and Deliverables: • Create 3D systems with intra and/or ectopic TLOs. • Improve viability and show that the 3D systems’ viability is for a minimum of 2 months with a preference for longer periods of 8-10 months and reproducibility is > 75%. • Characterize the interactions in the microenvironment - such as genotypic (chromatin accessibility, mutation, structural changes, etc.), epigenetic (methylation, histone modification, etc.) and phenotypic (gene expression, chemokines, cytokines, morphological, function, etc.) changes- among immune cells, endothelial cells and epithelial cells using physicochemical, biological, immunocytochemical/histochemical imaging methods. • Compare the 3D systems’ physicochemical and functional characteristics with the characteristics of TLO containing tissues/tumors from humans • Compare antigens displayed by the TLO-DCs in 3D systems with antigens displayed by the circulating migratory DCs in blood from experimental animals or cancer patients with respective cancer type(s) • Compare microenvironment interactions displayed by the 3D systems with the data from in vivo models • Demonstrate utility of the 3D systems for quickly collecting neoantigens in vitro: Identify antigens displayed by the dendritic cells (TLO resident or migratory dendritic cells) in the 3D system • Assess utility of the 3D systems for determining drug/immunotherapy response • Assess how the location and duration of TLO (intra or ectopic) alter infiltration and movement of TILs • Establish QC and QA parameters to increase reliability of the 3D systems and create a commercial prototype of the 3D tissue/tumor culture system • Submit final SOPs, QC/QA parameters, performance characteristics, and characterization and antigen data