PROJECT 3 SUMMARY/ABSTRACT The overall objective of Project 3 is to discover and exploit tumor cell extrinsic mechanisms of therapy resistance by focusing on the contribution and targeting the immunosuppressive tumor microenvironment (TME) created by the synergism between tumor-associated macrophages (TAM), cancer-associated fibroblasts (CAF), and neuroblastoma (NB) cells. Our overarching hypothesis is that TAM, CAF, and NB cells synergistically create an environment that enhances the ability of NB cells to escape targeted, cytotoxic, and immune therapies and that TGFβ-dependent and -independent pathways play prominent roles in promoting Adrenergic to Mesenchymal transition (AMT), producing a fibrotic ECM, and suppressing T and NK (Natural Killer) cells cytotoxic activities. We postulate that the TME is altered in its composition and function as tumors are treated and become increasingly rich in TAM and CAF leading to an inflammatory environment that suppress T and NK cell cytotoxic activities and leads to therapy resistance. This hypothesis is based on published and preliminary data demonstrating that TAM and CAF are enriched in NB tumors post-therapy, and at relapse. We have evidence that TAM and CAF synergistically activate in NB cells NFkB and the production of IL-6 in a TGFb-dependent mechanism and STAT3 by a TGFb and IL-6-independent mechanism. The activation of these two pathways induces AMT and drug-resistance. We also demonstrate that therapies with B7H3 Chimeric Antigen Receptor (CAR) T-cells can target B7H3-expressing TAM and have engineered TGFb resistant CAR T-cells. Aim 1, will determine the TME-dependent mechanisms of AMT and therapy resistance focusing on how TGFβ-dependent and -independent pathways promote AMT, resistance to chemotherapy and targeted therapies (with Project 1), and invasion in vitro and metastasis in vivo (with Project 2). We will examine how the ECM stimulated by TGFβ1 contributes to AMT and to NB cell migration and motility. Last, we will characterize the heterogeneous composition of the TME in serial human tumors with attention to spatial- temporal arrangements and identification of distinct TAM, CAF, NB subpopulations, ECM proteins, and activated pathways. The results of which will inform a clinically applicable multiplex immunofluorescence assay for inclusion in NANT trials. Aim 2 will translate these discoveries in pre-clinical tumor models by targeting TME-dependent pathways to inhibit AMT and enhance therapy response. We will test the therapeutic efficacy of TGFβ and JAK/STAT3 inhibition in combination with chemoimmunotherapy or including CAR T-cells using human NB lines and patient-derived xenotransplants in immunodeficient mice. We will assess the efficacy of TGFb-resistant B7H3-CAR T- in pre-clinical TME-dependent AMT in vitro and in vivo models (with Project 4). The most effective agent(s) will then be proposed for early phase clinical trials to the NANT (Core B). Thus Project 3 brings a unique contri...