PROJECT ABSTRACT Hepatocellular carcinoma (HCC) is a significant global health problem and is expected to become the third leading cause of cancer mortality in the United States (US). Among patients with early-stage HCC, approximately 70-80% of patients experience disease recurrence after surgical resection, and no systemic therapy is currently approved in the perioperative setting. Neoadjuvant immunotherapy (immunotherapy administered prior to a potentially curative HCC resection) aims to utilize the primary tumor as a source of antigens to enhance systemic anti-tumor immunity and prevent disease recurrence. Neoadjuvant studies also offer an opportunity to interrogate the mechanisms behind both tumor sensitivity and resistance to therapies by providing larger quantities of tumor to enable in-depth profiling of the tumor immune microenvironment (TiME) that is not possible with needle biopsies. We are conducting a neoadjuvant platform study to evaluate multiple programmed cell death protein- 1 (PD1)-based combinations in HCC. The three study arms are nivolumab (NIVO, anti-PD1), NIVO plus cabozantinib (CABO, a multi-kinase inhibitor of VEGFR-2, AXL, and c-MET), and NIVO plus relatlimab (RELA, anti-Lymphocyte Activating 3, LAG3). Our overarching hypothesis is that the addition of CABO and RELA to anti- PD1 will bypass barriers to recruitment and function of T cells in early-stage HCC. We will also investigate tertiary lymphoid structures (TLS) as a conserved mechanism of response to neoadjuvant anti-PD1-based immunotherapy, based on our preliminary data showing a strong association between the formation of TLS and major pathologic response in HCCs treated with anti-PD1 immunotherapy. In Aim 1, we will utilize high-parameter multiplex imaging mass cytometry (IMC) on pre-treatment and post-treatment surgical resection specimens to determine the additive effects of CABO and RELA on the density and spatial relationships of immune cells within the TiME. We will also evaluate whether the triple combination of CABO, anti-PD1, and anti-LAG3 enhances T effector (Teff) recruitment, function, and survival in preclinical models of HCC. In Aim 2, we will investigate the clonal dynamics of TLS that arise within anti-PD1 treated HCCs through T-cell receptor (TCR) and B-cell receptor (BCR) sequencing of individual lymphoid aggregates. We will determine the immunophenotype of TLS through spatial and functional analysis of TLS-associated T cells and B cells. In Aim 3, we will determine the cellular composition and organization of immunotherapy-associated TLS in three-dimensions by employing a new technology (CODA) that renders 3D reconstruction of 2D image stacks. This aim will provide the first multicellular structure-function analysis of TLS in immunotherapy-responsive HCC to understand if TLS are highly conserved within tumors and across tumors. The final deliverable for this project is understanding the mechanisms of response to promising anti-PD1 combinations, a...