ABSTRACT Systemic therapies for advanced stage cancer use immune checkpoint inhibitors to exploit patient's own immune system to generate a robust anti-tumor immune response. This approach is successfully applied for melanoma and lung cancer, but is less effective for hepatocellular carcinoma (HCC) patients. Since most HCC patients are diagnosed at intermediate or advanced stages when curative treatment options are limited, a novel immunotherapy driven treatment is highly desirable. The acidity of the tumor microenvironment driven by hyperglycolytic tumor metabolism represents a key mechanism of resistance and contribute to immune cell exhaustion, thereby helping cancer cells evade the local immune response. Our hypothesis is that tumors responding to immunotherapies have detectable features like normalization of tumor acidity and higher density of immune cells. These parameters, if measured together, can help predict which patients will respond to immunotherapy and/or understand why some patients do not. Imaging extracellular pH (pHe) of tumors along with assessing local immune cell recruitment is necessary to better monitor and guide liver cancer immunotherapies. However, to accurately co-localize the pHe information with the immune cells, high spatial resolution pHe imaging is needed. The goal of this proposal is to establish high resolution in vivo pHe imaging in mouse liver to investigate the effect of cryoablation on tumor aggressiveness in relation to recruitment of local immune cells. In Aim 1 we will establish and validate a high resolution pHe imaging technique in mouse liver using a multidrug resistance gene 2 knockout (Mdr2 KO) model of HCC on a cirrhotic background, where the chronic- inflammation-fibrosis mimics the important stages of liver injury seen in humans. Since repeated freezing of a tumor triggers release of antigens for an anti-tumor immune response, in Aim 2 we will determine if cryoablation can modulate the state of tumor's immunogenicity. Thus, cryoablation may provide an in situ immune system amplification instrument by converting an aggressive immuno-suppressive tumor into a more immuno-permissive tumor. The working hypothesis is that MRI-guided cryoablation will raise pHe by killing tumor cells, and will modulate tumor's degree of immunogenicity by releasing antigens. To investigate the effects of cryoablation, high resolution pHe images will be acquired in Mdr2 KO mice livers 1 day before and 7 days after cryoablation. We will use mice with multiple liver tumors and cryoablate only one to investigate both target and distal effects. Histopathology will be used to reveal intratumoral immune cell recruitment of activated T-cells, helper T-cells, effector T-cells, phagocytes and M2 macrophages. If successful, this approach will allow future investigations of synergistic anti-tumor effects achieved by combining cryoablation with immune checkpoint inhibitors for improved immunotherapeutic effects.