Overcoming Immune Exclusion in a Murine Model of Cirrhotic Hepatocellular Carcinoma via Cryoablation: Insights from Molecular Extracellular pH Imaging

Solid tumors, including hepatocellular carcinoma (HCC), arises most often in cirrhotic livers, where immune exclusion and metabolic reprogramming drive extracellular acidosis (the “Warburg effect”) and create an immunosuppressive tumor microenvironment (TME). This study applied a non-invasive MR Spectroscopic Imaging method called Biosensor Imaging of Redundant Deviation in Shifts (BIRDS) to quantify extracellular pH (pH _e ) dynamics in a mouse model of cirrhosis-associated HCC. Forty-two Mdr2⁻/⁻ mice received chronic carbon tetrachloride (CCl₄), inducing cirrhosis and HCC, confirmed by contrast-enhanced MR and histology. BIRDS revealed significantly lower tumor pH _e in untreated tumors (6.78 ± 0.3) compared with liver parenchyma (7.17 ± 0.02). Cryoablation induced tumor pH _e normalization (7.08 ± 0.03), coinciding with downregulation of metabolic markers and increased T-cell and macrophage infiltration. These results demonstrate that BIRDS enables non-invasive monitoring of the metabolic and immunologic response to cryoablation in HCC within cirrhotic livers. Cryoablation-induced re-normalization of tumor acidity, coupled with enhanced immune activity, suggests a favorable therapeutic outcome and establishes pH _e imaging as a tool for assessing treatment efficacy in acidic TMEs.