A High-Throughput Imaging Platform To Characterize Extracellular Ph In Organotypic Three-Dimensional In Vitro Models Of Liver Cancer

Given the extraordinary nature of tumor metabolism in hepatocellular carcinoma and its impact on oncologic treatment response, this study introduces a novel high-throughput extracellular pH (pH(e)) mapping platform using magnetic resonance spectroscopic imaging in a three-dimensional (3D) in vitro model of liver cancer. pH(e) mapping was performed using biosensor imaging of redundant deviation in shifts (BIRDS) on 9.4 T and 11.7 T MR scanners for validation purposes. 3D cultures of four liver cancer (HepG2, Huh7, SNU475, VX2) and one hepatocyte (THLE2) cell line were simultaneously analyzed (a) without treatment, (b) supplemented with 4.5 g/L d-glucose, and (c) treated with anti-glycolytic 3-bromopyruvate (6.25, 25, 50, 75, and 100 mu M). The MR results were correlated with immunohistochemistry (GLUT-1, LAMP-2) and luminescence-based viability assays. Statistics included the unpaired t-test and ANOVA test. High-throughput pH(e) imaging with BIRDS for in vitro 3D liver cancer models proved feasible. Compared with non-tumorous hepatocytes (pH(e) = 7.1 +/- 0.1), acidic pH(e) was revealed in liver cancer (VX2, pH(e) = 6.7 +/- 0.1; HuH7, pH(e) = 6.8 +/- 0.1; HepG2, pH(e) = 6.9 +/- 0.1; SNU475, pH(e) = 6.9 +/- 0.1), in agreement with GLUT-1 upregulation. Glucose addition significantly further decreased pH(e) in hyperglycolytic cell lines (VX2, HepG2, and Huh7, by 0.28, 0.06, and 0.11, respectively, all p < 0.001), whereas 3-bromopyruvate normalized tumor pH(e) in a dose-dependent manner without affecting viability. In summary, this study introduces a non-invasive pH(e) imaging platform for high-yield screening using a translational 3D liver cancer model, which may help reveal and target mechanisms of therapy resistance and inform personalized treatment of patients with hepatocellular carcinoma.