A high‐throughput imaging platform to characterize extracellular pH in organotypic three‐dimensional in vitro models of liver cancer

Abstract

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 (pHe) mapping platform using magnetic resonance spectroscopic imaging in a three‐dimensional (3D) in vitro model of liver cancer. pHe 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 μM). 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 pHe imaging with BIRDS for in vitro 3D liver cancer models proved feasible. Compared with non‐tumorous hepatocytes (pHe = 7.1 ± 0.1), acidic pHe was revealed in liver cancer (VX2, pHe = 6.7 ± 0.1; HuH7, pHe = 6.8 ± 0.1; HepG2, pHe = 6.9 ± 0.1; SNU475, pHe = 6.9 ± 0.1), in agreement with GLUT‐1 upregulation. Glucose addition significantly further decreased pHe 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 pHe in a dose‐dependent manner without affecting viability. In summary, this study introduces a non‐invasive pHe 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.

Transmembrane pH gradient imaging in rodent glioma models

“…40,41 Measurement of pH i with AACID was calibrated and validated in vivo previously using 31 P MRS. 34 Validation of pH e with BIRDS was performed previously in 3D cell cultures also using two different 31 P MRSbased methods. 64 In the future, additional validation of pH e may be obtained using the exogenous agent 3-aminopropylphosphonate. 23 In the current study, for the first time we report pH e imaging with BIRDS in mouse brains (Figure 3Aiii).…”

“…All infusions were performed using a syringe pump at a rate of 15 μL/min for a total infusion time of 90 min. The BIRDS technique using MRSI was extensively described previously 22,40,41,43,50,63,64 . BIRDS datasets were acquired using the surface RF coil for both transmission and acquisition.…”

“…The BIRDS technique using MRSI was extensively described previously. 22,40,41,43,50,63,64 BIRDS datasets were acquired using the surface RF coil for both transmission and acquisition. Selective excitation of H2, H3, and H6 protons of TmDOTP 5À was achieved using a dual-band 340-μs Shinnar-Le Roux (SLR) RF pulse.…”

Current Trends in Non-Invasive Imaging of Interactions in the Liver Tumor Microenvironment Mediated by Tumor Metabolism

“…Specifically, untreated VX2 tumors showed significantly lower pH e than the surrounding liver parenchyma. Within two weeks after TACE, tumor pH e gradually increased towards normal liver parenchyma pH e , suggesting that tumor pH e can serve as a functional biomarker for tumor responses to non-surgical therapies of liver cancer (Figures 1 and 2) [5,28].…”