Molecular Imaging of Extracellular Tumor pH to Reveal Effects of Locoregional Therapy on Liver Cancer Microenvironment

Abstract

Purpose: To establish magnetic resonance (MR)-based molecular imaging paradigms for the noninvasive monitoring of extracellular pH (pH_e) as a functional surrogate biomarker for metabolic changes induced by locoregional therapy of liver cancer.

Experimental design: Thirty-two VX2 tumor-bearing New Zealand white rabbits underwent longitudinal imaging on clinical 3T-MRI and CT scanners before and up to 2 weeks after complete conventional transarterial chemoembolization (cTACE) using ethiodized oil (lipiodol) and doxorubicin. MR-spectroscopic imaging (MRSI) was employed for pH_e mapping. Multiparametric MRI and CT were performed to quantify tumor enhancement, diffusion, and lipiodol coverage of the tumor posttherapy. In addition, incomplete cTACE with reduced chemoembolic doses was applied to mimic undertreatment and exploit pH_e mapping to detect viable tumor residuals. Imaging findings were correlated with histopathologic markers indicative of metabolic state (HIF-1α, GLUT-1, and LAMP-2) and viability (proliferating cell nuclear antigen and terminal deoxynucleotidyl-transferase dUTP nick-end labeling).

Results: Untreated VX2 tumors demonstrated a significantly lower pH_e (6.80 ± 0.09) than liver parenchyma (7.19 ± 0.03, P < 0.001). Upregulation of HIF-1α, GLUT-1, and LAMP-2 confirmed a hyperglycolytic tumor phenotype and acidosis. A gradual tumor pH_e increase toward normalization similar to parenchyma was revealed within 2 weeks after complete cTACE, which correlated with decreasing detectability of metabolic markers. In contrast, pH_e mapping after incomplete cTACE indicated both acidic viable residuals and increased tumor pH_e of treated regions. Multimodal imaging revealed durable tumor devascularization immediately after complete cTACE, gradually increasing necrosis, and sustained lipiodol coverage of the tumor.

Conclusions: MRSI-based pH_e mapping can serve as a longitudinal monitoring tool for viable tumors. As most liver tumors are hyperglycolytic creating microenvironmental acidosis, therapy-induced normalization of tumor pH_e may be used as a functional biomarker for positive therapeutic outcome.

Figure 1.

Experimental in vivo study design. In the horizontal direction, the flow chart illustrates the VX2 rabbit tumor model and multimodal imaging at sequential time points. In the vertical, imaging carried out at each time point is displayed according to the respective modality including spectroscopic pH-mapping, mpMRI including contrast-enhanced imaging and ADC-mapping, and CT with or without contrastad ministration. Briefly, 32 recipient rabbits were implanted into the left liver lobe with tumor chunks harvested from donor rabbits. Tumors were allowed to grow for 2 weeks until baseline imaging was performed. Seven rabbits were euthanized after multimodal imaging at base line and served as controls. The remaining 25 rabbits were treated with cTACE and received multimodal imaging 1 day later. Seven rabbits were euthanized after each imaging at 1 day and 1 week post-cTACE, respectively, and the remaining rabbits entered the next imaging cycle. A total of 11 rabbits were available for multimodal imaging at 2 weeks post-cTACE. After euthanasia, necropsy was performed and tumor and liver tissue were harvested for radiological–histopathologic comparison.

Figure 2.

Spectroscopic noninvasive pH_e mapping using BIRDS. A, Axial T₁ VIBE MR images were used for liver (blue)and tumor (red) delineation in a representative untreated VX2 tumor-bearing rabbit. B, Distribution of TmDOTP⁵⁻ peaks from BIRDS with CSI within the tumor and liver are displayed. C, Examples of spectra from H2, H3, and H6 protons of TmDOTP⁵⁻ from a liver and tumor voxel (black squares in B) demonstrating the pHe-dependent chemical shift of the peaks of each proton. D, The corresponding pH_e map was calculated using the chemical shifts of H2, H3, and H6 protons of TmDOTP⁵⁻ in each tumor and liver voxel, respectively, and illustrated with a color map overlay.

Figure 3.

Evolution of pH_e in liver cancer after cTACE. A, In the horizontal direction, images are arranged according to the time of acquisition in relation to the cTACE treatment. In the first row, CSI peaks are shown in red for each voxel, overlaid on the corresponding anatomic T₁ VIBE MR images. Mean pH_e of the example tumors shown in this figure were 6.77 ± 0.03 (control), 6.85 ± 0.02 (1 day), 6.94 ± 0.01 (1 week), 7.05 ± 0.01 (2 weeks), 6.81 ± 0.0 (undertreated), and 6.95 ± 0.03 (treated after incomplete cTACE). VX2 tumors are outlined in red with color map overlays illustrating tumor pH_e. Imaging findings were confirmed on histology of the tumor using H&E, HIF-1α, GLUT-1, and LAMP-2. Control tumors demonstrated a necrotic core and a viable rim with densely packed tumor cells and high expression levels of all metabolic targets, while they were nearly undetectable at 1 day and not detectable at 1 and 2 weeks post-cTACE. pH_e 2 weeks after incomplete cTACE indicated acidosis of undertreated regions determined as viable residuals on anatomic imaging and histology. In addition, treated regions in the same tumor revealed increased pH_e, which corresponded to disappearance of histological markers. B, Quantification of pH_e demonstrated significantly lower pH_e in the tumor and tumor edge compared with normal liver in animals that did not undergo cTACE. Longitudinal measurements revealed a gradual increase toward pH_e normalization in the tumor and the tumor edge at 1 day, 1 week, and 2 weeks post-cTACE, although a complete recovery to normalized pH_e was not achieved within this time frame. In normal liver, pH_e values remained stable at all time points. Significant differences are indicated by * between different time points (**, P < 0.01; ***, P < 0.001) and by # when compared with liver parenchyma at the same respective time point (#, P < 0.05; ##, P < 0.01; ###, P < 0.001).

Figure 4.

Evolution of multimodal multiparametric imaging biomarkers in liver cancer after cTACE. A, The figure illustrates representative changes of liver tumors on mpMRI and CT induced by cTACE. In the vertical direction, the imaging scans are arranged according to the time of acquisition. In the horizontal, images are arranged according to the modality including precontrast T₁-weighted (T_1w) images, DCE images in the arterial phase (15–20 seconds after contrast bolus injection), and ADC maps, all in axial plane, and axial as well as coronal CT images with (baseline) and without (post-cTACE) contrast administration. Untreated tumors demonstrated rim hyperenhancement on contrast-enhanced MRI and CT and a viable tumor rim (low ADC) and necrotic core (high ADC). Corresponding histology from the tumor rim revealed strong ubiquitous staining with the proliferation marker PCNA and low signal from TUNEL staining of cell death. Treatment with cTACE achieved durable devascularization and onset of necrosis as early as 1 day post-cTACE and sustained lipiodol deposition at all time points. These findings were histologically confirmed by disappearance of PCNA signal and high TUNEL signal in tumors post-cTACE. B, Box plots from volumetric quantification of arterial tumor enhancement (DCE), tumor cellularity (ADC), and lipiodol coverage (CT) of the tumor illustrate the longitudinal cTACE effects. Significant differences are indicated by * compared with baseline (**, P < 0.01;***, P < 0.001) and by # for comparison of incomplete cTACE with complete cTACE both at 2 weeks after treatment (#, P < 0.05; ##, P < 0.01).