Objective : The acidification of the tumor microenvironment subject to the hyperglycolytic cancer metabolism (“Warburg effect”) is a principle feature of carcinogenesis and associated with aggressive tumor growth and poor survival. Conventional transarterial chemoembolization (cTACE) is known to introduce variations of the tumor microenvironment driven by ischemia-induced effects on tumor metabolism and extracellular tumor pH (pHe). However, these cTACE-induced alterations vary between patients and thus, remain poorly understood. Hence, this study aimed to evaluate non-invasive pHe mapping for the functional characterization of the tumor microenvironment and monitoring of changes induced by cTACE in a translational animal model for liver cancer.
Methods : The experimental design was tailored to human-size imaging systems to facilitate a rapid translation into clinical practice. Twenty-six VX2 tumor-bearing rabbits were assigned to four groups for non-invasive pHe mapping on clinical 3T MRI scanners a) before treatment (control group; n=6) or b) immediately (1-2 days; n=5), c) one week (5-7 days; n=7), or d) two weeks (14-15 days; n=8) after cTACE. Additionally, multiparametric (mp)MRI was performed at baseline (n=26) and one day (n=7), one week (n=13), and two weeks (n=8) after cTACE for quantitative image analysis of cTACE effects on tumor perfusion (dynamic contrast-enhanced imaging [DCE]) and cellularity (apparent diffusion coefficient [ADC]). Rabbits with cTACE also had CT scans for volumetric lipiodol quantification one day (n=20), one week (n=12), and two weeks (n=7) after cTACE. Imaging findings from all time-points were correlated with histopathology. Statistical tests included Mann-Whitney-U and Wilcoxon t-test.
Results : pHe maps of untreated VX2 tumors indicate a lower pHe (6.80±0.08) as compared to liver parenchyma (pHe=7.19±0.03, p=0.002). With regards to longitudinal monitoring of therapeutic effects, a gradual increase towards normalization of tumor pHe reaching liver pHe was observed at one day (pHe=6.88±0.04, p=0.353), one week (pHe=6.91±0.04, p=0.012), and two weeks (pHe=7.02±0.04, p<0.001) post-cTACE indicating tumor response to treatment (Fig.1). Accordingly, histopathological markers indicative of glycolysis (GLUT-1) and chronic acidosis (LAMP-2) were found to be upregulated in untreated VX2 tumors. However, expression levels decreased immediately after cTACE and markers were not detectable anymore at one and two weeks after cTACE. Additionally, volumetric quantitative analysis of multimodal imaging showed sustained Lipiodol deposition in the tumor. Time evolution of mpMR imaging biomarkers demonstrated devascularization of tumors immediately after cTACE (p=0.016), which persisted at one (p<0.001) and two weeks (p=0.008) compared to baseline. Significant onset of necrosis became apparent immediately after treatment (p<0.001) compared to baseline and necrosis gradually increased over time (1 week p<0.001; two weeks p<0.001). Histopathological staining of proliferating cells (PCNA) and necrosis (TUNEL) confirmed these radiological findings.
Conclusions : As most liver tumors exhibit a hyperglycolytic metabolic phenotype, non-invasive pHe imaging of microenvironmental acidification may be used as an important quantitative functional indicator for metabolic activity in liver cancer. In turn, normalization of tumor pHe may serve as a biomarker for positive therapeutic outcome.