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 (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.

Molecular MRI of the Immuno-Metabolic Interplay in a Rabbit Liver Tumor Model: A Biomarker for Resistance Mechanisms in Tumor-targeted Therapy?

Background The immuno-metabolic interplay has gained interest for determining and targeting immunosuppressive tumor micro-environments that remain a barrier to current immuno-oncologic therapies in hepatocellular carcinoma. Purpose To develop molecular MRI tools to reveal resistance mechanisms to immuno-oncologic therapies caused by the immuno-metabolic interplay in a translational liver cancer model. Materials and Methods A total of 21 VX2 liver tumor-bearing New Zealand white rabbits were used between October 2018 and February 2020. Rabbits were divided into three groups. Group A (n = 3) underwent intra-arterial infusion of gadolinium 160 (160Gd)-labeled anti-human leukocyte antigen-DR isotope (HLA-DR) antibodies to detect antigen-presenting immune cells. Group B (n = 3) received rhodamine-conjugated superparamagnetic iron oxide nanoparticles (SPIONs) intravenously to detect macrophages. These six rabbits underwent 3-T MRI, including T1- and T2-weighted imaging, before and 24 hours after contrast material administration. Group C (n = 15) underwent extracellular pH mapping with use of MR spectroscopy. Of those 15 rabbits, six underwent conventional transarterial chemoembolization (TACE), four underwent conventional TACE with extracellular pH-buffering bicarbonate, and five served as untreated controls. MRI signal intensity distribution was validated by using immunohistochemistry staining of HLA-DR and CD11b, Prussian blue iron staining, fluorescence microscopy of rhodamine, and imaging mass cytometry (IMC) of gadolinium. Statistical analysis included Mann-Whitney U and Kruskal-Wallis tests. Results T1-weighted MRI with 160Gd-labeled antibodies revealed localized peritumoral ring enhancement, which corresponded to gadolinium distribution detected with IMC. T2-weighted MRI with SPIONs showed curvilinear signal intensity representing selective peritumoral deposition in macrophages. Extracellular pH-specific MR spectroscopy of untreated liver tumors showed acidosis (mean extracellular pH, 6.78 ± 0.09) compared with liver parenchyma (mean extracellular pH, 7.18 ± 0.03) (P = .008) and peritumoral immune cell exclusion. Normalization of tumor extracellular pH (mean, 6.96 ± 0.05; P = .02) using bicarbonate during TACE increased peri- and intratumoral immune cell infiltration (P = .002). Conclusion MRI in a rabbit liver tumor model was used to visualize resistance mechanisms mediated by the immuno-metabolic interplay that inform susceptibility and response to immuno-oncologic therapies, providing a therapeutic strategy to restore immune permissiveness in liver cancer. © RSNA, 2020 Online supplemental material is available for this article.

Neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios as predictors of tumor response in hepatocellular carcinoma after DEB-TACE.

OBJECTIVES: To investigate the predictive value of quantifiable imaging and inflammatory biomarkers in patients with hepatocellular carcinoma (HCC) for the clinical outcome after drug-eluting bead transarterial chemoembolization (DEB-TACE) measured as volumetric tumor response and progression-free survival (PFS). METHODS: This retrospective study included 46 patients with treatment-naïve HCC who received DEB-TACE. Laboratory work-up prior to treatment included complete and differential blood count, liver function, and alpha-fetoprotein levels. Neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) were correlated with radiomic features extracted from pretreatment contrast-enhanced magnetic resonance imaging (MRI) and with tumor response according to quantitative European Association for the Study of the Liver (qEASL) criteria and progression-free survival (PFS) after DEB-TACE. Radiomic features included single nodular tumor growth measured as sphericity, dynamic contrast uptake behavior, arterial hyperenhancement, and homogeneity of contrast uptake. Statistics included univariate and multivariate linear regression, Cox regression, and Kaplan-Meier analysis. RESULTS: Accounting for laboratory and clinical parameters, high baseline NLR and PLR were predictive of poorer tumor response (p = 0.014 and p = 0.004) and shorter PFS (p = 0.002 and p < 0.001). When compared to baseline imaging, high NLR and PLR correlated with non-spherical tumor growth (p = 0.001 and p < 0.001). CONCLUSIONS: This study establishes the prognostic value of quantitative inflammatory biomarkers associated with aggressive non-spherical tumor growth and predictive of poorer tumor response and shorter PFS after DEB-TACE. KEY POINTS: • In treatment-naïve hepatocellular carcinoma (HCC), high baseline platelet-to-lymphocyte ratio (PLR) and neutrophil-to-lymphocyte ratio (NLR) are associated with non-nodular tumor growth measured as low tumor sphericity. • High PLR and NLR are predictive of poorer volumetric enhancement-based tumor response and PFS after DEB-TACE in HCC. • This set of readily available, quantitative immunologic biomarkers can easily be implemented in clinical guidelines providing a paradigm to guide and monitor the personalized application of loco-regional therapies in HCC.

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

With the increasing understanding of resistance mechanisms mediated by the metabolic reprogramming in cancer cells, there is a growing clinical interest in imaging technologies that allow for the non-invasive characterization of tumor metabolism and the interactions of cancer cells with the tumor microenvironment (TME) mediated through tumor metabolism. Specifically, tumor glycolysis and subsequent tissue acidosis in the realms of the Warburg effect may promote an immunosuppressive TME, causing a substantial barrier to the clinical efficacy of numerous immuno-oncologic treatments. Thus, imaging the varying individual compositions of the TME may provide a more accurate characterization of the individual tumor. This approach can help to identify the most suitable therapy for each individual patient and design new targeted treatment strategies that disable resistance mechanisms in liver cancer. This review article focuses on non-invasive positron-emission tomography (PET)- and MR-based imaging techniques that aim to visualize the crosstalk between tumor cells and their microenvironment in liver cancer mediated by tumor metabolism.

Comparison of intrahepatic progression patterns of hepatocellular carcinoma and colorectal liver metastases following CT-guided high dose-rate brachytherapy.

INTRODUCTION: Given the metachronous and multifocal occurrence of hepatocellular carcinoma (HCC) and colorectal cancer metastases in the liver (CRLM), this study aimed to compare intrahepatic progression patterns after computed tomography (CT)-guided high dose-rate brachytherapy. PATIENTS AND METHODS: This retrospective analysis included 164 patients (114 HCC, 50 CRLM) treated with brachytherapy between January 2016 and January 2018. Patients received multiparametric magnetic resonance imaging (MRI) before, and about 8 weeks after brachytherapy, then every 3 months for the first, and every 6 months for the following years, until progression or death. MRI scans were assessed for local or distant intrahepatic tumor progression according to RECIST 1.1 and electronic medical records were reviewed prior to therapy. The primary endpoint was progression-free survival (PFS). Specifically, local and distant intra-hepatic PFS were assessed to determine differences between the intrahepatic progression patterns of HCC and CRLM. Secondary endpoints included the identification of predictors of PFS, time to progression (TTP), and overall survival (OS). Statistics included Kaplan-Meier analysis and univariate and multivariate Cox regression modeling. RESULTS: PFS was longer in HCC [11.30 (1.33-35.37) months] than in CRLM patients [8.03 (0.73-19.80) months, p = 0.048], respectively. Specifically, local recurrence occurred later in HCC [PFS: 36.83 (1.33-40.27) months] than CRLM patients [PFS: 12.43 (0.73-21.90) months, p = 0.001]. In contrast, distant intrahepatic progression occurred earlier in HCC [PFS: 13.50 (1.33-27.80) months] than in CRLM patients [PFS: 19.80 (1.43-19.80) months, p = 0.456] but without statistical significance. Multivariate Cox regression confirmed tumor type and patient age as independent predictors for PFS. CONCLUSION: Brachytherapy proved to achieve better local tumor control and overall PFS in patients with unresectable HCC as compared to those with CRLM. However, distant progression preceded local recurrence in HCC. As a result, these findings may help design disease-specific surveillance strategies and personalized treatment planning that highlights the strengths of brachytherapy. They may also help elucidate the potential benefits of combinations with other loco-regional or systemic therapies.

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

PURPOSE: To establish magnetic resonance (MR)-based molecular imaging paradigms for the noninvasive monitoring of extracellular pH (pHe) 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 pHe 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 pHe 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 pHe (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 pHe increase toward normalization similar to parenchyma was revealed within 2 weeks after complete cTACE, which correlated with decreasing detectability of metabolic markers. In contrast, pHe mapping after incomplete cTACE indicated both acidic viable residuals and increased tumor pHe 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 pHe 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 pHe may be used as a functional biomarker for positive therapeutic outcome.