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

Abstract

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.

Graphical abstract

Figure 1:

Diagram illustrates the experimental design of immune cell imaging in vivo. A, VX2 liver tumor chunks were injected into the hind leg of a donor rabbit. B, Tumor chunks were harvested and injected into the left liver of the recipient rabbit and allowed to grow for 14 days. C, Three rabbits received intra-arterial (IA) injections of gadolinium 160 (¹⁶⁰Gd)–labeled anti–human leukocyte antigen–DR isotope antibodies and, D, three were intravenously (IV) injected with superparamagnetic iron oxide nanoparticles (SPION). E, ¹⁶⁰Gd-conjugated antibodies target antigen-presenting immune cells (APC) in the peritumoral rim and SPIONs are phagocytosed by peritumoral macrophages. F, MRI was performed and imaging findings were confirmed with radiologic-pathologic correlation.

Figure 2:

In vivo molecular imaging of peritumoral antigen-presenting immune cell infiltrate using gadolinium 160 (¹⁶⁰Gd)–labeled anti–human leukocyte antigen–DR isotope (HLA-DR) antibodies. A, Baseline T1-weighted axial MRI scan of VX2 liver tumor (*). B, Peritumoral rim enhancement (arrows) on T1-weighted axial Dixon MRI scan (repetition time, 5.19 msec; echo times, 2.46 and 3.69 msec) obtained 24 hours after intra-arterial administration of ¹⁶⁰Gd-labeled anti–HLA-DR antibody indicates peritumoral immune cell infiltrate. * = tumor. C, Bright field image and, D, E, colored images from ex vivo imaging mass cytometry of tissue harvested from the rabbit in A and B after ablation of ¹⁶⁰Gd (¹⁶⁰Gd in green) confirm deposition of ¹⁶⁰Gd-labeled anti–HLA-DR antibody in the peritumoral rim. The box in D indicates the the area of magnification for E. L = liver, R = peritumoral rim, T = tumor.

Figure 3:

In vivo molecular imaging of superparamagnetic iron oxide nanoparticles (SPIONs) reveals macrophage infiltration in the peritumoral rim. A, Baseline T2-weighted fat-suppressed axial spin-echo MRI scan (repetition time msec/echo time msec, 1000/78) of VX2 liver tumor (*). B, T2-weighted axial and, C, coronal MRI scans obtained 24 hours after SPION administration show hypointense peritumoral rim (arrows) indicative of peritumoral retention of iron. * = tumor. D–F, Photomicrographs of iron (Prussian blue stain) reveal deposition of SPIONs primarily in the peritumoral rim at, D, ×1 and, E, ×5 magnification after phagocytosis by macrophages as seen at, F, ×20 magnification. The boxes in D and E indicate areas of magnification for E and F, respectively. The yellow lines in E outline the peritumoral rim. L = liver, R = peritumoral rim, T = tumor.

Figure 4:

Molecular imaging of cellular uptake of rhodamine-conjugated superparamagnetic iron oxide nanoparticles (SPIONs) in peritumoral macrophages. A, Baseline T2-weighted fat-suppressed axial spin-echo MRI scan (repetition time msec/echo time msec, 1000/78) of VX2 liver tumor (*). B, Hypointense peritumoral rim demarcation (arrows) on T2-weighted axial MRI scan obtained 24 hours after administration of rhodamine-conjugated SPIONs indicates peritumoral retention. * = tumor. C, Immunofluorescence image of rhodamine on tissue harvested from the same rabbit confirms deposition of SPIONs in the peritumoral rim (outlined by yellow lines). L = liver, R = peritumoral rim, T = tumor. D, Photomicrograph (hematoxylin-eosin [H&E] stain) of VX2 tumor and surrounding liver. E, F, Immunohistochemistry with, E, anti–human leukocyte antigen–DR isotope (HLA-DR) and, F, anti-CD11b antibodies reveals antigen-presenting immune cells and macrophages in the peritumoral rim, respectively.

Figure 5:

A, Images demonstrate that normalization of extracellular tumor pH (pH_e) neutralizes inherent tumor acidosis and restores immune permissiveness after conventional transarterial chemoembolization (TACE) (cTACE). Top, peaks from extracellular pH spectroscopy are shown in red, overlaid on the corresponding anatomic T1-weighted axial MRI scans (repetition time msec/echo time msec, 5.2/2.5). Color map overlays illustrate extracellular tumor pH. Middle, Hematoxylin-eosin (H&E) and, bottom, human leukocyte antigen–DR isotope (HLA-DR) receptor staining reveals peritumoral immune cell infiltration in acidic untreated tumors. While extracellular tumor pH changes after conventional TACE remained insignificant and immune cell infiltrates were similar to or decreased compared with untreated tumors, conventional TACE with bicarbonate (cTACE w/bicarb) significantly increased extracellular pH of the tumor and tumor edge, boosting peritumoral immune cell infiltration. B, Box-and-whisker plots indicate extracellular (pH_e) differences in control rabbits and those treated with conventional TACE (cTACE) and conventional TACE with bicarbonate (cTACE w/bicarb). Data are medians (lines in boxes) and 25th to 75th percentiles (bottom and top of boxes) and ranges (whiskers) (Mann-Whitney U, Kruskal-Wallis test).