An RNA-RNA crosstalk network involving HMGB1 and RICTOR facilitates hepatocellular carcinoma tumorigenesis by promoting glutamine metabolism and impedes immunotherapy by PD-L1+ exosomes activity.

Hepatocellular carcinoma (HCC) is the global leading cause of cancer-related deaths due to the deficiency of targets for precision therapy. A new modality of epigenetic regulation has emerged involving RNA-RNA crosstalk networks where two or more competing endogenous RNAs (ceRNAs) bind to the same microRNAs. However, the contribution of such mechanisms in HCC has not been well studied. Herein, potential HMGB1-driven RNA-RNA crosstalk networks were evaluated at different HCC stages, identifying the mTORC2 component RICTOR as a potential HMGB1 ceRNA in HBV+ early stage HCC. Indeed, elevated HMGB1 mRNA was found to promote the expression of RICTOR mRNA through competitively binding with the miR-200 family, especially miR-429. Functional assays employing overexpression or interference strategies demonstrated that the HMGB1 and RICTOR 3'untranslated regions (UTR) epigenetically promoted the malignant proliferation, self-renewal, and tumorigenesis in HCC cells. Intriguingly, interference against HMGB1 and RICTOR in HCC cells promoted a stronger anti-PD-L1 immunotherapy response, which appeared to associate with the production of PD-L1+ exosomes. Mechanistically, the HMGB1-driven RNA-RNA crosstalk network facilitated HCC cell glutamine metabolism via dual mechanisms, activating a positive feedback loop involving mTORC2-AKT-C-MYC to upregulate glutamine synthetase (GS) expression, and inducing mTORC1 signaling to derepress SIRT4 on glutamate dehydrogenase (GDH). Meanwhile, this crosstalk network could impede the efficacy of immunotherapy through mTORC1-P70S6K dependent PD-L1 production and PD-L1+ exosomes activity. In conclusion, our study highlights the non-coding regulatory role of HMGB1 with implications for RNA-based therapeutic targeting together with a prediction of anti-PD-L1 immunotherapy in HCC.

Ameliorative potential of manganese nanoparticles with low-level ionizing radiation against experimentally induced hepatocarcinogenesis.

Nanotechnology is a rich field with infinite possibilities of drug designs for cancer treatment. We aimed to biosynthesize manganese nanoparticles (Mn NPs) using Lactobacillus helveticus to investigate its anticancer synergistic effect with low-dose gamma radiation on HCC-induced rats. Diethylnitrosamine (DEN) (20 mg/kg BW, 5 times a week for 6 weeks) induced HCC in rats. Rats received Mn NPs (5 mg/kg BW/day) by gastric gavage over 4 weeks concomitant with single dose of gamma radiation (γ-R) (0.25 Gy). Characterization, cytotoxicity, and anticancer activity of Mn NPs were evaluated. DEN-induced significant liver dysfunction (alanine transaminase activity ALT, total proteins, and albumin levels) associated with significant increase in lipid peroxidation levels with reduction in super oxide dismutase activity. Furthermore, DEN intoxication is sponsored for remarkable increase in levels of Alfa-fetoprotein, tumor necrosis factor α, vascular endothelial growth factor, and transforming growth factor beta with remarkable decrease in caspase 3 and cytochrome c. Treatment with Mn NPs (4.98-11.58 nm) and single dose gamma radiation evoked significant repair in ALT, total protein, and albumin accompanied with balanced oxidative status, diminished inflammatory biomarkers, angiogenic factor, and growth factor with restoration in apoptotic factors. Mn NPs revealed obvious in vitro cytotoxic activity against HepG2 cell line in a dose-dependent manner. Our findings were well appreciated with the histopathological study. In conclusion, a new approach of the single or combined use of Mn NPs with low-dose γ-radiation regimens as promising paradigm for HCC treatment is recommended.

HOCl-Activated Aggregation of Gold Nanoparticles for Multimodality Therapy of Tumors.

Tumor microenvironment-responsive nanodrugs offer promising opportunities for imaging-guided precision therapy with reduced side effects. Considering that the antitumor effect is closely related to the size of the nanodrugs, it is particularly important to develop a therapeutic system with size adjustability in the tumor microenvironment, which is still a great challenge in the field of nanotheranostics. Herein, a reactive oxygen species (ROS)-activated aggregation strategy is reported for imaging-guided precision therapy of tumors. The ROS-activated nanoplatform is constructed based on gold nanoparticles (AuNPs) coated with an HOCl probe on its surface (namely, Au-MB-PEG NPs). The Au-MB-PEG NPs show high sensitivity toward HOCl, resulting in the modulation of surface charge and rapid aggregation of AuNPs, and simultaneous release of methylene blue as a photosensitizer for photodynamic therapy (PDT). In the tumor environment, the aggregated AuNPs ensure higher tumor accumulation and retention. Furthermore, the redshift of the absorption of aggregated AuNPs leads to activated photoacoustic imaging signals and photothermal therapy (PTT) under near-infrared irradiation. Au-MB-PEG NPs thus efficiently inhibit the tumor growth through combined PTT-PDT therapy. This work contributes to the design of stimuli-induced size-aggregation nanodrugs, thereby attaining advanced performance in cancer diagnosis and treatment.

Reduction of microwave ablation needle related metallic artifacts using virtual monoenergetic images from dual-layer detector spectral CT in a rabbit model with VX2 tumor.

The purpose of the study was to investigate the application of virtual monoenergetic images (VMIs) in reducing metal artifacts in rabbit VX2 liver cancer models treated with microwave ablation (MWA) therapy. A total of 31 VX2 liver cancer models that accepted CT-guided percutaneous microwave ablation were analyzed. Conventional images (CIs) with the most severe metallic artifacts and their corresponding energy levels from 40 to 200 keV with 10 keV increment of VMIs were reconstructed for further analysis. Objective image analysis was assessed by recording the attenuation (HU) and standard deviation of the most severe hyper/hypodense artifacts as well as artifact-impaired liver parenchyma tissue. Two radiologists visually evaluated the extent of artifact reduction, assessed data obtained by a diagnostic evaluation of liver tissues, and appraised the appearance of new artifacts according to the grade score. Statistical analysis was performed to compare the difference between CIs and each energy level of VMIs. For subjective assessment, reductions in hyperdense and hypodense artifacts were observed at 170-200 keV and 160-200 keV, respectively. The outcomes of the diagnostic evaluation of adjacent liver tissue were statistically higher at 140-200 keV for VMIs than for CIs. In terms of objective evaluation results, VMIs at 90-200 keV reduced the corrected attenuation of hyperdense and of artifact-impaired liver parenchyma compared with CIs (P < 0.001). When VMIs at 80-200 keV decreased the hypodense artifacts (P < 0.001). Therefore, we concluded that VMIs at 170-200 keV can obviously decrease the microwave ablation needle-related metal artifacts objectively and subjectively in rabbit VX2 liver cancer models.

The Apoptotic Effect of Trichinella spiralis Infection Against Experimentally Induced Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the sixth most common type of cancer. Prognosis of HCC remains unsatisfactory. Therefore, developing new therapeutic modalities is still mandatory. Tumor biotherapy is a novel concept developed as a therapeutic strategy for cancer treatment. There is a similarity between the regulatory mechanism of Trichinella spiralis nurse cell formation and tumor cell apoptosis signal regulation. OBJECTIVES: Induction of apoptosis by T. spiralis can represent a new strategy for tumor treatment. METHODS: Experimental animals were divided in four groups; negative control (GI), T. spiralis infected (GII), induced HCC (GIII) and HCC then infected with T. spiralis (GIV). The apoptotic effect of T. spiralis infection was assessed by histopathological and immunohistochemical staining of B-cell lymphoma 2 (Bcl-2). RESULTS: We found higher survival rate of rats and decreased weight of their livers with no nodules in HCC- T. spiralis group as compared to HCC group. Improvement of the dysplastic changes and increased apoptotic bodies which was confirmed by decreased expression of Bcl-2 reported in HCC- T. spiralis group. CONCLUSION: Trichinella-induced apoptosis can be a contributing mechanism of the anti-tumor effect of T. spiralis infection. Our results showed a certain level of decreased progression of the tumor in HCC-T. spiralis group as indicated by increased rate of apoptosis and subsequently had a positive impact on the survival of rats.
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Photoacoustic monitoring of oxygenation changes induced by therapeutic ultrasound in murine hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly vascular solid tumor. We have previously shown that ultrasound (US) therapy significantly reduces tumor vascularity. This study monitors US-induced changes in tumor oxygenation on murine HCC by photoacoustic imaging (PAI). Oxygen saturation and total hemoglobin were assessed by PAI before and after US treatments performed at different intensities of continuous wave (CW) bursts and pulsed wave (PW) bursts US. PAI revealed significant reduction both in HCC oxygen saturation and in total hemoglobin, proportional to the US intensity. Both CW bursts US (1.6 W/cm2) and the PW bursts US (0.8 W/cm2) significantly reduced HCC oxygen saturation and total hemoglobin which continued to diminish with time following the US treatment. The effects of US therapy were confirmed by power Doppler and histological examination of the hemorrhage in tumors. By each measure, the changes observed in US-treated HCC were more prevalent than those in sham-treated tumors and were statistically significant. In conclusion, the results show that US is an effective vascular-targeting therapy for HCC. The changes in oxygenation induced by the US treatment can be noninvasively monitored longitudinally by PAI without the use of exogenous image-enhancing agents. The combined use of PAI and the therapeutic US has potential for image-guided vascular therapy for HCC.

Intratumoral STING Agonist Injection Combined with Irreversible Electroporation Delays Tumor Growth in a Model of Hepatocarcinoma.

BACKGROUND/AIM: Irreversible electroporation (IRE) showed promising results for small-size tumors and very early cancers. However, further development is needed to evolve this procedure into a more efficient ablation technique for long-term control of tumor growth. In this work, we show that it is possible to increase the antitumor efficiency of IRE by simmultaneously injecting c-di-GMP, a STING agonist, intratumorally. MATERIALS AND METHODS: Intratumoral administration of c-di-GMP simultaneously to IRE was evaluated in murine models of melanona (B16.OVA) and hepatocellular carcinoma (PM299L). RESULTS: The combined therapy increased the number of tumor-infiltrating IFN-γ/TNF-α-producing CD4 and CD8 T cells and delayed tumor growth, as compared to the effect observed in groups treated with c-di-GMP or IRE alone. CONCLUSION: These results can lead to the development of a new therapeutic strategy for the treatment of cancer patients refractory to other therapies.

Liver metastasis restrains immunotherapy efficacy via macrophage-mediated T cell elimination.

Metastasis is the primary cause of cancer mortality, and cancer frequently metastasizes to the liver. It is not clear whether liver immune tolerance mechanisms contribute to cancer outcomes. We report that liver metastases diminish immunotherapy efficacy systemically in patients and preclinical models. Patients with liver metastases derive limited benefit from immunotherapy independent of other established biomarkers of response. In multiple mouse models, we show that liver metastases siphon activated CD8+ T cells from systemic circulation. Within the liver, activated antigen-specific Fas+CD8+ T cells undergo apoptosis following their interaction with FasL+CD11b+F4/80+ monocyte-derived macrophages. Consequently, liver metastases create a systemic immune desert in preclinical models. Similarly, patients with liver metastases have reduced peripheral T cell numbers and diminished tumoral T cell diversity and function. In preclinical models, liver-directed radiotherapy eliminates immunosuppressive hepatic macrophages, increases hepatic T cell survival and reduces hepatic siphoning of T cells. Thus, liver metastases co-opt host peripheral tolerance mechanisms to cause acquired immunotherapy resistance through CD8+ T cell deletion, and the combination of liver-directed radiotherapy and immunotherapy could promote systemic antitumor immunity.

Manipulation of focal Wnt activity via synthetic cells in a double-humanized zebrafish model of tumorigenesis.

The canonical Wnt signaling pathway is activated in numerous contexts, including normal and cancerous tissues. Here, we describe a synthetic cell-based therapeutic strategy that inhibits aberrant Wnt activity in specific focuses without interfering with the normal tissues in vivo. As a proof of principle, we generated a triple transgenic zebrafish liver cancer model that conditionally expressed human MET and induced ectopic Wnt signaling in hepatocytes. Then, we generated a customized synthetic Notch receptor (synNotch) cascade to express Wnt inhibitor DKK1 in Jurkat T cells and human peripheral blood mononuclear cells (PBMCs) after recognizing MET as antigen. After that, the synNotch PBMCs were sorted and microinjected into different tissues of the zebrafish model. In MET-expressing cancerous liver tissues, the injected cells expressed DKK1 and inhibited the local proliferation and Wnt activity; while in the yolk sac without MET, the injected cells remained inactive. Overall, our studies revealed the use of synthetic cells with antigen receptors to improve the spatiotemporal accuracy of anti-Wnt therapy, and proposed that the genetically humanized zebrafish model may serve as a small-scale and highly optically accessible platform for the functional evaluation of human synthetic cells.

Three-Dimensional Augmented Reality Visualization Informs Locoregional Therapy in a Translational Model of Hepatocellular Carcinoma.

PURPOSE: To evaluate the utility of visualizing preprocedural MR images in 3-dimensional (3D) space using augmented reality (AR) before transarterial embolization of hepatocellular carcinoma (HCC) in a preclinical model. MATERIALS AND METHODS: A total of 28 rats with diethylnitrosamine-induced HCCs > 5 mm treated with embolization were included in a prospective study. In 12 rats, 3D AR visualization of preprocedural MR images was performed before embolization. Procedural metrics including catheterization time and radiation exposure were compared vs a prospective cohort of 16 rats in which embolization was performed without AR. An additional cohort of 15 retrospective cases was identified and combined with the prospective control cohort (n = 31) to improve statistical power. RESULTS: A 37% reduction in fluoroscopy time, from 11.7 min to 7.4 minutes, was observed with AR when compared prospectively, which did not reach statistical significance (P = .12); however, when compared with combined prospective and retrospective controls, the reduction in fluoroscopy time from 14.1 min to 7.4 minutes (48%) was significant (P = .01). A 27% reduction in total catheterization time, from 42.7 minutes to 31.0 minutes, was also observed with AR when compared prospectively, which did not reach statistical significance (P = .11). No significant differences were seen in dose-area product or air kerma prospectively. CONCLUSIONS: Three-dimensional AR visualization of preprocedural imaging may aid in the reduction of procedural metrics in a preclinical model of transarterial embolization. These data support the need for further studies to evaluate the potential of AR in endovascular oncologic interventions.

Sodium Cholate Bile Acid-Stabilized Ferumoxytol-Doxorubicin-Lipiodol Emulsion for Transcatheter Arterial Chemoembolization of Hepatocellular Carcinoma.

PURPOSE: To develop bile acid-stabilized multimodal magnetic resonance (MR) imaging and computed tomography (CT)-visible doxorubicin eluting lipiodol emulsion for transarterial chemoembolization of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Ferumoxytol, a US Food and Drug Administration-approved iron oxide nanoparticle visible under MR imaging was electrostatically complexed with doxorubicin (DOX). An amphiphilic bile acid, sodium cholate (SC), was used to form a stable dispersion of ferumoxytol-DOX complex in lipiodol emulsion. Properties of the fabricated emulsion were characterized in various component ratios. Release kinetics of DOX were evaluated for the chemoembolization applications. Finally, in vivo multimodal MR imaging/CT imaging properties and potential therapeutic effects upon intra-arterial (IA) infusion bile acid-stabilized ferumoxytol-DOX-lipiodol emulsion were evaluated in orthotopic McA-Rh7777 HCC rat models. RESULTS: DOX complexed with ferumoxytol through electrostatic interaction. Amphiphilic SC bile acid at the interface between the aqueous ferumoxytol-DOX complexes and lipiodol enabled a sustained DOX release (17.2 ± 1.6% at 24 hours) at an optimized component ratio. In McA Rh7777 rat HCC model, IA-infused emulsion showed a significant contrast around tumor in both T2-weighted MR imaging and CT images (P = .044). Hematoxylin and eosin and Prussian blue staining confirmed the local deposition of IA-infused SC bile acid-stabilized emulsion in the tumor. The deposited emulsion induced significant increases in TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) stain-positive cancer cell apoptosis compared to those in a group treated with the nonstabilized emulsion. CONCLUSIONS: SC bile acid-stabilized ferumoxytol-DOX-lipiodol emulsion demonstrated sustained drug release and multimodal MR imaging/CT imaging capabilities. The new lipiodol-based formulation may enhance the therapeutic efficacy of chemoembolization in HCC.

Safety and Tolerability of Topotecan-Eluting Radiopaque Microspheres for Hepatic Chemoembolization in a Rabbit Preclinical Model.

PURPOSE: Topotecan is a camptothecin analogue with potential advantages over irinotecan for transarterial chemoembolization (TACE) of hepatic colorectal metastases including greater anti-neoplastic activity without enzymatic activation. The purpose of this study was to assess safety and tolerability of topotecan-loaded radiopaque microspheres (ROMTOP) administered by TACE in a rabbit model and to compare the in vitro elution of topotecan from microspheres to irinotecan. MATERIALS AND METHODS: Topotecan was loaded into radiopaque microspheres (70-150 µm, DC Bead LUMI™, Biocompatibles UK Ltd-Boston Scientific Corporation) to the maximum capacity of 80 mg/mL of microspheres. Six healthy New Zealand White rabbits underwent hepatic TACE with ROMTOP under fluoroscopic guidance until angiographic stasis. Assessment of toxicities included regular liver function tests and complete blood counts until euthanasia 28 days post-TACE. In vitro topotecan elution from the microspheres was assessed using an open-loop flow-through system and compared to irinotecan. RESULTS: The mean bead volume and topotecan dose delivered were 0.086 mL (0.076-0.105 mL) and 1.99 mg/kg (1.51-2.55 mg/kg), respectively. Aspartate aminotransferase and alanine aminotransferase were elevated post-embolization but resolved within 2 weeks. One rabbit died two days after TACE with pyloric duodenal perforation observed at necropsy, potentially due to non-target embolization. In vitro elution of topotecan from ROMTOP was complete in 10 h compared to 3 h for irinotecan-loaded microspheres. CONCLUSION: Selective embolization with ROMTOP was tolerated at a dose of 2 mg/kg (24 mg/m2) in rabbits. In vitro topotecan elution from microspheres was more prolonged compared to irinotecan.

Yttrium-90 Portal Vein Radioembolization in Sprague-Dawley Rats: Dose-Dependent Imaging and Pathological Changes in Normal Liver.

PURPOSE: Portal vein embolization (PVE) is an established neoadjuvant method to induce future liver remnant hypertrophy prior to surgical resection of hepatic tumors. The purpose of our study was to examine the feasibility of PVE with glass 90Y microspheres (Y90 PVE) in Sprague-Dawley rats. We tested the hypothesis that increased doses of Y90 PVE would increase target lobe fibrosis and atrophy. METHODS: Twenty-two rats were assigned to four groups for Y90 PVE to the right median lobe: very high- (273.8 MBq; n = 2), high- (99.9 MBq; n = 10), medium- (48.1 MBq; n = 5), and low-dose (14.8 MBq; n = 5). An untreated control group included seven rats. 90Y PET/CT of 90Y distributions confirmed lobar targeting. MRI volumes were measured at baseline, 2-, 4-, 8- and 12-weeks. Explanted hepatic lobes were weighed, sectioned, and stained for H&E and immunohistochemistry. Digitized slides allowed quantitative measurements of fibrosis (20 foci/slide). RESULTS: Ex vivo measurements confirmed 91-97% activity was localized to the target lobe (n = 4). The percent growth of the target lobe relative to baseline was - 5.0% (95% CI - 17.0-6.9%) for high-, medium dose rats compared to + 18.6% (95% CI + 7.6-29.7%) in the low-dose group at 12-weeks (p = 0.0043). Radiation fibrosis increased in a dose-dependent fashion. Fibrotic area/microsphere was 22,893.5, 14,946.2 ± 2253.3, 15,304.5 ± 4716.6, and 5268.8 ± 2297.2 µm2 for very high- (n = 1), high- (n = 4), medium- (n = 3), and low-dose groups (n = 5), respectively. CONCLUSION: Y90 PVE was feasible in the rat model, resulted in target lobe atrophy, and dose-dependent increases in hepatic fibrosis at 12 weeks. The onset of imaging-based volumetric changes was 8-12 weeks.

Hepatic Artery Embolization Enhances Expression of Programmed Cell Death 1 Ligand 1 in an Orthotopic Rat Hepatocellular Carcinoma Model: In Vivo and in Vitro Experimentation.

PURPOSE: To evaluate the effects of hepatic artery embolization (HAE) on the expression of programmed cell death 1 ligand 1 (PD-L1) in an orthotopic rat hepatocellular carcinoma (HCC) model. MATERIALS AND METHODS: A rat HCC model was established in Sprague-Dawley rats with the RH7777 cell line. Six animals each were assigned to receive HAE or sham treatment. Liver tissues were harvested 24 h after the procedure. Immunohistochemistry (IHC) was used to compare expression of PD-L1 and hypoxia-inducible factor (HIF)-1α in the intratumoral and peritumoral regions and normal liver tissue. In vitro cell culture study was performed for 24 h under normoxic and hypoxic conditions, and protein expression of PD-L1 and HIF-1α and the effects of HIF-1α inhibitors were assessed. RESULTS: IHC showed that PD-L1- and HIF-1α-positive areas were significantly larger in the HAE group vs the sham group in intratumoral (P = .006 and P < .001, respectively) and peritumoral regions (both P < .001). The expression of PD-L1 positively correlated with HIF-1α expression in the intratumoral region (r2 = 0.551; P < .001). In vitro cell culture study revealed that protein expression of PD-L1 and HIF-1α were significantly higher when cells were incubated under hypoxic vs normoxic conditions (P = .028 and P = .010, respectively). PD-L1 expression was suppressed significantly when the HIF-1α inhibitor rapamycin was added to the culture medium (P = .024). CONCLUSIONS: HAE enhances intratumoral and peritumoral PD-L1 expression in a rat HCC model. The HIF-1α pathway is a possible mechanism underlying increased intratumoral PD-L1 expression after HAE.

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.

Impact of focused ultrasound on the ethanol ablation of VX2 liver tumours in rabbits.

OBJECTIVES: In this study, a treatment combining ethanol ablation (EA) and focused ultrasound (FUS) was performed to investigate its synergistic ablation effect on normal liver and VX2 liver tumours in rabbits. METHODS: A total of 59 healthy New Zealand white rabbits were included. For normal liver ablation, 39 animals were treated with FUS alone (n = 12), EA alone (n = 12), EA+FUS combination treatment (n = 12), or the control treatment (n = 3). The other 20 rabbits with implanted VX2 liver tumours were treated with EA alone (n = 10) or EA+FUS (n = 10). For FUS, the liver was exposed to 1 MHz FUS with an intensity of 33.0 W/cm2 (ISPTA) for 20 s. The EA group received an injection of absolute ethanol in the liver or liver tumours. For EA+FUS combination therapy, FUS was focused at the EA injection site, and both methods were carried out at the same time. RESULTS: In normal liver tissues, the ablated volume treated by FUS combined with EA (1.46 ± 0.30 cm3) was approximately 3 times larger than that of EA alone (0.51 ± 0.17 cm3); in VX2 liver tumours, the tumour necrosis rate of the combination therapy was 90.27%, which was much higher than that of EA treatment (63.55%). CONCLUSION: The combination of EA and FUS could effectively increase the liver ablation volume and induce more complete tumour necrosis. KEY POINTS: • This study demonstrated a novel method for enhancing ethanol ablation and elucidated its potential to enhance percutaneous ethanol ablation (PEA) in a simple non-invasive way. • Ethanol excited by focused ultrasound (FUS) exposure tended to accumulate at the injection site, which could prevent ethanol from being washed out by the bloodstream. • The combination of EA and FUS could effectively increase the liver ablation volume and induce more complete tumour necrosis.

Feasibility of Combination Intra-arterial Yttrium-90 and Irinotecan Microspheres in the VX2 Rabbit Model.

PURPOSE: To evaluate the combination of 90Y radioembolization (Y90) and drug-eluting bead irinotecan (DEBIRI) microspheres in the VX2 rabbit model. MATERIALS AND METHODS: An initial dose finding study was performed in 6 White New Zealand rabbits to identify a therapeutic but subcurative dose of Y90. In total, 29 rabbits were used in four groups: Y90 treatment (n = 8), DEBIRI treatment (n = 6), Y90 + DEBIRI treatment (n = 7), and an untreated control group (n = 8). Hepatic toxicity was evaluated at baseline, 24 h, 72 h, 1 week, and 2 weeks. MRI tumor volume (TV) and enhancing tumor volume were assessed baseline and 2 weeks. Tumor area and necrosis were evaluated on H&E for pathology. RESULTS: Infused activities of 84.0-94.4 MBq (corresponding to 55.1-72.7 Gy) were selected based on the initial dose finding study. Infusion of DEBIRI after Y90 was technically feasible in all cases (7/7). Overall, 21/29 animals survived to 2 weeks, and the remaining animals had extrahepatic disease on necropsy. Liver transaminases were elevated with Y90, DEBIRI, and Y90 + DEBIRI compared to control at 24 h, 72 h, and 1 week post-treatment and returned to baseline by 2 weeks. By TV, Y90 + DEBIRI was the only treatment to show statistically significant reduction at 2 weeks compared to the control group (p = 0.012). The change in tumor volume (week 2-baseline) for both Y90 + DEBIRI versus control (p = 0.002) and Y90 versus control (p = 0.014) was significantly decreased. There were no statistically significant differences among groups on pathology. CONCLUSION: Intra-arterial Y90 + DEBIRI was safe and demonstrated enhanced antitumor activity in rabbit VX2 tumors. This combined approach warrants further investigation.

Antitumor Immunity Augmented by Combining Radiofrequency Ablation with Anti-CTLA-4 Therapy in a Subcutaneous Murine Hepatoma Model.

PURPOSE: To evaluate whether antitumor immunity is enhanced by combining radiofrequency (RF) ablation and anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) therapy and to evaluate its effect on untreated tumors. MATERIALS AND METHODS: First, 40 mice with tumors established in the bilateral flanks were randomly divided into 4 groups: the control group, the RF ablation-alone group, the anti-CTLA-4-alone group, and the RF ablation + anti-CTLA-4 group. In each group, 8 mice were used for untreated tumor evaluation and survival observation, and another 2 mice were killed for histopathologic study. Then, a rechallenge test was performed in another 32 mice to determine whether systemic antitumor immunity was established. RESULTS: Although the volume of the untreated tumors continued to increase until the end of the observation in all groups, tumor growth rates in the RF ablation + anti-CTLA-4 group were significantly smaller than tumor growth rates in the other 3 groups (all P < .05). The overall survival time of mice in the RF ablation + anti-CTLA-4 group was significantly longer than that of mice in the other 3 groups (all P < .05). Histopathologic studies of the untreated tumors showed more CD4-and CD8+ lymphocyte infiltration in mice from the RF ablation + anti-CTLA-4 group than in mice from the other 3 groups (all P < .05). After a tumor rechallenge, tumor rejection was apparent in 75% of the mice in the RF ablation + anti-CTLA-4 group, in 25% of the mice in the RF ablation group, and in 0% of the mice in the control and anti-CTLA-4 groups. CONCLUSIONS: This study demonstrated that RF ablation-induced systemic antitumor immunity was enhanced by the combined use of anti-CTLA-4 therapy in a multi-subcutaneous murine hepatoma model.

Effective control of tumor growth through spatial and temporal control of theranostic sodium iodide symporter (NIS) gene expression using a heat-inducible gene promoter in engineered mesenchymal stem cells.

Purpose: The tumor homing characteristics of mesenchymal stem cells (MSCs) make them attractive vehicles for the tumor-specific delivery of therapeutic agents, such as the sodium iodide symporter (NIS). NIS is a theranostic protein that allows non-invasive monitoring of the in vivo biodistribution of functional NIS expression by radioiodine imaging as well as the therapeutic application of 131I. To gain local and temporal control of transgene expression, and thereby improve tumor selectivity, we engineered MSCs to express the NIS gene under control of a heat-inducible HSP70B promoter (HSP70B-NIS-MSCs). Experimental Design: NIS induction in heat-treated HSP70B-NIS-MSCs was verified by 125I uptake assay, RT-PCR, Western blot and immunofluorescence staining. HSP70B-NIS-MSCs were then injected i.v. into mice carrying subcutaneous hepatocellular carcinoma HuH7 xenografts, and hyperthermia (1 h at 41°C) was locally applied to the tumor. 0 - 72 h later radioiodine uptake was assessed by 123I-scintigraphy. The most effective uptake regime was then selected for 131I therapy. Results: The HSP70B promoter showed low basal activity in vitro and was significantly induced in response to heat. In vivo, the highest tumoral iodine accumulation was seen 12 h after application of hyperthermia. HSP70B-NIS-MSC-mediated 131I therapy combined with hyperthermia resulted in a significantly reduced tumor growth with prolonged survival as compared to control groups. Conclusions: The heat-inducible HSP70B promoter allows hyperthermia-induced spatial and temporal control of MSC-mediated theranostic NIS gene radiotherapy with efficient tumor-selective and temperature-dependent accumulation of radioiodine in heat-treated tumors.

Targeting CAMKII to reprogram tumor-associated macrophages and inhibit tumor cells for cancer immunotherapy with an injectable hybrid peptide hydrogel.

Simultaneously targeted treatment of tumor cells and their surrounding growth-supporting immune cells is a promising strategy to reshape immunosuppressive tumor microenvironment (TME) and potentiate host innate and adaptive antitumor immune responses. Methods: We designed a series of melittin-(RADA)n hybrid peptide sequences with varying self-assembling motifs of RADA and screened out a melittin-(RADA)6 peptide that has an optimal gel-formation ability and in vitro antitumor activity. Results: The formed melittin-(RADA)6 (MR52) hydrogel scaffold could be loaded with a specific Ca2+/calmodulin-dependent protein kinase II (CAMKII) inhibitor, KN93, originally found to have both direct tumoricidal activity and macrophages-reprogramming ability, for potent immunotherapy against melanoma and hepatoma ascites in mice models. Our MR52 hydrogel has an interweaving nanofiber-like structure, possesses direct antitumor and controlled drug release properties, and promotes the enhanced intracellular uptake of loaded cargo. Compared to free KN93, the MR52-KN93 hydrogel (MRK) improved the killing effects and levels of immunogenic cell death (ICD) on tumor cells significantly. Due to the dual role of KN93, the injection of the MRK hydrogel retarded the growth of subcutaneous melanoma tumors dramatically and resulted in a high number of mature dendritic cells of draining lymph nodes, significantly enhancing the portion of cytotoxic T cells and reduced number of M2-like tumor-associated macrophages (TAMs) in tumors. Using a mouse model of malignant ascites (MAs), where traditional therapy was ineffective, we demonstrated that the MRK hydrogel treatment offered a significantly prolonged survival compared to controls. Following treatment with the MRK hydrogel, macrophages had elevated programmed cell death protein ligand-1 (PD-L1) expression, promising follow-up combined anti-PD-1 therapy that confers a cure rate of approximately 30% against MAs in mice models. Conclusion: Thus, the MRK hydrogel may serve as a prospective platform for antitumor applications.