Mesenchymal stromal cells (MSCs) hold promise in the treatment of liver disease. However, short survival time of MSCs after intrahepatic transplantation limits their value; therefore, understanding the basis of MSCs survival and rejection may increase their utility. This study was aimed at determining the role of intrahepatic natural killer (NK) cells on MSCs survival and their retention in the liver shortly after transplant.Human MSCs were labeled with the Luc2-mKate2 dual-fusion reporter gene (MSCs-R), and the residence time and survival of MSCs-R xenografts after intrahepatic transplantation were evaluated by in vivo bioluminescence imaging (BLI). Coculture of MSCs and NK cells was performed to assess cytotoxicity. To evaluate the role of NK cells in rejection of the xenografted cells, the fates of transplanted MSCs-R were then assessed in vivo by BLI after activation of intrahepatic NK cells.We observed a linear correlation between luciferase activity from live MSCs-R and cell number in vitro (R2 = 0.9956). In vivo, we observed a gradual decline in bioluminescent signals from transplanted MSCs-R over a region corresponding to the liver in both the control group and the NK-activated group. However, the survival time and retention of intrahepatic MSCs-R decreased more rapidly in the NK-activated group of mice compared to the control group. This indicated that activated NK cells accelerate the elimination of transplanted MSCs. Also, we found that the number of hepatic NK cells and the expression of NK activation markers significantly increased after intrahepatic delivery of MSCs. This suggested that resident NK cells, in a resting state, were activated by intrahepatic transplantation of human MSCs. Taken together, the data suggests that activated hepatic NK cells mediate, in part, rejection of the MSCs xenografts. Cytotoxicity assays showed that activated NK cells may inhibit the proliferation of MSCs and, to a certain extent, induce MSCs death.Human MSCs could be followed dynamically in vivo by BLI, and the role of murine hepatic NK cells, especially activated NK cells, could be inferred from the loss of signals from MSCs. This finding may have practical clinical implications in MSCs transplantation in treating liver disease.

The aim of this study is to evaluate the effect of overexpressing human hepatocyte growth factor (HGF) for mesenchymal stem cells (MSCs) in liver fibrosis regeneration and magnetic resonance (MR) tracking of MSCs in rat liver.MSCs were transfected with ad-HGF/ad-green fluorescent protein (GFP) and labeled with superparamagnetic iron oxide (SPIO). The characteristics of SPIO-HGF/MSCs were investigated. Prussian blue staining for iron assessment was conducted in vitro and in vivo. SPIO-HGF/MSCs (group A) or SPIO-GFP/MSCs (group B) were transplanted into a rat model of liver fibrosis, and MR imaging of the rat liver was performed. The signal to noise ratio (SNR) and R2* (1/T2*) value were measured. Prussian blue staining was performed to detect the in vivo distribution of MSCs, and liver Ki67 immunohistochemistry (IHC) staining was studied. The serum levels of HGF, alanine aminotransferase (ALT) and hyaluronic acid (HA) were determined.The positive rate of HGF transfection was 93.17 % and the HGF/MSCs were labeled with SPIO successfully (97.80 ± 1.06 %). Labeling of MSCs with SPIO did not alter cell proliferation in vitro. The signal intensity of liver T2* WI images decreased on day 1 after cell transplantation and recovered to pre-transplantation level on day 15 (group A) and day 13 (group B). The SNR of group A were significantly lower than that of group B (P = 0.006), and the R2* values of group A were significantly higher than those of group B (P < 0.001). The R2* value had a significantly negative correlation with SNR. There were more Prussian blue-positive cells in of group A were more than in group B in vivo. The positive rate of Ki67 was 16.11 ± 2.13 %, and the serum level of ALT/HA was decreased in group A.HGF transfection improved MSCs localization in the liver and aided liver repair. The R2* value might be a feasible index in addition to SNR to track the SPIO-MSC transplantation in the liver.

To investigate the survival benefit of transarterial chemoembolization (TACE) plus Iodine125 seed implantation (TACE-Iodine125) in hepatitis B-related HCC patients with portal vein tumour thrombus (PVTT) and the underlying prognostic factors.A retrospective matched cohort study was performed on consecutive HCC patients with PVTT from January 2011 to June 2014. Seventy patients (TACE-Iodine125 group) who underwent TACE-Iodine125 were compared with a historical case-matched control group of 140 patients (TACE group) who received TACE alone. The survival of patients and the underlying prognostic factors were analysed.The median survival times of the TACE-Iodine125 and TACE groups were 11.0 and 7.5 months, respectively (p < 0.001). The survival probability at 12, 24, and 36 months was 50 %, 14.5 %, and 14.5 % vs. 25 %, 9 %, and 5 % in the TACE-Iodine125 and TACE groups, respectively (p < 0.001). The PVTT responders had better survival than the PVTT non-responders (p < 0.001). For the PVTT non-responders, there were no differences in the survival curves between the groups (p = 0.353). Multivariate analysis showed that type III PVTT (p < 0.001) and APS (p < 0.001) were independent predictors of poor prognosis. In contrast, the treatment modality of TACE-Iodine125 (p < 0.001) and PVTT response (p = 0.001) were favourable prognostic features.TACE combined with Iodine125 seed implantation may be a good choice for selected HB-HCC patients with PVTT.• TACE-Iodine125 was more effective than TACE for patients with HCC-PVTT.• The TACE-Iodine125 procedure was safe.• TACE-Iodine125 was conditional for patients with HCC-PVTT.• TACE-Iodine125 resulted in a better PVTT response compared to TACE alone.• A good PVTT response is a favourable prognostic factor.

Delta-like ligand 4 (Dll4) expressed in tumor cells plays a key role to promote tumor growth of numerous cancer types. Based on a novel antihuman Dll4 monoclonal antibody (61B), we developed a 64Cu-labeled probe for positron emission tomography (PET) imaging of tumor Dll4 expression. In this study, 61B was conjugated with the 64Cu-chelator DOTA through lysine on the antibody. Human IgG (hIgG)-DOTA, which did not bind to Dll4, was also prepared as a control. The Dll4 binding activity of the probes was evaluated through the bead-based binding assay with Dll4-alkaline phosphatase. The resulting PET probes were evaluated in U87MG glioblastoma and HT29 colorectal cancer xenografts in athymic nude mice. Our results demonstrated that the 61B-DOTA retained (77.2 ± 3.7) % Dll4 binding activity of the unmodified 61B, which is significantly higher than that of hIgG-DOTA (0.06 ± 0.03) %. Confocal microscopy analysis confirmed that 61B-Cy5.5, but not IgG-Cy5.5, predominantly located within the U87MG and HT29 cells cytoplasm. U87MG cells showed higher 61B-Cy5.5 binding as compared to HT29 cells. In U87MG xenografts, 61B-DOTA-64Cu demonstrated remarkable tumor accumulation (10.5 ± 1.7 and 10.2 ± 1.2%ID/g at 24 and 48 h postinjection, respectively). In HT29 xenografts, tumor accumulation of 61B-DOTA-64Cu was significantly lower than that of U87MG (7.3 ± 1.3 and 6.6 ± 1.3%ID/g at 24 and 48 h postinjection, respectively). The tumor accumulation of 61B-DOTA-64Cu was significantly higher than that of hIgG-DOTA-64Cu in both xenografts models. Immunofluorescence staining of the tumor tissues further confirmed that tumor accumulation of 61B-Cy5.5 was correlated well with in vivo PET imaging data using 61B-DOTA-64Cu. In conclusion, 61B-DOTA-64Cu PET probe was successfully synthesized and demonstrated prominent tumor uptake by targeting Dll4. 61B-DOTA-64Cu has great potential to be used for noninvasive Dll4 imaging, which could be valuable for tumor detection, Dll4 expression level evaluation, and Dll4-based treatment monitoring.

Both experimental and initial clinical studies have shown the therapeutic potential of mesenchymal stem cells (MSCs) in liver disease. Noninvasive tracking of MSCs could facilitate its clinical translation. The purpose of this study was to optimize MSCs delivery dose and route in mice with acute liver injury using bioluminescence imaging (BLI) to track the cells.MSCs were labeled with the Luc2-mKate2 dual-fusion reporter gene (MSCs-R). The fate of MSCs-R was tracked through in vivo BLI after administration of different doses or delivery through different routes.When delivered via the superior mesenteric vein (SMV), the high-dose (1.0 × 106 and 5.0 × 105) group mice demonstrated high liver BLI signal but also had lethal portal vein embolization (PVE). By contrast, no PVE and its related death occurred in the low-dose (2.5 × 105) group mice. Thus, 2.5 × 105 is the optimal delivery dose. Three delivery routes, i.e., inferior vena cava (IVC), SMV, and intrahepatic (IH) injection, were also systematically compared. After IVC infusion, MSCs-R were quickly trapped inside the lungs, and no detectable homing to the liver and other organs was observed. By IH injection, lung entrapment was bypassed, but MSCs-R distribution was only localized in the injection region of the liver. By contrast, after SMV infusion, MSCs-R were dispersedly distributed and stayed as long as 7-day posttransplantation in the liver. The in vivo imaging results were further validated by ex vivo imaging, digital subtraction angiography (DSA), and tissue analysis. Therefore, SMV is the optimal MSCs delivery route for liver disease.Collectively, BLI, which could dynamically and quantitatively track cellular location and survival, is useful in determining MSCs transplantation parameters.

Following injurious stimuli, quiescent hepatic stellate cells (qHSCs) transdifferentiate into activated HSCs (aHSCs). aHSCs play pivotal roles in the onset and progression of liver fibrosis. Therefore, molecular imaging of aHSCs in liver fibrosis will facilitate early diagnosis, prognosis prediction, and instruction and evaluation of aHSC-targeted treatment. To date, several receptors, such as integrin αvβ3, mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-IIR), collagen type VI receptor (CVIR), platelet-derived growth factor receptor-β (PDGFR-β), vimentin, and desmin, have been identified as biomarkers of aHSCs. Corresponding ligands to these receptors have also been developed. This review will discuss strategies for developing aHSC-targeted imaging in liver fibrosis.

Accumulating experimental evidence indicates that overexpression of the oncogenic receptor tyrosine kinase, Axl, plays a key role in the tumorigenesis and metastasis of various types of cancer. The objective of this study is to design a novel imaging probe based on the monoclonal antibody, h173, for microPET imaging of Axl expression in human lung cancer. A bifunctional chelator, DOTA, was conjugated to h173, followed by radiolabeling with 64Cu. The binding of DOTA-h173 to the Axl receptor was first evaluated by a cell uptake assay and flow cytometry analysis using human lung cancer cell lines. The probe 64Cu-DOTA-h173 was further evaluated by microPET imaging, and ex vivo histology studies in the Axl-positive A549 tumors. In vitro cellular study showed that Axl probe, 64Cu-DOTA-h173, was highly immuno-reactive with A549 cells. Western blot analysis confirmed that Axl is highly expressed in the A549 cell line. For microPET imaging, the A549 xenografts demonstrated a significantly higher 64Cu-DOTA-h173 uptake compared to the NCI-H249 xenograft (a negative control model). Furthermore, 64Cu-DOTA-h173 uptake in A549 is significantly higher than that of 64Cu-DOTA-hIgG. Immuno-fluorescence staining was consistent with the in vivo micro-PET imaging results. In conclusion, 64Cu-DOTA-h173 could be potentially used as a probe for noninvasive imaging of Axl expression, which could collect important information regarding tumor response to Axl-targeted therapeutic interventions.Keywords: Axl receptor; lung cancer; monoclonal antibody; positron emission tomography (PET);

The tyrosine kinase receptor Axl is overexpressed in various types of cancer and correlated with cancer malignancy. Selective Axl blockade reduces tumor growth and metastasis. The purpose of this study was to examine whether the humanized anti-Axl antibody humanized 173 (h173) labeled with near-infrared fluorescence (NIRF) dye Cy5.5 could be applied as a molecular imaging probe for NIRF imaging of Axl expression in tumor models.NIRF dye Cy5.5 was conjugated to h173 or human normal immunoglobulin G (hIgG) control through amino groups. The resulting probes were evaluated in both A549 (Axl positive) and NCI-H249 (Axl negative) lung cancer xenografts through in vivo NIRF imaging. Ex vivo imaging and probe distribution assay were also carried out to confirm the in vivo imaging results.After conjugation, binding activity of h173-Cy5.5 was determined to be 97.75 % ± 2.09 % of the unmodified h173. In vitro fluorescence-activated cell sorting (FACS) and fluorescence microscopy analysis validated the specific binding of h173 toward Axl-positive A549 cells. h173-Cy5.5 was then applied to image Axl expression in vivo. In A549 (Axl positive) cancer xenografts, the tumor uptake of h173-Cy5.5 was significantly higher than that of the hIgG-Cy5.5 control (P < 0.05) at late time points (1, 2, 3, 4, and 7 days). On the contrary, in NCI-H249 (Axl negative) cancer xenografts, the tumor uptake of both hIgG-Cy5.5 and h173-Cy5.5 was low and showed no significant difference (P > 0.05) at all time points examined. Ex vivo imaging and immunofluorescence staining analysis further validated the in vivo imaging results.Collectively, all in vitro, in vivo, and ex vivo data suggested that h173-Cy5.5 could serve as a valid probe for Axl-targeted cancer imaging, which could therefore aid in tumor diagnosis, prognosis, and treatment monitoring.

Accumulating evidence suggests that EphB4 plays key roles in cancer progression in numerous cancer types. In fact, therapies focusing on EphB4 have become potentially important components of various cancer treatment strategies. However, tumor sensitivity to EphB4 suppression may not be uniform for different cancers. In this study, we developed near-infrared fluorescence (NIRF) probes for EphB4 targeted imaging, based on EphB4-specific humanized monoclonal antibody hAb47. NIRF dye Cy5.5 was introduced to hAb47 either through the reaction with amino groups (named hAb47-Cy5.5) or sulfhydryl groups (named hAb47-Cy5.5-Mal). The resulting probes were evaluated in both HT-29 xenograft and the mAb131 (anti-EphB4) treated models. Although these methods lead to modifications of both the heavy chain and light chain of the antibody, the majority of the EphB4 binding affinity was maintained (81.62 ± 2.08% for hAb47-Cy5.5 and 77.14 ± 2.46% for hAb47-Cy5.5-Mal, respectively). hAb47-Cy5.5 was then chosen for in vivo NIRF imaging of EphB4 expression. In HT29 colorectal tumor xenografts, hAb47-Cy5.5 demonstrated significantly higher tumor uptake compared with that of the hIgG-Cy5.5 control, which was further confirmed by immunofluorescent staining. Moreover, hAb47-Cy5.5 successfully imaged the decreased EphB4 expression (confirmed by Western blot) in EphB4-targeted immunotherapy using another EphB4-specific antibody, mAb131. Collectively, hAb47-Cy5.5 could be used as a specific NIRF contrast agent for noninvasive imaging of EphB4 expression, which may predict whether an individual tumor would likely respond to EphB4 targeted interventions, as well as monitor the therapeutic response.Keywords: antibody; EphB4 receptor; immunotherapy; near-infrared fluorescence (NIRF) imaging;

Accumulating evidence suggests that overexpression of the tyrosine kinase receptor EphB4, a mediator of vascular development, is a novel target for tumor diagnosis, prognosis and therapy. Noninvasive imaging of EphB4 expression could therefore be valuable for evaluating disease course and therapeutic efficacy at the earliest stages of anti-EphB4 treatment. In this study, we systematically investigated the use of anti-EphB4 antibody h131 (150 kDa) and its fragments (h131-F(ab′)2, 110 kDa; h131-Fab, 50 kDa) for near-infrared fluorescence (NIRF) imaging of EphB4 expression in vivo. h131-F(ab′)2 and h131-Fab were produced through pepsin and papain digestion of h131 respectively, whose purity was confirmed by FPLC and SDS–PAGE. After conjugation with Cy5.5, in vivo characteristics of h131, h131-F(ab′)2 and h131-Fab were evaluated in EphB4-positive HT29 tumor model. Although h131-Cy5.5 demonstrated highest tumor uptake among these probes, its optimal tumor uptake level was obtained at 2 days post injection (p.i.). For h131-Fab-Cy5.5, maximum tumor uptake was achieved at 4 h p.i. However, no significant difference was observed between h131-Fab-Cy5.5 and hIgG-Fab-Cy5.5, indicating the tumor accumulation was mainly caused by passive targeting. In contrast, h131-F(ab′)2-Cy5.5 demonstrated prominent tumor uptake at 6 h p.i. The target specificity was confirmed by hIgG-F(ab′)2-Cy5.5 control and immunofluorescent staining. Collectively, h131-F(ab′)2 exhibited prominent and specific tumor uptake at early time points, which suggests it is a promising agent for EphB4-targeted imaging.Keywords: antibody fragment; EphB4 receptor; F(ab′)2; Fab; near-infrared fluorescence (NIRF) imaging;

ObjectiveBudd-Chiari syndrome (BCS) is a rare and life-threatening disorder characterized by hepatic venous outflow obstruction. The management of BCS includes anticoagulation and thrombolysis, percutaneous transhepatic stent angioplasty (PTSA), and transjugular intrahepatic portosystemic shunt (TIPS), but the effect of these approaches varies greatly. The aim of our study was to retrospectively evaluate the medium-term effects of PTSA and TIPS of BCS secondary to hepatic venous outflow obstruction and to determine the critical factors affecting the efficacy.MethodsFrom June 2007 to June 2012, 18 patients (15 males and 3 females; mean age, 36 ± 9 years) with BCS (obstruction of the hepatic veins) treated by PTSA (n = 15) and TIPS (n = 3) were studied retrospectively. Clinical records were analyzed with respect to underlying disease, therapeutic interventions, complications, quality of life, and overall outcome.ResultsPercutaneous transhepatic interventional treatment was technically successful in all patients. In PTSA group, the primary and secondary stent patency rates were 80% and 86.7%, respectively. In the TIPS group, ascites resolved completely, and liver congestion and function were relieved greatly in all three patients. Hemodynamic features and clinical symptoms in patients with successful treatment improved significantly. Physical aspects evaluated by SF-36 were improved greatly at the end of follow-up.ConclusionsFor segmental stenosis or occlusion of hepatic vein caused by thrombosis or membranous webs, PTSA should be recommended as the first choice. TIPS should be applied for diffuse stenosis or occlusion in all the hepatic veins and branches. Standard anticoagulation may promote stent patency. Quality of life after interventional treatment was improved partially, and the mental aspects need to be further investigated.

PurposeTo evaluate the safety and feasibility of percutaneous transsplenic portal vein catheterization (PTSPC) by retrospective review of its use in patients with portal vein (PV) occlusion.Materials and MethodsFrom July 2004 to December 2010, 46 patients with a history of uncontrolled gastroesophageal variceal bleeding secondary to portal hypertension underwent endovascular PV interventions via a percutaneous transsplenic approach. All patients had occlusion of the main PV or central intrahepatic PV branches, which prevented the performance of a transhepatic approach. A vein within the splenic parenchyma was punctured under fluoroscopic guidance by referencing preoperative computed tomography images. PTSPC-related complications and clinical applications were analyzed.ResultsPTSPC was successfully performed in 44 of 46 patients (96%); two failures were caused by inaccessible small intrasplenic veins. PTSPC-related major bleeding complications occurred in three patients (6.5%), including large intraperitoneal hemorrhage in one patient and large splenic subcapsular hemorrhage in two patients. Two of the three patients developed hypotension, and one developed severe anemia. All three of the patients required blood transfusions. PTSPC-related minor bleeding complications occurred in six patients (13%) as a result of a small splenic subcapsular hemorrhage. In addition, three patients exhibited mild left pleural effusion, which subsided spontaneously 1 week later. All 44 patients successfully treated via PTSPC received gastroesophageal variceal embolization. Eight patients received PV stents, five for treatment of PV occlusion and three during transjugular intrahepatic portosystemic shunt placement.ConclusionsPTSPC is a safe and effective access for endovascular PV interventions in patients without a transhepatic window.