Kai-Wen Huang

Time-Evolution contrast of target MRI using high-stability antibody functionalized magnetic nanoparticles: an animal model

In this work, high-quality antibody functionalized Fe3O4 magnetic nanoparticles are synthesized. Such physical characterizations as particle morphology, particle size, stability, and relaxivity of magnetic particles are investigated. The immunoreactivity of biofunctionalized magnetic nanoparticles is examined by utilizing immunomagnetic reduction. The results show that the mean diameter of antibody functionalized magnetic nanoparticles is around 50 nm, and the relaxivity of the magnetic particles is 145 (mMċs)-1. In addition to characterizing the magnetic nanoparticles, the feasibility of using the antibody functionalized magnetic nanoparticles for the contrast medium of target magnetic resonance imaging is investigated. These antibody functionalized magnetic nanoparticles are injected into mice bearing with tumor. The tumor magnetic-resonance image becomes darker after the injection and then recovers 50 hours after the injection. The tumor magnetic-resonance image becomes the darkest at around 20 hours after the injection. Thus, the observing time window for the specific labeling of tumors with antibody functionalized magnetic nanoparticles was found to be 20 hours after injecting biofunctionalized magnetic nanoparticles into mice. The biopsy of tumor is stained after the injection to prove that the long-term darkness of tumor magnetic-resonance image is due to the specific anchoring of antibody functionalized magnetic nanoparticles at tumor.

Novel RNA oligonucleotide improves liver function and inhibits liver carcinogenesis in vivo

Hepatocellular carcinoma (HCC) occurs predominantly in patients with liver cirrhosis. Here we show an innovative RNA‐based targeted approach to enhance endogenous albumin production while reducing liver tumor burden. We designed short‐activating RNAs (saRNA) to enhance expression of C/EBPα (CCAAT/enhancer‐binding protein‐α), a transcriptional regulator and activator of albumin gene expression. Increased levels of both C/EBPα and albumin mRNA in addition to a 3‐fold increase in albumin secretion and 50% decrease in cell proliferation was observed in C/EBPα‐saRNA transfected HepG2 cells. Intravenous injection of C/EBPα‐saRNA in a cirrhotic rat model with multifocal liver tumors increased circulating serum albumin by over 30%, showing evidence of improved liver function. Tumor burden decreased by 80% (P = 0.003) with a 40% reduction in a marker of preneoplastic transformation. Since C/EBPα has known antiproliferative activities by way of retinoblastoma, p21, and cyclins, we used messenger RNA (mRNA) expression liver cancer‐specific microarray in C/EBPα‐saRNA‐transfected HepG2 cells to confirm down‐regulation of genes strongly enriched for negative regulation of apoptosis, angiogenesis, and metastasis. Up‐regulated genes were enriched for tumor suppressors and positive regulators of cell differentiation. A quantitative polymerase chain reaction (PCR) and western blot analysis of C/EBPα‐saRNA‐transfected cells suggested that in addition to the known antiproliferative targets of C/EBPα, we also observed suppression of interleukin (IL)6R, c‐Myc, and reduced STAT3 phosphorylation. Conclusion: A novel injectable saRNA‐oligonucleotide that enhances C/EBPα expression successfully reduces tumor burden and simultaneously improves liver function in a clinically relevant liver cirrhosis/HCC model. (Hepatology 2014;58:216–227)

Combined GM‐CSF and IL‐12 gene therapy synergistically suppresses the growth of orthotopic liver tumors

Immunotherapy is often effective only for small tumor burdens and, in many cases, is restricted to subcutaneous tumors. Here, we investigated the antitumor effects of combination therapy with GM‐CSF and IL‐12 on orthotopic liver tumors with intermediate or large tumor volumes, or on chemically‐induced multifocal liver tumors in animals. Adenoviruses encoding GM‐CSF or IL‐12 were injected intratumorally to animals bearing transplanted tumors, or injected via intrahepatic artery in animals with primary multifocal liver tumors induced by diethylnitrosamine. Our results demonstrated that IL‐12, but not GM‐CSF, monotherapy displayed significant therapeutic effects, whereas combination therapy with both cytokines displayed synergistic antitumor effects not only on transplanted tumor models with intermediate or large tumor loads, but also on carcinogen‐induced multifocal liver tumors. Effector cell analyses, revealed by in vivo cell subset depletion, flow cytometry analysis, and immunohistochemical staining of tumor infiltrates, indicated that NK cells were the prominent antitumor effectors for the IL‐12–mediated antitumor activity, whereas CD8+ T cells, NKT cells, and macrophages were more important than NK cells in the combination therapy–mediated antitumor effects. Both IL‐12 monotherapy and combination therapy could induce various types of effectors and high levels of IFN‐γ; however, the latter induced much higher levels than the former, which may explain why combination therapy is superior to IL‐12 monotherapy. Conclusion: Combination therapy with GM‐CSF and IL‐12 represents a promising immunotherapy strategy for treating orthotopic, widespread liver tumors. (HEPATOLOGY 2007;45:746–754.)

Transient Elastography to Assess Hepatic Fibrosis in Hemodialysis Chronic Hepatitis C Patients

BACKGROUND AND OBJECTIVES Although percutaneous liver biopsy (PLB) is the gold standard for staging hepatic fibrosis in hemodialysis patients with chronic hepatitis C (CHC) before renal transplantation or antiviral therapy, concerns exist about serious postbiopsy complications. Using transient elastography (TE, Fibroscan(®)) to predict the severity of hepatic fibrosis has not been prospectively evaluated in these patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS A total of 284 hemodialysis patients with CHC were enrolled. TE and aspartate aminotransferase-to-platelet ratio index (APRI) were performed before PLB. The severity of hepatic fibrosis was staged by METAVIR scores ranging from F0 to F4. Receiver operating characteristic curves were used to assess the diagnostic accuracy of TE and APRI, taking PLB as the reference standard. RESULTS The areas under curves of TE were higher than those of APRI in predicting patients with significant hepatic fibrosis (≥F2) (0.96 versus 0.84, P<0.001), those with advanced hepatic fibrosis (≥F3) (0.98 versus 0.93, P=0.04), and those with cirrhosis (F4) (0.99 versus 0.92, P=0.13). Choosing optimized liver stiffness measurements of 5.3, 8.3, and 9.2 kPa had high sensitivity (93-100%) and specificity (88-99%), and 87, 97, and 93% of the patients with a fibrosis stage of ≥F2, ≥F3, and F4 were correctly diagnosed without PLB, respectively. CONCLUSIONS TE is superior to APRI in assessing the severity of hepatic fibrosis and can substantially decrease the need of staging PLB in hemodialysis patients with CHC.

Characterization of tumors using high-Tc superconducting quantum interference device-detected nuclear magnetic resonance and imaging

The characterization of cancerous livers in rats using nuclear magnetic resonance and magnetic resonance imaging (NMR/MRI) based on high-Tc superconducting quantum interference devices (SQUIDs) is presented. The T1−1 were observed to be 6.5±0.5 s−1 for controlled livers and 2.85±0.2 s−1 for cancerous livers, which indicate that the T1 can be used to distinguish the cancerous tissues from controlled liver tissues. The intensity ratio for tap water, cancerous tissue, and controlled tissue, respectively, is 1:1.15:0.56 at TBp=1 s. The SQUID-detected NMR/MRI exhibits potential applications in research and clinics.

Gene Expression-Based Chemical Genomics Identifies Potential Therapeutic Drugs in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is an aggressive tumor with a poor prognosis. Currently, only sorafenib is approved by the FDA for advanced HCC treatment; therefore, there is an urgent need to discover candidate therapeutic drugs for HCC. We hypothesized that if a drug signature could reverse, at least in part, the gene expression signature of HCC, it might have the potential to inhibit HCC-related pathways and thereby treat HCC. To test this hypothesis, we first built an integrative platform, the “Encyclopedia of Hepatocellular Carcinoma genes Online 2”, dubbed EHCO2, to systematically collect, organize and compare the publicly available data from HCC studies. The resulting collection includes a total of 4,020 genes. To systematically query the Connectivity Map (CMap), which includes 6,100 drug-mediated expression profiles, we further designed various gene signature selection and enrichment methods, including a randomization technique, majority vote, and clique analysis. Subsequently, 28 out of 50 prioritized drugs, including tanespimycin, trichostatin A, thioguanosine, and several anti-psychotic drugs with anti-tumor activities, were validated via MTT cell viability assays and clonogenic assays in HCC cell lines. To accelerate their future clinical use, possibly through drug-repurposing, we selected two well-established drugs to test in mice, chlorpromazine and trifluoperazine. Both drugs inhibited orthotopic liver tumor growth. In conclusion, we successfully discovered and validated existing drugs for potential HCC therapeutic use with the pipeline of Connectivity Map analysis and lab verification, thereby suggesting the usefulness of this procedure to accelerate drug repurposing for HCC treatment.

Dual Therapeutic Effects of Interferon-α Gene Therapy in a Rat Hepatocellular Carcinoma Model With Liver Cirrhosis

Human hepatocellular carcinoma (HCC) often arises from a background of liver cirrhosis. Therefore, in order to develop therapeutic strategies for HCC, an animal model bearing multifocal liver tumors accompanied by liver cirrhosis is a preferred experimental setting. In this study, we developed a rapid and reproducible method for generating such a model in rats by weekly administration of diethylnitrosamine (DEN) at doses based on body weight (BW). By adjusting the duration of administration of DEN, the animals could be induced to develop HCC alone, or HCC and liver cirrhosis simultaneously. The latter model was used for evaluating the therapeutic effects of adenoviral delivery of interferon-alpha (IFN-alpha). Our results demonstrated that targeting of IFN-alpha expression to the liver significantly reduced liver tumor volume and ameliorated liver cirrhosis. Mechanistic studies revealed that IFN-alpha gene therapy induced immunomodulatory, antiproliferative, and proapoptotic activities that were effective in the control of tumor growth, and reduced the expressions of transforming growth factor-beta (TGF-beta) and tissue inhibitor of metalloproteinase-1 (TIMP-1), leading to amelioration of liver cirrhosis. These results suggest that IFN-alpha gene therapy is a promising strategy to treat HCC patients who have concomitant liver cirrhosis.Human hepatocellular carcinoma (HCC) often arises from a background of liver cirrhosis. Therefore, in order to develop therapeutic strategies for HCC, an animal model bearing multifocal liver tumors accompanied by liver cirrhosis is a preferred experimental setting. In this study, we developed a rapid and reproducible method for generating such a model in rats by weekly administration of diethylnitrosamine (DEN) at doses based on body weight (BW). By adjusting the duration of administration of DEN, the animals could be induced to develop HCC alone, or HCC and liver cirrhosis simultaneously. The latter model was used for evaluating the therapeutic effects of adenoviral delivery of interferon-α (IFN-α). Our results demonstrated that targeting of IFN-α expression to the liver significantly reduced liver tumor volume and ameliorated liver cirrhosis. Mechanistic studies revealed that IFN-α gene therapy induced immunomodulatory, antiproliferative, and proapoptotic activities that were effective in the control of tumor growth, and reduced the expressions of transforming growth factor-β (TGF-β) and tissue inhibitor of metalloproteinase-1 (TIMP-1), leading to amelioration of liver cirrhosis. These results suggest that IFN-α gene therapy is a promising strategy to treat HCC patients who have concomitant liver cirrhosis.

Targeted Delivery of C/EBPα -saRNA by Pancreatic Ductal Adenocarcinoma-specific RNA Aptamers Inhibits Tumor Growth In Vivo

The 5-year survival rate for pancreatic ductal adenocarcinoma (PDAC) remains dismal despite current chemotherapeutic agents and inhibitors of molecular targets. As the incidence of PDAC constantly increases, more effective multidrug approaches must be made. Here, we report a novel method of delivering antitumorigenic therapy in PDAC by upregulating the transcriptional factor CCAAT/enhancer-binding protein-α (C/EBPα), recognized for its antiproliferative effects. Small activating RNA (saRNA) duplexes designed to increase C/EBPα expression were linked onto PDAC-specific 2′-Fluropyrimidine RNA aptamers (2′F-RNA) - P19 and P1 for construction of a cell type–specific delivery vehicle. Both P19- and P1-C/EBPα-saRNA conjugates increased expression of C/EBPα and significantly suppressed cell proliferation. Tail vein injection of the saRNA/aptamer conjugates in PANC-1 and in gemcitabine-resistant AsPC-1 mouse-xenografts led to reduced tumor size with no observed toxicity. To exploit the specificity of the P19/P1 aptamers for PDAC cells, we also assessed if conjugation with Cy3 would allow it to be used as a diagnostic tool on archival human pancreatic duodenectomy tissue sections. Scoring pattern from 72 patients suggested a positive correlation between high fluorescent signal in the high mortality patient groups. We propose a novel aptamer-based strategy for delivery of targeted molecular therapy in advanced PDAC where current modalities fail.

Feasibility studies for assaying alpha-fetoprotein using antibody-activated magnetic nanoparticles

Some previous reports have already shown the characterizations of immunomagnetic reduction (IMR). The assay technology involves the utilities of biofunctionalized magnetic nanoparticles to label target biomolecules. However, the detection threshold and interference tests for IMR have not been investigated in detail. In this study, alpha-fetoprotein (AFP) was used as a target biomolecule. The signals for AFP solutions of various concentrations, or with interfering materials, were detected via IMR. These samples were also used for characterizing the detection threshold and interference with enzyme-linked immunosorbent assay (ELISA). The results of assaying AFP level with IMR and ELISA were compared. The detection threshold for assaying AFP with IMR was found to be 3 ng/mL, which is 15 times lower than that of ELISA, and definitely suppresses false negative. For the interfering materials noted commonly in serum such as hemoglobin, bilirubin, triglyceride, and vascular endothelial growth factor, there was no detectable interfering effect when assaying AFP with IMR. Several serum samples from normal people and liver-tumor-bearing patients were used for the detections of AFP concentration via IMR. These results reveal the feasibilities of assaying AFP in blood using IMR, as well as achieving high-sensitive and high-specific assay for AFP.

Combining antiangiogenic therapy with immunotherapy exerts better therapeutical effects on large tumors in a woodchuck hepatoma model

Cytokine and antiangiogenic gene therapies have proved effective in implanted hepatocellular carcinoma (HCC) models in which small tumor burdens were established in small rodents. These models, however, may not reflect human HCCs, which are frequently detected at a stage when tumors are large and multifocal. In addition, HCC in patients is often associated with viral hepatitis. To investigate the effectiveness of a mixture type of gene therapy strategy on large tumor burdens, we used the woodchuck model in which woodchuck hepatitis virus-induced HCCs are large and multifocal, simulating the conditions in humans. Adenoviruses encoding antiangiogenic factors (pigment epithelium-derived factor and endostatin) or cytokines (GM-CSF and IL-12) were delivered via the hepatic artery separately or in combination into woodchuck livers bearing HCCs. Our results showed that the mixture type of strategy, which contained two cytokines and two antiangiogenic factors, had better antitumor effects on large tumors as compared with monotherapy either with antiangiogenic or cytokine genes. The immunotherapy recruited significant levels of CD3+ T cells that infiltrated the tumors, whereas the antiangiogenesis-based therapy significantly reduced tumor vasculature. The mixture type of gene therapy achieved both effects. In addition, it induced high levels of natural killer cells and apoptotic cells and reduced the levels of immunosuppressive effectors in the tumor regions. Hence, antiangiogenic therapy may provide the advantage of reducing immune tolerance in large tumors, making them more vulnerable to the immune reactions. Our study implies that in the future, the combination therapy may prove effective for the treatment of patients with advanced HCC.