OBJECTIVE: The purpose of this study is to investigate the feasibility of using decellularized tissue ECM to culture circulating tumor cells (CTCs) collected from patients with metastatic solid tumor malignancies. BACKGROUND: Circulating tumor cells (CTCs) have been shown to recapitulate the heterogeneity and biology of patients’ cancers. However, there is a lack of technologies that can culture patient CTCs. Our group has previously shown that tumor cells cultured on decellularized tissue closely mimic the in vivo condition. Therefore, we hypothesized that we can utilized decellularized tissue as strata to culture CTCs. METHODS: Peripheral blood samples were collected from patients with metastatic disease who had not received any systemic therapy in the last six months or are progressing on systemic therapy. PMBC layer, which contains lymphocytes, monocytes, dendritic cells and CTCs (in metastatic patients) is separated from whole blood by Ficoll gradient. The cells are then mixed with decellularized lung or liver material and cultured using stemcell media. RESULTS: 26 subjects were enrolled in this study. We were able to generate cell colonies from 12 patients’ samples (either colonies on liver or lung strata). Of the patient samples that did not develop tumor colonies, 9/14 subjects had no metastatic tumors in either lung or liver. Colonies from the cultures were characterized by histological staining and SEM imaging. Of the samples that the tumor colonies were large enough for H&E, we observed phenotypes that are indicative of tumor cell colony. Ongoing work aim to further validate the CTC cultures using genomic sequencing and RNAse CONCLUSION: We showed decellularizd organ technology can be utilized to generate CTC cultures. Citation Format: Nikhila Reddy Sultanpuram, Isaiah Kim, Hossein Sendi, Nicole Kanpe, Tilden Hagen, Kyle Wagner, Andrew Wang. Culture of circulating tumor cells isolated from cancer patients with metastatic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 281.

Abstract Liver metastasis is a leading cause of cancer morbidity and mortality. Thus, there has been strong interest in the development of therapeutics that can effectively prevent liver metastasis. One potential strategy is to utilize molecules that have broad effects on the liver microenvironment, such as miR-122, a liver-specific miRNA that is a key regulator of diverse hepatic functions. Here we report the development of a nanoformulation miR-122 as a therapeutic agent for preventing liver metastasis. We engineered a galactose-targeted lipid calcium phosphate (Gal-LCP) nanoformulation of miR-122. This nanotherapeutic elicited no significant toxicity and delivered miR-122 into hepatocytes with specificity and high efficiency. Across multiple colorectal cancer liver metastasis models, treatment with Gal-LCP miR-122 treatment effectively prevented colorectal cancer liver metastasis and prolonged survival. Mechanistic studies revealed that delivery of miR-122 was associated with downregulation of key genes involved in metastatic and cancer inflammation pathways, including several proinflammatory factors, matrix metalloproteinases, and other extracellular matrix degradation enzymes. Moreover, Gal-LCP miR-122 treatment was associated with an increased CD8+/CD4+ T-cell ratio and decreased immunosuppressive cell infiltration, which makes the liver more conducive to antitumor immune response. Collectively, this work presents a strategy to improve cancer prevention and treatment with nanomedicine-based delivery of miRNA. Significance: Highly specific and efficient delivery of miRNA to hepatocytes using nanomedicine has therapeutic potential for the prevention and treatment of colorectal cancer liver metastasis.

Abstract Background: Metastatic tumors in the liver are 20 times more common than primary tumors and considered as a leading cause of mortality in several types of cancer. Therefore, making the liver (soil) inhospitable for tumor cells (seeds) to grow will have tremendous effects on outcome of multiple types of cancer. RNAi delivery -including miRNA delivery-shows promise for the development of novel molecular therapeutics that interfere with genes causing metastasis. Mir-122, which accounts for 70% of total miRNA population within the liver, is also known as a tumor suppressor miRNA within liver. We hypothesized that miR-122 delivery into hepatocytes can prevent development of metastatic colorectal cancer within mice liver. For testing this hypothesis, we used hepatic injection of ex-vivo engineered liver metastasis from human colorectal cancer cells to induce hepatic metastasis in nude mice and employed lipid calcium phosphate (LCP) nanoparticles for delivery of miR-122. Methods: LCP-miR122 and LCP-miRNC (negative control miRNA) were prepared mixing an emulsion of calcium which contained miRNA with a phosphate emulsion, followed by addition of DOPA to make the inner core of LCP. The outer leaflet of LCP contained DOTAP, Cholesterol and DSPE-PEG as. Galactose was conjugated at LCP surface for its targeted delivery to hepatocytes. For induction of hepatic metastasis, HT29-Luc cells were grown on liver biomatrix (BMX) for 7-10 days to acquire metastatic features and then harvested and injected into portal veins of nu/nu mice. Hepatic metastases formation was monitored weekly in vivo using bioluminescent imaging. LCP-miR122 or LCP-miRNC was systematically injected three times a week for 3 weeks starting 24 hours after tumor inoculations. Results: LCP particles were found to be 25-50 nm via dynamic light scattering analysis. When injected in vivo, LCP containing miR-122 was majorly distributed into liver (80%) and was detected in hepatocytes through confocal microscopy and target gene expression analysis. LCP-miR122 did not show any sign of hepatotoxicity or renal toxicity or any other pathological damage to other major organs. The injected conditioned HT29 cells formed metastatic foci within liver 1-3 weeks after portal vein injection as detected by bioluminescent imaging. Interestingly, we found that 67% of (6 out of 9) mice treated with LCP-miR122 did not show any sign of liver metastasis after 3 weeks compared with only 20% (2 out of 10) of mice treated with LCP containing negative control miRNA [OR=0.12, 95% CI:0.015-0.99; p=0.05]. Monitoring liver metastasis expansion on weekly basis, mice treated with LCP-miR122 showed a slight decrease in number of liver metastatic foci compared with control mice which showed a significant increase in number of liver metastatic foci during treatment [0.7 vs 3.8-fold; p=0.05]. Conclusion: These results suggest that targeted delivery of miR-122 into hepatocytes of patients prone to liver metastasis or harboring liver metastasis can prevent or regress hepatic metastasis. Citation Format: Hossein Sendi, Nikhila Sultanpuram, Jie Wang, Amanda Graboski, Liantao Li, Mengying Hu, Leaf Huang, Andrew Z. Wang. Targeted delivery of miR-122 into hepatocytes regresses liver metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5033.

Hepatotoxicity is a key concern in the clinical translation of nanotherapeutics because preclinical studies have consistently shown that nanotherapeutics accumulates extensively in the liver. However, clinical-stage nanotherapeutics have not shown increased hepatotoxicity. Factors that can contribute to the hepatotoxicity of nanotherapeutics beyond the intrinsic hepatotoxicity of nanoparticles (NPs) are poorly understood. Because of this knowledge gap, clinical translation efforts have avoided hepatotoxic molecules. By examining the hepatotoxicity of nanoformulations of known hepatotoxic compounds, it is demonstrated that nanotherapeutics are associated with lower hepatotoxicity than their small-molecule counterparts. It is also found that the reduced hepatotoxicity is related to the uptake of nanotherapeutics by macrophages in the liver. These findings can facilitate further development and clinical translation of nanotherapeutics.

We sought to determine if the baseline hepatic levels of miR-122, miR-29b, Claudin, Occludin, Protein Kinase R (PKR) or PKR activator (PRKRA) were correlated with HCV RNA or stage of fibrosis in patients with chronic hepatitis C (CHC). A total of 25 CHC patients (genotype 1) who were treatment naive at the time of sample collection enrolled in this study. By multivariate analysis, CLDN RNA was found as the single independent factor positively correlated with HCV RNA levels (p=0.003), while hepatic miR-29b levels was found as the single independent factor for predicting advanced stage of fibrosis (p=0.028). Conclusion: Our results highlight miR-29b and CLDN as novel predictors of advanced stage of liver fibrosis and baseline HCV RNA in CHC.

To investigate the role of miR-122 in the development and regression of non-alcoholic fatty liver disease (NAFLD) in vitro, we used multicellular 3D human liver organoids developed in our laboratory. These organoids consist of primary human hepatocytes, Kupffer cells, quiescent stellate cells and liver sinusoidal endothelial cells. They remain viable and functional for 4 weeks expressing typical markers of liver function such as synthesis of albumin, urea, and alpha-1 p450 drug metabolism. Before mixing, hepatic cells were transduced with lentivirus to inhibit miR122 expression (ABM, CA). Immediately after the organoids were fully formed (day 4) or after 1 or 2 weeks of additional incubation (days 11 or 18), the organoids were analyzed using fluorescent live/dead staining and ATP production; total RNA was extracted for qPCR gene expression profiling. Our results show that miR-122 inhibition in liver organoids leads to inflammation, necrosis, steatosis and fibrosis. This was associated with increase in inflammatory cytokines (IL6, TNF), chemokines (CCL2, CCL3) and increase in a subset of Matrix Metaloproteinases (MMP8, MMP9). An altered expression of key genes in lipid metabolism (i.e LPL, LDLR) and insulin signaling (i.e GLUT4, IRS1) was also identified.CONCLUSION:Our results highlight the role of miR-122 inhibition in liver inflammation, steatofibrosis and dysregulation of insulin signaling. Patients with NAFLD are known to have altered levels of miR-122, therefore we suggest that miR-122 mimics could play a useful role in reversing liver steatofibrosis and insulin resistance seen in patients with NAFLD.

BACKGROUND & AIMS:Analysis in silico suggests that occludin (OCLN), a key receptor for HCV, is a candidate target of miR-122; the most abundant hepatic micro RNA. We aimed to determine if miR-122 can decrease HCV entry through binding to the 3' UTR of OCLN mRNA.DESIGN:Huh7.5 cells were cotransfected with luciferase construct containing 3' UTR of OCLN (pLuc-OCLN) and with selected miRNAs (0-50 nM) and luciferase activity was measured. Huh7.5 cells were also infected by viral particles containing lenti-miR122 genome or control virus. After 48 h, the cells were infected with HCV pseudo-particles (HCVpp) and VSV pseudo-particles (VSVpp). After 72 h of infection, luciferase activity was measured and HCVpp activity was normalized to VSVpp activity.RESULTS:miR-122 binds to the 3'-UTR of OCLN and down-regulates its expression; cotransfection of miR-122 mimic with pLuc-OCLN resulted in a significant decrease in luciferase activity [by 55% (P < 0.01)], while a non-specific miRNA and a mutant miR-122 did not have any effect. miR-122 mimic significantly down-regulated [by 80% (P < 0.01)] OCLN protein in Huh7.5 cells. Accordingly, patients with chronic hepatitis C and higher levels of hepatic miR-122 have lower hepatic expression of OCLN. Immuno-fluorescence imaging showed a decrease in colocalization of OCLN and CLDN following miR-122 over-expression in HCV infected cells. Huh7.5 cells transiently expressing Lenti-miR122 system showed 42% (P < 0.01) decrease in HCV entry.CONCLUSION:This study uncovers a novel antiviral effect of miR-122 on human liver cells and shows that over-expression of miR-122 can decrease HCV entry into hepatocytes through down-regulation of OCLN.

Hepatitis B virus (HBV) continues to be a major cause of morbidity and mortality worldwide. It is estimated that about 350 million people throughout the world are chronically infected with HBV. Some of these people will develop hepatic cirrhosis with decompensation and/or hepatocellular carcinoma. For such patients, liver transplantation may be the only hope for cure or real improvement in quality and quantity of life. Formerly, due to rapidity of recurrence of HBV infection after liver transplantation, usually rapidly progressive, liver transplantation was considered to be contraindicated. This changed dramatically following the demonstration that hepatitis B immune globulin (HBIG), could prevent recurrent HBV infection. HBIG has been the standard of care for the past two decades or so. Recently, with the advent of highly active inhibitors of the ribose nucleic acid polymerase of HBV (entecavir, tenofovir), there has been growing evidence that HBIG needs to be given for shorter lengths of time; indeed, it may no longer be necessary at all. In this review, we describe genetic variants of HBV and past, present, and future prophylaxis of HBV infection during and after liver transplantation. We have reviewed the extant medical literature on the subject of infection with the HBV, placing particular emphasis upon the prevention and treatment of recurrent HBV during and after liver transplantation. For the review, we searched PubMed for all papers on the subject of "hepatitis B virus AND liver transplantation". We describe some of the more clinically relevant and important genetic variations in the HBV. We also describe current practices at our medical centers, provide a summary and analysis of comparative costs for alternative strategies for prevention of recurrent HBV, and pose important still unanswered questions that are in need of answers during the next decade or two. We conclude that it is now rational and cost-effective to decrease and, perhaps, cease altogether, the routine use of HBIG during and following liver transplantation for HBV infection. Here we propose an individualized prophylaxis regimen, based on an integrated approach and risk-assessment.

CCR5 is an important chemokine receptor involved in the recruitment of specific anti-viral immune cells (e.g., NK cells and T cytotoxic cells) to the liver. Previous studies indicated that the 32 mutation in CCR5 gene led to inactivation of CCR5. Several conflicting studies have suggested that this mutation may be associated with either recovery or persistence of HBV infection. The main purpose of this study was to compare the frequency of the 32 mutation within the CCR5 gene in a group of patients infected chronically with HBV with healthy individuals from South-East of Iran. Sixty patients with chronic HBV infection as well as 300 age-, and sex-match healthy individuals were enrolled in this study. Gap-PCR was applied to determine the frequency of CCR5 32 mutation in both groups. The results demonstrated that none of the patients infected with HBV carried the CCR5 32 mutation while, 3 (1%) of the healthy individuals were found to be heterozygotic for this mutation. The CCR5 32 mutation is not a prevalent mutation in either the patients infected chronically with HBV or their health counterparts in the South-East region of Iran. This may be attributed to either different genetic settings of the investigated population or lack of any significant correlation between this mutation and HBV pathogenicity. J. Med. Virol. 85: 964968, 2013. (c) 2013 Wiley Periodicals, Inc.

BACKGROUND:Escape mutations potentially allow viruses to avoid detection and clearance by the host immune system and may represent a mechanism through which infections may persist in some patients. The association of the mutations in the HBcAg gene with Hepatitis B asymptomatic carriers (ASC) has not been studied adequately. The current study was aimed to investigate HBcAg18-27 CTL epitope mutations in ASC patients in the South-Eastern region of Iran.METHODS:100 ASC patients were selected for this study and screened for HLA-A2 using flow cytometry. HBV-DNA was extracted from the HLA-A2 positive patients and the HBc gene was amplified using PCR. Direct double sequencing was performed to analyse mutations in the HBc gene of HBV isolates from patients with ASC.RESULTS:Overall, 25 (25%) of individuals were HLA-A2 positive. Direct double sequencing indicated no mutations in the HBcAg18-27 epitope. However, four mutations within the T helper and three mutations within the B cell epitopes of ASC patients were identified.CONCLUSIONS:The lack of mutations within the HBcAg18-27 epitope suggests that the antigenicity of this region is not altered in HBV isolates of our patients and therefore antigen presentation would occur normally to the patient's immune system through HLA-A2. However, in the course of this study we revealed some novel mutations within the T helper and B cell epitopes that may affect the efficiencies of immune response of ASC patients against these novel HBV epitopes.

Porphyria cutanea tarda (PCT) is the most common form of porphyria across the world. Unlike other forms of porphyria, which are inborn errors of metabolism, PCT is usually an acquired liver disease caused by exogenous factors, chief among which are excess alcohol intake, iron overload, chronic hepatitis C, oestrogen therapy and cigarette smoking. The pathogenesis of PCT is complex and varied, but hereditary or acquired factors that lead to hepatic iron loading and increased oxidative stress are of central importance. Iron loading is usually only mild or moderate in degree [less than that associated with full-blown haemochromatosis (HFE)] and is usually acquired and/or mutations in HFE. Among acquired factors are excessive alcohol intake and chronic hepatitis C infection, which, like mutations in HFE, decrease hepcidin production by hepatocytes. The decrease in hepcidin leads to increased iron absorption from the gut. In the liver, iron loading and increased oxidative stress leads to the formation of non-porphyrin inhibitor(s) of uroporphyrinogen decarboxylase and to oxidation of porphyrinogens to porphyrins. The treatment of choice of active PCT is iron reduction by phlebotomy and maintenance of a mildly iron-reduced state without anaemia. Low-dose antimalarials (cinchona alkaloids) are also useful as additional therapy or as alternative therapy for active PCT in those without haemochromatosis or chronic hepatitis C. In this review, we provide an update of PCT with special emphasis upon the important role often played by the hepatitis C virus.

The hepatic microRNA (miRNA), miR-122, is the most abundant miRNA within the liver, where it accounts for 70% of the total miRNA pool. It is known that miR-122, as an unusual host factor, increases the abundance of hepatitis C virus (HCV) RNA in HCV infection by binding directly to the 5’-UTR of the viral genome. Therefore, it has been suggested as a potential target for the treatment of hepatitis C. However, recent evidence shows that miR-122 decreases HBV replication through the inhibitory effect of p53 on HBV transcription, and consequently it acts as a tumor-suppressor through both a decrease in HBV replication and by directly targeting cyclin G1, as well as Wnt/beta-catenin, and NDRG3 pathways. This paper will briefly discuss the underlying mechanisms for the dual role of miR-122 in viral hepatitis, and explains why therapeutic applications of miR-122 may differ based on the underlying disease.

This article does not have abstract.

AIM:To determine the effect of Legalon-SIL (LS) on hepatitis C virus (HCV) core and NS5A expression and on heme oxygenase-1 (HMOX-1) and its transcriptional regulators in human hepatoma cells expressing full length HCV genotype 1b.METHODS:CON1 cells were treated with 50 μmol/L or 200 μmol/L LS. Cells were harvested after 2, 6 and 24 h. HCV RNA and protein levels were determined by quantitative real-time polymerase chain reaction and Western blotting, respectively.RESULTS:HCV RNA (core and NS5A regions) was decreased after 6 h with LS 200 μmol/L (P < 0.05). Both 50 and 200 μmol/L LS decreased HCV RNA levels [core region (by 55% and 88%, respectively) and NS5A region (by 62% and 87%, respectively) after 24 h compared with vehicle (dimethyl sulphoxide) control (P < 0.01). Similarly HCV core and NS5A protein were decreased (by 85%, P < 0.01 and by 65%, P < 0.05, respectively) by LS 200 μmol/L. Bach1 and HMOX-1 RNA were also downregulated by LS treatment (P < 0.01), while Nrf2 protein was increased (P < 0.05).CONCLUSION:Our results demonstrate that treatment with LS downregulates HCV core and NS5A expression in CON1 cells which express full length HCV genotype 1b, and suggests that LS may prove to be a valuable alternative or adjunctive therapy for the treatment of HCV infection.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Iron may play a comorbid role in HCV disease and other liver diseases. Although adjunctive treatment of HCV disease with iron reduction is popular in east Asia, especially Japan, it is less popular in the United States. However, iron reduction may be of importance, especially for certain HCV patient subgroups that have not responded to or cannot tolerate standard therapy. This review focuses on recent advances in understanding how iron may influence HCV infection and vice versa. A key is effects on levels of expression of the hepcidin gene in hepatocytes, because hepcidin has emerged as the key regulator of iron homeostasis. This review also summarizes prior clinical studies involving iron reduction therapy and clinical implications.