Renin-Angiotensin Inhibitor, Captopril, Attenuates Growth of Patient-Derived Colorectal Liver Metastasis Organoids.

The recurrence of colorectal liver metastasis (CRLM) following liver resection is common; approximately 40% of patients will experience tumor recurrence post-surgery. Renin-angiotensin inhibitors (RASis) have been shown to attenuate the growth and progression of CRLM in pre-clinical models following liver resection. This study examined the efficacy of the RASi captopril on patient-derived colorectal liver metastasis organoids. Patient-derived organoids (PDOs) were established using fresh samples of colorectal liver metastasis from appropriately consented patients undergoing liver resection. To mimic the regenerating liver post-CRLM liver resection, PDOs were cultured under hepatocyte regeneration conditions in vitro. CRLM PDOs were established from three patients' parent tissue. CRLM PDOs and parent tissue expressed markers of colorectal cancer, CDX2 and CK20, consistently. Furthermore, CRLM PDOs treated with captopril showed a dose dependent reduction in their expansion in vitro. In conclusion, CRLM PDOs recapitulate in vivo disease and displayed a dose-dependent response to treatment with captopril. RASis may be an additional viable treatment for patients with CRLM.

Epigenetic Silencing of RIPK3 in Hepatocytes Prevents MLKL-mediated Necroptosis From Contributing to Liver Pathologies.

BACKGROUND & AIMS: Necroptosis is a highly inflammatory mode of cell death that has been implicated in causing hepatic injury including steatohepatitis/ nonalcoholic steatohepatitis (NASH); however, the evidence supporting these claims has been controversial. A comprehensive, fundamental understanding of cell death pathways involved in liver disease critically underpins rational strategies for therapeutic intervention. We sought to define the role and relevance of necroptosis in liver pathology. METHODS: Several animal models of human liver pathology, including diet-induced steatohepatitis in male mice and diverse infections in both male and female mice, were used to dissect the relevance of necroptosis in liver pathobiology. We applied necroptotic stimuli to primary mouse and human hepatocytes to measure their susceptibility to necroptosis. Paired liver biospecimens from patients with NASH, before and after intervention, were analyzed. DNA methylation sequencing was also performed to investigate the epigenetic regulation of RIPK3 expression in primary human and mouse hepatocytes. RESULTS: Identical infection kinetics and pathologic outcomes were observed in mice deficient in an essential necroptotic effector protein, MLKL, compared with control animals. Mice lacking MLKL were indistinguishable from wild-type mice when fed a high-fat diet to induce NASH. Under all conditions tested, we were unable to induce necroptosis in hepatocytes. We confirmed that a critical activator of necroptosis, RIPK3, was epigenetically silenced in mouse and human primary hepatocytes and rendered them unable to undergo necroptosis. CONCLUSIONS: We have provided compelling evidence that necroptosis is disabled in hepatocytes during homeostasis and in the pathologic conditions tested in this study.

Assessment of HBV infection and inter-host cellular responses using intrahepatic cholangiocyte organoids.

Intrahepatic cholangiocyte organoids (ICOs) model was evaluated for host differences in hepatitis B virus (HBV) infection, cellular responses, antiviral, and immunomodulator responses. Twelve ICOs generated from liver resections and biopsies were assessed for metabolic markers and functional HBV entry receptor expression throughout differentiation. Structural changes relevant to HBV infection were characterized using histology, confocal, and electron microscopy examinations. Optimal ICO culture conditions for HBV infection using HepAD38 (genotype D) and plasma derived HBV (genotype B & C) were described. HBV infection was confirmed using HBcAg immunostaining, qRT-PCR (RNA, cccDNA, extracellular DNA), and ELISA (HBsAg and HBeAg). Drug response to antiviral and immunosuppressive agent, and cellular responses (interferon-stimulated genes [ISG]) to interferon-α and viral mimic (PolyI:C) were assessed. ICOs underwent metabolic and structural remodeling following differentiation. Optimal HBV infection was achieved in well-differentiated ICOs using spinoculation, with time and donor dependent increase in HBV RNA, cccDNA, extracellular DNA, HBeAg, and HBsAg. Donor dependent drug-responsiveness to entry inhibitor and JAK inhibitor was observed. Despite having a robust ISG response to interferon-α and PolyI:C, HBV infection in ICOs did not upregulate ISGs. Human ICOs support HBV infection and replication with donor dependent variation in viral dynamics and cellular responses. These features can be utilized for development of personalized drug testing platform for antivirals.

Assessment of hepatitis B virus infection and interhost cellular responses using intrahepatic cholangiocyte organoids.

The intrahepatic cholangiocyte organoids (ICOs) model was evaluated for host differences in hepatitis B virus (HBV) infection, cellular responses, antiviral and immunomodulator responses. Twelve ICOs generated from liver resections and biopsies were assessed for metabolic markers and functional HBV entry receptor expression throughout differentiation. Structural changes relevant to HBV infection were characterized using histology, confocal, and electron microscopy examinations. Optimal ICO culture conditions for HBV infection using HepAD38 (genotype D) and plasma-derived HBV (genotype B and C) were described. HBV infection was confirmed using HBcAg immunostaining, qRT-PCR (RNA, covalently closed circular DNA [cccDNA], extracellular DNA) and ELISA (HBsAg and HBeAg). Drug response to antiviral and immunosuppressive agent, and cellular responses (interferon-stimulated genes [ISG]) to interferon-α and viral mimic (PolyI:C) were assessed. ICOs underwent metabolic and structural remodeling following differentiation. Optimal HBV infection was achieved in well-differentiated ICOs using spinoculation, with time and donor-dependent increase in HBV RNA, cccDNA, extracellular DNA, HBeAg and HBsAg. Donor-dependent drug responsiveness to entry inhibitor and JAK inhibitor was observed. Despite having a robust ISG response to interferon-α and PolyI:C, HBV infection in ICOs did not upregulate ISGs. Human ICOs support HBV infection and replication with donor-dependent variation in viral dynamics and cellular responses. These features can be utilized for the development of personalized drug testing platform for antivirals.

Captopril, a Renin-Angiotensin System Inhibitor, Attenuates Tumour Progression in the Regenerating Liver Following Partial Hepatectomy.

(1) Liver regeneration following partial hepatectomy for colorectal liver metastasis (CRLM) has been linked to tumour recurrence. Inhibition of the renin−angiotensin system (RASi) attenuates CRLM growth in the non-regenerating liver. This study investigates whether RASi exerts an antitumour effect within the regenerating liver following partial hepatectomy for CRLM and examines RASi-induced changes in the tumour immune microenvironment; (2) CRLM in mice was induced via intrasplenic injection of mouse colorectal tumour cells, followed by splenectomy on Day 0. Mice were treated with RASi captopril (250 mg/kg/day), or saline (control) from Day 4 to Day 16 (endpoint) and underwent 70% partial hepatectomy on Day 7. Liver and tumour samples were characterised by flow cytometry and immunofluorescence; (3) captopril treatment reduced tumour burden in mice following partial hepatectomy (p < 0.01). Captopril treatment reduced populations of myeloid-derived suppressor cells (MDSCs) (CD11b+Ly6CHi p < 0.05, CD11b+Ly6CLo p < 0.01) and increased PD-1 expression on infiltrating hepatic tissue-resident memory (TRM)-like CD8+ (p < 0.001) and double-negative (CD4-CD8-; p < 0.001) T cells; (4) RASi reduced CRLM growth in the regenerating liver and altered immune cell composition by reducing populations of immunosuppressive MDSCs and boosting populations of PD-1+ hepatic TRMs. Thus, RASi should be explored as an adjunct therapy for patients undergoing partial hepatectomy for CRLM.

Air-Liquid-Interface Differentiated Human Nose Epithelium: A Robust Primary Tissue Culture Model of SARS-CoV-2 Infection.

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air-liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.

Frizzled7 Activates ß-Catenin-Dependent and ß-Catenin-Independent Wnt Signalling Pathways During Developmental Morphogenesis: Implications for Therapeutic Targeting in Colorectal Cancer.

Frizzled7 activates ß-catenin-dependent and ß-catenin-independent Wnt signalling pathways, is highly conserved through evolution from the ancient phylum hydra to man, plays essential roles in stem cells, tissue homeostasis and regeneration in the adult, and is upregulated in diverse cancers. Much of what is known about the core components of the Wnt signalling pathways was derived from studying the function of Frizzled7 orthologues in the development of lower organism. As we interrogate Frizzled7 signalling and function for therapeutic targeting in cancer, it is timely to revisit lower organisms to gain insight into the context dependent and dynamic nature of Wnt signalling for effective drug design.

Targeting Wnt Signaling for the Treatment of Gastric Cancer.

The Wnt signaling pathway is evolutionarily conserved, regulating both embryonic development and maintaining adult tissue homeostasis. Wnt signaling controls several fundamental cell functions, including proliferation, differentiation, migration, and stemness. It therefore plays an important role in the epithelial homeostasis and regeneration of the gastrointestinal tract. Often, both hypo- or hyper-activation of the pathway due to genetic, epigenetic, or receptor/ligand alterations are seen in many solid cancers, such as breast, colorectal, gastric, and prostate. Gastric cancer (GC) is the fourth commonest cause of cancer worldwide and is the second leading cause of cancer-related death annually. Although the number of new diagnoses has declined over recent decades, prognosis remains poor, with only 15% surviving to five years. Geographical differences in clinicopathological features are also apparent, with epidemiological and genetic studies revealing GC to be a highly heterogeneous disease with phenotypic diversity as a result of etiological factors. The molecular heterogeneity associated with GC dictates that a single 'one size fits all' approach to management is unlikely to be successful. Wnt pathway dysregulation has been observed in approximately 50% of GC tumors and may offer a novel therapeutic target for patients who would otherwise have a poor outcome. This mini review will highlight some recent discoveries involving Wnt signaling in GC.

HBV-related hepatocarcinogenesis: the role of signalling pathways and innovative ex vivo research models.

BACKGROUND: Hepatitis B virus (HBV) is the leading cause of liver cancer, but the mechanisms by which HBV causes liver cancer are poorly understood and chemotherapeutic strategies to cure liver cancer are not available. A better understanding of how HBV requisitions cellular components in the liver will identify novel therapeutic targets for HBV associated hepatocellular carcinoma (HCC). MAIN BODY: The development of HCC involves deregulation in several cellular signalling pathways including Wnt/FZD/ß-catenin, PI3K/Akt/mTOR, IRS1/IGF, and Ras/Raf/MAPK. HBV is known to dysregulate several hepatocyte pathways and cell cycle regulation resulting in HCC development. A number of these HBV induced changes are also mediated through the Wnt/FZD/ß-catenin pathway. The lack of a suitable human liver model for the study of HBV has hampered research into understanding pathogenesis of HBV. Primary human hepatocytes provide one option; however, these cells are prone to losing their hepatic functionality and their ability to support HBV replication. Another approach involves induced-pluripotent stem (iPS) cell-derived hepatocytes. However, iPS technology relies on retroviruses or lentiviruses for effective gene delivery and pose the risk of activating a range of oncogenes. Liver organoids developed from patient-derived liver tissues provide a significant advance in HCC research. Liver organoids retain the characteristics of their original tissue, undergo unlimited expansion, can be differentiated into mature hepatocytes and are susceptible to natural infection with HBV. CONCLUSION: By utilizing new ex vivo techniques like liver organoids it will become possible to develop improved and personalized therapeutic approaches that will improve HCC outcomes and potentially lead to a cure for HBV.

Wnt is necessary for mesenchymal to epithelial transition in colorectal cancer cells.

BACKGROUND: Metastasis underlies most colorectal cancer mortality. Cancer cells spread through the body as single cells or small clusters of cells that have an invasive, mesenchymal, nonproliferative phenotype. At the secondary site, they revert to a proliferative "tumor constructing" epithelial phenotype to rebuild a tumor. We previously developed a unique in vitro three-dimensional model, called LIM1863-Mph, which faithfully recapitulates these reversible transitions that underpin colorectal cancer metastasis. Wnt signaling plays a key role in these transitions and is initiated by the coupling of extracellular Wnt to Frizzled (FZD). Using the LIM1863-Mph model system we demonstrated that the Wnt receptor FZD7 is necessary for mesenchymal to epithelial transition (MET). Here we investigate the role of Wnt in MET. RESULTS: Wnt secretion is dependent on palmitoylation by Porcupine (PORC). A PORC inhibitor (IWP2) that prevents Wnt secretion, blocked the epithelial transition of mesenchymal LIM1863-Mph cells. Wnt gene array analysis identified several Wnts that are upregulated in epithelial compared with mesenchymal LIM1863-Mph cells, suggesting these ligands in MET. Wnt2B was the most abundant differentially expressed Wnt gene. Indeed, recombinant Wnt2B could overcome the IWP2-mediated block in epithelial transition of mesenchymal LIM1863-Mph cells. CONCLUSIONS: Wnt2B co-operates with Frizzled7 to mediate MET in colorectal cancer. Developmental Dynamics 247:521-530, 2018. © 2017 Wiley Periodicals, Inc.

Uncovering strain- and age-dependent innate immune responses to SARS-CoV-2 infection in air-liquid-interface cultured nasal epithelia.

Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, with the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly infected and uninfected bystander cells and heightened in Alpha-infection. Alpha highly infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control cells, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2.

Trabid patient mutations impede the axonal trafficking of adenomatous polyposis coli to disrupt neurite growth.

ZRANB1 (human Trabid) missense mutations have been identified in children diagnosed with a range of congenital disorders including reduced brain size, but how Trabid regulates neurodevelopment is not understood. We have characterized these patient mutations in cells and mice to identify a key role for Trabid in the regulation of neurite growth. One of the patient mutations flanked the catalytic cysteine of Trabid and its deubiquitylating (DUB) activity was abrogated. The second variant retained DUB activity, but failed to bind STRIPAK, a large multiprotein assembly implicated in cytoskeleton organization and neural development. Zranb1 knock-in mice harboring either of these patient mutations exhibited reduced neuronal and glial cell densities in the brain and a motor deficit consistent with fewer dopaminergic neurons and projections. Mechanistically, both DUB-impaired and STRIPAK-binding-deficient Trabid variants impeded the trafficking of adenomatous polyposis coli (APC) to microtubule plus-ends. Consequently, the formation of neuronal growth cones and the trajectory of neurite outgrowth from mutant midbrain progenitors were severely compromised. We propose that STRIPAK recruits Trabid to deubiquitylate APC, and that in cells with mutant Trabid, APC becomes hyperubiquitylated and mislocalized causing impaired organization of the cytoskeleton that underlie the neuronal and developmental phenotypes.

Assessment of Pre-Clinical Liver Models Based on Their Ability to Predict the Liver-Tropism of Adeno-Associated Virus Vectors.

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors. Multiple clinical trials have been undertaken for this target in the past 15 years; however, we are still to see market approval of the first liver-targeted adeno-associated virus (AAV)-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically and clinically predictive preclinical models. To this end, this study reports findings of a functional evaluation of 6 AAV vectors in 12 preclinical models of the human liver, with the aim to uncover which combination of models is the most relevant for the identification of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine.

BACKGROUND: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. METHODS: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid α-Galactosylceramide, or MF59® squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine. FINDINGS: Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59® adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59® increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5. INTERPRETATION: These results demonstrated that an RBD-Fc protein subunit/MF59® adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial. FUNDING: This work was supported by grants from the Medical Research Future Fund (MRFF) (2005846), The Jack Ma Foundation, National Health and Medical Research Council of Australia (NHMRC; 1113293) and Singapore National Medical Research Council (MOH-COVID19RF-003). Individual researchers were supported by an NHMRC Senior Principal Research Fellowship (1117766), NHMRC Investigator Awards (2008913 and 1173871), Australian Research Council Discovery Early Career Research Award (ARC DECRA; DE210100705) and philanthropic awards from IFM investors and the A2 Milk Company.

Myb controls intestinal stem cell genes and self-renewal.

Rapid advances have been made in the understanding of how the highly proliferative gastrointestinal tract epithelium is regulated under homeostasis and disease. The identification of putative intestinal stem cell (ISC) genes and the ability to culture ISC capable of generating all four lineages plus the architecture of small intestinal (SI) crypts has been transformative. Here, we show that transcription factor Myb governs ISC gene expression, particularly Lgr5. Lgr5 is associated with cells that have the capacity to generate all cell lineages in SI organoid cultures and colorectal cancer cells, which overexpress Myb. Furthermore, Wnt signaling and Myb cooperate in maximal Lgr5 promoter activation while hypomorphic Myb (plt4/plt4) mice have decreased Lgr5 expression. After ionizing radiation (IR), ISC genes are elevated; but in plt4/plt4 mice, this response is substantially subdued. ISC genes bmi-1 and olfm4 are expressed at subnormal levels in plt4/plt4 mice, and bmi-1 is induced with IR to half the level in mutant mice. dcamkl-1 and olfm4 failed to recover after IR in both wild-type (wt) and mutant mice. Although not considered as an ISC gene, cyclinE1 is nevertheless used to assist cells in the emergence from a quiescent state (an expectation of ISC following IR) and is overexpressed after IR in wt mice but does not change from a very low base in plt4/plt4 mice. Self-renewal assays using organoid cultures and inducible Myb knockout studies further highlighted the dependence of ISC on Myb consistent with role in other stem cell-containing tissues.

Use of Human Lung Tissue Models for Screening of Drugs against SARS-CoV-2 Infection.

The repurposing of licenced drugs for use against COVID-19 is one of the most rapid ways to develop new and alternative therapeutic options to manage the ongoing pandemic. Given circa 7817 licenced compounds available from Compounds Australia that can be screened, this paper demonstrates the utility of commercially available ex vivo/3D airway and alveolar tissue models. These models are a closer representation of in vivo studies than in vitro models, but retain the benefits of rapid in vitro screening for drug efficacy. We demonstrate that several existing drugs appear to show anti-SARS-CoV-2 activity against both SARS-CoV-2 Delta and Omicron Variants of Concern in the airway model. In particular, fluvoxamine, as well as aprepitant, everolimus, and sirolimus, has virus reduction efficacy comparable to the current standard of care (remdesivir, molnupiravir, nirmatrelvir). Whilst these results are encouraging, further testing and efficacy studies are required before clinical use can be considered.

Intestinal epithelial-specific PTEN inactivation results in tumor formation.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of phosphatidylinositol 3-kinase (PI3K) signaling that is frequently inactivated in colorectal cancer through mutation, loss of heterozygosity, or epigenetic mechanisms. The aim of this study was to determine the effect of intestinal-specific PTEN inactivation on intestinal epithelial homeostasis and tumorigenesis. PTEN was deleted specifically in the intestinal epithelium, by crossing PTEN(Lox/Lox) mice with villin(Cre) mice. PTEN was robustly expressed in the intestinal epithelium and maximally in the differentiated cell compartment. Targeted inactivation of PTEN in the intestinal epithelium of PTEN(Lox/Lox)/villin(Cre) mice was confirmed by genotyping, immunohistochemistry, and qPCR. While intestinal-specific PTEN deletion did not have a major effect on cell fate determination or proliferation in the small intestine, it did increase phosphorylated (p) protein kinase B (AKT) expression in the intestinal epithelium, and 19% of animals developed small intestinal adenomas and adenocarcinomas at 12 mo of age. These tumors demonstrated pAKT and nuclear ß-catenin staining, indicating simultaneous activation of the PI3K/AKT and Wnt signaling pathways. These findings demonstrate that, while PTEN inactivation alone has a minimal effect on intestinal homeostasis, it can facilitate tumor promotion upon deregulation of ß-catenin/TCF signaling, further establishing PTEN as a bona fide tumor suppressor gene in intestinal cancer.

Variable FZD7 expression in colorectal cancers indicates regulation by the tumour microenvironment.

Recent evidence shows that a sub-population of Wnt/beta-catenin target genes is specifically induced in different tissue contexts. FZD7 is a putative Wnt/beta-catenin target gene and although it is highly expressed in well-differentiated colorectal cancer tumour cells, its expression is decreased in de-differentiated tumour cells at the invasive front despite elevated Wnt/beta-catenin signalling in this area. This variable expression of FZD7 implicates additional regulation by the microenvironment; however, this has not been investigated. To begin to elucidate the role of extracellular matrix in regulating FZD7 expression, we generated a FZD7 promoter reporter and analysed FZD7 promoter activity in colorectal cancer cells grown on different matrices. We demonstrate that the FZD7 promoter is regulated by beta-catenin in colorectal cancer cells and observed decreased promoter activity in cells grown on fibronectin but not collagen I or collagen IV. Thus, expression of FZD7 in colorectal cancer may be regulated by fibronectin in the microenvironment.

Captopril, a Renin-Angiotensin System Inhibitor, Attenuates Features of Tumor Invasion and Down-Regulates C-Myc Expression in a Mouse Model of Colorectal Cancer Liver Metastasis.

(1) Background: Recent clinical and experimental data suggests that the liver's regenerative response following partial hepatectomy can stimulate tumor recurrence in the liver remnant. The Wnt/ß-catenin pathway plays important roles in both colorectal cancer carcinogenesis and liver regeneration. Studies have shown that the Wnt/ß-catenin pathway regulates multiple renin-angiotensin system (RAS) genes, whilst RAS inhibition (RASi) reduces tumor burden and progression. This study explores whether RASi attenuates features of tumor progression in the regenerating liver post-hepatectomy by modulating Wnt/ß-catenin signaling. (2) Methods: Male CBA mice underwent CRLM induction, followed one week later by 70% partial hepatectomy. Mice were treated daily with captopril, a RASi, at 250 mg/kg/day or vehicle control from experimental Day 4. Tumor and liver samples were analyzed for RAS and Wnt signaling markers using qRT-PCR and immunohistochemistry. (3) Results: Treatment with captopril reduced the expression of down-stream Wnt target genes, including a significant reduction in both c-myc and cyclin-D1, despite activating Wnt signaling. This was a tumor-specific response that was not elicited in corresponding liver samples. (4) Conclusions: We report for the first time decreased c-myc expression in colorectal tumors following RASi treatment in vivo. Decreased c-myc expression was accompanied by an attenuated invasive phenotype, despite increased Wnt signaling.

Investigating virus-host cell interactions: Comparative binding forces between hepatitis C virus-like particles and host cell receptors in 2D and 3D cell culture models.

Cell cultures have been successfully used to study hepatitis C virus (HCV) for many years. However, most work has been done using traditional, 2-dimensional (2D) cell cultures (cells grown as a monolayer in growth flasks or dishes). Studies have shown that when cells are grown suspended in an extra-cellular-matrix-like material, they develop into spherical, 'organoid' arrangements of cells (3D growth) that display distinct differences in morphological and functional characteristics compared to 2D cell cultures. In liver organoids, one key difference is the development of clearly differentiated apical and basolateral surfaces separated and maintained by cellular tight junctions. This phenomenon, termed polarity, is vital to normal barrier function of hepatocytes in vivo. It has also been shown that viruses, and virus-like particles, interact very differently with cells derived from 2D as compared to 3D cell cultures, bringing into question the usefulness of 2D cell cultures to study virus-host cell interactions. Here, we investigate differences in cellular architecture as a function of cell culture system, using confocal scanning laser microscopy, and determine differences in binding interactions between HCV virus-like particles (VLPs) and their cognate receptors in the different cell culture systems using atomic force microscopy (AFM). We generated organoid cultures that were polarized, as determined by localization of key apical and basolateral markers. We found that, while uptake of HCV VLPs by both 2D and 3D Huh7 cells was observed by flow cytometry, binding interactions between HCV VLPs and cells were measurable by AFM only on polarized cells. The work presented here adds to the growing body of research suggesting that polarized cell systems are more suitable for the study of HCV infection and dynamics than non-polarized systems.