ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3Gal1) synthesis of Siglec ligands mediates anti-tumour immunity in prostate cancer.

Immune checkpoint blockade has yet to produce robust anti-cancer responses for prostate cancer. Sialyltransferases have been shown across several solid tumours, including breast, melanoma, colorectal and prostate to promote immune suppression by synthesising sialoglycans, which act as ligands for Siglec receptors. We report that ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3Gal1) levels negatively correlate with androgen signalling in prostate tumours. We demonstrate that ST3Gal1 plays an important role in modulating tumour immune evasion through the synthesises of sialoglycans with the capacity to engage the Siglec-7 and Siglec-9 immunoreceptors preventing immune clearance of cancer cells. Here, we provide evidence of the expression of Siglec-7/9 ligands and their respective immunoreceptors in prostate tumours. These interactions can be modulated by enzalutamide and may maintain immune suppression in enzalutamide treated tumours. We conclude that the activity of ST3Gal1 is critical to prostate cancer anti-tumour immunity and provide rationale for the use of glyco-immune checkpoint targeting therapies in advanced prostate cancer.

Synthesis and preclinical evaluation of a 89Zr-labelled human single chain antibody for non-invasive detection of hepatic myofibroblasts in acute liver injury.

Synaptophysin is expressed on fibrogenic hepatic myofibroblasts. C1-3 is a single chain human antibody (scAb) that binds specifically to synaptophysin on hepatic myofibroblasts, providing a targeting vector for novel in vivo imaging agents of chronic liver disease. C1-3 and a negative control scAb, CSBD9, were radiolabelled with zirconium-89 via desferrioxamine chelation to enable non-invasive molecular imaging with positron emission tomography (PET). DFO-scAb conjugates were characterised by gel electrophoresis (SDS-PAGE) and MALDI-TOF spectrometry, and 89Zr-labelled with high radiolabelling efficiency (99%). [89Zr]Zr-DFO-C1-3 exhibited high in vitro stability (> 99%) in mouse and human sera over 3 days at 25 and 37 °C. Activated hepatic myofibroblasts incubated with [89Zr]Zr-DFO-C1-3 displayed significantly higher internalised activity (59.46%, P = 0.001) compared to the [89Zr]Zr-DFO-CSBD9 control, indicating synaptophysin-mediated uptake and high binding specificity of [89Zr]Zr-DFO-C1-3. Mice with CCl4-induced acute liver damage exhibited significantly higher liver uptake of [89Zr]Zr-DFO-C1-3, compared to controls, confirmed by both Cerenkov imaging and ex vivo gamma counting (4.41 ± 0.19%ID/g, P < 0.0001). CCl4-induced liver damage and the number of hepatic myofibroblasts was confirmed by αSMA staining of liver sections. These findings indicate that [89Zr]Zr-DFO-C1-3 has promising utility as a PET imaging agent for non-invasive detection of hepatic myofibroblasts following acute liver injury.

Harnessing neutrophil plasticity for HCC immunotherapy.

Neutrophils, until recently, have typically been considered a homogeneous population of terminally differentiated cells with highly conserved functions in homeostasis and disease. In hepatocellular carcinoma (HCC), tumour-associated neutrophils (TANs) are predominantly thought to play a pro-tumour role, promoting all aspects of HCC development and progression. Recent developments in single-cell technologies are now providing a greater insight and appreciation for the level of cellular heterogeneity displayed by TANs in the HCC tumour microenvironment, which we have been able to correlate with other TAN signatures in datasets for gastric cancer, pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). TANs with classical pro-tumour signatures have been identified as well as neutrophils primed for anti-tumour functions that, if activated and expanded, could become a potential therapeutic approach. In recent years, therapeutic targeting of neutrophils in HCC has been typically focused on impairing the recruitment of pro-tumour neutrophils. This has now been coupled with immune checkpoint blockade with the aim to stimulate lymphocyte-mediated anti-tumour immunity whilst impairing neutrophil-mediated immunosuppression. As a result, neutrophil-directed therapies are now entering clinical trials for HCC. Pharmacological targeting along with ex vivo reprogramming of neutrophils in HCC patients is, however, in its infancy and a greater understanding of neutrophil heterogeneity, with a view to exploit it, may pave the way for improved immunotherapy outcomes. This review will cover the recent developments in our understanding of neutrophil heterogeneity in HCC and how neutrophils can be harnessed to improve HCC immunotherapy.

The exercise IL-6 enigma in cancer.

Interleukin (IL)-6 elicits both anticancer and procancer effects depending on the context, which we have termed the 'exercise IL-6 enigma'. IL-6 is released from skeletal muscles during exercise to regulate short-term energy availability. Exercise-induced IL-6 provokes biological effects that may protect against cancer by improving insulin sensitivity, stimulating the production of anti-inflammatory cytokines, mobilising immune cells, and reducing DNA damage in early malignant cells. By contrast, IL-6 continuously produced by leukocytes in inflammatory sites drives tumorigenesis by promoting chronic inflammation and activating tumour-promoting signalling pathways. How can a molecule have such opposing effects on cancer? Here, we review the roles of IL-6 in chronic inflammation, tumorigenesis, and exercise-associated cancer prevention and define the factors that underpin the exercise IL-6 enigma.

Anti-Cholestatic Therapy with Obeticholic Acid Improves Short-Term Memory in Bile Duct-Ligated Mice.

Patients with cholestatic liver disease, including those with primary biliary cholangitis, can experience symptoms of impaired cognition or brain fog. This phenomenon remains unexplained and is currently untreatable. Bile duct ligation (BDL) is an established rodent model of cholestasis. In addition to liver changes, BDL animals develop cognitive symptoms early in the disease process (before development of cirrhosis and/or liver failure). The cellular mechanisms underpinning these cognitive symptoms are poorly understood. Herein, the study explored the neurocognitive symptom manifestations, and tested potential therapies, in BDL mice, and used human neuronal cell cultures to explore translatability to humans. BDL animals exhibited short-term memory loss and showed reduced astrocyte coverage of the blood-brain barrier, destabilized hippocampal network activity, and neuronal senescence. Ursodeoxycholic acid (first-line therapy for most human cholestatic diseases) did not reverse symptomatic or mechanistic aspects. In contrast, obeticholic acid (OCA), a farnesoid X receptor agonist and second-line anti-cholestatic agent, normalized memory function, suppressed blood-brain barrier changes, prevented hippocampal network deficits, and reversed neuronal senescence. Co-culture of human neuronal cells with either BDL or human cholestatic patient serum induced cellular senescence and increased mitochondrial respiration, changes that were limited again by OCA. These findings provide new insights into the mechanism of cognitive symptoms in BDL animals, suggesting that OCA therapy or farnesoid X receptor agonism could be used to limit cholestasis-induced neuronal senescence.

c-Rel-dependent Chk2 signaling regulates the DNA damage response limiting hepatocarcinogenesis.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. The NF-κB transcription factor family subunit c-Rel is typically protumorigenic; however, it has recently been reported as a tumor suppressor. Here, we investigated the role of c-Rel in HCC. APPROACH AND RESULTS: Histological and transcriptional studies confirmed expression of c-Rel in human patients with HCC, but low c-Rel expression correlated with increased tumor cell proliferation and mutational burden and was associated with advanced disease. In vivo , global ( Rel-/- ) and epithelial specific ( RelAlb ) c-Rel knockout mice develop more tumors, with a higher proliferative rate and increased DNA damage, than wild-type (WT) controls 30 weeks after N-diethylnitrosamine injury. However, tumor burden was comparable when c-Rel was deleted in hepatocytes once tumors were established, suggesting c-Rel signaling is important for preventing HCC initiation after genotoxic injury, rather than for HCC progression. In vitro , Rel-/- hepatocytes were more susceptible to genotoxic injury than WT controls. ATM-CHK2 DNA damage response pathway proteins were suppressed in Rel-/- hepatocytes following genotoxic injury, suggesting that c-Rel is required for effective DNA repair. To determine if c-Rel inhibition sensitizes cancer cells to chemotherapy, by preventing repair of chemotherapy-induced DNA damage, thus increasing tumor cell death, we administered single or combination doxorubicin and IT-603 (c-Rel inhibitor) therapy in an orthotopic HCC model. Indeed, combination therapy was more efficacious than doxorubicin alone. CONCLUSION: Hepatocyte c-Rel signaling limits genotoxic injury and subsequent HCC burden. Inhibiting c-Rel as an adjuvant therapy increased the effectiveness of DNA damaging agents and reduced HCC growth.

Claspin haploinsufficiency leads to defects in fertility, hyperplasia and an increased oncogenic potential.

Claspin is an adaptor protein required for ATR-dependent phosphorylation of CHK1 during S-phase following DNA replication stress. Claspin expression is highly variable in cancer, with low levels frequently correlating with poor patient survival. To learn more about the biological consequences of reduced Claspin expression and its effects on tumorigenesis, we investigated mice with a heterozygous knockout of the Clspn gene. Claspin haploinsufficiency resulted in reduced female fertility and a maternally inherited defect in oocyte meiosis I cell cycle progression. Furthermore, aged Clspn+/- mice developed spontaneous lymphoid hyperplasia and increased susceptibility to non-alcoholic fatty liver disease. Importantly, we demonstrate a tumour suppressor role for Claspin. Reduced Claspin levels result in increased liver damage and tumourigenesis in the DEN model of hepatocellular carcinoma. These data reveal that Clspn haploinsufficiency has widespread unanticipated biological effects and establishes the importance of Claspin as a regulatory node controlling tumorigenesis and multiple disease aetiologies.

CXCR2 inhibition enables NASH-HCC immunotherapy.

OBJECTIVE: Hepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy. DESIGN: Neutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry. RESULTS: Neutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoural XCR1+ dendritic cell activation and CD8+ T cell numbers which are associated with anti-tumoural immunity, this was confirmed by loss of therapeutic effect on genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8+ T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a protumour to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8+ T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs. CONCLUSION: CXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC.

Metabolic dysfunction and cancer in HCV: Shared pathways and mutual interactions.

HCV hijacks many host metabolic processes in an effort to aid viral replication. The resulting hepatic metabolic dysfunction underpins many of the hepatic and extrahepatic manifestations of chronic hepatitis C (CHC). However, the natural history of CHC is also substantially influenced by the host metabolic status: obesity, insulin resistance and hepatic steatosis are major determinants of CHC progression toward hepatocellular carcinoma (HCC). Direct-acting antivirals (DAAs) have transformed the treatment and natural history of CHC. While DAA therapy effectively eradicates the virus, the long-lasting overlapping metabolic disease can persist, especially in the presence of obesity, increasing the risk of liver disease progression. This review covers the mechanisms by which HCV tunes hepatic and systemic metabolism, highlighting how systemic metabolic disturbance, lipotoxicity and chronic inflammation favour disease progression and a precancerous niche. We also highlight the therapeutic implications of sustained metabolic dysfunction following sustained virologic response as well as considerations for patients who develop HCC on the background of metabolic dysfunction.

Neutrophils as potential therapeutic targets in hepatocellular carcinoma.

The success of atezolizumab plus bevacizumab treatment contributed to a shift in systemic therapies for hepatocellular carcinoma (HCC) towards combinations that include cancer immunotherapeutic agents. Thus far, the principal focus of cancer immunotherapy has been on interrupting immune checkpoints that suppress antitumour lymphocytes. As well as lymphocytes, the HCC environment includes numerous other immune cell types, among which neutrophils are emerging as an important contributor to the pathogenesis of HCC. A growing body of evidence supports neutrophils as key mediators of the immunosuppressive environment in which some cancers develop, as well as drivers of tumour progression. If neutrophils have a similar role in HCC, approaches that target or manipulate neutrophils might have therapeutic benefits, potentially including sensitization of tumours to conventional immunotherapy. Several neutrophil-directed therapies for patients with HCC (and other cancers) are now entering clinical trials. This Review outlines the evidence in support of neutrophils as drivers of HCC and details their mechanistic roles in development, progression and metastasis, highlighting the reasons that neutrophils are well worth investigating despite the challenges associated with studying them. Neutrophil-modulating anticancer therapies entering clinical trials are also summarized.

Sulfatase-2 from Cancer Associated Fibroblasts: An Environmental Target for Hepatocellular Carcinoma?

Introduction: Heparin sulphate proteoglycans in the liver tumour microenvironment (TME) are key regulators of cell signalling, modulated by sulfatase-2 (SULF2). SULF2 overexpression occurs in hepatocellular carcinoma (HCC). Our aims were to define the nature and impact of SULF2 in the HCC TME. Methods: In liver biopsies from 60 patients with HCC, expression and localization of SULF2 were analysed associated with clinical parameters and outcome. Functional and mechanistic impacts were assessed with immunohistochemistry (IHC), in silico using The Cancer Genome Atlas (TGCA), in primary isolated cancer activated fibroblasts, in monocultures, in 3D spheroids, and in an independent cohort of 20 patients referred for sorafenib. IHC targets included αSMA, glypican-3, ß-catenin, RelA-P-ser536, CD4, CD8, CD66b, CD45, CD68, and CD163. SULF2 impact of peripheral blood mononuclear cells was assessed by migration assays, with characterization of immune cell phenotype using fluorescent activated cell sorting. Results: We report that while SULF2 was expressed in tumour cells in 15% (9/60) of cases, associated with advanced tumour stage and type 2 diabetes, SULF2 was more commonly expressed in cancer-associated fibroblasts (CAFs) (52%) and independently associated with shorter survival (7.2 vs. 29.2 months, p = 0.003). Stromal SULF2 modulated glypican-3/ß-catenin signalling in vitro, although in vivo associations suggested additional mechanisms underlying the CAF-SULF2 impact on prognosis. Stromal SULF2 was released by CAFS isolated from human HCC. It was induced by TGFß1, promoted HCC proliferation and sorafenib resistance, with CAF-SULF2 linked to TGFß1 and immune exhaustion in TGCA HCC patients. Autocrine activation of PDGFRß/STAT3 signalling was evident in stromal cells, with the release of the potent monocyte/macrophage chemoattractant CCL2 in vitro. In human PBMCs, SULF2 preferentially induced the migration of macrophage precursors (monocytes), inducing a phenotypic change consistent with immune exhaustion. In human HCC tissues, CAF-SULF2 was associated with increased macrophage recruitment, with tumouroid studies showing stromal-derived SULF2-induced paracrine activation of the IKKß/NF-κB pathway, tumour cell proliferation, invasion, and sorafenib resistance. Conclusion: SULF2 derived from CAFs modulates glypican-3/ß-catenin signalling but also the HCC immune TME, associated with tumour progression and therapy resistance via activation of the TAK1/IKKß/NF-κB pathway. It is an attractive target for combination therapies for patients with HCC.

Key features of the environment promoting liver cancer in the absence of cirrhosis.

The prevalence of obesity and non-alcoholic fatty liver disease (NAFLD) associated hepatocellular carcinoma (HCC) is rising, even in the absence of cirrhosis. We aimed to develop a murine model that would facilitate further understanding of NAFLD-HCC pathogenesis. A total of 144 C3H/He mice were fed either control or American lifestyle (ALIOS) diet, with or without interventions, for up to 48 weeks of age. Gross, liver histology, immunohistochemistry (IHC) and RNA-sequencing data were interpreted alongside human datasets. The ALIOS diet promoted obesity, elevated liver weight, impaired glucose tolerance, non-alcoholic fatty liver disease (NAFLD) and spontaneous HCC. Liver weight, fasting blood glucose, steatosis, lobular inflammation and lipogranulomas were associated with development of HCC, as were markers of hepatocyte proliferation and DNA damage. An antioxidant diminished cellular injury, fibrosis and DNA damage, but not lobular inflammation, lipogranulomas, proliferation and HCC development. An acquired CD44 phenotype in macrophages was associated with type 2 diabetes and NAFLD-HCC. In this diet induced NASH and HCC (DINAH) model, key features of obesity associated NAFLD-HCC have been reproduced, highlighting roles for hepatic steatosis and proliferation, with the acquisition of lobular inflammation and CD44 positive macrophages in the development of HCC-even in the absence of progressive injury and fibrosis.

A Mammalian Target of Rapamycin-Perilipin 3 (mTORC1-Plin3) Pathway is essential to Activate Lipophagy and Protects Against Hepatosteatosis.

BACKGROUND AND AIMS: NAFLD is the most common hepatic pathology in western countries and no treatment is currently available. NAFLD is characterized by the aberrant hepatocellular accumulation of fatty acids in the form of lipid droplets (LDs). Recently, it was shown that liver LD degradation occurs through a process termed lipophagy, a form of autophagy. However, the molecular mechanisms governing liver lipophagy are elusive. Here, we aimed to ascertain the key molecular players that regulate hepatic lipophagy and their importance in NAFLD. APPROACH AND RESULTS: We analyzed the formation and degradation of LD in vitro (fibroblasts and primary mouse hepatocytes), in vivo and ex vivo (mouse and human liver slices) and focused on the role of the autophagy master regulator mammalian target of rapamycin complex (mTORC) 1 and the LD coating protein perilipin (Plin) 3 in these processes. We show that the autophagy machinery is recruited to the LD on hepatic overload of oleic acid in all experimental settings. This led to activation of lipophagy, a process that was abolished by Plin3 knockdown using RNA interference. Furthermore, Plin3 directly interacted with the autophagy proteins focal adhesion interaction protein 200 KDa and autophagy-related 16L, suggesting that Plin3 functions as a docking protein or is involved in autophagosome formation to activate lipophagy. Finally, we show that mTORC1 phosphorylated Plin3 to promote LD degradation. CONCLUSIONS: These results reveal that mTORC1 regulates liver lipophagy through a mechanism dependent on Plin3 phosphorylation. We propose that stimulating this pathway can enhance lipophagy in hepatocytes to help protect the liver from lipid-mediated toxicity, thus offering a therapeutic strategy in NAFLD.

Immunomodulatory Effects of Lenvatinib Plus Anti-Programmed Cell Death Protein 1 in Mice and Rationale for Patient Enrichment in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Lenvatinib is an effective drug in advanced HCC. Its combination with the anti-PD1 (programmed cell death protein 1) immune checkpoint inhibitor, pembrolizumab, has generated encouraging results in phase Ib and is currently being tested in phase III trials. Here, we aimed to explore the molecular and immunomodulatory effects of lenvatinib alone or in combination with anti-PD1. APPROACH AND RESULTS: We generated three syngeneic models of HCC in C57BL/6J mice (subcutaneous and orthotopic) and randomized animals to receive placebo, lenvatinib, anti-PD1, or combination treatment. Flow cytometry, transcriptomic, and immunohistochemistry analyses were performed in tumor and blood samples. A gene signature, capturing molecular features associated with the combination therapy, was used to identify a subset of candidates in a cohort of 228 HCC patients who might respond beyond what is expected for monotherapies. In mice, the combination treatment resulted in tumor regression and shorter time to response compared to monotherapies (P < 0.001). Single-agent anti-PD1 induced dendritic and T-cell infiltrates, and lenvatinib reduced the regulatory T cell (Treg) proportion. However, only the combination treatment significantly inhibited immune suppressive signaling, which was associated with the TGFß pathway and induced an immune-active microenvironment (P < 0.05 vs. other therapies). Based on immune-related genomic profiles in human HCC, 22% of patients were identified as potential responders beyond single-agent therapies, with tumors characterized by Treg cell infiltrates, low inflammatory signaling, and VEGFR pathway activation. CONCLUSIONS: Lenvatinib plus anti-PD1 exerted unique immunomodulatory effects through activation of immune pathways, reduction of Treg cell infiltrate, and inhibition of TGFß signaling. A gene signature enabled the identification of ~20% of human HCCs that, although nonresponding to single agents, could benefit from the proposed combination.

Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner.

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease.

Suppression of insulin-induced gene 1 (INSIG1) function promotes hepatic lipid remodelling and restrains NASH progression.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a silent pandemic associated with obesity and the metabolic syndrome, and also increases cardiovascular- and cirrhosis-related morbidity and mortality. A complete understanding of adaptive compensatory metabolic programmes that modulate non-alcoholic steatohepatitis (NASH) progression is lacking. METHODS AND RESULTS: Transcriptomic analysis of liver biopsies in patients with NASH revealed that NASH progression is associated with rewiring of metabolic pathways, including upregulation of de novo lipid/cholesterol synthesis and fatty acid remodelling. The modulation of these metabolic programmes was achieved by activating sterol regulatory element-binding protein (SREBP) transcriptional networks; however, it is still debated whether, in the context of NASH, activation of SREBPs acts as a pathogenic driver of lipotoxicity, or rather promotes the biosynthesis of protective lipids that buffer excessive lipid accumulation, preventing inflammation and fibrosis. To elucidate the pathophysiological role of SCAP/SREBP in NASH and wound-healing response, we used an Insig1 deficient (with hyper-efficient SREBPs) murine model challenged with a NASH-inducing diet. Despite enhanced lipid and cholesterol biosynthesis, Insig1 KO mice had similar systemic metabolism and insulin sensitivity to Het/WT littermates. Moreover, activating SREBPs resulted in remodelling the lipidome, decreased hepatocellular damage, and improved wound-healing responses. CONCLUSIONS: Our study provides actionable knowledge about the pathways and mechanisms involved in NAFLD pathogenesis, which may prove useful for developing new therapeutic strategies. Our results also suggest that the SCAP/SREBP/INSIG1 trio governs transcriptional programmes aimed at protecting the liver from lipotoxic insults in NASH.

Non-invasive synchronous monitoring of neutrophil migration using whole body near-infrared fluorescence-based imaging.

Advances in fluorescence imaging coupled with the generation of near infrared probes have significantly improved the capabilities of non-invasive, real-time imaging in whole animals. In this study we were able to overcome a limitation of in vivo fluorescence imaging and have established a dual cell tracking method where two different cell types can be monitored according to the spectral signature of the cell labelling fluorophore. Using a mouse model of acute liver injury, we have characterised the in vivo migration patterns of wild type and transgenic neutrophils with impaired chemotaxis. Here, we were able to demonstrate that IVIS provides a sensitive multiplexing technology to differentiate two different cell populations based on the spectral signature of the cell labelling fluorophores. This spectral unmixing methodology has the potential to uncover multidimensional cellular interactions involved in many diseases such as fibrosis and cancer. In vivo spectral un-mixing provides a useful tool for monitoring multiple biological process in real-time in the same animal.

Lipid Remodeling in Hepatocyte Proliferation and Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Hepatocytes undergo profound metabolic rewiring when primed to proliferate during compensatory regeneration and in hepatocellular carcinoma (HCC). However, the metabolic control of these processes is not fully understood. In order to capture the metabolic signature of proliferating hepatocytes, we applied state-of-the-art systems biology approaches to models of liver regeneration, pharmacologically and genetically activated cell proliferation, and HCC. APPROACH AND RESULTS: Integrating metabolomics, lipidomics, and transcriptomics, we link changes in the lipidome of proliferating hepatocytes to altered metabolic pathways including lipogenesis, fatty acid desaturation, and generation of phosphatidylcholine (PC). We confirm this altered lipid signature in human HCC and show a positive correlation of monounsaturated PC with hallmarks of cell proliferation and hepatic carcinogenesis. CONCLUSIONS: Overall, we demonstrate that specific lipid metabolic pathways are coherently altered when hepatocytes switch to proliferation. These represent a source of targets for the development of therapeutic strategies and prognostic biomarkers of HCC.