Andrew Graham Cox

Recent evidence has highlighted the importance of employing culture media designed to emulate the metabolic environment cells would be exposed to in vivo. Here, we utilize the physiologic medium Plasmax to examine the impact of nutrient availability on the human hepatocyte cell line, HepG2. Incubation of HepG2 cells in Plasmax suppressed a transcriptional program driven by Hepatocyte Nuclear Factor 4 (HNF4A), a master regulator of hepatocyte identity, leading to a dedifferentiated phenotype. Given that HepG2 cells were originally isolated from a patient with hepatoblastoma, this suggested reversion to the native state in physiologic medium. Importantly, exclusion of iron from Plasmax reinstated the HNF4A-driven transcriptional program. These studies suggest a relationship between iron availability and the fidelity of hepatocyte cell fate and highlight the importance of more faithfully recapitulating in vivo metabolite availability in vitro.

Chronic exposure of local fibroblasts to the reactive tumour niche results in the emergence of cancer-associated fibroblasts (CAFs), which can be classed into multiple subtypes with distinct functional and molecular characteristics. Of these subtypes, myofibroblastic CAFs (myCAFs), contribute to tumour progression primarily by depositing and remodelling extracellular matrix (ECM) within the tumour microenvironment (TME). ECM production is known to be a metabolically demanding process, but specific metabolic pathways that are reprogrammed during myCAF activation are poorly understood. Here, we show that cholesterol biosynthesis is elevated in myCAFs relative to normal fibroblasts. We find that cholesterol is specifically enriched in the myCAF endoplasmic reticulum (ER) membrane and is associated with augmented ER mass and enhanced ER secretory function. Importantly, we demonstrate that pharmacological or genetic inhibition of cholesterol biosynthesis plays a critical role in regulating the ECM-secreting and ECM remodelling function of myCAFs. Moreover, we show that inhibition of cholesterol biosynthesis in myCAFs markedly constrains mammary tumour metastasis in vivo . These studies suggest that cholesterol biosynthesis represents a metabolic vulnerability that can be exploited to normalise myCAF-mediated ECM dysfunction in the TME.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays a role in the regulation of redox homeostasis and cellular metabolism. Activating mutations in the NRF2 pathway have been identified in approximately 15% of liver cancer patients. However, the mechanisms by which NRF2 promotes liver tumorigenesis are poorly understood. Employing a transgenic zebrafish model with hepatocyte-specific, inducible expression of a clinically relevant constitutively active NRF2 mutant (NRF2 ^T80K ), we show that constitutive activation of NRF2 drives hepatocyte to cholangiocyte transdifferentiation. Importantly, we demonstrate that NRF2 affects liver cell plasticity in a cell-autonomous, evolutionarily conserved, and reversible manner. Utilizing an epigenetic-focused chemical screen, the BRG1/BRM inhibitor FHD-286 was identified as a potent suppressor of NRF2-driven transdifferentiation. Overall, our study reveals a novel role for NRF2 in the regulation of liver cell plasticity during tumour initiation and identifies a therapeutic approach to overcome the oncogenic activity of NRF2.

Cancer-associated cachexia is a severe metabolic syndrome marked by dramatic loss of adipose and muscle mass. Although preclinical models have advanced our understanding of cachexia, there are still no approved therapies due to the limited insights into the mechanisms underlying tissue wasting. Here, we utilise a YAP-driven model of liver cancer in zebrafish, which rapidly develops cachexia, to uncover an evolutionarily conserved role for bile acid disruption in the onset of cachexia. Spatial transcriptomic analysis revealed that YAP induces a bi-lineage cholangiocarcinoma phenotype, which was associated with bile acid dysregulation. Mechanistically, we establish that both bile acid synthesis (via CYP7A1) and signalling through the bile acid receptor TGR5 are essential for cachexia induction. Notably, we find that the promotion of bile acid excretion with odevixibat ameliorates cachexia. Together, our findings reveal an evolutionarily conserved mechanism by which YAP promotes cachexia and suggest a potential therapeutic strategy to treat the syndrome.

This Protocol Extension describes adapting an existing cryoinjury protocol to study heart regeneration in zebrafish ¹ . We recently characterized a novel hepatic cryoinjury model that induces localized necrosis, inflammation, and fibrosis, which resolves as the liver regenerates. Here, we present a detailed description of the hepatic cryoinjury procedure. One of the key advantages of this model is that it allows direct comparison of healthy and injured liver parenchyma within individual animals. Our hepatic cryoinjury model provides a rapid and reproducible platform to investigate the molecular and cellular mechanisms underlying fibrosis and liver regeneration. This Protocol Extension aims to extend the reach of the cryoinjury approach for deployment in studying liver regeneration.

Recent evidence has highlighted the importance of employing culture media designed to emulate the metabolic environment Here, we utilize the physiological medium Plasmax to examine the impact of nutrient availability on the human hepatocyte cell line, HepG2. Incubation of HepG2 cells in Plasmax suppressed a transcriptional program driven by Hepatocyte Nuclear Factor 4 (HNF4A), a master regulator of hepatocyte identity. Given that HepG2 cells were originally isolated from a patient with hepatoblastoma, this suggests reversion to the native state in physiological media. Importantly, exclusion of iron from Plasmax reinstated the HNF4A-driven transcriptional program. These studies suggest a relationship between iron availability and the fidelity of hepatocyte cell fate and highlight the importance of more faithfully recapitulating in vivo metabolite availability in vitro .

The optical transparency of zebrafish larvae enables visualization of subcellular structures in intact organs, and these vertebrates are widely used to study lipid biology and liver disease. Lipid droplet (LD) presence is a prevalent feature of healthy cells, but, under conditions such as nutrient excess, toxicant exposure or metabolic imbalance, LD accumulation in hepatocytes can be a harbinger of more severe forms of liver disease. We undertook a comprehensive analysis of approaches useful to investigate LD distribution and dynamics in physiological and pathological conditions in the liver of zebrafish larvae. This comparative analysis of the lipid dyes Oil Red O, Nile Red, LipidTox and LipidSpot, as well as transgenic LD reporters that rely on EGFP fusions of the LD-decorating protein perilipin 2 (PLIN2), demonstrates the strengths and limitations of each approach. These protocols are amenable to detection methods ranging from low-resolution stereomicroscopy to confocal imaging, which enables measurements of hepatic LD size, number and dynamics at cellular resolution in live and fixed animals. This resource will benefit investigators studying LD biology in zebrafish disease models.

The liver is a remarkable organ that can regenerate in response to injury. Depending on the extent of injury, the liver can undergo compensatory hyperplasia or fibrosis. Despite decades of research, the molecular mechanisms underlying these processes are poorly understood. Here, we developed a new model to study liver regeneration based on cryoinjury. To visualise liver regeneration at cellular resolution, we adapted the CUBIC tissue-clearing approach. Hepatic cryoinjury induced a localised necrotic and apoptotic lesion characterised by inflammation and infiltration of innate immune cells. After this initial phase, we observed fibrosis, which resolved as regeneration re-established homeostasis in 30 days. Importantly, this approach enables the comparison of healthy and injured parenchyma within an individual animal, providing unique advantages to previous models. In summary, the hepatic cryoinjury model provides a fast and reproducible method for studying the cellular and molecular pathways underpinning fibrosis and liver regeneration.