A Developmentally Prometastatic Niche to Hepatoblastoma in Neonatal Liver mediated by the Cxcl1/Cxcr2 Axis

Background and Rationale: Hepatoblastoma (HB) is the most common pediatric liver cancer. Its predominant occurrence in very young children led us to investigating whether the neonatal liver provides a protumorigenic niche to HB development. Methods: HB development was compared between orthotopic transplantation models established in postnatal day 5 and 60 mice (P5Tx and P60Tx models). Single-cell RNA-sequencing was performed using tumor and liver tissues from both models and the top candidate cell types and genes identified are investigated for their roles in HB cell growth, migration, and survival. Results: We found that various HB cell lines including HepG2 cells were consistently and considerably more tumorigenic and metastatic in the P5Tx model than in the P60Tx models. Sc-RNAseq of the P5Tx and P60Tx HepG2 models revealed that the P5Tx tumor was more hypoxic and had a larger number of activated hepatic stellate cells (aHSCs) in the tumor-surrounding liver which express significantly higher levels of Cxcl1 than those from the P60Tx model. We found these differences were developmentally present in normal P5 and P60 liver. We showed that the Cxcl1/Cxcr2 axis mediated HB cell migration and was critical to HB cell survival under hypoxia. Treating HepG2 P60Tx model with recombinant CXCL1 protein induced intrahepatic and pulmonary metastasis and CXCR2 knockout in HepG2 cells abolished their metastatic potential in the P5Tx model. Lastly, we showed that in metastatic HB patient tumors there was a similar larger population of aHSCs in the tumor-surrounding liver than in localized tumors, and tumor hypoxia was uniquely associated with HB patient prognosis among pediatric cancers. Conclusion: We demonstrated that the neonatal liver provides a prometastatic niche to HB development via the Cxcl1/Cxcr2 axis.

Changes in PGC-1α-Dependent Mitochondrial Biogenesis Are Associated with Inflexible Hepatic Energy Metabolism in the Offspring Born to Dexamethasone-Treated Mothers

In the present study we investigated the participation of hepatic peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) in the metabolic programming of newborn rats exposed in utero to dexamethasone (DEX). On the 21st day of life, fasted offspring born to DEX-treated mothers displayed increased conversion of pyruvate into glucose with simultaneous upregulation of PEPCK (phosphoenolpyruvate carboxykinase) and G6Pase (glucose-6-phosphatase). Increased oxidative phosphorylation, higher ATP/ADP ratio and mitochondrial biogenesis and lower pyruvate levels were also found in the progeny of DEX-treated mothers. On the other hand, the 21-day-old progeny of DEX-treated mothers had increased hepatic triglycerides (TAG) and lower CPT-1 activity when subjected to short-term fasting. At the mechanistic level, rats exposed in utero to DEX exhibited increased hepatic PGC-1α protein content with lower miR-29a-c expression. Increased PGC-1α content was concurrent with increased association to HNF-4α and NRF1 and reduced PPARα expression. The data presented herein reveal that changes in the transcription machinery in neonatal liver of rats born to DEX-treated mothers leads to an inflexible metabolic response to fasting. Such programming is hallmarked by increased oxidative phosphorylation of pyruvate with impaired FFA oxidation and hepatic TAG accumulation.

Absence of CD11a Expression Identifies Embryonic Hematopoietic Stem Cell Precursors via Competitive Neonatal Transplantation Assay

Hematopoietic stem cells (HSCs) are defined by their self-renewal, multipotency, and bone marrow (BM) engraftment abilities. How HSCs emerge during embryonic development remains unclear, but are thought to arise from hemogenic endothelium through an intermediate precursor called “pre-HSCs.” Pre-HSCs have self-renewal and multipotent activity, but lack BM engraftability. They can be identified functionally by transplantation into neonatal recipients, or by in vitro co-culture with cytokines and stroma followed by transplantation into adult recipients. While pre-HSCs express markers such as Kit and CD144, a precise surface marker identity for pre-HSCs has remained elusive due to the fluctuating expression of common HSC markers during embryonic development. We have previously determined that the lack of CD11a expression distinguishes HSCs in adults as well as multipotent progenitors in the embryo. Here, we use a neonatal transplantation assay to identify pre-HSC populations in the mouse embryo. We establish CD11a as a critical marker for the identification and enrichment of pre-HSCs in day 10.5 and 11.5 mouse embryos. Our proposed pre-HSC population, termed “11a- eKLS” (CD11a- Ter119- CD43+ Kit+ Sca1+ CD144+), contains all in vivo long-term engrafting embryonic progenitors. This population also displays a cell-cycle status expected of embryonic HSC precursors. Furthermore, we identify the neonatal liver as the likely source of signals that can mature pre-HSCs into BM-engraftable HSCs.

Neonatal thyroxine activation modifies epigenetic programming of the liver

The type 2 deiodinase (D2) in the neonatal liver accelerates local thyroid hormone triiodothyronine (T3) production and expression of T3-responsive genes. Here we show that this surge in T3 permanently modifies hepatic gene expression. Liver-specific Dio2 inactivation (Alb-D2KO) transiently increases H3K9me3 levels during post-natal days 1–5 (P1–P5), and results in methylation of 1,508 DNA sites (H-sites) in the adult mouse liver. These sites are associated with 1,551 areas of reduced chromatin accessibility (RCA) within core promoters and 2,426 within intergenic regions, with reduction in the expression of 1,363 genes. There is strong spatial correlation between density of H-sites and RCA sites. Chromosome conformation capture (Hi-C) data reveals a set of 81 repressed genes with a promoter RCA in contact with an intergenic RCA ~300 Kbp apart, within the same topologically associating domain (χ2 = 777; p < 0.00001). These data explain how the systemic hormone T3 acts locally during development to define future expression of hepatic genes.

Neonatal Selenoenzyme Expression Is Variably Susceptible to Duration of Maternal Selenium Deficiency

Maternal selenium (Se) deficiency is associated with decreased neonatal Se levels, which increases the risk for neonatal morbidities. There is a hierarchy to selenoprotein expression after Se deficiency in adult rodents, depending on the particular protein and organ evaluated. However, it is unknown how limited Se supply during pregnancy impacts neonatal selenoprotein expression. We used an Se-deficient diet to induce perinatal Se deficiency (SeD), initiated 2–4 weeks before onset of breeding and continuing through gestation. Neonatal plasma, liver, heart, kidney, and lung were collected on the day of birth and assessed for selenoproteins, factors required for Se processing, and non-Se containing antioxidant enzymes (AOE). Maternal SeD reduced neonatal circulating and hepatic glutathione peroxidase (GPx) activity, as well as hepatic expression of Gpx1 and selenophosphate synthetase 2 (Sps2). In contrast, the impact of maternal SeD on hepatic thioredoxin reductase 1, hepatic non-Se containing AOEs, as well as cardiac, renal, and pulmonary GPx activity, varied based on duration of maternal exposure to SeD diet. We conclude that the neonatal liver and circulation demonstrate earlier depletion in selenoenzyme activity after maternal SeD. Our data indicate that prolonged maternal SeD may escalate risk to the neonate by progressively diminishing Se-containing AOE across multiple organs.

NEONATAL THYROXINE ACTIVATION MODIFIES EPIGENETIC PROGRAMMING OF THE LIVER

In the neonatal liver, a peak of type 2 deiodinase (D2) activity accelerates local T3 production and the expression of thyroid hormone (TH)-responsive genes. Here we show that this acute increase in T3 signaling permanently modifies hepatic gene expression. Liver-specific Dio2 inactivation (Alb-D2KO) transiently increased H3K9me3 levels during post-natal days 1-5 (P1-P5) in discrete chromatin areas, and methylation of 1,508 DNA sites (H-sites) that remained in the adult mouse liver. These sites were associated with 1,551 areas of reduced chromatin accessibility (RCA; Atac-seq) within core promoters and 2,426 within intergenic regions, with reduction in the expression of 1,525 genes (RNA-seq). There was strong correlation between H-sites and RCA sites (r=0.85; p<0.0002), suggesting a cause-effect relationship. The analysis of chromosome conformation capture (Hi-C) data revealed a set of 57 repressed genes that have a promoter RCA in close contact with an intergenic RCA ~300 Kbp apart, including Foxa2 that plays an important role during development. Thus, the post-natal surge in hepatic D2 activity and TH-signaling prevents discrete DNA methylation and modifies the transcriptome of the adult mouse. This explains how the systemic T3 hormone acts locally during development to define future chromatin accessibility and expression of critically relevant hepatic genes.

The Effect of Prolonged Antenatal IVIG Treatment on the Development of Gestational Alloimmune Liver Disease in the Neonate

Introduction: Gestational Alloimmune Liver Disease (GALD) is a rare disease characterized by subacute fetal liver injury and often accompanied by hepatic and extrahepatic iron deposition. Findings include hypoglycemia, coagulopathy, hypoalbuminemia, elevated serum ferritin, elevated alpha-fetoprotein, and ascites. Extrahepatic hemosiderin deposition is often seen in salivary glands. Previously, the mortality rate was close to 80% with all patients needing liver transplantation. With maternal IVIG treatment and changes in neonatal treatment, there is now less than 20% mortality with infrequent need for liver transplantation. Diagnosis of GALD is typically done on a postmortem analysis. Case Description: A term female infant was born via scheduled c-section to a 32 year old G2P1000 mother who had been receiving weekly IVIG during this pregnancy due to the death of her first child at 4 days of life. Autopsy of that female baby demonstrated extensive neuropathological changes, liver steatosis, iron depletion, and ascites, consistent with GALD. Following delivery of our current patient, there was an elevated alpha-fetoprotein (greater than 80,000), decreased fibrinogen, and coagulopathy with peak international normalized ratio of 1.6. The patient received fresh frozen plasma and IVIG on day of life 1 with improvement of these levels. Complete blood count, liver function tests, and ammonia were within normal limits. An MRI of the liver demonstrated normal size, morphology, and normal iron levels based on T2 relaxometry. A buccal biopsy did not demonstrate extrahepatic iron deposition. MRI of the brain showed significant stenosis of the right transverse and sigmoid sinus relating to dural venous sinus thrombosis. There was no evidence of parenchymal infarction and no evidence of iron deposition. At this time, enoxaparin was initiated. The patient was discharged home on day of life nine on enoxaparin therapy. Discussion: There are few reported cases of patients with GALD, especially after maternal IVIG treatment. This case report exemplifies the effect of antenatal IVIG infusions during subsequent pregnancies in women with a history of GALD in prior children. This effect is protective, evidenced by lack of liver injury noted in this patient. This supports the use of immunotherapy during pregnancy to prevent recurrence of alloimmune injury. References: 1. Is exchange transfusion a possible treatment for neonatal hemochromatosis? Giuseppina Timpani-Francesca Foti-Antonino Nicolò-Pier Nicotina-Emanuele Nicastro-Raffaele Iorio - Journal of Hepatology - 2007 2. Medical and surgical treatment of neonatal hemochromatosis: Single center experience-Thomas Heffron-Todd Pillen-David Welch-Massimo Asolati-Gregory Smallwood-Phil Hagedorn-Carlos Fasola-David Solis-John Rodrigues-Jill Depaolo-James Spivey-Enrique Martinez-Stuart Henry-Rene Romero - Pediatric Transplantation - 2007 3. Neonatal Hemochromatosis: A Congenital Alloimmune Hepatitis - Peter Whitington - Seminars in Liver Disease - 2007 4. Neonatal hemochromatosis: The importance of early recognition of liver failure Pankaj Vohra-Cindy Haller-Sukru Emre-Margret Magid-Ian Holzman-Ming Ye-Elizaveta Iofel-Benjamin Shneider - The Journal of Pediatrics - 2000 5. Neonatal Liver Cirrhosis Without Iron Overload Caused by Gestational Alloimmune Liver Disease. Debray-François Guillaume de Halleux- Virginie Guidi-Ornella Detrembleur-Nancy Gaillez-Stéphanie Rausin-Léon Goyens-Philippe Pan-Xiaomin Whitington Peter-Pediatrics-2012 6. Neonatal Liver Failure and Congenital Cirrhosis due to Gestational Alloimmune Liver Disease: A Case Report and Literature Review Oscar Roos Mariano da Rocha Carolina-Renata Rostirola Guedes- Carlos Oscar Kieling- Marina Rossato Adami- Carlos Thadeu Schmidt Cerski- Sandra Maria Conçalvez Vieria - Hindawi - 2017 Image: (A) MRI liver showing normal appearance without evidence of hemochromatosis (B) MRI brain showing no evidence of iron deposition within the brain parenchyma (C) MRV head showing right transverse and sigmoid venous sinus thrombosis Figure Disclosures No relevant conflicts of interest to declare.