Increased Prevalence of Alpha-1-Antitrypsin Deficiency in Patients with Biliary Tract Cancer and Its Associated Clinicopathological Features

Alpha-1 antitrypsin deficiency (A1ATD) is underdiagnosed and associated with liver diseases. Here, we genotyped 130 patients with biliary tract cancer (BTC) scheduled for liver resection and found A1ATD in 10.8% of the patients. A1ATD was found in all BTC subtypes, and patients had similar clinical features as non-A1ATD BTC, not permitting their identification using clinical routine liver tests. In intrahepatic cholangiocarcinoma (iCCA), the abundance of A1AT protein was increased in the tumor and appeared to be influenced by the genomic alterations. On the one hand, BTC with A1ATD had lower perineural invasion at histopathology and displayed a longer survival, suggesting that a deficiency in this protein is associated with a less aggressive phenotype. On the other hand, iCCA with high A1AT expression had more advanced tumor staging and enriched pathways for complement system and extracellular matrix interactions, indicating that A1AT protein might contribute to a more aggressive phenotype. With increased awareness, screening, and basic studies, A1ATD could represent one more layer of stratification for future targeted therapies in BTC

Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins

<div><p>Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human hepatocytes, and that recombinant laminin is a promising xeno-free and chemical defined strategy for preservation of hepatocyte specific function <i>in vitro</i>.</p></div

dsDNA concentrations and mRNA expression of Ki67.

<p>The dsDNA concentrations (A) and mRNA expression of Ki67 (B) were evaluated at day six after seeding. No significant differences in dsDNA or Ki67 expression could be noted between different laminin isoforms, Collagen and EHS. The results from five replicates are expressed as mean ± SEM.</p

Characteristics of hepatocytes donors.

<p>Characteristics of hepatocytes donors.</p

Viability of cultured hepatocytes.

<p>Hepatocyte viability during the culture was assessed at day six after seeding by MTT assay. Each laminin isoform supports hepatocyte viability during culture for at least 6 days, and no statistical differences between each laminin, EHS and Collagen were seen. The results are from five replicates and are expressed as mean ± SEM.</p

Morphology of primary human hepatocytes seeded on laminin, EHS, or Collagen.

<p>Isolated primary human hepatocytes were seeded on laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521, EHS, or Collagen pre-coated plates. Hepatocytes cultured on EHS coated plates migrated and formed three-dimensional structures. Hepatocytes cultured on laminins and collagen showed a hexagonal shape in a flat cell monolayer. Isolated human hepatocytes could be incubated on laminins for up to six days without obvious morphological changes.</p

Characteristics of hepatocytes and liver tissue donors for western blot analysis.

<p>Characteristics of hepatocytes and liver tissue donors for western blot analysis.</p

Metabolic activity of isolated hepatocyte.

<p>The mRNA expression of CYP3A4 (A), HNF4a (B), G6P (C), and MRP2 (D) of isolated hepatocytes cultured on laminin were assessed. Hepatocytes cultured on laminins demonstrated no significant differences in mRNA expressions of CYP3A4, HNF4a, G6P, or MRP2 as compared to those of Collagen or EHS. The results are from five replicates and are expressed as mean ± SEM.</p

Productions and mRNA expressions of human albumin and A1AT.

<p>Production and mRNA expression of human albumin (Fig 5A) and human alpha-1-antitrypsin (A1AT; Fig 5B) were evaluated by ELISA and RT-PCR at six days after seeding. Hepatocytes cultured on laminin maintained the production and mRNA expressions of albumin and A1AT as compared to those of hepatocytes on EHS or Collagen. No significant differences in the productions and mRNA expressions of human albumin and human A1AT could be noted among different laminin isoforms, Collagen and EHS. The results from five replicates are expressed as mean ± SEM.</p

Expression of laminin in human liver and isolated hepatocyte.

<p>Expression of laminin was determined in protein samples from liver tissue (LT) and isolated hepatocytes (HC) by western blot analysis. (A; Laminin-α1, B; Laminin-α2, C; Laminin-α3, D; Laminin-α4, E; Laminin-α5, F; Laminin-β1, G; Laminin-β2, H; Laminin-β3, I; Laminin-γ1, J; Laminin-γ2, K; β-actin) Phenotypes of laminins, α2, α3, α4, β1, β3, γ1, and γ2 were detected both in liver tissue and isolated hepatocytes, and α1 and α5 were absent in both. Laminin chain of β2 was inconclusive due to multiple bands. Sample number (#1–#6) is corresponding to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161383#pone.0161383.t002" target="_blank">Table 2</a>. Values were normalized to the level of β-actin in each lane.</p