Characterization of an Immortalized Hepatic Stellate Cell Line Established From Metallothionein-Null Mice

Miura, Nobuhiko; Kanayama, Yoshiharu; Nagai, Wakako; Hasegawa, Tatsuya; Seko, Yoshiyuki; Kaji, Toshiyuki; Naganuma, Akira

doi:10.2131/jts.31.391

Cited by 8 publications

(6 citation statements)

References 27 publications

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“…Cell homogeneity was confirmed by direct microscopic observations, while cells were designated as fibroblastic following confirmation of mesenchymal marker vimentin expression, as well as absence of epithelial marker cytokeratin 19 expression. In addition, normal fibroblasts were characterized as pancreatic stellate cells following confirmation of uptake and storage of vitamin A, by auto-fluorescence stemming from retinyl acetate (Sigma) containing droplets [24]. The resultant (PaSCs) and pancreatic tumor-associated fibroblasts (which were confirmed to have lost the vitamin A droplets) were used for self-derived matrix characterization.…”

Section: Methodsmentioning

confidence: 99%

FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells

et al. 2011

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BackgroundAlterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions.We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer.MethodsWe generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses.ResultsWe observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated.ConclusionCancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and β1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking β1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions.

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Section: Methodsmentioning

confidence: 99%

FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells

et al. 2011

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“…Cd concentration was determined at the mass numbers of 111 m/z. of the liver were analyzed using high performance liquid chromatography-ICP-MS (HPLC/ICP-MS) (Miura et al, 2006). Liver samples which obtained from 2, 6, 12, and 24 hr after the single injection of CdCl 2 as described -lowed by ultracentrifugation at 105,000 x g for 1 hr at 4°C.…”

Section: Accumulation and Distribution Of CD In The Liver After Cdcl mentioning

confidence: 99%

Mechanisms of cadmium-induced chronotoxicity in mice

et al. 2013

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-Biological defense factors show diurnal variations in their expression levels or activities. These variations can induce the different sensitivity to external toxicants of a day. We reported earlier that mice showed clear diurnal variation of cadmium (Cd)-induced toxicity, i.e., chronotoxicity. In this report, we investigated additional new evidences for the cadmium (Cd)-induced chronotoxicity, and considered the mechanisms contributed to this chronotoxicity. Male C57BL/6J mice were injected with CdCl 2 (6.4 mg/kg, one shot) intraperitoneally at 6 different time points of a day (zeitgeber time (ZT); ZT2, ZT6, ZT10, ZT14, ZT18 or ZT22) followed by monitoring the mortality until 14 days after the injection. We observed extreme difference in survival numbers: surprisingly, all mice died at ZT2 injection while all mice survived at ZT18 injection. Moreover, in non-lethal dose of Cd (4.5 mg/kg), the values of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) used as indexes of hepatotoxicity markedly increased at ZT6 injection while mostly unchanged at ZT18 injection. To consider the mechanisms of this extreme diurnal variation, we examined biochemical studies and concluded that the diurnal variation was not caused by the differences in hepatic Cd level, basal hepatic metallothionein (MT) level, and induction level or induction speed of hepatic MT. We suggested that one of the candidate determination factors was glutathione. We believe that the "chronotoxicology" for metal toxicity may be classic, yet new viewpoint

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“…In agreement with this former characterization and the proposed ancestry from HSC, we could confirm the expression of typical HSC markers including Fibronectin, α-smooth muscle actin (α-SMA), collagen type I, and Vimentin. Moreover, the cells were negative for SV40T that was used for immortalization of many other continuous HSC lines, such as LX-1, LX-2, HSC-T6, SV68-IS, A640-IS, IMS/MT (-), IMS/N, and Col-GFP HSC ( Figure 5 ) [ 10 , 15 , 16 , 17 , 18 , 19 ].…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, Western blot analysis showed that the cells are capable of expressing typical HSC markers such as Fibronectin, Vimentin, and α-SMA, while expectedly lacking expression of SV40T ( Figure 5 ). These biochemical features underpin that the cells are of HSC origin and lack SV40T that was used for immortalization of many other HSC lines (e.g., LX-1, LX-2, HSC-T6, SV68-IS, A640-IS, IMS/MT (-), IMS/N, and Col-GFP HSC) [ 10 , 15 , 16 , 17 , 18 , 19 ].…”

Section: Discussionmentioning

confidence: 99%

Rat Hepatic Stellate Cell Line CFSC-2G: Genetic Markers and Short Tandem Repeat Profile Useful for Cell Line Authentication

Nanda

Schröder

Steinlein

et al. 2022

Cells

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Hepatic stellate cells (HSCs) are also known as lipocytes, fat-storing cells, perisinusoidal cells, or Ito cells. These liver-specific mesenchymal cells represent about 5% to 8% of all liver cells, playing a key role in maintaining the microenvironment of the hepatic sinusoid. Upon chronic liver injury or in primary culture, these cells become activated and transdifferentiate into a contractile phenotype, i.e., the myofibroblast, capable of producing and secreting large quantities of extracellular matrix compounds. Based on their central role in the initiation and progression of chronic liver diseases, cultured HSCs are valuable in vitro tools to study molecular and cellular aspects of liver diseases. However, the isolation of these cells requires special equipment, trained personnel, and in some cases needs approval from respective authorities. To overcome these limitations, several immortalized HSC lines were established. One of these cell lines is CFSC, which was originally established from cirrhotic rat livers induced by carbon tetrachloride. First introduced in 1991, this cell line and derivatives thereof (i.e., CFSC-2G, CFSC-3H, CFSC-5H, and CFSC-8B) are now used in many laboratories as an established in vitro HSC model. We here describe molecular features that are suitable for cell authentication. Importantly, chromosome banding and multicolor spectral karyotyping (SKY) analysis demonstrate that the CFSC-2G genome has accumulated extensive chromosome rearrangements and most chromosomes exist in multiple copies producing a pseudo-triploid karyotype. Furthermore, our study documents a defined short tandem repeat (STR) profile including 31 species-specific markers, and a list of genes expressed in CFSC-2G established by bulk mRNA next-generation sequencing (NGS).

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Characterization of an Immortalized Hepatic Stellate Cell Line Established From Metallothionein-Null Mice

Cited by 8 publications

References 27 publications

FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells

FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells

Mechanisms of cadmium-induced chronotoxicity in mice

Rat Hepatic Stellate Cell Line CFSC-2G: Genetic Markers and Short Tandem Repeat Profile Useful for Cell Line Authentication

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