Joerg Schrader

Matrix stiffness modulates proliferation, chemotherapeutic response, and dormancy in hepatocellular carcinoma cells

There is increasing evidence that the physical environment is a critical mediator of tumor behavior. Hepatocellular carcinoma (HCC) develops within an altered biomechanical environment, and increasing matrix stiffness is a strong predictor of HCC development. The aim of this study was to establish whether changes in matrix stiffness, which are characteristic of inflammation and fibrosis, regulate HCC cell proliferation and chemotherapeutic response. Using an in vitro system of “mechanically tunable” matrix‐coated polyacrylamide gels, matrix stiffness was modeled across a pathophysiologically relevant range, corresponding to values encountered in normal and fibrotic livers. Increasing matrix stiffness was found to promote HCC cell proliferation. The proliferative index (assessed by Ki67 staining) of Huh7 and HepG2 cells was 2.7‐fold and 12.2‐fold higher, respectively, when the cells were cultured on stiff (12 kPa) versus soft (1 kPa) supports. This was associated with stiffness‐dependent regulation of basal and hepatocyte growth factor–stimulated mitogenic signaling through extracellular signal‐regulated kinase, protein kinase B (PKB/Akt), and signal transducer and activator of transcription 3. β1‐Integrin and focal adhesion kinase were found to modulate stiffness‐dependent HCC cell proliferation. Following treatment with cisplatin, we observed reduced apoptosis in HCC cells cultured on stiff versus soft (physiological) supports. Interestingly, however, surviving cells from soft supports had significantly higher clonogenic capacity than surviving cells from a stiff microenvironment. This was associated with enhanced expression of cancer stem cell markers, including clusters of differentiation 44 (CD44), CD133, c‐kit, cysteine‐X‐cysteine receptor 4, octamer‐4 (CXCR4), and NANOG. Conclusion: Increasing matrix stiffness promotes proliferation and chemotherapeutic resistance, whereas a soft environment induces reversible cellular dormancy and stem cell characteristics in HCC. This has implications for both the treatment of primary HCC and the prevention of tumor outgrowth from disseminated tumor cells. (HEPATOLOGY 2011;)

Increased angiogenesis protects against adipose hypoxia and fibrosis in metabolic disease-resistant 11β-hydroxysteroid dehydrogenase type 1 (HSD1)deficient mice

Background: Adipose hypertrophy limits fat cell oxygenation, promotes scarring, and associates with increased local glucocorticoid regeneration (higher 11βHSD1 enzyme). Results: 11βHSD1 knock-out mice have reduced scarring and better vascularization and oxygenation in their adipose tissue. Conclusion: Elevated adipose 11βHSD1 contributes to obesity pathogenesis by suppressing adipose angiogenesis. Significance: Enhancement of adipose oxygenation and vascularization is a novel therapeutic modality for 11βHSD1 inhibitors. In obesity, rapidly expanding adipose tissue becomes hypoxic, precipitating inflammation, fibrosis, and insulin resistance. Compensatory angiogenesis may prevent these events. Mice lacking the intracellular glucocorticoid-amplifying enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1−/−) have “healthier” adipose tissue distribution and resist metabolic disease with diet-induced obesity. Here we show that adipose tissues of 11βHSD1−/− mice exhibit attenuated hypoxia, induction of hypoxia-inducible factor (HIF-1α) activation of the TGF-β/Smad3/α-smooth muscle actin (α-SMA) signaling pathway, and fibrogenesis despite similar fat accretion with diet-induced obesity. Moreover, augmented 11βHSD1−/− adipose tissue angiogenesis is associated with enhanced peroxisome proliferator-activated receptor γ (PPARγ)-inducible expression of the potent angiogenic factors VEGF-A, apelin, and angiopoietin-like protein 4. Improved adipose angiogenesis and reduced fibrosis provide a novel mechanism whereby suppression of intracellular glucocorticoid regeneration promotes safer fat expansion with weight gain.

Relaxin modulates human and rat hepatic myofibroblast function and ameliorates portal hypertension in vivo.

Active myofibroblast (MF) contraction contributes significantly to the increased intrahepatic vascular resistance that is the primary cause of portal hypertension (PHT) in cirrhosis. We sought proof of concept for direct therapeutic targeting of the dynamic component of PHT and markers of MF activation using short‐term administration of the peptide hormone relaxin (RLN). We defined the portal hypotensive effect in rat models of sinusoidal PHT and the expression, activity, and function of the RLN‐receptor signaling axis in human liver MFs. The effects of RLN were studied after 8 and 16 weeks carbon tetrachloride intoxication, following bile duct ligation, and in tissue culture models. Hemodynamic changes were analyzed by direct cannulation, perivascular flowprobe, indocyanine green imaging, and functional magnetic resonance imaging. Serum and hepatic nitric oxide (NO) levels were determined by immunoassay. Hepatic inflammation was assessed by histology and serum markers and fibrosis by collagen proportionate area. Gene expression was analyzed by quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR) and western blotting and hepatic stellate cell (HSC)‐MF contractility by gel contraction assay. Increased expression of RLN receptor (RXFP1) was shown in HSC‐MFs and fibrotic liver diseases in both rats and humans. RLN induced a selective and significant reduction in portal pressure in pathologically distinct PHT models, through augmentation of intrahepatic NO signaling and a dramatic reduction in contractile filament expression in HSC‐MFs. Critical for translation, RLN did not induce systemic hypotension even in advanced cirrhosis models. Portal blood flow and hepatic oxygenation were increased by RLN in early cirrhosis. Treatment of human HSC‐MFs with RLN inhibited contractility and induced an antifibrogenic phenotype in an RXFP1‐dependent manner. Conclusion: We identified RXFP1 as a potential new therapeutic target for PHT and MF activation status. (Hepatology 2014;59:1492‐1504)

Persistence of functional hepatocyte-like cells in immune-compromised mice

Background: Human embryonic stem cells (hESCs) can be efficiently differentiated to hepatocyte‐like cells (HLCs) in vitro and demonstrate many of the functions and gene expression found in the adult liver.

Establishment of the First Well-differentiated Human Pancreatic Neuroendocrine Tumor Model

Clinical options for systemic therapy of neuroendocrine tumors (NET) are limited. Development of new drugs requires suitable representative in vitro and in vivo model systems. So far, the unavailability of a human model with a well-differentiated phenotype and typical growth characteristics has impaired preclinical research in NET. Herein, we establish and characterize a lymph node–derived cell line (NT-3) from a male patient with well-differentiated pancreatic NET. Neuroendocrine differentiation and tumor biology was compared with existing NET cell lines BON and QGP-1. In vivo growth was assessed in a xenograft mouse model. The neuroendocrine identity of NT-3 was verified by expression of multiple NET-specific markers, which were highly expressed in NT-3 compared with BON and QGP-1. In addition, NT-3 expressed and secreted insulin. Until now, this well-differentiated phenotype is stable since 58 passages. The proliferative labeling index, measured by Ki-67, of 14.6% ± 1.0% in NT-3 is akin to the original tumor (15%–20%), and was lower than in BON (80.6% ± 3.3%) and QGP-1 (82.6% ± 1.0%). NT-3 highly expressed somatostatin receptors (SSTRs: 1, 2, 3, and 5). Upon subcutaneous transplantation of NT-3 cells, recipient mice developed tumors with an efficient tumor take rate (94%) and growth rate (139% ± 13%) by 4 weeks. Importantly, morphology and neuroendocrine marker expression of xenograft tumors resembled the original human tumor. Implications: High expression of somatostatin receptors and a well-differentiated phenotype as well as a slow growth rate qualify the new cell line as a relevant model to study neuroendocrine tumor biology and to develop new tumor treatments. Mol Cancer Res; 16(3); 496–507. ©2018 AACR.

OC-157 Physiological changes in matrix stiffness modulate hepatic progenitor cell morphology, proliferation and differentiation

Introduction Liver injury is associated with changes in the biochemical and physical properties of the extracellular matrix (ECM). Hepatic progenitor cell (HPC) activation occurs in the context of severe liver injury. ECM stiffness has been shown to direct differentiation in mesenchymal stem cells. However, the effect of mechanical factors, such as ECM stiffness on HPC responses is poorly characterised. We examined the effect of ECM stiffness on HPC proliferation and differentiation. Methods Experiments were undertaken using a murine HPC line (BMOL) and primary murine HPCs. Cell culture experiments were performed using a system of laminin-coated polyacrylamide (PA) gel supports of variable stiffness. The stiffness of the PA supports (expressed as shear modulus) was altered across a physiological range (1–12 kPa) corresponding to values encountered in normal and fibrotic livers. Results Increasing matrix stiffness is associated with enhanced cell spreading. BMOL cells cultured on stiff (12kPa) supports develop prominent actin stress fibres. The projected surface area (mean±SEM) of BMOL cells culture on soft (1kPa) supports was 378±21 μm2 compared to 687±47 μm2 for BMOL cells cultured on stiff (12 kPa) supports (p<0.001). Cell proliferation (Ki67 positivity) increased as a function of increasing matrix stiffness. The proliferative index (PI) of BMOL cells cultured on 2.5 kPa and 12 kPa supports was 7.1-fold (p<0.01) and 11.8-fold higher (p<0.001), respectively, than cells cultured on 1kPa supports. Similarly, in experiments with primary cells, the PI of murine HPCs was 1.7-fold higher (p<0.05) when cells were cultured on stiff (12 kPa) vs soft (1 kPa) supports. Quantitative PCR revealed that BMOL cells cultured on soft (1 kPa) supports up-regulate hepatocyte markers, including; albumin (1.5-fold, p<0.01) and CYP7A1 (1.6-fold, p<0.01), and down-regulate the HPC/biliary marker cytokeratin-19 (0.6-fold, p<0.01), relative to cells on stiff (12kPa) supports. There was no significant change in expression of the biliary epithelial cell markers aquaporin-1 and γ-glutamyl-transferase. Conclusion Physiological changes in ECM stiffness lead to alterations in HPC morphology, proliferation and differentiation. Increased ECM stiffness (as would be encountered in an injured or fibrotic liver) promotes HPC proliferation and expression of the HPC/biliary marker cytokeratin-19. In contrast, a low-stiffness environment is associated with a reduction in cell proliferation and up-regulation of hepatocyte-specific markers. These results suggest that mechanical factors, such as ECM stiffness might regulate HPC responses following liver injury. Competing interests None declared.

P38 Matrix stiffness regulates proliferation, differentiation and chemotherapeutic responsiveness in hepatocellular carcinoma

Introduction The majority (80%) of hepatocellular carcinomas (HCC) develop within the context of advanced liver fibrosis and cirrhosis. Recent studies with ultrasound elastography have demonstrated that increased liver stiffness is a strong predictor of HCC. Aim To establish whether alterations in matrix stiffness regulate the phenotype and chemotherapeutic response of HCC cells. Method Experiments were conducted using a system of ligand-coated polyacrylamide gels of variable stiffness. Matrix stiffness (expressed as shear modulus) was modelled across a physiologically-relevant range (1–12 kPa), corresponding to values encountered in normal and fibrotic livers. Experiments were conducted in two HCC cells lines (Huh7/ HepG2). Results In each cell type, there was a consistent morphological response to changes in matrix stiffness. There was increased cell spreading on stiff gels in association with both stress-fibre and mature focal adhesion formation. Increasing matrix stiffness promoted cellular proliferation. The proliferative index (assessed by Ki67 staining) of Huh7 and HepG2 cells was 2.7-fold (p<0.001) and 12.2-fold (p<0.001) higher, respectively, when the cells were cultured on stiff (12 kPa) vs soft (1 kPa) supports. Cells cultured on soft supports developed a quiescent (dormant) phenotype with marked reduction in cyclinD1/ D3 expression, without upregulation of p21/p27. We postulated that altered sensitivity to mitogenic growth factors mediates the stiffness-dependent regulation of proliferation. Matrix stiffness modulated both the magnitude and time-course of mitogenic signalling in response to HGF, with lower baseline and HGF-induced FAK, ERK and STAT3 pathway activation. Increasing stiffness results in upregulation of mesenchymal markers (including N-cadherin and vimentin), consistent with mesenchymal shift, and down-regulation of differentiated hepatocyte markers (including albumin, α-1-antitrypsin and HNF4). Following treatment with cisplatin, cells cultured on soft supports were more susceptible to apoptosis (PARP/ Caspase-3 cleavage). However, in both Huh7 and HepG2 cells, surviving cells from soft supports had 2.2-fold (p<0.05) and 2.4-fold (p<0.001) higher clonogenic capacity respectively, than surviving cells from stiff supports. This was associated with upregulation of cancer stem cell markers (Oct4, NANOG, CD44, CD133, c-kit and CXCR4). Conclusion HCC is a tumour that develops within an altered biomechanical niche. Increasing matrix stiffness regulates HCC mitogenic signalling, proliferation, differentiation and chemotherapeutic resistance. However, a soft microenvironment (as may be encountered by disseminated tumour cells) promotes stem cell characteristics following chemotherapy. This provides a possible explanation for the failure of systemic chemotherapy both in relation to treatment of primary HCCs and the eradication of disseminated tumour cells that give rise to metastases. The selective targeting of the cytoskeleton represents a potentially novel approach to the treatment of HCC.