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.
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