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. 2023 Jan 13;13(1):748.
doi: 10.1038/s41598-023-27997-3.

Influence of extracellular matrix composition on tumour cell behaviour in a biomimetic in vitro model for hepatocellular carcinoma

Carlemi Calitz  1 , Jenny Rosenquist  2 , Oliver Degerstedt  3 , Jaafar Khaled  1 , Maria Kopsida  1 , Mårten Fryknäs  4 , Hans Lennernäs  3 , Ayan Samanta  2 , Femke Heindryckx  5
Affiliations

Influence of extracellular matrix composition on tumour cell behaviour in a biomimetic in vitro model for hepatocellular carcinoma

Carlemi Calitz et al. Sci Rep. .

Abstract

The tumor micro-environment (TME) of hepatocellular carcinoma (HCC) consists out of cirrhotic liver tissue and is characterized by an extensive deposition of extracellular matrix proteins (ECM). The evolution from a reversible fibrotic state to end-stage of liver disease, namely cirrhosis, is characterized by an increased deposition of ECM, as well as changes in the exact ECM composition, which both contribute to an increased liver stiffness and can alter tumor phenotype. The goal of this study was to assess how changes in matrix composition and stiffness influence tumor behavior. HCC-cell lines were grown in a biomimetic hydrogel model resembling the stiffness and composition of a fibrotic or cirrhotic liver. When HCC-cells were grown in a matrix resembling a cirrhotic liver, they increased proliferation and protein content, compared to those grown in a fibrotic environment. Tumour nodules spontaneously formed outside the gels, which appeared earlier in cirrhotic conditions and were significantly larger compared to those found outside fibrotic gels. These tumor nodules had an increased expression of markers related to epithelial-to-mesenchymal transition (EMT), when comparing cirrhotic to fibrotic gels. HCC-cells grown in cirrhotic gels were also more resistant to doxorubicin compared with those grown in fibrotic gels or in 2D. Therefore, altering ECM composition affects tumor behavior, for instance by increasing pro-metastatic potential, inducing EMT and reducing response to chemotherapy.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Liver stiffness determined via parallel plate rheology in different 3D hydrogels and liver samples derived from a DEN-induced model for HCC). (A) Storage modulus of 3D hydrogels resembling a cirrhotic and fibrotic liver, grown with cells on day 1, 11 and 21. (B) Storage modulus of 3D hydrogels resembling a cirrhotic and fibrotic liver, seeded without cells, measured on day 1, 11 and 21. (C) Storage modulus of 3D hydrogels compared to the liver biopsies derived from a DEN-induced mouse model for HCC at day 1 and (D) day 21 of culture (n = 3, error bars = SD, ****p < 0.0001; ***p < 0.001, **p < 0.01, *p < 0.05).
Figure 2
Figure 2
Viability, albumin, and urea content of cells grown in the 3D hydrogels resembling a fibrotic and cirrhotic liver. (A) Proliferation of HepG2 and (B) Huh7 cells. (C) Albumin normalised to total protein content of HepG2 and (D) Huh7 cells. (E) Urea normalised to total protein for HepG2 and (F) Huh7 cells. (n = 3, error bars = SD, ***p < 0.001, **p < 0.01, *p < 0.05).
Figure 3
Figure 3
(A) Photomicrographs of tumour HepG2 spheroids forming in a 3D fibrotic and cirrhotic hydrogel after 14 and 21 days in culture. (B) Surface area of tumour nodules formed on day 14 and 21 in both fibrotic and cirrhotic environments. (C) Photomicrographs of Huh7 cells outside the hydrogel after 14 and 21 days in culture (dashed green line marks edge of gels, n = 150, error bars = SD, **p < 0.001, Scale bar = 100 µm).
Figure 4
Figure 4
mRNA expression of EMT markers. (A) mRNA-expression of ACTA2 (B) CDH1 (C) CDH2 (D) EPCAM (E) SNAI1 and (F) POUF1 in HepG2 and Huh7 cells isolated from cells that appeared outside the gels and from cells that remained within the hydrogels (n = 3, error bars = SD, ****p < 0.0001, **p < 0.01, *p < 0.05).
Figure 5
Figure 5
Effect of microenvironment on drug response to doxorubicin, sorafenib and nitazoxanide. (A) Percentage cell viability of HepG2 cells grown in 2D and 3D 96 h after treatment with Doxorubicin (1 µM), sorafenib or Nitazoxinide (10 µM). (B) Percentage cell viability of Huh7 cells grown in 2D and 3D 96 h after treatment with Doxorubicin (1 µM), sorafenib or Nitazoxinide (10 µM). (C) mRNA expression of ABCB1 in HepG2 and Huh7 cells grown in 2D and 3D. All values are normalised to the respective untreated control groups. (n = 3, error bars = SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Figure 6
Figure 6
(A) Permeability of doxorubicin (1 mM) in fibrotic and cirrhotic gels without cells and with HepG2 or Huh7 cells. Doxorubicinol (B) and Doxorubicin (C) tumour concentration (nmol/g) in collected fibrotic and cirrhotic gels without cells or with HepG2 or Huh7 cells. (n = 3, error bars = SD.
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