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Distribution of Collagen in Colorectal Liver Metastasis and Normal Liver tissue

ABSTRACT: We determined whether collagen is upregulated in colorectal liver metastasis tissue (CRLM). We previously showed that naturally occurring peptides of collagen type I were elevated in the urine of patients with colorectal liver metastasis (CRLM). CRLM tissue was compared to healthy adjacent liver tissue (controls) using high resolution mass spectrometry. Twenty-two different collagen alpha chains were identified, 19 of which were significantly upregulated (P<0.05) in CRLM tissue compared with control tissue. We observed expression of four collagen alpha chains which were absent or low expressed in healthy colon and control tissue, but were highly present in CRC and CRLM tissues analyzed. This expression pattern was also observed for six non-collagen colon specific proteins. Two of these proteins (CDH17 and PPP1R1B/DARP-32) were not known to be differently expressed in CRLM in comparison to healthy liver tissue. Furthermore, 16 of 20 identified proteins related to collagen synthesis were upregulated, indicating increased synthesis of collagen in CRLM. Cross-validation of the mass spectrometry data with immunohistochemistry for collagen type XII confirmed the significant upregulation of collagen type XII in CRLM. This study shows that the majority of collagen types are upregulated in CRLM compared to control tissue most likely as a result of an increased collagen turnover and changes in the collagen supramolecular structure. This research provides further understanding of morphologic changes in extracellular matrix of CRLM and the finding of proteins and peptides that might be specific for tumor metastasis in liquid biopsies.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Liver, Colon

DISEASE(S): Colon Cancer

SUBMITTER: Nick van Huizen

LAB HEAD: Theo M. Luider

PROVIDER: PXD008383 | Pride | 2018-11-21

REPOSITORIES: Pride

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Project description:The liver is the most common site for colorectal cancer (CRC) metastasis and new tissue culture models are needed to study hepatic metastasis from CRC as none of the current models mimic the biological, biochemical and structural characteristics of the metastatic microenvironment. Decellularization provides a novel approach for the study of cancer extracellular matrix (ECM) as decellularized scaffolds retained their tissue-specific features and biological properties. In the present study we created a 3D model of CRC and matched CRC liver metastasis (CRLM) using patient-derived decellularized ECM scaffolds seeded with HT-29 CRC cell line. Here we show increased HT-29 cell proliferation and migration capability when cultured in cancer-derived scaffolds compared to same-patient healthy colon and liver tissues. CRLM scaffolds also induced activation of epithelial-mesenchymal transition (EMT) with cancer cells showing loss of E-cadherin expression and increased Vimentin expression. EMT was confirmed by gene expression profiling, with the most represented biological processes in CRLM-seeded scaffolds involving demethylation, deacethylation, cellular response to stress metabolic processes, response to oxygen level and to starvation. The complex 3D culture environment reduced the HT-29 cell response to anti-CRC drug 5-FU when used at standard IC50 determined in 2D cultures. In conclusion, our 3D culture system with patient-derived tissue-specific decellularized ECM better recapitulates the metastatic microenvironment compared to conventional 2D culture conditions and represents a relevant approach for the study of liver CRC metastasis formation and progression. Transcriptomic analysis was performed comparing the global gene expression profiles of HT29 cells grown on CRLM and HL scaffolds. The transcriptomic profile of each 3D subtype was compared with HT29 cells grown in plastic. Hierarchical clustering analysis revealed that the gene expression signature of recellularized CRLM scaffolds was more comparable to recellularized HL scaffolds, than to HT29 cells grown in 2D. Gene set enrichment analysis (GSEA) of the differently expressed genes (DEG) up-regulated in recellularized CRLM scaffolds compared to HT29 grown in 2D revealed that genes involved in demethylation, deacethylation and metabolic process belong to the most enriched biological pathways in repopulated-CRLM scaffolds. Comparing the DEG between recellularized CRLM scaffolds vs HT29 grown in 2D with a series of a-priori defined gene-set, called Hallmark, we obtained concordant results with the “HYPOXIA” and “EPITHELIAL_MESENCHYMAL_TRANSITION” pathway, enriched in recellularized CRLM, supporting the idea that 3D culture models are the ability to re-create a complex environment in terms of oxygen tension, nutrients, and metabolic gradient, similarly to the in vivo environment.

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Distribution of Collagen in Colorectal Liver Metastasis and Normal Liver tissue

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