ABSTRACT: The role of different cells in the tumor microenvironment (TME) is critical to the metastatic process. Phenotypic transformation of the liver cells is one of the most important stages of hepatic metastasis progression of colorectal cancer (CRC). Our aim was to identify the major molecules (i.e. genes, miRNAs and proteins) involved in this process. We isolated and performed whole genome analysis of gene, miRNA and proteins expression in three types of liver cells (Ito cells, Kupffer cells and liver sinusoidal endothelial cells) from the TME of a murine model of CRC liver metastasis. We selected the statistically significant differentially expressed molecules using the Student's t-test with Benjamini-Hochberg correction. and performed functional statistically-significant enrichment analysis of differentially expressed molecules with hypergeometric distribution using the curated collection of molecular signatures, MSigDB. To build a gene-miRNA-protein networks centered in Brca1, we developed software that collects miRNA targets from the union of the TargetScan, MicroCosm, mirTarBase and miRWalk databases. This was used to select miRNAs targeting Brca1. We validated the most relevant miRNAs with real-time quantitative PCR. To investigate BRCA1 protein expression, we built tissue microarrays (TMAs) from hepatic metastases of 34 CRC patients. Using integrated omics analyses we observed that Brca1 gene is among the twenty transcripts simultaneously up-regulated in all three types of TME liver cells during metastasis. Further analysis revealed that Brca1 is the last BRCA1-associated genome surveillance complex (BASC) gene activated in the TME. We confirmed this finding in human reanalyzing transcriptomics datasets from 184 patients from non-tumor colorectal tissue, primary colorectal tumor and colorectal liver metastasis of the GEO database. We found that the sequence of cell activation during metastasis is Endothelial->Ito->Kupffer. Immunohistochemical analysis of human liver metastases showed BRCA1 protein was co-localized in Ito, Kupffer and endothelial cells in 81.8% of early or synchronous metastases. However, in the greater part of the metachronous liver metastases this protein was not expressed in any of these TME cells. These results suggest a possible role of the co-expression of BRCA1 in Ito, Kupffer and sinusoidal endothelial cells in the early occurrence of CRC liver metastases, and point to BRCA1 as a potential TME biomarker. Animals: Balb/c mice (6- to 8-week-old males) were obtained from Charles River Laboratories Spain SA (Barcelona, Spain). All procedures were approved by the Ethical Committee for Animal Experimentation of the University of the Basque Country (EHU/UPV) in accordance with institutional, national and international guidelines regarding the protection and care of animal use for scientific purposes. Mice were kept in the animal facility of EHU/UPV and had access to standard chow and water ad libitum. Colorectal cancer cells: Murine CRC C26 cells (ATCC, Manassas, VA) syngeneic with Balb/c mice were grown under standard conditions in RPMI medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 mg/ml) and amphotericin B (0.25 mg/ml), all purchased from Life technologies, Carlsbad, CA. Control and tumor-activated hepatic cell isolation and culture: Control and tumor-activated primary cultures of hepatic cells, LSECs, Ito cells and Kupffer cells were isolated from livers with CRC metastasis, or from healthy controls. Balb/c mice were anesthetized with isofluorano and underwent surgical incisions on their left broadside. Mice were inoculated into the spleen at the incision sites using 2x106 of C26 colon carcinoma cells. Control mice were inoculated with PBS. Fourteen days later, all mice were sacrificed and the liver cells removed and purified by differential centrifugation. In brief, the mice were perfused with Clostridium histolyticum collagenase P (Sigma-Aldrich, St. Louis, MO, USA) through the cava vein and the obtained cell suspension twice centrifuged resulting in a parenchymal (PC)-enriched pellet and a non-PC-enriched supernatant. The non-PC-enriched supernatant was layered on Percoll gradients (25% on top of 50% w/v) to obtain LSECs and on Percoll gradients (33% on top of 50% w/v) to obtain Ito cells. After centrifugation the interphase between the two density cushions was collected and contained purified non-PC enriched LSECs in the first gradient and Ito cells in the second one. Both solutions contained Kupffer cells which were further separated by adherence assays. All cell fractions were washed and cultured with RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS and used in different experiments a maximum of 24 hours after isolation (Life technologies, Carlsbad, CA). Omics analysis: 100ng of total RNA from each fraction was labeled and hybridized onto Agilent mouse RNA and miRNA microarrays, Release 18.0 (Agilent Technologies, USA) following the standard Agilent Protocol with RNA and miRNA Complete Labelling and Hybridization Kits, including Agilent RNA Spike-ins. Results were scanned using an Agilent Microarray Scanner G2565CA. Scanned TIFF image files were processed using Agilent Feature Extraction Software (v10.7.3.1) to extract the raw data. All omics data were normalized with the quantile method.
