Project description:(1) Background & Aims: The roles of different cells in the tumor microenvironment (TME) are critical to the metastatic process. The phenotypic transformation of the liver cells is one of the most important stages of the 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. (2) Methods: We isolated and performed whole-genome analysis of gene, miRNA, and protein 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 network centered in Brca1, we developed a software package (miRDiana) that collects miRNA targets from the union of the TargetScan, MicroCosm, mirTarBase, and miRWalk databases. This was used to search for 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. (3) Results: Using integrated omics analyses, we observed that the 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 most probable sequence of cell activation during metastasis is Endothelial→Ito→Kupffer. Immunohistochemical analysis of human liver metastases showed the 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. (4) Conclusions: 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.

Project description: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.

Project description: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). miRNA microarray analysis : The purified total RNA was analysed using Agilent mouse miRNA microarrays. Release 18.0, 8x60K (v18) microarray slides (Agilent Technologies, USA), with 1,200 mouse miRNAs represented, were used. Briefly, 100 ng of total RNA was labelled and hybridized following the standard Agilent Protocol for the miRNA Microarray System with a miRNA Complete Labelling and Hybridization Kit, including Agilent miRNA Spike-ins, and the results were scanned using an Agilent Microarray Scanner G2565CA (Agilent Technologies, USA). The scanned TIFF image files were processed using Agilent Feature Extraction Software vs 10.7.3.1 to extract raw data and obtain QC reports. The microarray raw data were normalized using the quantile method.

Project description:Emerging evidence indicates that gene products implicated in human cancers often cluster together in "hot spots" in protein-protein interaction (PPI) networks. Additionally, small sub-networks within PPI networks that demonstrate synergistic differential expression with respect to tumorigenic phenotypes were recently shown to be more accurate classifiers of disease progression when compared to single targets identified by traditional approaches. However, many of these studies rely exclusively on mRNA expression data, a useful but limited measure of cellular activity. Proteomic profiling experiments provide information at the post-translational level, yet they generally screen only a limited fraction of the proteome. Here, we demonstrate that integration of these complementary data sources with a "proteomics-first" approach can enhance the discovery of candidate sub-networks in cancer that are well-suited for mechanistic validation in disease. We propose that small changes in the mRNA expression of multiple genes in the neighborhood of a protein-hub can be synergistically associated with significant changes in the activity of that protein and its network neighbors. Further, we hypothesize that proteomic targets with significant fold change between phenotype and control may be used to "seed" a search for small PPI sub-networks that are functionally associated with these targets. To test this hypothesis, we select proteomic targets having significant expression changes in human colorectal cancer (CRC) from two independent 2-D gel-based screens. Then, we use random walk based models of network crosstalk and develop novel reference models to identify sub-networks that are statistically significant in terms of their functional association with these proteomic targets. Subsequently, using an information-theoretic measure, we evaluate synergistic changes in the activity of identified sub-networks based on genome-wide screens of mRNA expression in CRC. Cross-classification experiments to predict disease class show excellent performance using only a few sub-networks, underwriting the strength of the proposed approach in discovering relevant and reproducible sub-networks.

Project description:Background Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix in the pulmonary interstitium and progressive functional decline. We hypothesized that integration of multi-omics data would identify clinically meaningful molecular endotypes of IPF. Methods The IPF-PRO Registry is a prospective registry of patients with IPF. Proteomic and transcriptomic (including total RNA [toRNA] and microRNA [miRNA]) analyses were performed using blood collected at enrollment. Molecular data were integrated using Similarity Network Fusion, followed by unsupervised spectral clustering to identify molecular subtypes. Cox proportional hazards models tested the relationship between these subtypes and progression-free and transplant-free survival. The molecular subtypes were compared to risk groups based on a previously described 52-gene (toRNA expression) signature. Biological characteristics of the molecular subtypes were evaluated via linear regression differential expression and canonical pathways (Ingenuity Pathway Analysis [IPA]) over-representation analyses. Results Among 232 subjects, two molecular subtypes were identified. Subtype 1 (n = 105, 45.3%) and Subtype 2 (n = 127, 54.7%) had similar distributions of age (70.1 +/- 8.1 vs. 69.3 +/- 7.6 years; p = 0.31) and sex (79.1% vs. 70.1% males, p = 0.16). Subtype 1 had more severe disease based on composite physiologic index (CPI) (55.8 vs. 51.2; p = 0.002). After adjusting for CPI and antifibrotic treatment at enrollment, subtype 1 experienced shorter progression-free survival (HR 1.79, 95% CI 1.28,2.56; p = 0.0008) and similar transplant-free survival (HR 1.30, 95% CI 0.87,1.96; p = 0.20) as subtype 2. There was little agreement in the distribution of subjects to the molecular subtypes and the risk groups based on 52-gene signature (kappa = 0.04, 95% CI= -0.08, 0.17), and the 52-gene signature risk groups were associated with differences in transplant-free but not progression-free survival. Based on heatmaps and differential expression analyses, proteins and miRNAs (but not toRNA) contributed to classification of subjects to the molecular subtypes. The IPA showed enrichment in pulmonary fibrosis-relevant pathways, including mTOR, VEGF, PDGF, and B-cell receptor signaling. Conclusions Integration of transcriptomic and proteomic data from blood enabled identification of clinically meaningful molecular endotypes of IPF. If validated, these endotypes could facilitate identification of individuals likely to experience disease progression and enrichment of clinical trials. Trial registration NCT01915511 Supplementary Information The online version contains supplementary material available at 10.1186/s12931-023-02435-0.

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An Integrative Omics Approach Reveals Involvement of BRCA1 in Hepatic Metastatic Progression of Colorectal Cancer

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