Project description:In this study we want present a bank of metastatic colorectal cancer (mCRC) Patient Derived Organoids (PDOs) obtained from Patient Derived Xenografts (PDXs). These models are annotated with different omics to advance our understanding of CRC. We wanted to create a resource for the scientific community to assess the predictive reliability of these preclinical models. We performed comparative analyses between PDOs and matched PDXs to assess the similarities of these two platforms regarding molecular profiles and transcriptional classification. Moreover, we analyzed how these models respond to Cetuximab, a chimeric monoclonal antibody, normally given to patients after chemotherapy, that inhibits EGFR. After having assessed models’ reliability with Cetuximab, we aimed at identifying potential synergistic drugs to individuate new possible therapeutic prospects.
Project description:We performed in-vivo selection of human patient derived colorectal cancer xenografts. 4 independent highly-liver metastatic sub-lines (lvm PDXs) were generated. Those lvm PDXs were harvested with the corresponding parental PDX tumors from mice and then we performed MACS mice cell depletion followed by FACS sorting to obtain human EpCAM positive and mice MHC negatitve populations. total RNA was extracted from those ex-vivo tumors and high-throuput sequencing was performed
Project description:Three separate experiments were carried out using MeDIP-seq and cfMeDIP-seq for methylome analysis. For the first experiment, different starting amounts of HCT116 cell line DNA, sheared to mimic cell-free DNA, were analyzed using MeDIP-seq and cfMeDIP-seq. In the second experiment the limit of detection of cfMeDIP-seq was tested using varying dilutions of colorectal cancer cell line DNA (HCT116) with multiple myeloma cell line DNA (MM1.S). For both cell line DNA samples, the DNA was sheared to mimic cell-free DNA. In the final experiment, we tested the enrichment of human ctDNA using cfMeDIP-seq performed on plasma collected from patient-derived xenografts (PDXs) generated in mice from two colorectal cancer patients.
Project description:Metastatic colorectal cancer (mCRC) relies on the detachment of aggressive malignant cells from the primary tumor into the bloodstream, these Circulating Tumor Cells (CTC) being the principal source of the further metastasis. Here, we approached the massive molecular profiling of the CTC population isolated from mCRC patients. Clinically, the presence of CTC is associated with poor prognosis and there exists a clear necessity for more specific and efficient chemotherapies in the treatment of mCRC. Immunoisolation of CTC from these patients combined with a whole transcriptome amplification and hybridization onto gene expression arrays, lead us to specifically describe for the first time a CTC population with adhesive and migratory characteristics, in addition to a modulated expression of genes related to cell death and survival. Furthermore, bioinformatic analysis provided with an armamentarium of highly specific and sensitive valuable markers that should impact on the management and follow-up of mCRC patients. Finally, molecular profiling of CTC resulted in the identification of new therapeutic targets, like TGF-β1 impairing, specifically targeting the CTC population which should improve the efficacy in the eradication and prevention of CRC metastasis. In conclusion, molecular profiling of CTC represents an innovative and promising approach in the clinical management of mCRC patients. Circulating Tumor Cells (CTC) were immunoisolated from six metastatic colorectal patients (mCRC). Moreover, blood from three healthy donors were obtained, immunoisolating non-specific cellularity background. RNA was extracted and globally amplified with a Whole Transcriptome Amplification system. Samples were hybridized onto agilent arrays and CTC specific genes were obtained. Gene networks were built with IPA software and a gene ontology analysis was performed. Eleven candidate genes were validated by real time PCR. Finally, TGFB1 was evaluated as a potential drugable target against CTC population.
Project description:In this study, we aimed to determine the characteristics and clinical significance of the TCR repertoire in patients with unresectable metastatic colorectal cancer (mCRC).
Project description:Genome wide DNA methylation profiling of primary (PR) and liver metastasis (LM) colorectal cancer (CRC). The Illumina Infinium MethylationEPIC Beadchip was used to obtain DNA methylation profiles across approximately 850,000 CpGs in human and xenogratfs derived samples. Raw data were processed using R minfi package. The main goal of this study is to develop a methylation-based classifier and predictor for metastatic CRC (mCRC), in order to find specific patients stratification for anti-EGFR targeted therapy (cetuximab) and bring the promise to shed new light on the role of methylation in driving colorectal cancer progression in different groups of patients.