Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

RNAseq for B16-OVA and 4T1 murine cancer cell lines

ABSTRACT: RNA Sequencing of the murine breast cancer cell line 4T1 and of the murine melanoma cell line B16-ova was carried out with the aim of establishing the complete transcriptome of these cell lines. Because these are cancer cells and we expect a lot of aberrant splicing, we carried out de novo assembly (genome guided) of the transcripts. We also ran deep Mass Spectrometry proteomics analysis on the same cell lines, aiming to determine which aberrant transcripts carry over to the protein expression level. Further, we tested these cancer specific protein epitopes (putative neoantigens) for immunogenicity using mouse models. Finally, the putative neoantigens that showed good immunogenic potential were used in tumor growth control experiments with mice engrafted with the two tumor cell lines. In these experiments we tested whether cancer vaccines based on individual neoantigen peptides (MHC-I) restricted the growth of the tumor compared to adequate controls. The overall aim of the project is to validate the ability of our multi-omics/bioinformatics pipeline to identify and deliver neoantigens that can be used to suppress tumor growth.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Mus musculus

SUBMITTER: Rui Miguel Mamede Branca

PROVIDER: E-MTAB-14130 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Tumor cell invasion and metastasis are hallmarks of malignancy. Despite recent advances in the understanding of lymphatic spread, the mechanisms by which tumors metastasize to sentinel/distant lymph nodes and beyond are poorly understood. To gain new insights into this complex process, we established a highly metastatic melanoma cell line (B16F1-variant) by in vivo passaging the B16 parental cell line through the lymphatic system. Here, we characterized morphology, rate of cell proliferation, colony formation, migration, tumorogenicity, lymph flow, and capacities to induce tumor- and sentinel lymph node- lymphangiogenesis. Furthermore, microarray-based comparative analysis bewteen parental and passaged cell lines was performed to identify specific gene expression profiles. The most differentially expressed gene was SPP (osteopontin), a secreted glycophosphoprotein which is known to be involved in cancer metastasis. Overexpression of osteopontin in B16 F1-variant was confirmed by Western blot and quantitative RT-PCR. Treatment of cultured lymphatic endothelial cells (LEC) with osteopontin promoted cell migration mediated by the integrin α9 pathway. Our results identify osteopontin as a novel lymphangiogenic factor. B16 and B16 variants were cultured in DMEM supplemented with 20% fetal bovine serum (FBS; Invitrogen, Grand Island, NY) supplemented with antibiotic-antimycotic solution. Every B16 variant was cultured in duplicates. Total cellular RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA) extracted with chloroform, precipitated with isopropanol, washed with 70% ethanol, and dissolved in DNase-free/RNase-free distilled water. The concentration of RNA was measured using NanoDrop ND-1000 spectrophotometer (Witec AG, Littau, Switzerland) and RNA quality was assessed using 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Digoxigenin-UTP–labeled cRNA was generated and amplified from 500ng of total RNA using the NanoAmp RT-IVT Labeling Kit (Applied Biosystems, Foster City, CA) following the manufacturer's protocol, and was hybridized to Applied Biosystems Mouse Genome Survey Microarrays V2.0. Chemiluminescence detection, image acquisition, and analysis were performed using the Chemiluminescence Detection Kit (Applied Biosystems) and the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer following the manufacturer's protocol. A total of two biological replicates were generated for each B16 variant and each cell line data consisted of pooled RNA of two diffferent passage numbers.

Project description:Bromodomains (BRDs) have emerged as compelling targets for cancer therapy. The development of selective and potent BET inhibitors and their significant activity in diverse tumor models has rapidly translated into clinical studies and has motivated drug development efforts targeting non-BET BRDs. However, the complex multidomain/subunit architecture of bromodomain protein complexes complicates predictions of consequences of their pharmacological targeting. To address this issue we developed a promiscuous bromodomain inhibitor (bromosporine, BSP) that broadly targets BRDs (including BETs) with nanomolar affinity, creating a tool for the identification of cellular processes and diseases where BRDs have a regulatory function. As a proof of principle we studied the effect of BSP in leukemic cell-lines known to be sensitive to BET inhibition and found as expected strong anti-proliferative activity. Comparison of the modulation of transcriptional profiles by BSP at short inhibitor exposure resulted in a BET inhibitor signature but no significant additional changes in transcription that could account for inhibition of other BRDs. Thus, non-selective targeting of BRDs identified BETs, but not other BRDs, as master regulators of a context dependent primary transcription response. Cells were seeded the day prior to treatment at 2x10^5 per ml. Treatments were performed so that a final concentration of 0.1 % DMSO (Cat.#D1435; Sigma) was achieved and cells were incubated with DMSO or 500 nM test compound (BSP, JQ1, LP99, GSK2801, iCBP112 or OF1) for 6 h prior to isolation of RNA. Total RNA was isolated using a standard TRIzol (Invitrogen) protocol and prepared using RNeasy columns (Cat.#74106 plus; Qiagen). RNA was quantified using a Nanodrop spectrophotometer (model ND1000; Thermo Fisher) and integrity assessed on a BioAnalyzer (2100; Agilent Laboratories, USA). All samples had a RNA Integrity Number (RIN) â¥ 9. Biological triplicates were performed for each condition tested.

Dataset Information

RNAseq for B16-OVA and 4T1 murine cancer cell lines

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets