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Transcription profiling of human melanoma-endothelial cells co-cultured vs controls to investigate cellular communication

ABSTRACT: Average life expectancy for patients with metastatic melanoma is less than 6 months, and only a handful of treatment options are available. If the disease can be stopped before it spreads to other organs, life expectancy is greatly increased. The goal of this project is to identify possible regulators of melanoma metastasis by determining genes whose expression is modulated when the cells are grown in contact with endothelial cells. Identification of genes involved in this cell-cell communication could have therapeutic implications. Experiment Overall Design: Using a lentiviral system, melanoma cell line 1205Lu was transduced with GFP, and HUVEC (human umbillical vein endothelial cells) cells at passage 2 were transduced with RFP. These labeled cells were plated at near confluency either alone (controls) or mixed at a 1:1 ratio (co-cultures) for 48 hours in endothelial growth medium (EGM-2, Cambrex). The labeled cells were then sorted by FACS and RNA was collected from all 4 cultures. There are a total of four samples: GFP-1205Lu cells grown alone, GFP-1205Lu cells separated from co-culture with RFP-HUVEC cells, RFP-HUVEC cells grown alone, and RFP-HUVEC cells separated from co-culture with GFP-1205Lu cells. The JHMI Microarray Core was responsible for sample processing and initial data processing using Affymetrix-recommended protocols (Affymetrix GeneChip Expression Analysis Technical Manual, 2004).

ORGANISM(S): Homo sapiens

SUBMITTER: Keefe Megan Jean

PROVIDER: S-ECPF-GEOD-8699 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:Myocardial infarction (MI) is one of the most common among cardiovascular diseases. Endothelial cells (ECs) are considered to have protective effects on cardiomyocytes (CMs) under stress conditions such as MI; however, the paracrine CM-EC crosstalk and the resulting endogenous cellular responses that could contribute to this protective effect are not thoroughly investigated. Here we created biomimetic synthetic tissues containing CMs and human induced pluripotent stem cell (hiPSC)-derived ECs (iECs), which showed improved cell survival compared to single cultures under conditions mimicking the aftermath of MI, and performed high-throughput RNA-sequencing to identify target pathways that could govern CM-iEC crosstalk and the resulting improvement in cell viability. Our results showed that single cultured CMs had different gene expression profiles compared to CMs co-cultured with iECs. More importantly, this gene expression profile was preserved in response to oxidative stress in co-cultured CMs while single cultured CMs showed a significantly different gene expression pattern under stress, suggesting a stabilizing effect of iECs on CMs under oxidative stress conditions. Furthermore, we have validated the in vivo relevance of our engineered model tissues by comparing the changes in the expression levels of several key genes of the encapsulated CMs and iECs with in vivo rat MI model data and clinical data, respectively. We conclude that iECs have protective effects on CMs under oxidative stress through stabilizing mitochondrial complexes, suppressing oxidative phosphorylation pathway and activating pathways such as the drug metabolism-cytochrome P450 pathway, Rap1 signaling pathway, and adrenergic signaling in cardiomyocytes pathway. STATEMENT OF SIGNIFICANCE:Heart diseases are the leading cause of death worldwide. Oxidative stress is a common unwanted outcome that especially occurs due to the reperfusion following heart attack or heart surgery. Standard methods of in vivo analysis do not allow dissecting various intermingled parameters, while regular 2D cell culture approaches often fail to provide a biomimetic environment for the physiologically relevant cellular phenotypes. In this research, a systematic genome-wide transcriptome profiling was performed on myocardial cells in a biomimetic 3D hydrogel-based synthetic model tissue, for identifying possible target genes and pathways as protecting regulators against oxidative stress. Identification of such pathways would be very valuable for new strategies during heart disease treatment by reducing the cellular damage due to reperfusion injury.

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Transcription profiling of human melanoma-endothelial cells co-cultured vs controls to investigate cellular communication

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