PXD003048

PXD003048 is an original dataset announced via ProteomeXchange.

Title	Cell-specific analysis of tumour-endothelial contact-intiated signalling - PROTEOMICS
Description	Metastasizing tumor cells exit the vascular system through dynamic interactions with endothelial cells that line the internal surface of vessels. While extravasation is a key event within the metastatic cascade, the signals regulating tumor cell adhesion to the endothelium and their subsequent transendothelial migration are poorly understood. Here, we combined Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and phosphoproteomic analysis to identify cell-specific signaling pathways regulated between interacting breast cancer cells and endothelial cells (see PRIDE repository PXD001558). Further co-culture experiments were performed alongside the phosphoproteomic analysis in order to control for the protein quantity. These are presented here. Using SILAC, cell-specific labels were introduced into MDA-MB-231-LM2 cells (Human Breast cancer) and HUVECs (Human endothelial cells) to ensure each cell type had a distinct and traceable phosphoproteome when tumor and endothelial cells were co-cultured. To probe regulatory signaling events triggered in cancer cells following contact with endothelial cells, we labeled LM2 cells with medium or heavy isotopomers of arginine and lysine (Arg+6 Da, Lys+4 Da and Arg+10 Da, Lys+8 Da respectively). Heavy-labeled LM2 cells were collected by enzyme-free cell dissociation buffer, thereby preserving membrane proteins and adhesion receptors, and seeded onto a monolayer of light-labeled (Arg+0 Da, Lys+0 Da) HUVECs. Following 15 min of co-culture, non-adherent LM2 cells were gently removed and cancer cells that had attached to the endothelial layer were lysed together with the HUVECs. In parallel, medium-labeled LM2 cells were collected under the same conditions and maintained as suspension cells in monoculture to represent circulating tumor cells prior to any contact with the endothelium. These were then added to the harvested LM2-HUVEC co-culture in a 1:1 ratio of heavy:medium-labeled cells to provide a point of reference. Based on the SILAC labeling of the different cell populations, light-labeled peptides were assigned to HUVECs, medium-labeled peptides to monocultured LM2 cells in suspension, and heavy-labeled peptides to LM2 cells that had made contact with HUVECs. As such, the heavy/medium ratio for each peptide was used to quantify phosphorylation-dependent signaling changes occurring specifically in the LM2 cells upon contact with HUVECs. Conversely, to elucidate signaling events in HUVECs that were initiated by contacting cancer cells, light-labeled LM2 cells were seeded on top of a confluent monolayer of heavy-labeled HUVECs, while medium-labeled HUVECs were maintained in monoculture. Following 15 min of co-culture, the unattached LM2 cells were removed. Cells were lysed, mixed in a 1:1 ratio of heavy:medium HUVECs, followed by membrane fractionation and phosphoproteomic analysis as described above. A variation in phospho-peptide quantity could be due to the experimental difficulties in mixing heavy and medium labels in a 1:1 ratio or a quick regulation of the protein quantity through modification of its expression/degradation balance. Thus, we performed relative quantification of non-phosphorylated peptides in parallel with the phosphoproteomic analysis to account for changes in total protein abundance (data presented here) or correct for experimental bias. Cytoplasmic fractions were analyzed by LC-MS/MS before TiO2 enrichment and their median log2(H/M) were used for normalization of the phosphoproteomic dataset. In order to increase the number of proteins quantified in the LM2 cells, we performed a supplementary co-culture experiment and fractionated the peptides by SDS-PAGE. In order to get a confident relative quantification of Ephrin type-A receptor 2 (EPHA2), we performed 2 independent experiments followed by EPHA2 IP and LC-MSMS.
HostingRepository	PRIDE
AnnounceDate	2017-10-24
AnnouncementXML	Submission_2017-10-24_05:53:21.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Marie Locard-Paulet
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; acetylated residue; deamidated residue; iodoacetamide derivatized residue
Instrument	LTQ Orbitrap Velos; Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2015-10-13 05:56:34	ID requested
1	2016-02-12 04:26:26	announced
2	2017-06-20 05:43:43	announced	Updated project metadata.
⏵ 3	2017-10-24 05:53:22	announced	Updated project metadata.

Locard-Paulet M, Lim L, Veluscek G, McMahon K, Sinclair J, van Weverwijk A, Worboys JD, Yuan Y, Isacke CM, J, ø, rgensen C, Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration. Sci Signal, 9(414):ra15(2016) [pubmed]

curator keyword: Biomedical

submitter keyword: MDA-MB-231-LM2, HUVEC, "contact-initiated signaling", EPHA2

Claus Jorgensen
contact affiliation	Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, Manchester M20 4BX, United Kingdom
contact email	Claus.Jorgensen@cruk.manchester.ac.uk
lab head
Marie Locard-Paulet
contact affiliation	Institute of Pharmacology and Structural Biology
contact email	marie.locard@ipbs.fr
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/02/PXD003048

PRIDE project URI

• PRIDE
  1. PXD003048
     1. Label: PRIDE project
     2. Name: Cell-specific analysis of tumour-endothelial contact-intiated signalling - PROTEOMICS