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Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and their interacting stromal fibroblast cells

ABSTRACT: Communication and interaction between carcinoma associated fibroblast (CAF) cells and tumor epithelium cells plays a critical role in cancer initiation and progression. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. In this study, we employed a SILAC labeling based co-culture system coupled with mass spectrometry analysis to identify and quantify the early phospho-signaling transductions induced by the crosstalk between CAFs and tumor cells. We performed three-state SILAC labeling for MDA-MB-231 and 82T. 9 million light labeled 82-L cells were co-cultured with medium labeled MDA-MB-231-M cells for 30 minutes. 9 million heavy labeled 82T-H cells were individually cultured in heavy SILAC media with for 30 minutes. Both individually cultured and co-cultured cells were quickly harvested with 9 M urea lysis buffer and mixed together. Mixed lysates were sonicated, reduced, alkylated and digested by trypsin. Tryptic peptides were desalted using SepPak C18 cartridge and followed by phosphotyrosine peptide enrichment using immunoaffinity purification with pY100 antibody. The enriched phosphopeptides were analyzed using a reverse-phase liquid chromatography system interfaced with an LTQ-Orbitrap Velos mass spectrometer. The Proteome Discoverer (v2.0; Thermo Fisher Scientific) suite was used for quantitation and database searches. The tandem mass spectrometry data were searched using SEQUEST search algorithm against a Human RefSeq database supplemented with frequently observed contaminants. The probability of phosphorylation sites was determined from the PhosphoRS algorithm.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture, Fibroblast

DISEASE(S): Breast Cancer

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD003544 | Pride | 2018-10-26

REPOSITORIES: Pride

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Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts.

Wu Xinyan X Zahari Muhammad Saddiq MS Renuse Santosh S Sahasrabuddhe Nandini A NA Chaerkady Raghothama R Kim Min-Sik MS Fackler Mary Jo MJ Stampfer Martha M Gabrielson Edward E Sukumar Saraswati S Pandey Akhilesh A

Clinical proteomics 20180615

<h4>Background</h4>Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells.<h4>Methods</h4>In this stu ...[more]

PMID: 29946230

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