Dataset Information

Improved phosphoproteomic analysis for phosphosignaling and active-kinome profiling in Matrigel-embedded spheroids and patient-derived organoids.

ABSTRACT: Many attempts have been made to reproduce the three-dimensional (3D) cancer behavior. For that purpose, Matrigel, an extracellular matrix from Engelbreth-Holm-Swarm mouse sarcoma cell, is widely used in 3D cancer models such as scaffold-based spheroids and patient-derived organoids. However, severe ion suppression caused by contaminants from Matrigel hampers large-scale phosphoproteomics. In the present study, we successfully performed global phosphoproteomics from Matrigel-embedded spheroids and organoids. Using acetone precipitations of tryptic peptides, we identified more than 20,000 class 1 phosphosites from HCT116 spheroids. Bioinformatic analysis revealed that phosphoproteomic status are significantly affected by the method used for the recovery from the Matrigel, i.e., Dispase or Cell Recovery Solution. Furthermore, we observed the activation of several phosphosignalings only in spheroids and not in adherent cells which are coincident with previous study using 3D culture. Finally, we demonstrated that our protocol enabled us to identify more than 20,000 and nearly 3,000 class 1 phosphosites from 1.4?mg and 150??g of patient-derived organoid, respectively. Additionally, we were able to quantify phosphosites with high reproducibility (r?=?0.93 to 0.95). Our phosphoproteomics protocol is useful for analyzing the phosphosignalings of 3D cancer behavior and would be applied for precision medicine with patient-derived organoids.

SUBMITTER: Abe Y

PROVIDER: S-EPMC6065387 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Improved phosphoproteomic analysis for phosphosignaling and active-kinome profiling in Matrigel-embedded spheroids and patient-derived organoids.

Abe Yuichi Y Tada Asa A Isoyama Junko J Nagayama Satoshi S Yao Ryoji R Adachi Jun J Tomonaga Takeshi T

Scientific reports 20180730 1

Many attempts have been made to reproduce the three-dimensional (3D) cancer behavior. For that purpose, Matrigel, an extracellular matrix from Engelbreth-Holm-Swarm mouse sarcoma cell, is widely used in 3D cancer models such as scaffold-based spheroids and patient-derived organoids. However, severe ion suppression caused by contaminants from Matrigel hampers large-scale phosphoproteomics. In the present study, we successfully performed global phosphoproteomics from Matrigel-embedded spheroids an ...[more]

PMID: 30061712

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Project description:BackgroundCholangiocarcinoma is a rare but lethal cancer of the biliary tract. Its first-line treatment is currently restricted to chemotherapy, which provides limited clinical benefit. Kinase inhibitors targeting oncogenic intracellular signaling have changed the treatment paradigm of cancer over the last decades. However, they are yet to be widely applied in cholangiocarcinoma therapy. Cholangiocarcinoma has marked molecular heterogeneity, which complicates the discovery of new treatments and requires patient stratification. Therefore, we investigated whether a commercial kinome profiling platform could predict druggable targets in cholangiocarcinoma.MethodsKinase activity in patient-derived cholangiocarcinoma organoids, non-tumorous adjacent tissue-derived and healthy donor-derived intrahepatic cholangiocyte organoids was determined using the PamChip® phosphotyrosine kinase microarray platform. Kinome profiles were compared and correlated with RNA sequencing and (multi-)kinase inhibitor screening of the cholangiocarcinoma organoids.ResultsKinase activity profiles of individual cholangiocarcinoma organoids are different and do not cluster together. However, growth factor signaling (EGFR, PDGFRβ) and downstream effectors (MAPK pathway) are more active in cholangiocarcinoma organoids and could provide potential druggable targets. Screening of 31 kinase inhibitors revealed several promising pan-effective inhibitors and compounds that show patient-specific efficacy. Kinase inhibitor sensitivity correlated to the activity of its target kinases for several inhibitors, signifying them as potential predictors of response. Moreover, we identified correlations between drug response and kinases not directly targeted by those drugs.ConclusionsIn conclusion, kinome profiling is a feasible method to identify druggable targets for cholangiocarcinoma. Future studies should confirm the potential of kinase activity profiles as biomarkers for patient stratification and precision medicine.

Dataset Information

Improved phosphoproteomic analysis for phosphosignaling and active-kinome profiling in Matrigel-embedded spheroids and patient-derived organoids.

Publications

Improved phosphoproteomic analysis for phosphosignaling and active-kinome profiling in Matrigel-embedded spheroids and patient-derived organoids.

Similar Datasets

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