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3D imaging of colorectal cancer organoids identifies responses to Tankyrase inhibitors

ABSTRACT: We generated 3D tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. We demonstrated that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation, highlighting the value of phenotypic readouts as a quantitative method to assess drug-induced effects in a relevant preclinical model.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Homo Sapiens

SUBMITTER: Cardiff University

PROVIDER: PRJEB37607 | EVA | 2020-04-29

REPOSITORIES: EVA

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Project description:Using a three-pronged MS-based proteomic approach, we assessed the mode-of-action of four clinically-relevant tyrosine kinase inhibitors in an epithelial cancer cell line. Here we defined the: (I) semi-quantitative tyrosine kinome; (II) drug interactome; and (III) drug-induced effects on the tyrosine signalome. We first examined the entire proteome and kinome profile expressed in the A431 cell line, and determined the relative abundance of all possible protein targets of the tyrosine kinase inhibitors. We therefore utilized a ‘shot-gun’ approach that involved a multidimensional separation-based proteomic strategy. To ensure maximal protein identification, we reduced sample complexity by strong cation exchange (SCX) chromatography, and analyzed all fractions by LC-MS/MS. To determine the direct targets of the four selected TKIs, we subjected the proteins extracted from the A431 cancer cell line to drug-coupled affinity matrices. By comparing the pull-down results from a lysate with a second identical lysate to which 20 uM of the inhibitor had been added to block binding to the beads, we were able to identify selective binding interactors using LC-MS/MS. This differential affinity pull-downs approach allowed us to also quantitatively evaluate the binding affinity of the tyrosine kinases to each of the drugs. To evaluate the global tyrosine phosphorylation dynamics that occurs in A431 cells upon treatment with each of the four inhibitors, phosphotyrosine peptides were enriched and identified by quantitative mass spectrometry. For this purpose, we treated the cells with each inhibitor for 2 h at appropriate concentrations adopted from reported studies. Cells were lysed, and the proteins digested with the proteases Lys-C and trypsin. To allow quantitation of the phosphorylated peptides originating from the three different peptide pools (control/imatinib/dasatinib or control/bosutinib/nilotinib), we used stable isotope dimethyl labeling. Subsequently, tyrosine-phosphorylated peptides were enriched by immunoprecipitation and analyzed by LC-MS/MS.

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3D imaging of colorectal cancer organoids identifies responses to Tankyrase inhibitors

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