Project description:Phosphoproteomics can provide insights into cellular signaling dynamics. To achieve robust quantitative phosphoproteomics profiling from minute amounts of sample, we here develop a global phosphoproteomics strategy based on data-independent acquisition (DIA) mass spectrometry and high-quality hybrid spectral libraries derived from data-dependent acquisition (DDA) and DIA data. Benchmarking the method using 166 synthetic phosphopeptides shows high sensitivity (<0.1 ng), accurate site localization and reproducible quantification (~5% median coefficient of variation). As a proof-of-concept, we use lung cancer cell lines and patient-derived tissue to construct a hybrid phosphoproteome spectral library covering 159,524 phosphopeptides (88,107 phosphosites). Based on this library, our single-shot streamlined DIA workflow quantifies 36,350 phosphosites (19,755 class 1) in cell line samples within 2 hours. Application to drug-resistant cells and patient-derived lung cancer tissues delineates site-specific phosphorylation events associated with resistance and tumor progression, showing that our workflow enables the characterization of phosphorylation signaling with deep coverage, high sensitivity and low between-run missing values.
Project description:Patient-derived cancer cells (PDCs) were established by three-dimensional (3D) spheroid culture from testicular germ cell tumor (GCT) specimens. Microarray expression analysis revealed that cancer stem-like cell-related genes were upregulated in 3D culture condition compared with two-dimensional (2D) culture condition.
Project description:Spheroids, near spherical multicellular aggregates, are one of the most common types of threedimensional (3D) cell cultures. Despite decades of implementation of spheroid technology in various fields of life science and medical research, no minimal information (MI) guidelines are available to cope with heterogeneity and to stimulate transparency. To cope with this unmet need, we assembled an international consortium to develop the MISpheroID knowledgebase (https://www.mispheroid.org) and interrogation revealed heterogeneity and lack of transparency in published spheroid-related experiments. This steered us to empirically evaluate the impact of cell line, culture medium type, spheroid formation method and spheroid size on complementary spheroid metrics (RNA fingerprints, presence of cell death, ATP content, glucose to lactate conversion, secreted protein signatures, circularity, size and cancer therapy response). We measured media-induced transcriptional variation in lung cancer (A549), colorectal cancer (HCT116), ovarium cancer (SKOV3) and glioblastoma (U87MG) spheroids using RNA sequencing (RNA-seq). These spheroids were formed in ultra-low attachment plates and cultured in 6 different media types (DMEM high glucose, DMEM/F12, RPMI1640, DMEM low glucose, EMEM and MEM) for 5 (HCT116) or 7 (A549, SKOV3 and U87MG) days. RNA extraction was performed on 2 spheroids per condition using the miRNeasy micro kit (217084, Qiagen, Hilden, Germany). RNA-sequencing libraries were prepared from purified RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria) according to the manufacturer's instructions, using 27.5ng of RNA that was DNase treated using HL-dsDNA (Arcticzymes, TromsØ, Norway). The individual libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Pleasanton, CA, US) and equimolarly pooled. The pool concentration was measured with Qubit and 1.4pM with 1% PhiX was sequenced on a NextSeq 500 (Illumina, San Diego, CA, US) using a high-output 1x75 run. Reads were mapped to the human genome using Tophat and gene expression counts were generated using HTSeq. This data was used to perform Principal Component Analysis (PCA) and Gene Set Enrichment Analysis (GSEA).
Project description:Here we performed phosphoproteomic, kinome examination of surgical specimens and patient-derived cancer cell lines.Kinomics and phosphoproteomics identified changes in the activity of a number of identical protein kinases in PDAC tumors and tissue culture cells. This allowed us to use these kinases as small molecular inhibitor targets for successful in vitro PDAC cell eradication.
Project description:Mesothelioma is an aggressive cancer of the mesothelial layer associated with an extensive fibrotic response. The latter is in large part mediated by cancer-associated fibroblasts which mediate tumour progression and poor prognosis. However, understanding of the crosstalk between cancer cells and fibroblasts in this disease is mostly lacking. Here, using co-cultures of patient-derived mesothelioma cell lines and lung fibroblasts, we demonstrate that fibroblast activation is a self-propagated process producing a fibrotic extracellular matrix (ECM) and triggering drug resistance in mesothelioma cells. Following characterisation of mesothelioma cells/fibroblasts signalling crosstalk, we identify several FDA-approved targeted therapies as far more potent than standard-of-care Cisplatin/Pemetrexed in ECM-embedded co-culture spheroid models. In particular, the SRC family kinase inhibitor, Saracatinib, extends overall survival well beyond standard-of-care in a mesothelioma genetically-engineered mouse model. In short, we lay the foundation for the rational design of novel therapeutic strategies targeting mesothelioma/fibroblast communication for the treatment of mesothelioma patients.
Project description:Cancer stem-like cells (CSCs) are considered as a tempting target due to their role in tumor progression. Although recent studies attempted to obtain and analyze patient-derived CSCs ex vivo, there are limitations in supplying diverse and large quantity of CSCs. Previously, functional polymer thin film (PTF) platform which induces cancer cells transformation into tumorigenic CSCs was invented. Here, we verified the functionality of polymer X, new PTF with a streamlined production process. Polymer X induced tumor spheroid formation and the spheroids showed increase in the expression of CSC-related genes. STAT3 phosphorylation was upregulated by fibronectin-integrin 5α-JAK2 axis and maintained by the cytosolic LMO2/LBD1 complex. In addition, STAT3 signaling was critical in spheroid formation on polymer X. By allowing the generation of CSCs from cancer cells, the PTF platform will enable to overcome the previous limitation of patient-derived CSC.
Project description:To examine the role of Efp in endometrial cancer, patient-derived cells were treated with siRNA targeting Efp (siEfp) or control siRNA (siControl). siRNA-mediated Efp silencing resulted in suppressed spheroid proliferation and altered gene expression profile, featuring downregulation of genes related to cell cycle and inflammation/immune responses. This study provides an insight into alternative endometrial cancer therapeutic strategies targeting Efp.
Project description:Phosphoproteomics requires better separation of phosphopeptides to boost the coverage of the phosphoproteome. We argue that an alternative separation method that produces orthogonal phosphopeptide separation to the widely used LC needs to be considered. Capillary zone electrophoresis (CZE) is one important alternative because CZE and LC are orthogonal for phosphopeptide separation and because the migration time of peptides in CZE can be accurately predicted. In this work, we coupled strong cation exchange (SCX)-reversed-phase LC (RPLC) to CZE-MS/MS for large-scale phosphoproteomics of the colon carcinoma HCT116 cell line. The CZE-MS/MS-based platform identified 11,555 phosphopeptides. The phosphopeptide dataset is at least 100% larger than that from previous CZE-MS/MS studies and will be a valuable resource for building a model for predicting the migration time of phosphopeptides in CZE. Preliminary investigations demonstrated that predicted and observed electrophoretic mobility of phosphopeptides containing one phosphoryl group had good linear correlations (R2≥0.94). Adding one phosphoryl group on a peptide decreased its electrophoretic mobility dramatically because the phosphoryl group reduced the peptide’s charge by roughly 1 based on our experimental data. Phosphopeptides tended to migrate significantly slower than unphosphopeptides in the CZE separation capillary and phosphorylated and unphosphorylated forms of peptides were separated well by CZE. The CZE-MS/MS and LC-MS/MS were complementary in large-scale phosphopeptide identifications and produced different phosphosite motifs from the HCT116 cell line. The data highlight the value of CZE-MS/MS for phosphoproteomics as a complementary separation approach for not only improving the phosphoproteome coverage but also providing more insight into the phosphosite motifs.
Project description:Defining molecular features that can predict the recurrence of colorectal cancer (CRC) for stage II-III patients remains challenging in cancer research. Most available clinical samples are Formalin-Fixed and Paraffin-Embedded (FFPE). NanoString nCounter® and Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA) are the two platforms marketed for high-throughput gene expression profiling for FFPE tissue samples. In this study, to identify an optimal platform for the gene expression profiling of FFPE CRC samples, we evaluated the expression of 516 genes from published frozen tissue-derived prognostic signatures in 42 CRC patient samples measured by these two platforms. Based on HTA platform-derived data, we identified both gene (99 individual genes, FDR < 0.05) and gene set (four of the six reported multi-gene signatures with sufficient information for evaluation, P < 0.05) expression differences associated with survival outcomes. Using nCounter platform-derived data, only one of the six multi-gene signatures (P < 0.05) but no individual gene was associated with survival outcomes. Therefore, the HTA appears to provide a more robust gene expression dataset using genes from published gene signatures. Our study indicated that sufficiently high quality RNA could be obtained from FFPE tumor tissues to detect frozen tissue-derived prognostic gene expression signatures for CRC patients.