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BackgroundCancer-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.MethodsIn this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF this has already been defined above cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC-MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells.ResultsWe discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication.ConclusionsOur study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.

Simple Summary: AXL is a receptor tyrosine kinase belonging to the TAM (Tyro3, Axl and Mer) family. The AXL protein plays an important role in promoting cancer development, such as proliferation, migration, invasion and survival of cancer cells. In this study, we used mass spectrometry-based proteomics to quantify the cancer signaling regulated by AXL activation. Our study identified more than 1000 phosphotyrosine sites and discovered that activation of AXL can upregulate multiple cancer-promoting and cell migration/invasion-related signaling pathways. We also observed significant crosstalk as evidenced by rapid phosphorylation of multiple receptor tyrosine kinases and protein tyrosine phosphatases, including PTPN11 and PTPRA, upon GAS6 stimulation. These discoveries should serve as a potentially useful resource for studying AXL functions as well as for the development of effective therapeutic options to target AXL. Overexpression and amplification of AXL receptor tyrosine kinase (RTK) has been found in several hematologic and solid malignancies. Activation of AXL can enhance tumor-promoting processes such as cancer cell proliferation, migration, invasion and survival. Despite the important role of AXL in cancer development, a deep and quantitative mapping of its temporal dynamic signaling transduction has not yet been reported. Here, we used a TMT labeling-based quantitative proteomics approach to characterize the temporal dynamics of the phosphotyrosine proteome induced by AXL activation. We identified >1100 phosphotyrosine sites and observed a widespread upregulation of tyrosine phosphorylation induced by GAS6 stimulation. We also detected several tyrosine sites whose phosphorylation levels were reduced upon AXL activation. Gene set enrichment-based pathway analysis indicated the activation of several cancer-promoting and cell migration/invasion-related signaling pathways, including RAS, EGFR, focal adhesion, VEGFR and cytoskeletal rearrangement pathways. We also observed a rapid induction of phosphorylation of protein tyrosine phosphatases, including PTPN11 and PTPRA, upon GAS6 stimulation. The novel molecules downstream of AXL identified in this study along with the detailed global quantitative map elucidating the temporal dynamics of AXL activation should not only help understand the oncogenic role of AXL, but also aid in developing therapeutic options to effectively target AXL. [ABSTRACT FROM AUTHOR]

Detection of illicit drugs in the environmental samples has been challenged as the consumption increases globally. Current review examines the recent developments and applications of sample preparation techniques for illicit drugs in solid, liquid, and gas samples. For solid samples, traditional sample preparation methods such as liquid-phase extraction, solid-phase extraction, and the ones with external energy including microwave-assisted, ultrasonic-assisted, and pressurized liquid extraction were commonly used. The sample preparation methods mainly applied for liquid samples were microextraction techniques including solid-phase microextraction, microextraction by packed sorbent, dispersive solid-phase extraction, dispersive liquid-liquid microextraction, hollow fiber-based liquid-phase microextraction, and so on. Capillary microextraction of volatiles and airborne particulate sampling were primarily utilized to extract illicit drugs from gas samples. Besides, the paper introduced recently developed instrumental techniques applied to detect illicit drugs. Liquid chromatograph mass spectrometry and gas chromatograph mass spectrometry were the most widely used methods for illicit drugs samples. In addition, the development of ambient mass spectrometry techniques, such as desorption electrospray ionization mass spectrometry and paper spray mass spectrometry, created potential for rapid in-situ analysis. [ABSTRACT FROM AUTHOR]

Delays between tissue collection and tissue fixation result in ischemia and ischemia-associated changes in protein phosphorylation levels, which can misguide the examination of signaling pathway status. To identify a biomarker that serves as a reliable indicator of ischemic changes that tumor tissues undergo, we subjected harvested xenograft tumors to room temperature for 0, 2, 10 and 30 minutes before freezing in liquid nitrogen. Multiplex TMT-labeling was conducted to achieve precise quantitation, followed by TiO2 phosphopeptide enrichment and high resolution mass spectrometry profiling. LC-MS/MS analyses revealed phosphorylation level changes of a number of phosphosites in the ischemic samples. The phosphorylation of one of these sites, S82 of the heat shock protein 27 kDa (HSP27), was especially abundant and consistently upregulated in tissues with delays in freezing as short as 2 minutes. In order to eliminate effects of ischemia, we employed a novel cryogenic biopsy device which begins freezing tissues in situ before they are excised. Using this device, we showed that the upregulation of phosphorylation of S82 on HSP27 was abrogated. We thus demonstrate that our cryogenic biopsy device can eliminate ischemia-induced phosphoproteome alterations, and measurements of S82 on HSP27 can be used as a robust marker of ischemia in tissues. [ABSTRACT FROM AUTHOR]