1. |
- Beal, Jacob, et al.
(author)
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Robust estimation of bacterial cell count from optical density
- 2020
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In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Journal article (peer-reviewed)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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2. |
- Tian, Chenxi, et al.
(author)
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Cancer cell-derived matrisome proteins promote metastasis in pancreatic ductal adenocarcinoma
- 2020
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In: Cancer Research. - : The American Association for Cancer Research. - 0008-5472 .- 1538-7445. ; 80:7, s. 1461-1474
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Journal article (peer-reviewed)abstract
- The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains poor despite decades of effort. The abundant extracellular matrix (ECM) in PDAC comprises a major fraction of the tumor mass and plays various roles in promoting resistance to therapies. However, nonselective depletion of ECM has led to poor patient outcomes. Consistent with that observation, we previously showed that individual matrisome proteins derived from stromal cells correlate with either long or short patient survival. In marked contrast, those derived from cancer cells correlate strongly with poor survival. Here, we studied three cancer cell-derived matrisome proteins that are significantly overrepresented during PDAC progression, AGRN (agrin), SER-PINB5 (serine protease inhibitor B5), and CSTB (cystatin B). Using both overexpression and knockdown experiments, we demonstrate that all three are promoters of PDAC metastasis. Furthermore, these proteins operate at different metastatic steps. AGRN promoted epithelial-to-mesenchymal transition in primary tumors, whereas SERPINB5 and CSTB enhanced late steps in the metastatic cascade by elevating invadopodia formation and in vivo extravasation. All three genes were associated with a poor prognosis in human patients and high levels of SERPINB5, secreted by cancer cells and deposited in the ECM, correlated with poor patient prognosis. This study provides strong evidence that cancer cell-derived matrisome proteins can be causal in promoting tumorigenesis and metastasis and lead to poor patient survival. Therefore, compared with the bulk matrix, mostly made by stromal cells, precise interventions targeting cancer cell-derived matrisome proteins, such as AGRN, SERPINB5, and CSTB, may represent preferred potential therapeutic targets. Significance: This study provides insights into the biological roles of cancer cell-derived matrisome proteins in PDAC and supports the notion that these proteins are protumorigenic and better therapeutic targets.
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3. |
- Tian, Chenxi, et al.
(author)
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Proteomic analyses of ECM during pancreatic ductal adenocarcinoma progression reveal different contributions by tumor and stromal cells
- 2019
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In: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 116:39, s. 19609-19618
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Journal article (peer-reviewed)abstract
- Pancreatic ductal adenocarcinoma (PDAC) has prominent extracellular matrix (ECM) that compromises treatments yet cannot be nonselectively disrupted without adverse consequences. ECM of PDAC, despite the recognition of its importance, has not been comprehensively studied in patients. In this study, we used quantitative mass spectrometry (MS)-based proteomics to characterize ECM proteins in normal pancreas and pancreatic intraepithelial neoplasia (PanIN)- and PDAC-bearing pancreas from both human patients and mouse genetic models, as well as chronic pancreatitis patient samples. We describe detailed changes in both abundance and complexity of matrisome proteins in the course of PDAC progression. We reveal an early upregulated group of matrisome proteins in PanIN, which are further upregulated in PDAC, and we uncover notable similarities in matrix changes between pancreatitis and PDAC. We further assigned cellular origins to matrisome proteins by performing MS on multiple lines of human-to-mouse xenograft tumors. We found that, although stromal cells produce over 90% of the ECM mass, elevated levels of ECM proteins derived from the tumor cells, but not those produced exclusively by stromal cells, tend to correlate with poor patient survival. Furthermore, distinct pathways were implicated in regulating expression of matrisome proteins in cancer cells and stromal cells. We suggest that, rather than global suppression of ECM production, more precise ECM manipulations, such as targeting tumor-promoting ECM proteins and their regulators in cancer cells, could be more effective therapeutically.
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