National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN)
Title
Discovery Proteomics
Abstract
Cancer phosphoproteomics can provide useful insights regarding kinases that can be potentially targeted for therapeutic applications. Compared to surgical tissues and patient-derived models, rapidly cryo-preserved endoscopic biopsy is an ideal material for clinical phosphoproteomics in terms of the freshness and reflection of original patient condition. However, endoscopic biopsy samples do not contain enough protein amount for conventional deep-phosphoproteomics. We have developed a method which is enough sensitive to evaluate the phosphoproteomic status in endoscopic biopsies from patients with gastric cancer. We conducted a phosphoproteomic analysis using paired endoscopic biopsy specimens of pre- and post-treatment tumors (Ts) and non-tumor adjacent tissues (NATs) obtained from 4 HER2-positive gastric cancer patients who received trastuzumab-based treatment and from pre-treatment Ts and NATs of 4 HER2-negative gastric cancer patients. Our analysis revealed molecular changes by HER2 positivity and treatment. An inhibitory signature of the ErbB signaling was observed in the post-treatment HER2-positive tumor group compared with the pre-treatment HER2-positive tumor group. Phosphoproteomic profiles obtained by a case-by-case review using paired pre- and post-treatment HER2-positive tumor could be utilized to discover predictive or resistant biomarkers. Furthermore, these data nominated therapeutic kinase targets which were exclusively activated in the patient unresponded to the treatment. In summary, the pioneering approach in this study suggests that a phosphoproteomic analysis of endoscopic biopsy specimens provides information on dynamic molecular changes which can individually characterize biologic features upon drug treatment and identify therapeutic targets in stage IV gastric cancer.
Biological organs and tissues contain diverse cell types and tremendous cell-to-cell heterogeneity that dictates a multitude of functions for, e.g., homeostasis and response to the physiological environment. Molecular measurements at the single-cell level can reveal mechanisms and unique features of cell populations, differentiation, impacts of microenvironments, and rare cell populations in complex cellular systems that are typically masked from bulk-scale measurements. However, most of single-cell measurements are focused on the RNA transcripts, which poorly correlate with proteins, the main function determinant. In this presentation, I will briefly introduce a microfluidic sample preparation method, nanoPOTS (nanodroplet processing in one pot for trace samples), to enable sensitive proteomic analysis of single cells. I will highlight the value of reaction miniaturization for low-input samples, as well as sample multiplexing approaches for high throughput single-cell analysis. I will introduce several highly sensitive MS acquisition methods to allow the detection of low-abundant proteins for both label free and multiplexing workflow. Finally, I will conclude with our effort to improve the spatial proteomics, which aims to directly measure proteome abundance in tissue sections. For example, our recent study indicated up to 4000 proteins can be detected at a near-single-cell level (50 µm) on a pancreas tissue section.
There are reports revealed dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using longitudinal analysis. We analyzed the dynamic of severe SARS-CoV-2 infection by conducting single-cell RNA sequencing (scRNA-seq) of sequential sample of lung tissue obtained from SARS-CoV-2 infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) driven by the epithelial cell cytoketain-18 (K18) promoter as a model of severe COVID-19 disease. As the severity of COVID-19 disease increases, distinct changes in T cell proportions and characteristics were observed in lung microenvironment. Interestingly, when we identified alveolar epithelial cell types and states, the accumulation of DATPs and impaired differentiation of AT1 cell appeared higher in severe group (5Low) compared to moderate group (5High). We found that COVID-19 disease severity correlates with increased signaling of TGF between the myeloid fraction and the alveolar epithelial cells. Our results provide a dynamics of host responses and lung repair responses over the course of severe COVID-19 and important information to identify therapeutic target and diagnostic test
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