Electrophoretic cytopathology resolves ERBB2 forms with single-cell resolution
| Autor: | Chi-Chih Kang, Courtney Schiffman, Haiyan Huang, Toby M. Ward, Amy E. Herr, Mark D. Pegram, Jessica Bockhorn |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Protein isoform Gene isoform Cancer Research Proteolysis Cell Biology lcsh:RC254-282 Article 03 medical and health sciences 0302 clinical medicine Western blot medicine skin and connective tissue diseases neoplasms medicine.diagnostic_test lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens Molecular biology 3. Good health Blot 030104 developmental biology medicine.anatomical_structure Oncology Cytopathology 030220 oncology & carcinogenesis biology.protein Antibody |
| Zdroj: | npj Precision Oncology, Vol 2, Iss 1, Pp 1-10 (2018) NPJ Precision Oncology |
| ISSN: | 2397-768X |
| Popis: | In addition to canonical oncoproteins, truncated isoforms and proteolysis products are implicated in both drug resistance and disease progression. In HER2-positive breast tumors, expression of truncated HER2 isoforms resulting from alternative translation and/or carboxy-terminal fragments (CTFs) resulting from proteolysis (collectively, t-erbB2) have been associated with shortened progression-free survival of patients. Thus, to advance clinical pathology and inform treatment decisions, we developed a high-selectivity cytopathology assay capable of distinguishing t-erbB2 from full-length HER2 expression without the need for isoform-specific antibodies. Our microfluidic, single-cell western blot, employs electrophoretic separations to resolve full-length HER2 from the smaller t-erbB2 in each ~28 pL single-cell lysate. Subsequently, a pan-HER2 antibody detects all resolved HER2 protein forms via immunoprobing. In analysis of eight breast tumor biopsies, we identified two tumors comprised of 15% and 40% t-erbB2-expressing cells. By single-cell western blotting of the t-erbB2-expressing cells, we observed statistically different ratios of t-erbB2 proteins to full-length HER2 expression. Further, target multiplexing and clustering analyses scrutinized signaling, including ribosomal S6, within the t-erbB2-expressing cell subpopulation. Taken together, cytometric assays that report both protein isoform profiles and signaling state offer cancer classification taxonomies with unique relevance to precisely describing drug resistance mechanisms in which oncoprotein isoforms/fragments are implicated. Breast cancer: A precision test for truncated oncoproteins in single cells A single-cell western blot provides a simple way of studying whether a cancer-related protein found in breast tumors has transcriptionally or post-translationally modified to a truncated form, which is implicated in resistance to targeted therapies. The microfluidic assay, developed by Amy E. Herr from the University of California, Berkeley, USA, and colleagues at Stanford University, involves first dissociating a breast tumor, then isolating each tumor cell in cell-sized well on a microscope slide. Isolated in the well, each cell is broken up and HER2 proteins are size-separated via single-cell electrophoresis, before being immobilized and finally detected with a fluorescent probe that binds to both the full-length and truncated HER2 proteins. Herr’s team assayed eight patient samples and identified two tumor samples with mixed populations of full-length and truncated HER2 proteins, a finding with therapeutic and prognostic relevance for patients. |
| Databáze: | OpenAIRE |
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