Electrophoretic cytopathology resolves ERBB2 forms with single-cell resolution

Abstract

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.

Microparticle Delivery of Protein Markers for Single‐Cell Western Blotting from Microwells

“…Run‐to‐run reproducibility in electromigration is monitored using protein markers, providing a quality control indicator . Such protein markers to assess technical variation in electromigration would be invaluable for quantifying proteoform expression that drives drug resistance in single cancer cells …”

“…Our laboratory has developed and utilized an “open” microfluidic device format comprising an array of microwells cast in a thin layer of polyacrylamide gel to perform electrophoretic analyses of isolated mammalian cells, including circulating tumor cells and breast cancer cells . In addition to cell isolation, each microwell acts as a reactor for cell preparation (i.e., imaging, staining, chemical lysis), with each cell lysate then subjected to electrophoretic analysis (i.e., protein PAGE, isoelectric focusing) in the surrounding polyacrylamide gel layer.…”

“…The microparticle‐delivery maintains locally high protein marker concentrations by eliminating diffusion of markers into both the gel network and the microwells prior to cell lysis. Single‐cell western blotting measures targets that may not be discernable using an immunoassay alone . For example, protein isoforms are involved in pathological processes including cancer, autoimmune diseases, diabetes, and heart hypertrophy .…”

“…Single‐cell western blotting measures targets that may not be discernable using an immunoassay alone . For example, protein isoforms are involved in pathological processes including cancer, autoimmune diseases, diabetes, and heart hypertrophy . However, distinguishing isoforms has been challenging; thus, the exact roles of most isoforms are hence poorly defined .…”

3D projection electrophoresis for single-cell immunoblotting

“…We attribute the ~15% discrepancy to possible diffusion barrier on the microwell wall surface, arising from either the presence of Rhinohide in the gel matrix, or the presence of residual GelSlick or dichlorodimethylsilane used during gel fabrication. One important consideration for Z-directional projection electrophoresis is that, in contrast to other single-cell protein separation assays such as scWB 23,30,[54][55][56][57][58] and scIEF [59][60][61] , there is no actual protein 'loss' in in the Z direction projection platform -only dilution. While other electrophoretic cytometry assays have a fluid layer or lid gel above the thin separation gel, into which protein can diffuse and is lost, in the projection electrophoresis device most (if not all) protein is mobilized into the bulk of the 3D gel when an electric field is applied to initiate PAGE.…”