Abstract 352: HER2 protein isoform heterogeneity investigated by single-cell western blotting

Abstract

Abstract Accounting for ∼20% of breast cancers, half of metastatic HER2-positive breast cancer patients treated with trastuzumab plus chemotherapy will experience progression of disease within one year. Trastuzumab treatment, though associated with improvement in median overall survival, still sees drug resistance as an unmet challenge. Differential response to therapy may arise from tumor heterogeneity. Further complicating the situation is the fact that HER2/erbB2 biology is complex. HER2 is not simply one full-length protein (p185) overexpressed on tumor cell surfaces, but also includes truncated isoforms, t-erbB2 (e.g., 95 and 110 kDa isoforms) lacking the extracellular domain targeted by trastuzumab (and pertuzumab and T-DM1). The t-erbB2 isoforms are implicated in tumorigenesis, metastasis, and therapeutic response. Given existing pathology and life sciences tools, the high heterogeneity and need for single cell resolution presents a tremendous challenge. We introduce here a novel microfluidic device to quantitatively assess HER2 protein isoforms in heterogeneous tumor biopsies with single-cell resolution. The single-cell western blot (scWB) provides both the sensitivity and specificity needed to detect t-erbB2 isoforms owing to use of a single-cell electrophoretic protein separation followed by immunoprobing. The assay further provides enough sensitivity to obviate the need for cell pooling, which obscures cell-to-cell heterogeneity. We assay ∼1000's of single cells on one microscope slide in a 4-6 hr workflow. First, we validated that the scWB can separate full length (p185) from truncated (p95) HER2 in single p185- and p95-genetically engineered CHO cells. The p95 was resolved from p185 protein in a short ∼1 mm separation lane (resolution = 0.97±0.03, n>500 cells). Next, we assessed a HER2-positive breast cancer cell line (BT474) and found <3% t-erbB2 expressing cells. To further investigate the effect of t-erbB2 in HER2-related signaling, we examined phospho-proteins including p-HER2, p-HER3, p-ERK Thr187/Tyr185, p-ERK Thr202/Tyr204, p-rs6 from each t-erbB2 expressing BT474 cell. Protein activation in t-erbB2 expressing cells was similar to cells expressing full length HER2. This first-in-kind study demonstrates a powerful single-cell resolution multiplexing capability including for phospho-proteins. We are now applying the novel scWB to measure t-erbB2 in HER2-positive and triple positive (ER/PR/HER2) breast tumor biopsies and will report our results at AACR. Taken together, the microfluidic single-cell resolution western provides a sensitive, quantitative, and multiplexed tool to assay clinically-relevant oncoprotein isoforms that are currently difficult or impossible to assay. Correlating isoform expression ratios with clinical outcomes may confer a more comprehensive understanding of drug resistance responses derived from oncoprotein isoform profiles within heterogeneous cancer cell populations. Citation Format: Ginny (Chi-Chih) Kang, Toby M. Ward, Jessica Bockhorn, Mark D. Pegram, Amy E. Herr. HER2 protein isoform heterogeneity investigated by single-cell western blotting. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 352.