Single molecule localization microscopy coupled with touch preparation for the quantification of trastuzumab-bound HER2

Abstract

All breast cancers are assessed for levels of human epidermal growth factor receptor 2 (HER2). Fluorescence in situ hybridization (FISH) and immunohistochemistry are currently used to determine if a patient is eligible for anti-HER2 therapy. Limitations of both tests include variability and relatively long processing times. Additionally, neither test determines whether HER2 contains the extracellular domain. While truncated in some tumors, this domain is required for binding of the therapeutic antibody trastuzumab. Here, trastuzumab was used to directly detect HER2 with quantitative single molecule localization microscopy (qSMLM). In proof of concept studies, our new method rapidly quantified both HER2 density and features of nano-organization. In cultured cells, the method was sensitive to subtle variations in HER2 expression. To assess patient samples, we combined qSMLM with tissue touch preparation (touch prep-qSMLM) and examined large areas of intact membranes. For cell lines and patient samples, HER2 copy numbers from FISH showed a significant positive correlation with detected densities from qSMLM and trended with HER2 cluster occupancy.

Single molecule characterization of individual extracellular vesicles from pancreatic cancer

“…Where applicable, primary Abs were fluorescently labelled with AF647 N ‐hydroxysuccinimidyl (NHS) ester (Thermo; A20006). We used optimized Ab labelling conditions [25] to obtain approximately one dye per Ab. The degree of labelling was calculated with a NanoDrop for each batch of labelled Abs.…”

Membrane therapy using DHA suppresses epidermal growth factor receptor signaling by disrupting nanocluster formation

“…Similarly, we cannot rule out the contribution of a DHA bioactive metabolite, although to date there is no evidence that secreted lipid metabolites (in general) have membrane-altering properties. Therefore, additional work is needed to determine if DHA impacts the nanoscale architecture of other EGFR mutants that arise from anti-EGFR therapies ( 92 ), including the clinically relevant EGFR family member HER2 ( 93 , 94 ).…”

A Review of Super-Resolution Single-Molecule Localization Microscopy Cluster Analysis and Quantification Methods

“…Another recent work that utilized machine learning to detect clustered and unclustered (background) molecules was proposed by Tobin et al. 103 Williamson et al. 136 proposed a supervised machine-learning method that is capable of classifying all the localizations from microscopy datasets into clustered or non-clustered classes.…”

“… 110 2018 proteins within centrioles and procentrioles in KE37 cells STORM 3D 32–65 30–60K NR NR DBSCAN 2 × 2 ✓ ✓ 8 ✓ SPARTAN Tobin et al. 103 2018 trastuzumab (HER2 receptor) in breast cancer cell lines (BT-474, SK-BR-3, MDA-MB-468) dSTORM 2D NR 20–40K 230–360 locs/ μm 2 17–23 cells density pair correlation 20 × 20 ✓ 6 ✓ ClusterOccupancy Nino et al. 108 2019 nuclear pore complex in U-2 human OS cells dSTORM 3D NR NR 2 × 2 b NR density-based; DBSCAN 2 × 2 b ✓ 2 ✓ FOCAL3D Paul et al.…”

“…82,88,96 Many other correlation functions have been used to quantify the localization clustering for post-processing SMLM data. 82,88,96,99,[102][103][104] The proposed correlation functions are used to analyze biological clusters rather than the biologically irrelevant pseudoclusters (also known as nanoclusters). For example, Schnitzbauer et al 102 derived a cross-correlation function for the localization coordinates inspired by the translational cross-correlation function in pixel-based image representation.…”