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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.

SUBMITTER: Tobin SJ

PROVIDER: S-EPMC6181918 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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

Tobin Steven J SJ Wakefield Devin L DL Jones Veronica V Liu Xueli X Schmolze Daniel D Jovanović-Talisman Tijana T

Scientific reports 20181011 1

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 a ...[more]

PMID: 30310083

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Project description:IntroductionThe progression-free survival of patients with HER2-positive metastatic breast cancer is significantly extended by a combination of two monoclonal antibodies, trastuzumab and pertuzumab, which target independent epitopes of the extracellular domain of HER2. The improved efficacy of the combination over individual antibody therapies targeting HER2 is still being investigated, and several molecular mechanisms may be in play: the combination downregulates HER2, improves antibody-dependent cell mediated cytotoxicity, and/or affects the organization of surface-expressed antigens, which may attenuate downstream signaling.MethodsBy combining protein engineering and quantitative single molecule localization microscopy (qSMLM), here we both assessed and optimized clustering of HER2 in cultured breast cancer cells.ResultsWe detected marked changes to the cellular membrane organization of HER2 when cells were treated with therapeutic antibodies. When we compared untreated samples to four treatment scenarios, we observed the following HER2 membrane features: (1) the monovalent Fab domain of trastuzumab did not significantly affect HER2 clustering; (2) individual therapy with either trastuzumab or (3) pertuzumab produced significantly higher levels of HER2 clustering; (4) a combination of trastuzumab plus pertuzumab produced the highest level of HER2 clustering. To further enhance this last effect, we created multivalent ligands using meditope technology. Treatment with a tetravalent meditope ligand combined with meditope-enabled trastuzumab resulted in pronounced HER2 clustering. Moreover, compared to pertuzumab plus trastuzumab, at early time points this meditope-based combination was more effective at inhibiting epidermal growth factor (EGF) dependent activation of several downstream protein kinases.DiscussionCollectively, mAbs and multivalent ligands can efficiently alter the organization and activation of the HER2 receptors. We expect this approach could be used in the future to develop new therapeutics.

Dataset Information

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

Publications

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

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