Liquid-phase electron microscopy of molecular drug response in breast cancer cells reveals irresponsive cell subpopulations related to lack of HER2 homodimers

Peckys, Diana B.; Korf, Ulrike; Wiemann, Stefan; Jonge, Niels de

doi:10.1091/mbc.e17-06-0381

Cited by 20 publications

(30 citation statements)

References 72 publications

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“…As was found in a previous study, trastuzumab-induced HER2 uptake predominantly takes place in bulk/ruffled cancer cells, while flat/resting cells without membrane ruffles (examples are marked with an asterisks in Fig. 1A,B) do not exhibit significant uptake 20 . A set of experiments with different timings and controls was performed to determine the difference between flat- and ruffled membrane areas in the drug-induced HER2 clearance from the plasma membrane.…”

Section: Resultssupporting

confidence: 81%

Mathematical modeling of drug-induced receptor internalization in the HER2-positive SKBR3 breast cancer cell-line

Fehling-Kaschek

Peckys

Kaschek

et al. 2019

Sci Rep

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About 20% of breast cancer tumors over-express the HER2 receptor. Trastuzumab, an approved drug to treat this type of breast cancer, is a monoclonal antibody directly binding at the HER2 receptor and ultimately inhibiting cancer cell growth. The goal of our study was to understand the early impact of trastuzumab on HER2 internalization and recycling in the HER2-overexpressing breast cancer cell line SKBR3. To this end, fluorescence microscopy, monitoring the amount of HER2 expression in the plasma membrane, was combined with mathematical modeling to derive the flux of HER2 receptors from and to the membrane. We constructed a dynamic multi-compartment model based on ordinary differential equations. To account for cancer cell heterogeneity, a first, dynamic model was expanded to a second model including two distinct cell phenotypes, with implications for different conformational states of HER2, i.e. monomeric or homodimeric. Our mathematical model shows that the hypothesis of fast constitutive HER2 recycling back to the plasma membrane does not match the experimental data. It conclusively describes the experimental observation that trastuzumab induces sustained receptor internalization in cells with membrane ruffles. It is also concluded that for rare, non-ruffled (flat) cells, HER2 internalization occurs three orders of magnitude slower than for the bulk, ruffled cell population.

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Section: Resultssupporting

confidence: 81%

Mathematical modeling of drug-induced receptor internalization in the HER2-positive SKBR3 breast cancer cell-line

Fehling-Kaschek

Peckys

Kaschek

et al. 2019

Sci Rep

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“…Since the g ( r ) for Fig. 3c exhibits a clear peak at ~ 20 nm, an inter-label distance of 20 nm thus appeared with a higher likelihood than random occurrence, indicating the presence of HER2 homodimers, as was also found in HER2 overexpressing SKBR3 cells (Peckys et al 2015; Peckys et al 2017). The g ( r ) also shows that pair distances beyond the peak value do not return to 1 but remain higher, which presumably reflects the curved topography of the cell surface.…”

Section: Resultsmentioning

confidence: 65%

“…It has been postulated that the excessive presence of HER2 molecules also leads to the formation of HER2 homodimers, which in turn activate the HER2 signalling pathway. But as we have shown earlier at the single-cell level, the overexpression of HER2 does not always lead to the formation of HER2 homodimers in rare subpopulations of resting, or quiescent cancer cells (Peckys et al 2015; Peckys et al 2017). This might possibly play a role in the context of primary resistance to Trastuzumab, as found for instance in ~ 20% of early stage breast cancer patients, and ~ 70% of patients with metastatic disease (Wilken and Maihle 2010).…”

Section: Discussionmentioning

confidence: 73%

Visualisation of HER2 homodimers in single cells from HER2 overexpressing primary formalin fixed paraffin embedded tumour tissue

Peckys

Hirsch

Gaiser

et al. 2019

Mol Med

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Background HER2 is considered as one of the most important, predictive biomarkers in oncology. The diagnosis of HER2 positive cancer types such as breast- and gastric cancer is usually based on immunohistochemical HER2 staining of tumour tissue. However, the current immunohistochemical methods do not provide localized information about HER2’s functional state. In order to generate signals leading to cell growth and proliferation, the receptor spontaneously forms homodimers, a process that can differ between individual cancer cells. Materials and methods HER2 overexpressing tumour cells were dissociated from formalin-fixed paraffin-embedded (FFPE) patient’s biopsy sections, subjected to a heat-induced antigen retrieval procedure, and immobilized on microchips. HER2 was specifically labelled via a two-step protocol involving the incubation with an Affibody-biotin compound followed by the binding of a streptavidin coated quantum dot (QD) nanoparticle. Cells with membrane bound HER2 were identified using fluorescence microscopy, coated with graphene to preserve their hydrated state, and subsequently examined by scanning transmission electron microscopy (STEM) to obtain the locations at the single molecule level. Label position data was statistically analysed via the pair correlation function, yielding information about the presence of HER2 homodimers. Results Tumour cells from two biopsies, scored HER2 3+, and a HER2 negative control sample were examined. The specific labelling protocol was first tested for a sectioned tissue sample of HER2-overexpressing tumour. Subsequently, a protocol was optimized to study HER2 homodimerization in single cells dissociated from the tissue section. Electron microscopy data showed membrane bound HER2 in average densities of 201–689 proteins/μm 2 . An automated, statistical analysis of well over 200,000 of measured protein positions revealed the presence of HER2 homodimers in 33 and 55% of the analysed images for patient 1 and 2, respectively. Conclusions We introduced an electron microscopy method capable of measuring the positions of individually labelled HER2 proteins in patient tumour cells from which information about the functional status of the receptor was derived. This method could take HER2 testing a step further by examining HER2 homodimerization directly out of tumour tissue and may become important for adjusting a personalized antibody-based drug therapy. Electronic supplementary material The online version of this article (10.1186/s10020-019-0108-z) contains supplementary material, which is available to authorized users.

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“…In this study, we examined the spatial distribution of HER2 in large areas ($1000 mm 2 ) of the intact plasma membrane for two different cell lines. Our aim was to study the receptor distribution beyond its mere presence as homodimers found in membrane-ruffled areas versus monomers in flat areas of the plasma membrane (3); the distribution was found to alter upon incubation with the antibody-based drug trastuzumab (6). For the detection of membrane-bound HER2, we applied specific labeling with affibodies and quantum dots (QDs) suitable for correlative fluorescence and electron microscopy (7).…”

Section: Introductionmentioning

confidence: 99%

Linear Chains of HER2 Receptors Found in the Plasma Membrane Using Liquid-Phase Electron Microscopy

Parker

Trampert

Tinnemann

et al. 2018

Biophysical Journal

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The spatial distribution of the human epidermal growth factor 2 (HER2) receptor in the plasma membrane of SKBR3 and HCC1954 breast cancer cells was studied. The receptor was labeled with quantum dot nanoparticles, and fixed whole cells were imaged in their native liquid state with environmental scanning electron microscopy using scanning transmission electron microscopy detection. The locations of individual HER2 positions were determined in a total plasma membrane area of 991 mm 2 for several SKBR3 cells and 1062 mm 2 for HCC1954 cells. Some of the HER2 receptors were arranged in a linear chain with interlabel distances of 40 5 7 and 32 5 10 nm in SKBR3 and HCC1954 cells, respectively. The finding was tested against randomly occurring linear chains of six or more positions, from which it was concluded that the experimental finding is significant and did not arise from random label distributions. Because the measured interlabel distance in the HER2 chains is similar to the 36-nm helix-repetition distance of actin filaments, it is proposed that a linking mechanism between HER2 and actin filaments leads to linearly aligned oligomers.

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Liquid-phase electron microscopy of molecular drug response in breast cancer cells reveals irresponsive cell subpopulations related to lack of HER2 homodimers

Cited by 20 publications

References 72 publications

Mathematical modeling of drug-induced receptor internalization in the HER2-positive SKBR3 breast cancer cell-line

Mathematical modeling of drug-induced receptor internalization in the HER2-positive SKBR3 breast cancer cell-line

Visualisation of HER2 homodimers in single cells from HER2 overexpressing primary formalin fixed paraffin embedded tumour tissue

Linear Chains of HER2 Receptors Found in the Plasma Membrane Using Liquid-Phase Electron Microscopy

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