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Local variations of HER2 dimerization in breast cancer cells discovered by correlative fluorescence and liquid electron microscopy - PubMed

  • ️Thu Jan 01 2015

Local variations of HER2 dimerization in breast cancer cells discovered by correlative fluorescence and liquid electron microscopy

Diana B Peckys et al. Sci Adv. 2015.

Abstract

The formation of HER2 homodimers plays an important role in breast cancer aggressiveness and progression; however, little is known about its localization. We have studied the intra- and intercellular variation of HER2 at the single-molecule level in intact SKBR3 breast cancer cells. Whole cells were visualized in hydrated state with correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM). The locations of individual HER2 receptors were detected using an anti-HER2 affibody in combination with a quantum dot (QD), a fluorescent nanoparticle. Fluorescence microscopy revealed considerable differences of HER2 membrane expression between individual cells and between different membrane regions of the same cell (that is, membrane ruffles and flat areas). Subsequent ESEM of the corresponding cellular regions provided images of individually labeled HER2 receptors. The high spatial resolution of 3 nm and the close proximity between the QD and the receptor allowed quantifying the stoichiometry of HER2 complexes, distinguishing between monomers, dimers, and higher-order clusters. Downstream data analysis based on calculating the pair correlation function from receptor positions showed that cellular regions exhibiting membrane ruffles contained a substantial fraction of HER2 in homodimeric state. Larger-order clusters were also present. Membrane areas with homogeneous membrane topography, on the contrary, displayed HER2 in random distribution. Second, HER2 homodimers appeared to be absent from a small subpopulation of cells exhibiting a flat membrane topography, possibly resting cells. Local differences in homodimer presence may point toward functional differences with possible relevance for studying metastasis and drug response.

Keywords: HER2 homodimers; SKBR3 cells; breast cancer; correlative fluorescence- and electron microscopy; electron microscopy in liquid; membrane ruffles; resting cells.

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Figures

Fig. 1
Fig. 1. Schematic representation of HER2 labeling with affibody-QD probes and correlative microscopy of whole cells in hydrated state.

(A) The biotinylated affibody (blue) directed against HER2 (red) binds to a single epitope on the extracellular part of the membrane receptor. The single biotin moiety of the affibody binds a streptavidin (green)–conjugated QD. QDs can emit bright fluorescence signals, and their electron-dense core can be detected with electron microscopy. (B) Cells were grown on a silicon nitride (SiN) membrane supported by a silicon chip. A microchip with QD-labeled and fixed cells was positioned upside down in a saline-filled glass-bottom dish. Fluorescence imaging was performed with an oil immersion lens on an inverted bright-field microscope. (C) For ESEM, the same microchip was positioned upright on a cooled stage and kept in a saturated water vapor atmosphere. The hydrated cells were covered with a thin layer of purified water. Contrast was obtained on the QDs using the STEM detector located underneath the sample.

Fig. 2
Fig. 2. Correlative light and electron microscopic overview images of affibody-QD–labeled HER2 on SKBR3 human breast cancer cells.

(A) Fluorescence overview image of the central region of a microchip, showing several dozen cells. Individual cells exhibit a high degree of heterogeneity in their morphology and HER2 membrane expression. The location of the SiN membrane window is indicated as a rectangular dashed outline. A differential interference contrast (DIC) image of the window area is overlaid. The DIC image provides an impression of the three-dimensional topography. Scale bar, 100 μm. Cells from which ESEM-STEM images were recorded are indicated by numbers. (B) Higher-resolution fluorescence image, recorded with a 63× oil immersion lens, of the cells in the upper half of the window area, marked as a solid line rectangle in (A). The fluorescence signal of cell #2 indicated a high level of HER2 membrane expression, whereas cell #5 showed only weak HER2 membrane expression. Cell #4 concentrated HER2 on membrane ruffles. The boxed area is shown in Fig. 3A. (C) DIC image of the same region as in (B), depicting the membrane topography in greater detail. (D) ESEM-STEM image from the same region as shown in (B) and (C) (×1000 magnification). Scale bars, 20 μm.

Fig. 3
Fig. 3. Correlative light and electron microscopic images showing the distribution of QD-labeled HER2 on an SKBR3 cell.

(A) Selected area of the fluorescence image in Fig. 2B. This region contained membrane ruffles as shown in the DIC image of Fig. 2C. (B) ESEM-STEM image at the same area, recorded at ×15,000 magnification. (C) Pair correlation function g(x) (line) determined from the 122 detected label positions in the rectangular region in (B). The dashed line at unity serves as a guide to the eye for a random distribution. (D) STEM image recorded of the boxed region shown in (B) at ×75,000 magnification. The localization of individual HER2 receptors became visible as the bright, bullet-shaped QDs. (E) Automatically detected labels were outlined in light green. Numerous pairs of HER2 were observed (two examples are indicated by arrowheads). Scale bars, 2 μm (A and B); 200 nm (D and E).

Fig. 4
Fig. 4. Cluster analysis of HER2 proteins in 11 SKBR3 cells.

(A) The pair correlation function g(x) of a total of 14,171 labels exhibited a peak at 20 nm, indicating HER2 dimerization. Larger-sized clusters of up to several hundreds of nanometers were also observed. The curves of randomly dispersed QDs and a simulation (Simu) of random data were included as reference. (B) HER2 pairs were absent in cellular areas with a homogeneous membrane topography, contrasting g(x) in the ruffled areas. (C) HER2 does not appear clustered in the two analyzed flat cells. Clustering was only observable in cells with membrane ruffles.

Fig. 5
Fig. 5. Correlative light and electron microscopy of a cell with ruffles.

(A) Fluorescence image of a selected region at the edge of cell #11. (B) Same area as in (A) but imaged with ESEM-STEM. Several membrane ruffles are visible as structures of increased brightness. (C) An ESEM-STEM image was recorded at the location #1 in (B) of a region with homogeneous membrane topography, without ruffles. The g(x) function indicated a random distribution of 209 automatically detected QD label positions. (D) Region #2 in (B) contained membrane ruffles. The curve of g(x) determined from 417 label positions in the ESEM-STEM image in the area contained a peak at 20 nm and also pointed toward clustering at larger distances. Scale bars, 10 μm.

Fig. 6
Fig. 6. Correlative light and electron microscopy of a typical flat cell.

Flat cell is indicated with an asterisk. (A) A selected area of the fluorescence image showed a lower HER2 labeling density in this cell compared to adjacent cells. The dashed line indicated the edge of the SiN window. (B) DIC shows that the cell has a ~10-μm flat and smooth outer rim without membrane ruffles or protrusions. (C) Overview ESEM-STEM image in which the flat rim was clearly visible. (D) A selected region [indicated by the rectangle in (C)] of a high-resolution ESEM-STEM image showed scattering of QD labels. (E) The curve of g(x) obtained from the 188 QDs in this cellular region indicated a random distribution of HER2 around unity. Scale bars, 20 μm (A and B); 5 μm (C); 500 nm (D).

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