Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

Abstract

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine.

Keywords: Smurf2; cancer research; chromatin organization; chromatin radiation response; fluorescent nano-probes; receptor conformation changes; super-resolution localization microscopy; γ-H2AX phosphorylation sites.

Figure 1

Schematic description of results obtained from localization microscopy. Blue points represent the measured frequencies of distances usually obtained from several cells of a specimen; the red curve represents the calculated envelope. The data points presented in this figure were taken from different specimens and different labels, showing the structural characteristics being described. (A) Distribution indicating a random arrangement of molecules; (B) structured organization with a characteristic dimension indicated by the maximum of the theoretical curve (red point); (C) organized structure implemented in randomly-distributed molecules.

Figure 2

(A) Image section of a breast cancer cell (MCF7 cell line) after specific labelling of ErbB3: Widefield image (left), image obtained from localization microscopy data (right). The inserts are magnifications of a section of 2 μm × 2 μm. (B) Relative frequency distributions of receptor to receptor distances within the detected clusters of ErbB2 (left) and ErbB3 (right); for comparison see Figure 1B: (a) non-treated control; (b) after neuregulin-1 treatment of ErbB3; (c) after neuregulin-1 stimulation of ErbB3 and trastuzumab treatment of ErbB2; (d) after neuregulin-1 stimulation of ErbB3 and combined treatment of ErbB2 with trastuzumab and pertuzumab. For each curve, 21–25 cells were analyzed and merged. The number of points showed a variety of about 30–35% between the individual cells.

Figure 3

(A) Relative frequencies of the pair-wise distances (in nm) of heterochromatin tags (H3K9me3) labelled with a secondary antibody, carrying rhodamine red X. The curves obtained from 20 cells each (time-stacks of 3500 frames per cell) indicate compaction regions (≤100 nm) in a more randomly organized environment for wild-type U2OS cells (a). Treatment with etoposide resulted in a relaxed configuration for wild-type U2OS cells (b) and Smad specific ubiquitin protein ligase 2 (Smurf2) over-expressing U2OS cells (c). For comparison and explanation of the curves in Figure 3A, see Figure 1C; (B) example of an image of a cell nucleus from a U2OS cell, reconstructed from the loci matrix of heterochromatin tags (Scale bar: 1 µm).

Figure 4

Frequency of pair-wise distances (in nm) of GFP tags for three U2OS cell line modifications: (a) cell line with GFP over-expression; (b) cell line with Smurf2-GFP over-expression; (c) cell line with Smurf2 CG-GFP over-expression. For each curve, 20 cells were measured with 3500 frames per cell. For comparison and interpretation of the curves see Figure 1C.

Figure 5

Image section of a cell nucleus of a breast cancer cell line (SKBr3) after staining of heterochromatin (green) and γH2AX foci (red) by fluorescent specific antibodies. The cells were irradiated by 1 Gy photon-radiation and the images were acquired 60 minutes after irradiation during activated repair. (A) wide-field fluorescence image, scale bar: 2 µm ; (B) localization microscopy image of the same image section, scale bar: 2 µm; (C) enlarged inserts of A separated in the color channels red and green, image size 2 µm × 2 µm; (D) enlarged inserts of B separated in the color channels red and green, image size 2 µm × 2 µm.

Figure 6

Normalized frequencies of the signal distances of fluorescence-labelled antibodies to γH2AX phosphorylation sites (upper graphs) and heterochromatin H3K9me3 methylation sites (lower graphs), as determined by localization microscopy for different repair times after exposure to 3.9 Gy ionizing radiation. The differently colored curves were obtained from 20–25 cells from aliquots that were fixed at certain time points after irradiation (see insert in the lower figure). For comparison and interpretation of the curve shapes see Figure 1; for instance, for the pink curves see Figure 1A; for the green curve in the lower figure see Figure 1C; for the light blue curve in the upper image see Figure 1B.

Figure 7

Average number of measured ALU elements relative to the radiation exposure dose. The data were detected after specimen fixation 30 min after irradiation with 6 MeV photons. The measurement data is fitted by a linear curve (N(D) = aD + b) with the parameters: a = −12,143, and b = 24,345. The error bars reflect the variations between the individual cells.

Figure 8

Visualization of the point matrix after two-color localization microscopy data acquisition. ALU COMBO-FISH oligonucleotides are labeled in red; heterochromatin methylation sites simultaneously labeled by antibodies (H3K9me3) are shown in green. The right image shows the merged visualization. The enlarged inserts demonstrate the exclusive arrangement of ALU sequences and heterochromatin target sites. In the whole image, only one (blue point) locus suggests a co-localization, which may be due to optical overlap (scale bar 1 µm for the merged image).