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.

Antioxidative stress of Chitosan-Gallium Nanoparticles and low doses of γ-irradiation on mice bearing Ehrlich carcinoma

“…Researchers have considered the impacts of low-dose radiation, given as a single dose or fractionated or prolonged over time, with regard to altering cancer progression or resolution as well as their impacts on the systemic immune system [23] .It was found that low-dose radiation increases cellular antioxidant activity; facilitates DNA damage repair; reduces malignant transformation and mutagenesis, and stimulates immune surveillance [24]. The cell nucleus and DNA are susceptible to several stressors[25, 26,27,28] and can be greatly harmed with relatively low doses of ionizing radiation [29,30]. As adverse effects of ionizing radiation on (cancer) cells are mainly mediated by fragmentation of nuclear chromatin via inserting double strand breaks (DSBs) into the DNA molecule [31].…”

COMBO-FISH: A Versatile Tool Beyond Standard FISH to Study Chromatin Organization by Fluorescence Light Microscopy

“…For each series of SMLM experiments, typically 50 cell nuclei were registered. An in-house software (see [31,39,54,65,77,78] for detailed descriptions) was used for the localization of signal points from blinking events in raw image stacks. The resulting data matrix containing the coordinates of detected signals was subject to further computational analysis.…”

“…Thereby, mega base-pair sized topologically associating domains (TADs) and subdomains therein (subTADs) [26][27][28][29] have been identified as structural units of the threedimensional genome [30]. In parallel, modern super-resolution microscopy technologies [31][32][33][34][35][36][37][38][39] have been developed, enabling analyses at the single cell level of cellular nanostructures and molecular interactions on the order of magnitude of single antibodies, histones, receptors, etc. (i.e.…”

“…(i.e. in the order of 10 nm) by in situ nano-tagging [31]. In this respect, the identification of representative structural parameters as markers for the nano-scale organization of genomic elements in chromatin context is among the central tasks [10,40,41].…”

“…centromere 9 [56], receptor tyrosine kinase 2 (HER2/NEU) [61], breakpoint cluster region (BCR) [62,63], ABL proto-oncogene 1 (ABL) [62][63][64], and genomic Alu elements [31,65]). Oligo-nucleotide labelling localization microscopy [39,66,67] of Alu elements revealed structural changes in chromatin after irradiation [65] and was discovered as a novel dosimetric marker for low [31] to high [65] dose ranges. Results from nanoscopy of COMBO-FISH labelled trinucleotide expansions in the FMR1 gene promoter represent microscopic counting of repetitive units, which could be addressed as first steps towards realization of in situ optical sequencing [54].…”