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.

Complex functioning of the genome in the cell nucleus is controlled at different levels: (a) the DNA base sequence containing all relevant inherited information; (b) epigenetic pathways consisting of protein interactions and feedback loops; (c) the genome architecture and organization activating or suppressing genetic interactions between different parts of the genome. Most research so far has shed light on the puzzle pieces at these levels. This article, however, attempts an integrative approach to genome expression regulation incorporating these different layers. Under environmental stress or during cell development, differentiation towards specialized cell types, or to dysfunctional tumor, the cell nucleus seems to react as a whole through coordinated changes at all levels of control. This implies the need for a framework in which biological, chemical, and physical manifestations can serve as a basis for a coherent theory of gene self-organization. An international symposium held at the Biomedical Research and Study Center in Riga, Latvia, on 25 July 2022 addressed novel aspects of the abovementioned topic. The present article reviews the most recent results and conclusions of the state-of-the-art research in this multidisciplinary field of science, which were delivered and discussed by scholars at the Riga symposium.

In cancer, vulnerable breast epithelium malignance tendency correlates with number and activation of ErbB receptor tyrosine kinases. In the presented work, we observe ErbB receptors activated by irradiation-induced DNA injury or neuregulin- $1\mathsf{\beta }$ application, or alternatively, attenuated by a therapeutic antibody using high resolution fluorescence localization microscopy. The gap junction turnover coinciding with ErbB receptor activation and co-transport is simultaneously recorded. DNA injury caused by 4 Gray of 6 MeV photon $\mathsf{\gamma }$ -irradiation or alternatively neuregulin- $1\mathsf{\beta }$ application mobilized ErbB receptors in a nucleograde fashion—a process attenuated by trastuzumab antibody application. This was accompanied by increased receptor density, indicating packing into transport units. Factors mobilizing ErbB receptors also mobilized plasma membrane resident gap junction channels. The time course of ErbB receptor activation and gap junction mobilization recapitulates the time course of non-homologous end-joining DNA repair. We explain our findings under terms of DNA injury-induced membrane receptor tyrosine kinase activation and retrograde trafficking. In addition, we interpret the phenomenon of retrograde co-trafficking of gap junction connexons stimulated by ErbB receptor activation.

Cancer studies suggest that the spatial localization of connexin43 (Cx43) could play an important role during tumor genesis and the formation of metastasis. Cx43 has been shown to be upregulated in cancer cells; thereby a shift from Cx43 normal localization in gap junctions in the cell membrane towards a primarily cytoplasmic localization was observed in many studies. So far neither the spatial arrangements of Cx43 in breast cancer cells nor the effects of treatment outcome (ionizing radiation and antibody therapy) on the spatial arrangements of Cx43, have been microscopically studied on the nanoscale. This has brought up the idea to study the micro- and nanoscaled spatial Cx43 arrangements in a model of breast cancer-related cell types, i.e., SkBr3 breast cancer cells, BJ fibroblasts, and primary human internal mammary artery endothelial cells (HIMAECs). The cells were treated with neuregulin1 (NRG1), trastuzumab (Herceptin), or 6MeV-photon irradiation at a dose of 4 Gy. NRG1 stimulates further NRG1 release in the tumor endothelium that may lead to an enhanced tumor protective effect whereas Herceptin, used in antibody treatment, works in an antagonistic fashion to NRG1. After fluorescent labelling with specific antibodies, the molecular positions of Cx43 in the perinuclear cytosol and in the cell periphery at the membrane were determined for the three treatment related applications (NRG1, trastuzumab, 4 Gy irradiation) using confocal laser scanning microscopy (CLSM) and single molecule localization microscopy (SMLM). These techniques enable investigations of Cx43 enrichment and topological arrangements of Cx43 molecules from the micro-scale of a whole cell to the nano-scale of single molecules. In SkBr3 cells with and without radiation treatment high density accumulations were detected which seem to be diluted after NRG1 and trastuzumab treatment although the SMLM distance frequency distributions did not significantly vary. In BJ fibroblasts and HIMAECs differences between periphery and perinuclear cytosol were observed after the different treatment processes. HIMAECs showed significant Cx43 accumulation after NRG1, trastuzumab, and radiation treatment in the perinuclear region whereas in the periphery radiation has less influence as compared to the control. BJ cells were reacting to the treatments by Cx43 accumulations in the perinuclear region but also in the periphery. In conclusion, it was shown that by using CLSM and super-resolution SMLM, treatment effects on the spatial and thus functional arrangements of Cx43 became detectable for investigations of tumor response mechanisms.

The present work addresses the question of to what extent a geometrical support acts as a physiological determining template in the setup of artificial cardiac tissue. Surface patterns with alternating concave to convex transitions of cell size dimensions were used to organize and orientate human-induced pluripotent stem cell (hIPSC)-derived cardiac myocytes and mouse neonatal cardiac myocytes. The shape of the cells, as well as the organization of the contractile apparatus recapitulates the anisotropic line pattern geometry being derived from tissue geometry motives. The intracellular organization of the contractile apparatus and the cell coupling via gap junctions of cell assemblies growing in a random or organized pattern were examined. Cell spatial and temporal coordinated excitation and contraction has been compared on plain and patterned substrates. While the α-actinin cytoskeletal organization is comparable to terminally-developed native ventricular tissue, connexin-43 expression does not recapitulate gap junction distribution of heart muscle tissue. However, coordinated contractions could be observed. The results of tissue-like cell ensemble organization open new insights into geometry-dependent cell organization, the cultivation of artificial heart tissue from stem cells and the anisotropy-dependent activity of therapeutic compounds.

DNA double-strand breaks (DSBs) are known to be the most serious lesions in irradiated cells. Several protein pathways exist for repair. The mechanisms by which cells determine a specific pathway for repair remain poorly understood. DSB induction and repair can be spatiotemporally monitored by ionizing radiation-induced foci (IRIFs) and the formation of repair complexes. IRIF analyses revealed that DSB formation, repair and misrepair are strongly dependent on the radiation characteristics and the microarchitecture of the chromatin environment. However, the IRIF nano-architecture remains unknown, as does its impact on the decision-making process and follow-up protein recruitment. New insights into the relationship between the physical properties of radiation, environmental chromatin architecture, IRIF architecture and DSB repair mechanisms are presented using single-molecule localization microscopy. Ripley distance statistics and persistent homology calculations have shifted our ability to analyze chromatin and IRIF architectures from imaging to topology and structure calculations. We discuss these approaches for cancer treatment-relevant irradiation processes on selected cell systems and consider whether this “structuromics” can enhance our knowledge about the radiation response.