Spatial Arrangements of Connexin43 in Cancer Related Cells and Re-Arrangements under Treatment Conditions: Investigations on the Nano-Scale by Super-Resolution Localization Light Microscopy

Abstract

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.

Synapses and neural communication in neuropathological conditions

“…From this perspective, researchers have been able to identify cancer cell lineages with these characteristics 42 . In fact, identifying cells with aberrant Cx43 expression allow experts to develop new treatment options for brain tumors and block GJs to prevent chemoresistance 43 . New approaches have been developed to identify tumor genesis response mechanisms.…”

Advanced image-free analysis of the nano-organization of chromatin and other biomolecules by Single Molecule Localization Microscopy (SMLM)

“…Another example that illustratively demonstrates the fundamental importance of architecture-based regulations is the self-formation of lipid membranes with embedded clusters of receptor proteins, driven by minimum entropy of the system [58] , [59] . Cell activities overexpressing receptors [60] , ligand binding to receptors [61] , or cell irradiation—all these and other processes provoke changes in the receptor cluster formation and induce incorporation of the receptor molecules into the cytosol followed by molecular trafficking [61] , [62] , [63] .…”

Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach

“…[ 5 , 6 ]. Such damage may dramatically impact intracellular processes such as energy metabolism, DNA replication or protein synthesis (see, for example, [ 4 , 7 , 8 , 9 , 10 ]). Hence, the cells would never properly function if they would not have developed efficient strategies and mechanisms to repair all types of DNA damage.…”

“…Sci. 2021, 22, 3636 2 of 25 replication or protein synthesis (see, for example, [4,[7][8][9][10]). Hence, the cells would never properly function if they would not have developed efficient strategies and mechanisms to repair all types of DNA damage.…”