Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles.

BACKGROUND: Disruption of Ca2+ homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca2+ kinetics and gene expression involved in the regulation of Ca2+ signaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1. METHODS: CaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca2+ concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca2+ regulation ([Ca2+]i) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl3, and GdCl3. Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca2+ signaling through RNA sequencing. RESULTS: EA.hy926 cells, at increasing Ca2+ concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca2+-dependent morphological changes in EA.hy926's actin filaments, microtubules, and cell-cell junctions. Spectrofluorometric Ca2+ kinetics showed higher amplitudes in Ca2+ responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca2+]i changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca2+ ions induced a significantly higher [Ca2+]i rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca2+-related gene expression, highlighting upregulation of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca2+ import and downregulated export genes in EA.hy926. CONCLUSIONS: Our finding show that significant differences in CaEP response and [Ca2+]i regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca2+]i changes, which may be linked to overexpression of Ca2+-related genes and an inability to mitigate changes in [Ca2+]i. The study offers a bioinformatic basis for selecting EC models based on research objectives.

Imaging of Extravasation of Splenocytes in the Dorsal Skinfold Window Chamber.

Infiltration of immune cells into the tumor is one of the major drivers of antitumor immune response, which can direct the outcome of anticancer therapies. In mice, implantation of dorsal skinfold window chamber (DSWC) combined with intravital confocal fluorescence microscopy allows real-time observation of splenocyte extravasation and infiltration into tumors. Here, we describe a detailed procedure of the DSWC implantation, splenocyte isolation and fluorescent labeling, intravenous injection of labeled splenocytes, and imaging of splenocyte extravasation into tumors using confocal fluorescence microscopy.

Effect of electrochemotherapy on myogenesis of mouse C2C12 cells in vitro.

Electrochemotherapy (ECT) is a local ablative therapy for the treatment of different skin and subcutaneous tumors and certain tumors in internal organs. Skeletal muscle represents a major tumor- surrounding tissue, exposed to side effects of ECT. At the cellular level, side-effects of ECT on skeletal muscle and underlying mechanisms have not been examined yet. Thus, we aimed to determine the effect of ECT in the mouse muscle cell line C2C12 during in vitro myogenesis. We evaluated the electroporation efficiency and viability of C2C12 myotubes at increasing voltages (200-1300 V/cm) using propidium iodide (PI). Permeabilization of PI into the cells was voltage-dependent accounting up to 97 % efficiency at the highest voltage. High cell viability and myotube integrity were maintained until 4 days after electroporation. ECT with the cytostatic drugs bleomycin and cisplatin decreased the viability of C2C12 myoblasts and myotubes in a dose-dependent manner. However, myoblasts were more sensitive to ECT than myotubes. Increased secretion of IL-6, observed 3 days after ECT, confirming its effects on early myogenesis. Only minor effects of ECT were observed in treated myotubes. These results contribute to the safety profile of ECT in tumor treatment.

Estimating quantitative physiological and morphological tissue parameters of murine tumor models using hyperspectral imaging and optical profilometry.

Understanding tumors and their microenvironment are essential for successful and accurate disease diagnosis. Tissue physiology and morphology are altered in tumors compared to healthy tissues, and there is a need to monitor tumors and their surrounding tissues, including blood vessels, non-invasively. This preliminary study utilizes a multimodal optical imaging system combining hyperspectral imaging (HSI) and three-dimensional (3D) optical profilometry (OP) to capture hyperspectral images and surface shapes of subcutaneously grown murine tumor models. Hyperspectral images are corrected with 3D OP data and analyzed using the inverse-adding doubling (IAD) method to extract tissue properties such as melanin volume fraction and oxygenation. Blood vessels are segmented using the B-COSFIRE algorithm from oxygenation maps. From 3D OP data, tumor volumes are calculated and compared to manual measurements using a vernier caliper. Results show that tumors can be distinguished from healthy tissue based on most extracted tissue parameters ( p < 0.05 ). Furthermore, blood oxygenation is 50% higher within the blood vessels than in the surrounding tissue, and tumor volumes calculated using 3D OP agree within 26% with manual measurements using a vernier caliper. Results suggest that combining HSI and OP could provide relevant quantitative information about tumors and improve the disease diagnosis.

Structural Diversity of Sense and Antisense RNA Hexanucleotide Repeats Associated with ALS and FTLD.

The hexanucleotide expansion GGGGCC located in C9orf72 gene represents the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). Since the discovery one of the non-exclusive mechanisms of expanded hexanucleotide G4C2 repeats involved in ALS and FTLD is RNA toxicity, which involves accumulation of pathological sense and antisense RNA transcripts. Formed RNA foci sequester RNA-binding proteins, causing their mislocalization and, thus, diminishing their biological function. Therefore, structures adopted by pathological RNA transcripts could have a key role in pathogenesis of ALS and FTLD. Utilizing NMR spectroscopy and complementary methods, we examined structures adopted by both guanine-rich sense and cytosine-rich antisense RNA oligonucleotides with four hexanucleotide repeats. While both oligonucleotides tend to form dimers and hairpins, the equilibrium of these structures differs with antisense oligonucleotide being more sensitive to changes in pH and sense oligonucleotide to temperature. In the presence of K+ ions, guanine-rich sense RNA oligonucleotide also adopts secondary structures called G-quadruplexes. Here, we also observed, for the first time, that antisense RNA oligonucleotide forms i-motifs under specific conditions. Moreover, simultaneous presence of sense and antisense RNA oligonucleotides promotes formation of heterodimer. Studied structural diversity of sense and antisense RNA transcripts not only further depicts the complex nature of neurodegenerative diseases but also reveals potential targets for drug design in treatment of ALS and FTLD.