Abstract
Three-dimensional (3D) cell cultures represent small avascular tumors in vitro and simulate some of the biological characteristics of solid tumors, enhancing the evaluation of anticancer drug efficacy. Automated image analysis can be used for the assessment of tumor growth and documentation of changes in the size parameters of 3D tumor spheroids following anticancer treatments such as electrochemotherapy. The objective of this article is to assess the effect of various electroporation (EP) conditions (500–750 Volts/cm, 8–20 pulses, 100 µs pulse duration, 5 Hz repetition rate) combined with different bleomycin concentrations (1–2.5 ug/mL) on normal epithelial (HCjE-Gi) and conjunctival melanoma (CRMM1, CRMM2) 3D-cell cultures, through an automated image analysis and a comparison with standard histological assays. A reduction in tumor mass with loss of cell definition was observed after ECT (750 Volts/cm with eight pulses and 500 Volts/cm with 20 pulses) with bleomycin (1 μg/mL and 2.5 μg/mL) in the histological and immunohistochemical analyses of 3D CRMM1 and CRMM2 spheroids, whereas an increase in volume and a decrease in sphericity was documented in the automated image analysis and 3D visualization of both melanoma cell lines. For all other treatment conditions and for the HCjE-Gi cell line, no significant changes to their morphological features were observed. Image analysis with integrated software tools provides an accessible and comprehensive platform for the preliminary selection of homogenous spheroids and for the monitoring of drug efficacy, implementing the traditional screening methods.
Highlights
Cancer cells cultured in a two-dimensional (2D) environment offer a simple in vitro model to evaluate various chemical and biological compounds during investigation of novel candidate anticancer drugs [1,2,3,4]
Open-source automated image analysis software toolboxes, such as ImageJ, ANAlyse SPheroids (AnaSP), Reconstruction and Visualization from a Single Projection (ReViSP) are widely used for the assessment of various sized parameters of 3D tumor spheroids, and thereby tumor response at each step of a drug screen
We present the application of ECT in Conjunctival melanoma (CM) 3D-spheroid models using various drug concentrations and electric field parameters
Summary
Cancer cells cultured in a two-dimensional (2D) environment offer a simple in vitro model to evaluate various chemical and biological compounds during investigation of novel candidate anticancer drugs [1,2,3,4] Despite their cost-effectiveness and reproducibility, monolayers grown in culture flasks or petri dishes do not reproduce the development of normal or tumor tissues nor their complex spatial organization and interactions [5,6]. Open-source automated image analysis software toolboxes, such as ImageJ, AnaSP, ReViSP are widely used for the assessment of various sized parameters of 3D tumor spheroids, and thereby tumor response at each step of a drug screen Such software applications allow the analysis of tumor growth after anticancer treatments, such as electrochemotherapy (ECT), with or without a cytostatic drug
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