A Novel Computer-Assisted Approach to evaluate Multicellular Tumor Spheroid Invasion Assay

Abstract

Multicellular tumor spheroids (MCTSs) embedded in a matrix are re-emerging as a powerful alternative to monolayer-based cultures. The primary information gained from a three-dimensional model is the invasiveness of treatment-exposed MCTSs through the acquisition of light microscopy images. The amount and complexity of the acquired data and the bias arisen by their manual analysis are disadvantages calling for an automated, high-throughput analysis. We present a universal algorithm we developed with the scope of being robust enough to handle images of various qualities and various invasion profiles. The novelty and strength of our algorithm lie in: the introduction of a multi-step segmentation flow, where each step is optimized for each specific MCTS area (core, halo, and periphery); the quantification through the density of the two-dimensional representation of a three-dimensional object. This latter offers a fine-granular differentiation of invasive profiles, facilitating a quantification independent of cell lines and experimental setups. Progression of density from the core towards the edges influences the resulting density map thus providing a measure no longer dependent on the sole area size of MCTS, but also on its invasiveness. In sum, we propose a new method in which the concept of quantification of MCTS invasion is completely re-thought.

The impact of temozolomide and lonafarnib on the stemness marker expression of glioblastoma cells in multicellular spheroids

“…Overall, such a heterogenous MCTS model can be utilized to investigate the impact of cellular cues on the drug response of GBM cells. However, we note the following limitations of our study: (a) An established GBM cell line U87‐MG was used in this study which has been commonly used in MCTS models 56–60 . However, the histopathological and biologic profile of these cells have been reported to be different from the original tumor due to prolonged passaging, immortalization, and culture conditions 61,62 .…”

Therapeutic anti-glioma effect of the combined action of PCSK inhibitor with the anti-tumoral factors secreted by Poly (I:C)-stimulated macrophages

“…The implemented algorithm uses local fluctuations of the image intensity for automated estimation of the invasion magnitude. It is robust enough to handle micrographs of different generation methods and various qualities without the concept of an invasive front of the spheroids [ 26 , 42 ]. Data of areas are normalized for each day to the relative size of day zero and transformed into the percentage of invasion.…”

Location of neonatal microglia drives small extracellular vesicles content and biological functions in vitro

“…The implemented algorithm uses local fluctuations of the image intensity for an automated estimation of the invasion magnitude. It is robust enough to handle micrographs of different generation methods and various qualities without the concept of an invasive front of the spheroids [46]. Invasion and spheroid areas are normalized for each day to the invasion and spheroid areas measured at day 0.…”