Radiomic analysis of 3D spheroids using 2D brightfield images

• Radiomic analysis of 3D spheroids using standard 2D brightfield microscopy images. • Description of AnaSP 3.0, an open-source software for spheroid image analysis. • AnaSP 3.0 can assess spheroid sphericity, cell count, and necrotic core percentage. • New grey-level features extracted not from binary masks but from grey-level spheroid images. • Co-culture spheroids from osteosarcoma and mesenchymal cells. Radiomics is a field of quantitative imaging involving the extraction of features from medical images transforming them into mineable data for diagnostic, prognostic, and predictive purposes. In this work, we focused on the analysis of 2D brightfield images acquired with standard microscopes today available in all biological laboratories. We analysed images of 3D multicellular spheroids, an in vitro model commonly used in Regenerative Medicine and Oncology. Besides describing Analysis of Spheroid version 3.0 ( AnaSP 3.0 ), a new release of an open-source software suite specifically designed for segmenting and extracting features from 2D brightfield spheroid images. To validate the tool, spheroids generated with U-shape bottom multi-well plates and different concentrations of mesenchymal stem cells and osteosarcoma cell lines have been used. We demonstrated how advanced features included in AnaSP 3.0 , i.e. sphericity, volume, and image gradient, may be used to indirectly assess the spheroidization of the spheroids over time, the number of cells composing the spheroids, and the extension of a possible central necrotic core. AnaSP 3.0 is freely available at https://sourceforge.net/p/anasp . Compared to the previous versions, it includes a new algorithm for volume estimation and introduces new modules to extract advanced features using the original grey-level image values, rather than just relying on binary masks.