BACKGROUND Three-dimensional tumour spheroid models are increasingly used in cancer research. However, imaging for quantification analysis of spheroids in drug testing has remained primarily based on 2D methodologies. Imaging 3D structures in 2D can cause discrepancies and introduce interpretive bias. We previously described a novel method to create high-resolution 3D spheroid images using histopathological sectioning. We compare for the first time 3D histopathological images and data analysis of glioma spheroid models in drug studies to commonly used imaging techniques, highlighting the advantages of 3D imaging over 2D methodologies. METHODS U251 glioma spheroids embedded in collagen were untreated or treated with inhibitors 6-Bromoindirubin-3’-oxime (BIO) or MI-192. Spheroids were imaged at 24-hour time points over 72 hours using light-microscopy and confocal laser-microscopy and then subsequently embedded in paraffin and sectioned at 5µm. Slides were H&E stained and serially scanned at 20x magnification. Custom software was used to digitally reconstruct the spheroid in 3D. For details at the single cellular level, 70µm spheroids were also imaged at 72 hours at high-magnification using an Instant Structured illumination microscope (iSIM). RESULTS Superior detail of core cellular components and migratory single cells was achieved by 3D histopathological reconstruction compared to all other methods. Migration indices were similar to 3D confocal-generated reconstructions demonstrating promoted migration with MI-192 and reduced migration with BIO. Detailed morphological analysis of migratory cells was achieved at 40x optical magnification using our 3D histology method. However, greater morphological detail was obtained using high-magnification iSIM. Failure to capture migratory cells in the z-plane using 2D techniques lead to considerable differences between 3D and 2D imaging data. CONCLUSION We have shown that 3D histopathological image reconstruction is a preferable method for cell migration and morphological analyses of 3D cancer spheroid models. This method can be used for high-resolution analysis of both core and migratory cells.
|Number of pages||1|
|Publication status||Published - Nov 2018|
|Event||23rd Annual Scientific Meeting and Education Day of the Society for Neuro-Oncology - New Orleans, United States|
Duration: 15 Nov 2018 → 18 Nov 2018
Conference number: 23
https://www.soc-neuro-onc.org/SNO/2018Annual/2018AnnualHome.aspx (Link to Event Information)
Samarasekara, J., Esteves, F., Curd, A., Hale, M., Magee, D., Treanor, D., Short, S. C., & Bruning-Richardson, A. (2018). A novel 3D high-resolution histopathological image reconstruction method versus common 2D and 3D imaging methodologies for application in cancer spheroid research: Which is better? Neuro-Oncology, 20(S6), 271. https://doi.org/10.1093/neuonc/noy148.1125