Multimodal Imaging of 3D Cell Aggregates

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Authors

PREISLER Jan MACHÁLKOVÁ Markéta ADAMOVÁ Barbora NAVRÁTILOVÁ Jarmila STIBOREK Marek MELIORISOVÁ Stanislava KROUPA Jiří HOUŠKA Pavel MICHÁLEK Jan ŠTĚPKA Karel RADASZKIEWICZ Katarzyna Anna PRUŠKA Adam KANICKÝ Viktor KOZUBEK Michal ŠMARDA Jan

Year of publication 2021
MU Faculty or unit

Faculty of Science

Citation
Description Spheroids, 3D cell aggregates are popular for studies of drug uptake and diffusion. Proliferating and apoptotic cells in the spheroids can be visualized using immunofluorescence microscopy (IFM). Distribution of drugs, which often do not exhibit specific fluorescence, has to be revealed using another technique, such as matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). To evaluate the efficacy of a potential anti-cancer drug perifosine within spheroids of colorectal carcinoma, images of drug abundance from MALDI MSI, and fluorescently visualized proliferating and/or apoptotic cells must be precisely colocalized and carefully compared. Approximately 1-mm spheroids (HT-29 cell line) treated with perifosine were imaged with MALDI MS, the matrix was washed away and cell viability was inspected with IFM. Alternatively, laser ablation coupled to inductively coupled plasma mass spectrometry (LA - ICP MS) was applied for more sensitive viability visualization using immunostaining with 20-nm gold nanoparticles as the tags. The overlay of MALDI MS and IFM spheroid images required fiducial-based coregistration because of the lack of any morphological features in the spheroids. The spatial relationship between the MALDI MS and IFM intensities was evaluated based on the respective intensities along equidistant layers, or “peels” of the entire spheroid, starting from the spheroid boundary. The accurate colocalization of MALDI MS and IFM maps and subsequent peeling data analysis showed a limited penetration of perifosine into spheroids within 24 hours and allowed differentiation between apoptosis resulting from hypoxia/nutrient deprivation and drug exposure. As expected, viability assays from IFM and LA - ICP MS for markers of cell proliferation and apoptosis revealed a higher signal of the apoptotic marker in the spheroid core and a higher signal of proliferative cells on the spheroid edge. We thank to CSF (21-12262S) and GAMU (MUNI/G/0974/2016).
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