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Anal Chem. 2019 Nov 5;91(21):13475-13484. doi: 10.1021/acs.analchem.9b02462. Epub 2019 Oct 17.

Drug Penetration Analysis in 3D Cell Cultures Using Fiducial-Based Semiautomatic Coregistration of MALDI MSI and Immunofluorescence Images.

Author information

1
Department of Chemistry, Faculty of Science and Central European Institute of Technology (CEITEC) , Masaryk University , Kamenice 5 , 625 00 Brno , Czech Republic.
2
Department of Experimental Biology, Faculty of Science , Masaryk University , Kamenice 5 , 625 00 Brno , Czech Republic.
3
Centre for Biomedical Image Analysis, Faculty of Informatics , Masaryk University , Botanická 68a , 602 00 Brno , Czech Republic.
4
Center for Biological and Cellular Engineering, International Clinical Research Center , St. Anne's University Hospital , Pekařská 53 , 656 91 Brno , Czech Republic.

Abstract

In this paper, we present an easy-to-follow procedure for the analysis of tissue sections from 3D cell cultures (spheroids) by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and laser scanning confocal microscopy (LSCM). MALDI MSI was chosen to detect the distribution of the drug of interest, while fluorescence immunohistochemistry (IHC) followed by LSCM was used to localize the cells featuring specific markers of viability, proliferation, apoptosis and metastasis. The overlay of the mass spectrometry (MS) and IHC spheroid images, typically without any morphological features, required fiducial-based coregistration. The MALDI MSI protocol was optimized in terms of fiducial composition and antigen epitope preservation to allow MALDI MSI to be performed and directly followed by IHC analysis on exactly the same spheroid section. Once MS and IHC images were coregistered, the quantification of the MS and IHC signals was performed by an algorithm evaluating signal intensities along equidistant layers from the spheroid boundary to its center. This accurate colocalization of MS and IHC signals showed limited penetration of the clinically tested drug perifosine into spheroids during a 24 h period, revealing the fraction of proliferating and promigratory/proinvasive cells present in the perifosine-free areas, decrease of their abundance in the perifosine-positive regions, and distinguishing between apoptosis resulting from hypoxia/nutrient deprivation and drug exposure.

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