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

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.

Light sheet fluorescence microscopy guided MALDI-imaging mass spectrometry of cleared tissue samples

“…Also the application of external fiducial masks, i.e., external (physical) position markers which are added to the cleared tissue, and which can unambiguously be identified in the (virtual) 3D-LSFM sample reconstruction as well as in subsequently generated histological sections and MALDI-IMS images, might be useful for the precise excision of LSFM-defined ROI section planes from the cleared 3D tissue sample, as well as for the accurate fusion (“back-matching”) of 2D-MALDI-MSI images with the corresponding 3D-LSFM data. Such external fiducial masks have for example previously successfully been applied for the correlation of mass spectrometry imaging and confocal Raman microscopy 23 or laser scanning confocal microscopy 26 in studies of three-dimensional cell cultures.…”

“…Furthermore, MALDI-MSI analyses can also be combined with a variety of other imaging modalities, providing additional morphological and/or biochemical information from the identical tissue section, or sample 14 , 17 . Previously reported multimodal (MALDI)-MSI analysis approaches e.g., include the combination with autofluorescence-microscopy 21 , 22 , Raman spectroscopy 23 , magnetic resonance imaging (MRI) 24 , 25 , histology, immunohistochemistry, and laser scanning confocal microscopy 26 . Using appropriate alignment methods 22 , 24 , 27 , the images acquired by MALDI-MSI and additional imaging modalities can be exactly overlaid and thus directly be related to e.g., distinct morphological features of the examined tissue, such as different structural compartments, cell types, pathological alterations, etc.…”

Lactic Acidosis Interferes With Toxicity of Perifosine to Colorectal Cancer Spheroids: Multimodal Imaging Analysis

“…Finally, all the spheroids were collected in cryomolds with warm gelatine and frozen at −80°C. Next, the sectioned tissue was processed for the perifosine analysis (MALDI MS) followed by cleaved caspase 8 determination (IHC) (briefly described in the Supplementary Data Sheet 1), full description in (51). Unless otherwise stated, experiments were performed under floating conditions.…”

“…MALDI MSI settings and the workflow for detection of the perifosine spatial distribution in 3D were fully described by Machaĺkováet al, 2019 (51). For an overview, see the Supplementary Data Sheet 1, Methodology.…”

“…The same threshold was used for all samples to be compared. Semiautomatic co-registration of MALDI MSI and IHC signals and the "peeling" analysis were also described by our group recently (50,51).…”