MS imaging of multicellular tumor spheroids and organoids as an emerging tool for personalized medicine and drug discovery

Abstract

MS imaging (MSI) is a powerful tool in drug discovery because of its ability to interrogate a wide range of endogenous and exogenous molecules in a broad variety of samples. The impressive versatility of the approach, where almost any ionizable biomolecule can be analyzed, including peptides, proteins, lipids, carbohydrates, and nucleic acids, has been applied to numerous types of complex biological samples. While originally demonstrated with harvested organs from animal models and biopsies from humans, these models are time consuming and expensive, which makes it necessary to extend the approach to 3D cell culture systems. These systems, which include spheroid models, prepared from immortalized cell lines, and organoid cultures, grown from patient biopsies, can provide insight on the intersection of molecular information on a spatial scale. In particular, the investigation of drug compounds, their metabolism, and the subsequent distribution of their metabolites in 3D cell culture systems by MSI has been a promising area of study. This review summarizes the different ionization methods, sample preparation steps, and data analysis methods of MSI and focuses on several of the latest applications of MALDI-MSI for drug studies in spheroids and organoids. Finally, the application of this approach in patient-derived organoids to evaluate personalized medicine options is discussed.

Keywords: MS; cell culture; imaging; pharmacology; tumor therapy.

Figure 1

Combination of analytical images of spheroid sections showing the concentric arrangement of cell proliferation, viability, and the chemical micromilieu. This figure was reprinted with permission from Ref. (14). Copyright (2010) Elsevier B.V.

Figure 2

General workflow of MALDI-MSI on MCTS. MCTSs are collected when they grow to a stable size. (For HCT 116 in culture for 6000 initial cells, it is about 1 mm in diameter at day 14–20. (16)) The harvested MCTSs are washed with PBS to remove the cell culture media and then are embedded in gelatin. The samples are stored in −80 °C and cryosectioned to 12-μm thick slices. The sections are thaw mounted onto ITO slides. A matrix nebulizer can be used to spray the matrix solution onto the slides homogeneously, and MALDI-MSI can be performed. ITO, indium–tin oxide; MCTS, multicellular tumor spheroid; MSI, MS imaging.

Figure 3

Workflow of the washing steps for proteins MSI before matrix desorption. Example taken from Ref. (56), to improve the signal of proteins, tissue samples were washed in 70% EtOH first, followed by 90% EtOH and buffer solution at last. MSI, MS imaging.

Figure 4

Data processing and data analysis of MSI data. MSI data are preprocessed after acquisition. This process includes several steps including spectra normalization, baseline correction, smoothing, and recalibration. Next, MSI data will undergo compression and representation, which reduces the computational load. Then data analysis could be completed, including peak picking and other statistical analyses, such as classification, principal component analysis (PCA), spatial segmentation, and others. Machine learning is a popular method used in these analyses. Postprocessing includes magnification and coregistration of the images. Adapted with permission from Ref. (29). Copyright (2018) American Chemical Society. MSI, MS imaging.

Figure 5

Supervised machine learning process to analytes MSI data of the spheroids After collecting MSI data in a matrix containing “samples” and “features,” cardinal is used to perform machine learning on these features to classify different samples into “control” or “treated.” Reprinted with permission from Ref. (82). Copyright (2020) American Chemical Society. MSI, MS imaging.

Figure 6

Image of FOLFIRI-treated HCT 116 spheroids.A, localization of irinotecan and folinic acid within 24-h treatment. B, a metabolite of folinic acid was detected in the proliferating layer of the spheroids. Reprinted with permission from Ref. (115). Copyright (2018) American Chemical Society. FOLFIRI, FOL: folinic acid/leucovorin; F: 5-fluorouracil (5-FU); and IRI: irinotecan.

Figure 7

Mapping of endogenous metabolites in MCF-7 breast cancer spheroids by MALDI-MSI. Elemental formula of the metabolites assigned can be mapped onto the hexosamine biosynthetic pathway (HBP). N-acetyl neuraminic acid (sialic acid) is formed by the end product of the HBP. Adapted with permission from Ref. (18). Copyright (2019) American Chemical Society. CMP, cytidine monophosphate; CMP-NeuAc, cytidine monophosphate N-acetylneuraminic acid; GlcNAc-1-P, N-acetyl glucosamine-1-phosphate; ManNAc-6-P, N-acetylmannosamine-6-phosphate; MSI, MS imaging; NeuNAc, N-acetylneuraminic acid; Neu9NAc-9-P, N-acetylneuraminic acid-9-phosphate; UDP, uridine diphosphate; UDP-GlcNAc, uridine diphosphate-N-acetyl glucosamine; UMP, uridine monophosphate; UTP, uridine triphosphate.

Figure 8

Analysis of BKM120 and dabrafenib penetration by MALDI–MSI. BBB organoids were incubated with 10 μM of either drug for 24 h. MALDI-MSI ion images show successful detection of BKM120 accumulation within the organoids (green; top row; m/z = 411.1751 ± 0.001). Dabrafenib was not detected within the organoids (bottom row; m/z = 520.1083 ± 0.001). Dashed lines on the scanned images delineate the positions of the BBB sphere tissue sections. This figure is adapted from Ref. (116) under a Creative Commons Attribution 4.0 license (https://creativecommons.org/licenses/by/4.0/legalcode). BBB, blood–brain barrier; MSI, MS imaging.