Role of MALDI-MSI in combination with 3D tissue models for early stage efficacy and safety testing of drugs and toxicants

Figures & data

Table 1. Table includes a summary of literature reporting the analysis of 3D culture models with MSI discussed within review. Table is not limited to all publications of 3D culture models in combination with MSI

Paper	Year Published	Disease	3D Model	3D Method	MSI Method	Spatial Resolution	Summary
Li and Hummon. Anal. Chem. [30]	2011	HCT 116 colon carcinoma	Spheroids	Aggregation	MALDI	75 µm	MSI detected specific regional distributions of proteins across the 3D culture structures and highlighted a central necrotic region. Proteins including Histone H4 and cytochrome C identified in spheroids, validated by LC MS/MS.
Ahlf Wheatcraft, Liu and Hummon. J. Vis. Exp. [31]	2014	HCT 116 colon carcinoma	Spheroids	Aggregation	MALDI	unreported	Spheroid-MSI workflow protocol. Includes the culture, preparation and analysis of spheroid samples by MALDI-MSI.
Hiraide et al., Sci. Rep. [32]	2016	HCT 116 & DLD-1 colon carcinoma. Cancer tissue-originated spheroids (CTOs) – primary cells.	Spheroids	Aggregation followed by Cellmatrix type I-A scaffold gel	(AP) MALDI	5 µm laser spot size	Tandem MSI identified specific lipid species arachidonic acid containing PI (18:0/20:4) within the outer region in spheroid culture. Phospholipid associated with metastatic behavior.
Liu, Weaver and Hummon. Anal Chem. [33]	2013	HCT 116 colon carcinoma	Spheroids	Aggregation	MALDI	75 µm	Drug penetration of irinotecan mapped over a 72 h time-dependent course, moving from outer region of spheroid into the core. Detection of drug metabolites within spheroid validated by nanoLC MS/MS.
Li and Hummon. Sci. Rep. [34]	2016	HCT 116 colon carcinoma	Spheroids	Aggregation	MALDI	75 µm	MSI of platinum-based drugs and their metabolites displayed heterogeneous distributions within spheroids by derivatization. UPLC MS/MS quantification.
Lukowski, Weaver and Hummon. Sci. Rep. [35]	2017	HCT 116 colon carcinoma	Spheroids	Aggregation	MALDI	75 µm	Demonstration of doxorubicin-encased liposomes delivery into spheroids. Detection of doxorubicin and active metabolites throughout spheroid by 72 h.
Feist et al., Anal. Chem. [36]	2017	HCT 116 colon carcinoma	Spheroids	Aggregation	MALDI	75 µm	Proteomic imaging of histone peptides and their posttranslational modifications determined the response to epigenetic drug within serial sections of spheroids, generating samples from core, the mid and external areas.
LaBonia et al., Anal. Chem. [37]	2018	HCT 116 colon carcinoma	Spheroids	Aggregation	MALDI	75 µm	Proteomic changes from a dynamic flow dosing with FOLFIRI treatment investigated and quantified by iTRAQ analysis. Heterogeneous distribution of drugs and active metabolites throughout spheroid.
Liu et al., Anal. Chem. [38]	2018	HT-29 & DLD-1 colon carcinoma Colorectal cancer	Spheroids Cancer organoids	Aggregation Patient biopsies	MALDI	70 µm	On-tissue reduction and alkylation method to detect time-dependent penetration and distribution of monoclonal antibody, Cetuximab. Different localizations of immuno-drug determined between two cell lines. Detection in organoid cultures also demonstrated.
Flint et al., Anal Chem. [43]	2020	HCT 116 colon carcinoma	“Aggregoid”	Alginate scaffold followed by aggregation	DESI, imaging mass cytometry, LA-ICP-MSI	30 µm 1 µm 6 µm (laser spot)	Characterization of a novel aggregated spheroid model by metabolites, proteins and metals determined phenotypic regions of the tumor microenvironment for pre-clinical applications.
Avery et al., Xenobiotica [47]	2011	Healthy human epidermal skin	Skin organoid	Straticell-RHE-EPI-001 Reconstruct-ed Human Epidermis	MALDI	150 µm	Demonstrated a MALDI-MSI method of an artificial skin model analysis by detection of imipramine absorption.
Francese et al., Anal. Chem. [48]	2013	Healthy living skin equivalent (LSE)	Skin organoid	Evocutis “LabSkin”	MALDI	50 µm	The evaluation of curcumin as a matrix for MALDI across different applications, lung tissue, artificial skin and fingermark. Lipid imaging identified epidermis and dermis regions within the LSE. Detection of acitretin distribution within LSE.
Harvey et al., Proteomics [49]	2016	Healthy and psoriatic LSE	Skin organoid	Innovenn “Labskin”	MALDI	100 μm	Absorption of acitretin within psoriatic LSE by MALDI-MSI determined a larger depth of penetration in psoriatic model compared to healthy LSE.
Russo et al., Anal. Chem. [50]	2018	Healthy living skin equivalent (LSE)	Skin organoid	Innovenn “Labskin”	MALDI	60 μm	Quantitation of terbinafine hydrochloride absorption into epidermal region of LSE by MALDI-MSI. Regions of LSE identified by detection of abundant lipid species. Penetration enhancer DMI increased the concentration of drug. Validated by LC MS/MS.
Bergmann et al., Nat. Proto. [52]	2018	Blood-brain-barrier (BBB)	Organoid	Co-culture of endothelial cells, pericytes and astrocytes by low-adhesion conditions.	MALDI	unreported	Development of a BBB organoid platform for drug analysis by fluorescent microscopy and MALDI MSI. Analysis of BKM120 and dabrafenib permeability with BBB organoids.
Johnson et al., J. Mass Spec. [53]	2020	Pancreatic cancer	Cancer organoid	Patient biopsies	MALDI Orbitrap	75 μm	Method development of organoid cultures into geltain microarrays for high-throughput MSI analysis. Organized array of organoids in the same z-axis for multi-sample imaging.
Liu et al., J. Am. Soc. Mass Spectrom. [41]	2018	Colorectal cancer	Cancer organoid	Patient biopsies	MALDI	35 μm	Heterogeneous distribution of irinotecan and its active metabolites within organoid cultures demonstrated metabolic variability in complex tissue samples.
David et al., ACS Medicinal Chemistry Letters. [42]	2018	MCF-7 breast cancer	Cancer organoid	Mice xenograft	MALDI TOF	unreported	Ex vivo method of visualizing peptides and small molecules in breast cancer tumor explants as an alternative to spheroid cultures. Detection of small molecule drug (4-hydroxytamoxifen) and peptide drug (cyclosporin A) as proof-of-concept.

Figure 1. Distribution of metabolites regulating cancer growth and survival within the HCC827 lung adenocarcinoma aggregoid central section by DESI-MSI. Ion density maps of metabolites outlining the core and the outer regions of the aggregoid on the image to highlight hypoxic and proliferative areas, respectively. Mean intensity plotted on bar graph against the core and outer regions. Scale bar 200 μm. Intermediates of the glycolysis reaction: (a) pyruvate, m/z 87.00880 and (b) lactate, m/z 89.02440. Glutaminolysis reaction: (c) glutamine, m/z 145.06190 and (d) glutamate, m/z 146.04590. TCA cycle: (e) citrate, m/z 191.01980; (f) malate, m/z 133.01430; and (g) succinate, m/z 117.01940. [Flint et al., 2020, Reference [43]]

Figure 2. (a) MALDI-MSI of the deuterated Terbinafine (Terbinafine-d7) source generated fragment ion in red (m/z 148) superimposed with choline headgroup in blue (m/z 184) and ceramide fragment peak in green (m/z 264). (b) Hematoxylin & eosin stained optical image of the sublimated section after MALDI-MSI (4× magnification). Calibration curve (n = 3) generated using (c) the average intensity of m/z 141 (no normalization) and (d) the ratio average intensity of m/z 141/148. Normalization to the internal standard m/z 148 improved the linearity of the calibration curve. [Russo et al., 2019, Reference [50]]

Figure 3. Ex vivo small intestine tissue from a Quasi Vivo 600 Liquid-Liquid Interphase in vitro system. The small intestinal tissue, with the apical layer facing upwards, was treated with 0.5 mg/mL Atorvastatin over a 6-hour period to investigate drug absorption. (a) A scanned image of the intestinal tissue section taken using a Super Coolscan 5000 ED Film Scanner. (b) A MALDI-MS image showing cholesterol [Chol+H-H₂O]⁺ at m/z 369 in red. (c) A MALDI- MS image showing the Peyer’s patches at m/z 389 in blue. (d) A MALDI-MS image showing cholesterol [Chol+H-H₂O]⁺ at m/z 369 in red and Peyer’s patches in blue; overlapping ions are shown in pink. (e) A MALDI-MS image showing the sodium adduct of atorvastatin at m/z 581 in green. (f) A MALDI-MS image showing the sodium adduct of atorvastatin at m/z 581 in green and cholesterol [Chol+H-H₂O]⁺ at m/z 369 in red. (g) A MALDI-MS image showing sodium adduct of atorvastatin (m/z 581) in green and Peyer’s patches (m/z 389) in blue. (g) A MALDI-MS image showing the sodium adduct of atorvastatin at m/z 581 in green, cholesterol [Chol+H-H₂O]⁺ at m/z 369 in red and Peyer’s patches in blue; overlapping blue and red ions are shown in pink [Credit: C. Spencer, data not published]

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LaBonia GJ, Ludwig KR, Mousseau CB, et al. iTRAQ quantitative proteomic profiling and MALDI–MSI of colon cancer spheroids treated with combination chemotherapies in a 3D printed fluidic device. Anal Chem. 2018;90:1423.

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