Small Drug Molecules MALDI-MS Imaging on Colorectal Tumor Organoids

Significance of the Topic

The ability to visualize and quantify the distribution of chemotherapeutic agents and their metabolites within three-dimensional tumor models is crucial for understanding drug efficacy and resistance mechanisms. Patient-derived colorectal tumor organoids (CTOs) recapitulate tumor heterogeneity and vascular architecture, making them valuable platforms for preclinical evaluation of drug penetration, uptake, and metabolism. Conventional optical imaging techniques can track metabolic activity but cannot directly map small drug molecules and their biotransformation products in situ. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI) addresses this gap by enabling spatially resolved detection of parent drugs and metabolites at micrometer resolution.

Objectives and Overview of the Study

This study aimed to demonstrate the application of MALDI-TOF MSI on Bruker’s ultrafleXtreme platform to:

• Detect and spatially resolve irinotecan and its active (SN-38) and inactive (SN-38G) metabolites in CTOs.
• Assess the influence of treatment duration (6 vs. 24 hours) and drug concentration (20 vs. 40 µM) on tissue penetration and metabolic conversion.
• Employ multivariate data analysis to elucidate co-localization patterns between parent compound and metabolites.

Methodology and Instrumentation

A published CTO culture and treatment protocol was followed to generate organoids ranging from 50 to 500 µm. For time-course experiments, CTOs (n=5) were exposed to 20.6 µM irinotecan for 6 or 24 hours; concentration studies involved treatment with 20 or 40 µM for 72 hours (n=3). Vehicle controls were maintained in DMSO. Post-treatment, organoids were embedded in gelatin, cryosectioned at 12 µm thickness, and located via H&E or immunofluorescence staining. Consecutive sections were prepared for MSI.
An internal standard (irinotecan-d10) and the matrix super-DHB were applied sequentially using a TM Sprayer under optimized parameters for solvent composition, temperature, flow rate, number of passes, and drying time. MALDI-TOF MSI was performed on the ultrafleXtreme in positive reflector mode (m/z 200–1000) at 1 kHz laser repetition rate and 35 µm spatial resolution. Calibration used peptide standards on unsprayed slide areas. FlexImaging and SCiLS Lab software facilitated spatial segmentation and region-specific ion analysis.

Main Results and Discussion

• Time-dependent Penetration: Irinotecan signals (m/z 587.3) exhibited limited penetration at 6 hours, with markedly deeper and more homogeneous distribution at 24 hours.
• Metabolite Detection: Both SN-38 (m/z 393.1) and SN-38G (m/z 569.2) were visualized within the organoid sections, confirming in situ biotransformation.
• Co-localization Analysis: Spatial correlation between irinotecan and SN-38G (inactive metabolite) was stronger than that with SN-38, suggesting variable metabolic rates linked to heterogeneous cell populations.
• Concentration Effects: Increasing drug concentration from 20 to 40 µM resulted in higher parent compound uptake, while SN-38 abundance increased to a lesser extent, indicating metabolic saturation or rate-limiting enzymatic activity.

Practical Benefits and Applications of the Method

• Provides direct, label-free imaging of drug and metabolite distributions in complex 3D tumor models.
• Enables quantitative, spatially resolved pharmacokinetic and pharmacodynamic assessments.
• Supports optimization of dosing strategies by revealing penetration barriers and metabolic hotspots.

Future Trends and Potential Applications

The integration of high-resolution MALDI-MSI with complementary modalities (e.g., fluorescence, immunohistochemistry) and multi-omics approaches will enhance understanding of drug action in organoids. Advances in instrumentation may push spatial resolution below 10 µm, facilitating single-cell analyses. Expansion to other therapeutic classes and tumor types will broaden translational impact. Automated data analytics and machine learning will further refine interpretation of complex spatial correlations.

Conclusion

The application of MALDI-TOF MSI on the ultrafleXtreme system effectively maps irinotecan and its metabolites within CTOs, revealing distinct temporal and concentration-dependent penetration and metabolic patterns. These insights are instrumental for optimizing chemotherapeutic regimens and underscore the value of MSI in preclinical oncology research.

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