MULTIMODAL IMAGING OF PHOTOSENSITIZERS IN 3D TUMOR CELL MODELS

Importance of Topic

Photodynamic therapy (PDT) is an emerging cancer treatment that relies on photosensitizers (PS) activated by light to generate reactive oxygen species, inducing tumor cell apoptosis. Understanding how PS molecules penetrate and distribute within tumor tissue is critical for optimizing therapeutic efficacy. Combining molecular and elemental imaging techniques provides complementary insights into PS localization and concentration in three-dimensional (3D) tumor models.

Study Objectives and Overview

This work investigates the spatial distribution and quantification of two porphyrin-based photosensitizers—mTHPP and its palladium-tagged analogue mTHPP-Pd—in 3D tumor spheroids derived from HT29 human colon carcinoma cells. The study aims to demonstrate how matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) can be combined to map molecular integrity and elemental content of PS in thin tissue sections.

Methodology

Stable 3D tumor spheroids were cultured using the liquid overlay technique and incubated with 5 µM PS for 24 hours. Spheroids were embedded in OCT, cryosectioned at 5–14 µm thickness, and prepared for two imaging workflows:

• MALDI-MSI sample prep: OCT removal with ammonium acetate buffer, CHCA matrix sublimation using iMLayer™ (20 min, 250 °C).
• LA-ICP-MS sample prep: Sections placed on microscope slides, external calibration with gelatin standards spiked with Pd (0.1–100 µg/g).

Used Instrumentation

• MALDI-MSI: iMScope Trio with 355 nm Nd:YAG laser, 1000 Hz pulse rate, 100 shots/pixel, 3.5 kV sample voltage, 1.9 kV detector voltage.
• LA-ICP-MS: LSX-213 G2+ Nd:YAG 213 nm laser (20 Hz, 7 µm spot, 21 µm/s), helium carrier, coupled to Shimadzu ICPMS-2030 (collision mode, He 6 ml/min), nickel cones, plasma 1.2 kW.

Main Results and Discussion

MALDI-MSI of mTHPP-treated spheroids revealed a ring-shaped distribution localized in the outer cell layers, consistent with limited tissue penetration. Phosphatidylcholines (PC 32:1, PC 34:1, PC 36:1) served as tissue markers and correlated with spheroid morphology. Intact PS molecules were detected without significant degradation.

Combined analysis of mTHPP-Pd sections showed Pd signal up to 10 µg/g (average 1.9 µg/g) confined to the spheroid periphery. MALDI-MSI could no longer detect the Pd-tagged PS due to altered protonation, but could image endogenous phospholipids. Overlaying Pd maps with PC 34:1 distributions confirmed PS localization relative to tissue structure.

Benefits and Practical Applications

The dual imaging approach merges molecular specificity from MALDI-MSI with quantitative elemental mapping by LA-ICP-MS. This allows precise localization and concentration measurements of PS in 3D tumor models, supporting the design of more effective PDT drugs. The methods can be adapted for other metal-tagged compounds and complex biological matrices.

Future Trends and Opportunities

Advances may include:

• Development of novel PS tags enabling simultaneous molecular and elemental detection.
• Integration with higher-resolution imaging and three-dimensional reconstruction.
• Automation and machine-learning algorithms for large-scale bioimaging data analysis.
• Translation to in vivo models and clinical biopsy assessment.

Conclusion

The presented multimodal imaging workflow effectively characterizes photosensitizer penetration and retention in 3D tumor spheroids. By combining MALDI-MSI and LA-ICP-MS on the same tissue sections, researchers gain complementary molecular and quantitative elemental insights that are essential for optimizing photodynamic therapy agents.

Reference

• [1] J. Friedrich et al., Nat. Protoc. 4 (2009) 309–324.
• [2] A.-C. Niehoff et al., Metallomics 6 (2014) 77–81.