Tracking free-toxin distribution in xenografts after in-vivo ADC dosing by MALDI Imaging

Significance of the topic

Antibody-drug conjugates (ADCs) are emerging as a potent class of targeted anticancer therapeutics that rely on selective delivery of cytotoxic payloads to antigen-expressing tumor cells. Tracking the spatial release of the toxin component within tissues is crucial for assessing drug safety, efficacy, and off-target effects.

Objectives and study overview

This study aimed to map the in-vivo distribution of free toxin released from an ADC in mouse xenograft tumors following a single 15 mg/kg dose. By comparing treated and control groups, the work sought to demonstrate the capability of MALDI magnetic resonance mass spectrometry imaging (MRMS-MSI) combined with SCiLS™ Lab software for label-free, spatially resolved detection of toxin species.

Methodology

An in-vivo DMPK study was conducted in CB17-SCID mice bearing human multiple myeloma (NCI-H929) xenografts. Mice received a single intravenous ADC dose (15 mg/kg) or no treatment (control) and were sacrificed after 48 hours. Tumors were harvested, snap-frozen, sectioned at 10 μm, and thaw-mounted on ITO-coated slides. Sections were desiccated, scanned for histology, and prepared for MALDI analysis using 2,5-dihydroxybenzoic acid matrix deposition (35 mg/mL in 50/50 acetonitrile/water with 0.1% TFA) via an HTX M5 Sprayer.

Used instrumentation

• Mass spectrometer: solariX XR 7 T MRMS with 7.0 T cryomagnet and smartbeam-II laser (355 nm).
• Imaging software: SCiLS™ Lab 2021a for raw data visualization and TIC normalization.
• Cryostat: Leica CM1950 for tissue sectioning.
• Slide scanner: Aperio CS2 (20×, reduced to 10% size, TIF LZW format).

Main results and discussion

High-resolution MALDI-MSI enabled unambiguous detection of the free toxin as protonated [M+H]+ and alkali adducts [M+Na]+ and [M+K]+ with mass errors below 1 ppm. Signals were exclusive to ADC-treated xenografts and absent in controls. Ion images revealed heterogeneous intratumoral distribution, suggesting areas of necrosis or limited vascular access. Reproducibility studies across three sections per sample showed consistent spatial patterns and relative intensities, underscoring the method's robustness.

Benefits and practical applications

• Label-free spatial mapping of drug and metabolites without radiolabels.
• Preservation of tissue architecture for correlative histology.
• High mass accuracy permits clear distinction between payload and conjugated forms.
• Applicability in preclinical DMPK to optimize ADC design and dosing.

Future trends and opportunities

Integration of MALDI-MSI with immunohistochemistry (e.g., hypoxia, vasculature, antigen markers) could clarify mechanisms behind heterogeneous payload distribution. Adoption of isotopically labeled internal standards may improve quantitative comparisons. Advances in high-throughput MSI and data analysis pipelines will further accelerate ADC development and personalized therapeutic strategies.

Conclusion

MALDI-MRMS imaging on a solariX XR platform demonstrates powerful, label-free assessment of free toxin distribution in xenograft tumors post-ADC dosing. The approach offers sub-ppm mass accuracy, spatial resolution, and reproducibility, providing valuable insights for ADC optimization and preclinical evaluation.

References

1. Schulz S, Becker M, Groseclose MR, Schadt S, Hopf C (2019). Advanced MALDI mass spectrometry imaging in pharmaceutical research and drug development. Curr. Opin. Biotechnol. 55, 51–59.
2. Abu Sammour D, et al. (2019). Quantitative Mass Spectrometry Imaging Reveals Mutation Status-independent Lack of Imatinib in Liver Metastases of Gastrointestinal Stromal Tumors. Sci. Rep. 9, 1–9.