Evaluating the utility of axial TOF MALDI Imaging–based HiPLEX IHC in CNS tumor analysis

Significance of the topic

The integration of high-plex, spatially resolved protein detection into routine neuropathology promises to transform diagnostic precision for central nervous system (CNS) tumors and metastatic lesions. Axial time-of-flight (TOF) MALDI imaging combined with HiPLEX IHC mass‑tag technology enables multiplexed, label‑based visualization of many antibodies directly on tissue while preserving histological context. This approach addresses clinical challenges such as identification of cancer of unknown primary (CUP) and detailed characterization of tumor microenvironments, where spatial co‑localization of cell types and markers is critical for diagnosis and therapeutic decision making.

Objectives and study overview

This multi‑site study evaluated the utility, reproducibility and tissue‑level interpretability of axial TOF MALDI imaging–based HiPLEX IHC applied to brain metastases from breast cancer in the clinical context of Cancer of Unknown Primary. The study aimed to:

• Assess site‑to‑site reproducibility of monoplex versus multiplex (10‑plex) MALDI‑IHC measurements.
• Compare MALDI‑IHC results to conventional diaminobenzidine (DAB) IHC and standard histology (H&E) annotations.
• Demonstrate computational workflows for correlative analysis using the M2aia framework and its Python interface.

Methodology

The experimental workflow combined HiPLEX IHC staining with axial TOF MALDI mass spectrometry imaging (MSI). Key methodological elements included:

• Application of photocleavable mass‑tagged antibodies (Miralys probes) to tissue sections enabling simultaneous detection of multiple targets.
• Acquisition of mass spectra across tissue with an axial TOF MALDI imaging instrument to record mass tags as distinct peaks corresponding to individual antibodies.
• Multiplex experiments up to 10 antibodies per run and comparison to monoplex acquisitions to evaluate spectral separation and signal fidelity.
• Correlative annotation using H&E images to define regions such as tumor, necrosis, immune infiltrates, brain parenchyma, blood vessels and stroma.
• Computational processing and multimodal data integration using M2aia and pyM2aia for visualization, alignment and region‑based quantitation (median intensity per region, global scaling).

Instrumentation used

The study used axial TOF MALDI mass spectrometry imaging platforms and benchtop mass spectrometers provided by Bruker for spatially resolved acquisition. Photocleavable mass‑tag imaging probes (Miralys) from AmberGen were used for antibody labeling. Data processing and visualization relied on M2aia software and its Python interface pyM2aia for memory‑efficient handling of 2D/3D multimodal MSI datasets.

Main results and discussion

Key findings from the multi‑site evaluation were:

• Good site‑to‑site reproducibility: Comparative analyses between participating centers showed consistent spectral patterns and antibody distributions for both monoplex and 10‑plex acquisitions, supporting cross‑site robustness of the MALDI‑IHC workflow.
• Clear spectral separation in multiplex data: Average mass spectra from 10‑plex runs exhibited well‑separated peaks across the mass range, allowing unambiguous assignment of individual antibody mass tags despite multiplexing.
• Concordance with conventional IHC and histology: Spatial localization and relative intensities of antibody signals in annotated tissue regions matched expectations from DAB‑IHC and H&E‑based tissue classification (tumor, necrosis, immune cells, parenchyma, vessels, stroma).
• Tissue type‑specific antibody signatures: Distinct marker patterns were observed across anatomical and pathological compartments, demonstrating the method's ability to resolve cellular and microenvironmental heterogeneity.

Discussion points:

• The combination of photocleavable mass tags and axial TOF detection allows high multiplexing without the spectral crowding common to fluorescence approaches, at least at the demonstrated 10‑plex level.
• M2aia/pyM2aia provided effective pipelines for multimodal registration and region‑wise quantitation; such computational tools are essential for routine, reproducible cross‑site analyses.
• Limitations include the need for continued standardization of staining and acquisition protocols across sites, potential differences in ionization efficiency between tags, and the requirement for careful calibration to maintain quantitative comparability.

Benefits and practical applications

This axial TOF MALDI HiPLEX IHC approach offers several practical advantages:

• High‑plex, spatially resolved protein mapping compatible with standard histology workflows, enabling direct correlation with morphological features.
• Improved multiplex capacity compared with chromogenic IHC, reducing tissue consumption and enabling richer phenotypic profiling on single sections.
• Robust cross‑site reproducibility that supports multi‑center studies and prospective clinical validation efforts.
• Potential to aid diagnosis in challenging cases such as CUP by revealing marker panels and microenvironmental clues indicative of tissue of origin.

Future trends and potential applications

Anticipated developments and opportunities include:

• Scaling to higher plexity: development of additional orthogonal mass tags and optimized acquisition strategies to expand beyond 10‑plex while maintaining spectral separation.
• Standardization and clinical validation: larger multi‑center clinical studies to define diagnostic panels, performance metrics and regulatory pathways for clinical adoption.
• Integration with machine learning: use of deep learning on multimodal MSI + histology datasets (facilitated by pyM2aia) to automate pattern recognition and predictive biomarker discovery.
• 3D and multimodal expansion: combining MSI proteomics with lipidomics, transcriptomics and volumetric imaging to characterize tumor architecture and invasion routes.
• Operational improvements: streamlined sample prep, automated data pipelines and inter‑laboratory quality control standards to accelerate translational uptake.

Conclusion

This multi‑site evaluation demonstrates that axial TOF MALDI imaging–based HiPLEX IHC can deliver reproducible, high‑quality multiplexed protein maps that are concordant with conventional IHC and histology. The approach is well suited for detailed characterization of CNS tumors and metastatic lesions and holds promise for improving diagnostic workflows in complex clinical scenarios such as Cancer of Unknown Primary. Continued technical optimization, computational standardization and larger clinical validation studies are the next steps toward routine diagnostic and research use.

References

• Pouyiourou M., et al. Rethinking cancer of unknown primary: from diagnostic challenge to targeted treatment. Nature Reviews Clinical Oncology. 2025;22(10):781–799.
• Müller E., et al. Exploring the Aβ plaque microenvironment in Alzheimer’s disease model mice by multimodal lipid-protein-histology imaging on a benchtop mass spectrometer. Pharmaceuticals. 2025;18(2):252.
• Cordes J., et al. M2aia—Interactive, fast, and memory-efficient analysis of 2D and 3D multi-modal mass spectrometry imaging data. GigaScience. 2021;10(7):giab049.
• Cordes J., et al. pyM2aia: Python interface for mass spectrometry imaging with focus on deep learning. Bioinformatics. 2024;40(3):btae133.