Multiomics analysis for advanced tumor typing of lung cancer using 116plex MALDI HiPLEX-IHC and released N-glycans on the neofleX

Importance of the Topic

Spatially resolved molecular tumor typing is a critical emerging technology in clinical research for precise cancer diagnosis and biomarker discovery. MALDI HiPLEX-IHC leverages photocleavable mass‐tagged antibodies to visualize spatial protein expression across hundreds of markers in tissue sections, offering a comprehensive approach beyond smaller panels. Integrating this with released N‐glycan imaging further enriches molecular characterization and may reveal glycosylation patterns relevant to tumor biology.

Objectives and Study Overview

This study presents a proof‐of‐concept application of a 116‐plex MALDI HiPLEX-IHC panel on lung adenocarcinoma and squamous cell carcinoma tissues compared against healthy lung controls. The aim was to demonstrate unbiased, multiplexed spatial profiling of protein markers commonly used in clinical settings, to distinguish tumor subtypes and healthy tissue, and to integrate N‐glycan imaging for multiomics insights.

Methodology and Instrumentation

A total of 116 antibodies conjugated with photocleavable mass tags were applied to tissue sections of adenocarcinoma, squamous cell carcinoma, and healthy lung. Data acquisition was performed on a neofleX Imaging Profiler in positive ion mode with 30 µm pixel size. Post‐imaging, on‐tissue PNGase F digestion released N‐glycans for MALDI imaging of glycan species. Finally, tissues were stained with H&E to correlate morphology. Data processing and segmentation analyses were carried out using SCiLS Lab 2025a.

• neofleXTM Imaging Profiler (MALDI MS Imaging)
• On‐tissue PNGase F digestion (GlycoPath protocols)
• SCiLS Lab version 2025a (data analysis)

Main Results and Discussion

Unsupervised segmentation reproducibly differentiated tumor regions from healthy lung and separated adenocarcinoma from squamous carcinoma across duplicate measurements. Key protein markers—TTF-1 and Napsin A—showed strong spatial specificity for adenocarcinoma, while structural and metabolic markers such as HIF-1α, VEGFA/VEGFR-1, GLUT-1, and LDHA highlighted tumor hypoxia, angiogenesis, metabolic adaptation, and stromal cooperation. Proliferation marker Ki-67 was elevated in both tumor types. Multiomic integration revealed distinct N-glycan patterns: Hex6dHex1HexNAc7 enriched in adenocarcinoma and Hex5dHex3HexNAc5 in squamous carcinoma, both co‐localized with CEA/CD66e positive cells, suggesting glycosylation changes may influence metastatic potential.

Benefits and Practical Applications

• Simultaneous spatial profiling of over 100 protein markers in a single experiment.
• Broad coverage addresses multiple clinical research questions without repeated assays.
• Multiomics workflow combining protein and glycan imaging delivers a richer molecular picture for biomarker discovery.

Future Trends and Applications

Advancements are expected in integrating other molecular classes such as lipids and metabolites to build a fully multimodal spatial omics platform. Automation and AI‐driven image segmentation will enhance throughput and precision. Refinement of glycan analysis protocols may uncover novel glyco‐biomarkers for prognosis. Ultimately, translation into routine clinical diagnostics could guide personalized therapeutic strategies based on comprehensive spatial molecular maps.

Conclusion

The 116‐plex MALDI HiPLEX-IHC combined with released N‐glycan imaging demonstrates a robust, reproducible, and unbiased method for high‐resolution tumor subtyping and multiomics profiling. This approach holds significant promise for advancing precision oncology research and clinical diagnostics.

Reference

1. Yagnik G, Liu Z, Rothschild KJ, Lim MJ (2021) J Am Soc Mass Spectrom. 32(4), 977-988.
2. Turner BM, Cagle PT, Sainz IM, Fukuoka J, Shen SS, Jagirdar J (2012) Arch Pathol Lab Med. 136(2), 163-171.
3. Reticker-Flynn NH, Bhatia SN (2015) Cancer Discov. 5(2), 168-181.