Development of an custom near single-cell spatial transcriptomics platform using photolithography to study cellular heterogeneity induced by targeted therapies in neuroblastoma

BACKGROUND: High-risk neuroblastoma accounts for 15% of pediatric cancer mortality, with survival rates stagnating at 50%. While spatial transcriptomics (ST) offers a path to understanding the tumor microenvironment and therapy resistance, current commercial platforms are either cost-prohibitive, low resolutions and/or lack sensitivity required for precious clinical samples.

AIMS: To develop a high-resolution, cost-effective ST platform (approximately €100/array) tailored for investigating the spatial effects of innovative targeted therapies in neuroblastoma.

METHODS: Our platform utilizes custom-printed microarrays featuring four subarrays, each containing ± 85,000 spots (13.6 x 13.6 µm) with a 1 µm interspot distance. This architecture provides high spatial resolution across a 10 x 14 mm capture area. To validate the workflow, we performed on-slide fluorescent first and second-strand cDNA synthesis using mouse olfactory bulb as a structural reference tissue. We also assessed the quality of spatial barcodes by cleaving the capture probes and sequencing them.

RESULTS: Barcode quality was adequate with 70% assignment rate at 99% precision. Proof-of-concept experiments successfully demonstrated transcript capture, with fluorescent footprinting revealing the distinct anatomical layers of the mouse olfactory bulb. This confirms the platform’s ability to maintain spatial integrity during enzymatic steps. The platform achieves a >10-fold cost reduction compared to commercial alternatives. Ongoing work focuses on library preparation optimization, integration with arrayed tumor cuboids for high-throughput drug screening, and modifications such as targeted ST for higher sensitivity (at lower cost) and long-read ST.

CONCLUSIONS: Our platform democratizes spatial profiling for pediatric oncology. By combining affordability with near single-cell resolution, our platform enables deeper investigation into neuroblastoma heterogeneity and supports the development of more effective precision medicine strategies.