Trekker enables high-sensitivity, single-cell spatial profiling of the FFPE lung SCC tumor microenvironment without segmentation or deconvolution

Squamous cell carcinoma (SCC) is a highly heterogeneous malignancy characterized by complex cellular architecture, variable patterns of immune infiltration, and diverse stromal interactions that influence progression, metastatic potential, and therapeutic response. A detailed understanding of the SCC tumor microenvironment (TME) is therefore essential for identifying biomarkers, resolving resistant niches, and guiding precision oncology strategies. Although single-cell sequencing captures cellular diversity, it lacks spatial context, while most spatial transcriptomics platforms depend on complex protocols, proprietary reagents, and computationally intensive segmentation, deconvolution, or binning steps that limit true single-cell resolution-especially in FFPE clinical samples. To address these challenges, we present Trekker, a flexible, next-generation single-cell spatial transcriptomics platform that delivers spatial context with the simplicity of standard snRNA-seq. Using a Slide-tag-inspired spatial tagging approach (Russel et al., 2023), Trekker™ indexes nuclei directly within intact tissue sections and is compatible with same-section H&E staining for direct histopathological correlation. The workflow requires no specialized instrumentation and integrates seamlessly with a fixed snRNA-seq protocol, enabling high-sensitivity, single-nucleus expression and spatial data without modifying established laboratory practices. By applying Trekker to FFPE human lung squamous cell carcinoma specimens, we generated high-resolution spatial maps of malignant cells, stromal elements, and diverse immune populations. Because each nucleus carries its own spatial barcode, downstream analysis bypasses cell segmentation, unmixing, and binning, allowing users to analyze the data using familiar single-cell bioinformatics pipelines. This streamlined approach supports robust characterization of SCC architecture, tumor-immune interaction zones, and spatially informed differential gene expression. By combining ease of implementation, FFPE flexibility, reduced computational complexity, and high-sensitivity single-cell spatial resolution, Trekker provides an accessible and powerful platform for cancer researchers. This technology enables deeper, more intuitive exploration of SCC tumor ecosystems and has the potential to accelerate biomarker discovery and therapeutic development.