Longitudinal spatial multi-omics delineates tumor microenvironment remodeling across sequential EGFR-TKIs in EGFR-mutant NSCLC.

BACKGROUND: Resistance to therapy is a frequent occurrence in patients with epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) who are treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs). However, spatial information on how the tumor microenvironment (TME) changes within the same patient from baseline to resistance to first- and then third-generation EGFR-TKIs is scarce. Here, we used rare consecutive re-biopsy samples to build an exploratory, longitudinal, and spatially resolved atlas, aiming to describe within-patient TME remodeling patterns as a resource for hypothesis generation. METHODS: A patient-matched cohort with serial samples obtained at baseline (T0), after first-generation EGFR-TKI resistance (T1), and after third-generation EGFR- TKI resistance (T2) was established. Using GeoMx Digital Spatial Profiling (DSP), tumor-enriched and stroma-enriched areas of interest (AOIs) were segmented, and paired RNA and protein profiles were quantified. We tracked temporal changes in compartment-specific heterogeneity and immune remodeling and explored associations between early remodeling and subsequent T790M acquisition. RESULTS: We analyzed the data from 15 samples of 6 patients. Tumor- and stroma-enriched compartments were not only transcriptionally but also protein-wise consistently distinct. Spatial heterogeneity was relatively stable from T0 to T1 but increased from T1 to T2, with changes in the stroma-enriched compartments accounting for most of the increase. Early remodeling featured loss of T cell activation programs, reduced neutrophil signatures, increased myeloid remodeling, and impairment of antigen presentation. Longitudinal analyses suggested a biphasic immune trajectory, with early myeloid remodeling and late stromal checkpoint reprogramming. Early spatial remodeling patterns showed potential differences between samples that later acquired T790M and those that did not. CONCLUSIONS: This study provides preliminary evidence for dynamic TME changes during sequential EGFR-TKI therapy in EGFR-mutant NSCLC. It suggests a late, stroma-associated expansion of spatial heterogeneity and stage-dependent immune remodeling. Additionally, it offers an initial spatial atlas and generates testable, time-aware hypotheses for future validation.