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

Abstract 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-biopsies to build a longitudinal, compartment-resolved spatial atlas that captures within-patient TME remodeling across sequential EGFR-TKIs. Methods A patient-matched biopsy cohort with serial samples obtained at baseline (T0), after resistance to first-generation EGFR-TKI (T1), and after resistance to third-generation EGFR-TKI (T2) was created. 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 almost entirely stable from T0 to T1 and increased substantially from T1 to T2, with changes in the stroma-enriched compartments accounting for most of the increase. Early remodeling was characterized by loss of T cell activation programs, reduced neutrophil signatures, increased myeloid remodeling, and impairment of antigen presentation. Longitudinal analyses pointed to a biphasic immune trajectory, with early myeloid remodeling and late stromal checkpoint reprogramming. Importantly, early spatial remodeling patterns were different in samples, which later acquired T790M. Conclusions This study shows how the TME changes dynamically during sequential EGFR-TKI therapy in EGFR-mutant NSCLC. It reveals a late, stroma-driven expansion of spatial heterogeneity and stage-dependent immune remodeling, and provides a longitudinal reference that supports testable, time-aware hypotheses for future validation and functional studies.