Spatial stromal multi‑omics predicts EGFR‑TKI resistance via exosome‑mediated metabolic reprogramming before genomic drift

Hypothesis

Spatial stromal multi‑omics can forecast EGFR‑TKI resistance in NSCLC by detecting exosome‑mediated metabolic reprogramming in cancer‑associated fibroblasts (CAFs) before any tumor‑genomic alteration appears.

Background

Multi‑omics integration reveals resistance mechanisms invisible to genomics, capturing transcriptomic, proteomic, metabolomic and epigenomic shifts that drive tumor adaptation [1] [2]. In EGFR‑mutant NSCLC, resistance often arises through bypass signaling such as MET activation, a process traced from mutation to phenotypic change via proteogenomic workflows [3]. Critically, the tumor microenvironment confers resistance through epigenetic remodeling of stromal cells like CAFs and tumor‑associated macrophages (TAMs) [5]. Despite these advances, clinical multi‑omics remains largely baseline and tumor‑centric, lacking prospective, spatial reads of stromal evolution under therapeutic pressure [9] [10].

Mechanistic Insight

We propose that CAFs under EGFR‑TKI stress release exosomes enriched in specific miRNAs (e.g., miR‑21) and metabolic enzymes (e.g., LDHA, GLUT1) that remodel the redox state of neighboring cancer cells. This exosomal cargo raises cytosolic NADH/NAD⁺, stabilizes HIF‑1α independently of hypoxia, and fuels a glycolytic‑dependent bypass cascade that activates MET/PI3K‑AKT signaling. Because these stromal changes precede measurable tumor‑genomic shifts, spatial multi‑omics of the CAF niche can serve as an early warning system.

Testable Prediction

In a prospective cohort of EGFR‑mutant NSCLC patients receiving first‑generation EGFR‑TKI, serial minimally invasive biopsies (or fine‑needle aspirates) will be taken from the tumor core and the invasive stromal front every 2 weeks. Each sample will undergo:

• Spatial transcriptomics (10x Visium) and proteomics (CODEX or MIBI) to map CAF activation states.
• Spatial metabolomics (MALDI‑IMS) for lactate, NAD⁺/NADH ratios.
• Isolation of extracellular vesicles from interstitial fluid for miRNA‑seq and enzyme activity assays.

Parallel plasma will be monitored for ctDNA EGFR T790M and MET amplification using ultrasensitive PCR.

We predict that a statistically significant increase in CAF‑derived exosomal miR‑21 and LDHA activity, coupled with a spatial rise in stromal NADH/NAD⁺ ratio, will occur a median of 6 weeks before ctDNA‑detectable resistance mutations. Patients lacking this stromal signature will either remain sensitive or develop resistance through alternative, genomically detectable routes.

Falsifiability

If longitudinal spatial multi‑omics fails to show any consistent stromal exosomal or metabolic shift that anticipates resistance—i.e., the timing and magnitude of CAF changes are random with respect to ctDNA emergence—the hypothesis is falsified. Conversely, observation of the predicted stromal lead‑time would support the model and justify early stromal‑targeted interventions (e.g., LDHA inhibitors or exosome release blockers) to delay or prevent resistance emergence.