Targeting the CAF-LPL-STAT3 axis mitigates osimertinib resistance associated with EGFR downregulation

Acquired resistance to osimertinib in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) arises from both tumor cell-intrinsic pathways and tumor microenvironmental cues. Cancer-associated fibroblasts (CAFs) are critical stromal components that drive therapeutic resistance, yet the complete molecular and metabolic cascade linking CAFs to EGFR downregulation and osimertinib insensitivity remains unclear. This work aimed to clarify how CAF-derived signals trigger epithelial-to-mesenchymal transition (EMT), lipid metabolic rewiring, and loss of EGFR expression to confer osimertinib resistance.

We first performed ATP viability assays on mixed tumor tissues from 6 patients, then adopted a validation cohort of 12 clinical specimens. Flow sorting isolated viable CD45^-EpCAM⁺ tumor cells for osimertinib sensitivity testing, and correlations among tumor cell drug damage, EGFR-positive cell ratio and CAF-enriched stromal fraction were analyzed. Next, cellular mechanism research was carried out: H1975 and PC9 cells were co-cultured with CAFs in a non-contact Transwell system to detect EGFR expression and drug response. We distinguished transcriptional inhibition from mRNA/protein degradation via mRNA stability, protein half-life and EGFR promoter luciferase assays. Quantitative phosphoproteomics, gene interference, ChIP-qPCR and promoter mutation experiments clarified the STAT3-ZEB1 regulatory axis. Secretome proteomics screened CAF-secreted FSTL1, and gene knockdown plus BLT receptor inhibition verified the FSTL1-AKT-LPL-arachidonic acid/LTB4 signaling pathway; exogenous LTB4 treatment was also used to assess its effect on STAT3 mRNA and total protein expression. Finally, in vivo efficacy verification was conducted on tumor xenografts mixed with H1975 cells and CAFs treated with tripchlorolide (T4) combined with osimertinib to evaluate combination anti-tumor activity.

Clinical specimen results: In the 12-patient cohort, osimertinib-induced tumor cell damage positively correlated with EGFR positivity and inversely with CAF-enriched stromal fraction. CAF co-culture reduced EGFR expression and osimertinib sensitivity and triggered EMT in H1975/PC9 cells. Further mechanistic exploration revealed the transcriptional regulatory mechanism: CAFs suppressed EGFR promoter activity without altering EGFR mRNA or protein stability. ZEB1 bound the EGFR promoter E-box to silence EGFR, with STAT3 acting upstream of ZEB1. Subsequent assays uncovered the CAF lipid metabolic signaling axis: CAF-derived FSTL1 activated AKT to elevate LPL, boosting arachidonic acid and LTB4. LPL depletion or BLT inhibition blocked STAT3-ZEB1 signaling, while exogenous LTB4 rescued this pathway; LTB4 treatment also increased STAT3 mRNA and total protein abundance, indicating that LTB4 signaling regulates STAT3 expression in addition to its phosphorylation. Finally, the in vivo therapeutic effect was validated: T4 suppressed STAT3 signaling and potentiated osimertinib anti-tumor activity in CAF-bearing xenografts.

This study characterizes a CAF-associated stromal-metabolic signaling program in EGFR-mutant NSCLC. The FSTL1-AKT-LPL-LTB4-STAT3-ZEB1 cascade transcriptionally suppresses EGFR expression and induces an EGFR-low tumor-cell state associated with reduced osimertinib sensitivity; LTB4-associated signaling increases both STAT3 phosphorylation and STAT3 expression. Modulating this stromal-metabolic axis may improve osimertinib response, although the therapeutic conclusions remain preclinical.