EACR26-0697

KRAS/LKB1 co-mutated NSCLC-derived Extracellular Vesicles trigger TLR4-dependent IL-6 induction to drive inflammatory fibroblast reprogramming

C. Borzi¹, R. Petracca¹, A. Tomassetti¹, C. Moscheni², E. Manenti¹, C. Ansermino^3,4, R. Mortarini¹, A. Andolfo³, G. Sozzi¹, M. Moro¹

¹Fondazione IRCCS Istituto Nazionale dei Tumori, Experimental Oncology, Milan, Italy
²Università deglu Studi di Milano, Biomedical and Clinical Sciences, Milan, Italy
³IRCCS Ospedale San Raffaele, Proteomics and Metabolomics Facility, Milan, Italy
⁴Università del Piemonte Orientale, Department of Health Sciences, Novara, Italy

Introduction:

KRAS/LKB1 co-mutated (KL) non-small cell lung cancers (NSCLCs) are aggressive, treatment-resistant tumors characterized by an immunosuppressive, "cold" tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are key orchestrators of this TME, contributing to immune exclusion and therapeutic resistance. However, the molecular mechanisms regulating fibroblast phenotypic and functional modulation in the KL context remain poorly understood. This study investigates whether extracellular vesicles (EVs) released by KL NSCLC cells act as primary mediators in reprogramming lung fibroblasts.

Material and method:

EVs were isolated via ultracentrifugation from isogenic H1299 NSCLC clones (WT: wt-KRAS/wt-LKB1; KL: G12C-KRAS-/del-LKB1) and characterized by NTA, TEM, Western blot and Nano-LC-MS. Functional assays were performed on normal (CCD-19Lu) and cancer-associated (CAF154hTERT) lung fibroblasts. EV uptake was evaluated by flow cytometry and confocal microscopy, while fibroblast reprogramming was assessed through RT-qPCR, RNA-seq (GSEA analysis), and ELISA. The TLR4 inhibitor TAK-242 was used to investigate the molecular mechanism. Findings were validated through in silico datasets (CPTAC, E-MTAB-6149), in vivo Patient-Derived Xenograft (PDX) models, and IHC analysis of an internal patient cohort.

Result and discussion:

KL co-mutations significantly increased EV secretion and altered their protein cargo without affecting fibroblast uptake. EVs derived from KL cells (KL-EVs) triggered an inflammatory response in fibroblasts, primarily characterized by upregulation of IL-6, followed by additional pro-inflammatory mediators (CCL2, IL-8, CXCL1, and CXCL3). GSEA revealed that KL-EV-treated fibroblasts acquire a transcriptional profile consistent with inflammatory cancer-associated fibroblasts (iCAFs). This elevated stromal IL-6 expression was consistently observed across in silico data, PDX models, and clinical samples. Mechanistically, pre-treatment of fibroblasts with TAK-242 significantly prevented the KL-EV-induced upregulation of IL-6, identifying the TLR4 signaling axis as the initial driver of this inflammatory communication.

Conclusion:

These data identify tumor-derived EVs as primary drivers of iCAF-like reprogramming in KL NSCLC. By elucidating the TLR4-dependent induction of IL-6, this study provides new mechanistic insights into the stromal contribution to KL tumor aggressiveness and highlights potential therapeutic targets within the TME.

Acknowledgement:

Supported by AIRC (IG-27141) and Italian Ministry of Health (Ricerca Corrente). We thank Dr. M. Barella for IHC analysis and Dr. E. Caiola for providing KL H1299 cells.