# Modulation of Cancer-Associated Fibroblasts via the miR-624-5p/FAP Axis Drives Progression and Metastasis in Non-Small Cell Lung Cancer

## TL;DR

This study identifies a new regulatory pathway involving miR-624-5p and FAP in cancer-associated fibroblasts that promotes non-small cell lung cancer metastasis.

## Contribution

The discovery of miR-624-5p as a novel upstream regulator of FAP in cancer-associated fibroblasts offers a new therapeutic target for NSCLC.

## Key findings

- miR-624-5p is significantly downregulated in cancer-associated fibroblasts.
- miR-624-5p directly targets FAP to suppress fibroblast activation and tumor metastasis.
- 68Ga-FAPI-04 PET/CT imaging shows FAP overexpression correlates with advanced NSCLC and metastasis.

## Abstract

In the tumor microenvironment, cancer-associated fibroblasts (CAFs) facilitate metastasis and contribute to the poor prognosis of non-small cell lung cancer (NSCLC). Fibroblast activation protein (FAP) serves as a key mediator of CAF activity, yet its upstream regulatory mechanisms remain unclear. In this study, we utilized 68Ga-FAP inhibitor (FAPI)-04 PET/CT imaging to visualize FAP expression in NSCLC patients, revealing its overexpression in advanced disease and correlation with metastatic progression. Using primary CAFs and normal fibroblasts isolated from human NSCLC tissues, we confirmed through functional assays that CAFs promote NSCLC metastasis. Importantly, through mechanistic investigation, we identified miR-624-5p as a significantly downregulated miRNA in CAFs. We further demonstrated that miR-624-5p directly targets FAP, thereby suppressing CAF activation and NSCLC metastasis. Our findings reveal a novel miR-624-5p/FAP regulatory axis in CAF activation and highlight its potential as a therapeutic target for restraining NSCLC progression.

Background: Cancer-associated fibroblasts (CAFs) are key mediators of metastatic progression in non-small cell lung cancer (NSCLC). Fibroblast activation protein (FAP) serves as the hallmark of CAF activation. However, the upstream regulation of FAP remains elusive, limiting stroma-targeted therapy development. Methods: 68Ga-FAP inhibitor (FAPI)-04 PET/CT imaging was performed on 61 NSCLC patients to evaluate the clinical significance of FAP. CAFs and normal fibroblasts (NFs) were isolated from patient tissues. Bioinformatic analysis and qRT-PCR were employed to screen and validate miRNAs. Functional assays (CCK-8, collagen contraction, wound healing, transwell co-culture) were utilized to investigate the role of miR-624-5p in regulating fibroblast activation and the effects on the metastatic potential of NSCLC cells. The targeting relationship between miR-624-5p and FAP was validated using FISH, dual-luciferase assay, and Western blotting. Results: 68Ga-FAPI-04 uptake was higher in advanced NSCLC (p < 0.001) and correlated with tumor size, lymph node metastases, and distant metastases (p < 0.05). Isolated primary CAFs significantly enhanced the migration and invasion of A549 and PC9 cells compared to NFs (p < 0.001). We identified miR-624-5p as a significantly downregulated miRNA in CAFs (p < 0.001). Functionally, miR-624-5p overexpression inhibited CAF proliferation and collagen contraction (p < 0.01) and reduced the proliferation, migration, and invasion capabilities of A549 and PC9 cells (p < 0.001). Mechanistically, miR-624-5p bound to FAP mRNA and negatively regulated FAP expression (p < 0.001), thus suppressing CAF activation and tumor metastasis. Conclusions: Our findings establish miR-624-5p as a novel upstream regulator that suppresses FAP expression, consequently inhibiting CAF activation and its pro-metastatic function. Targeting the miR-624-5p/FAP axis represents a promising therapeutic strategy for NSCLC metastasis.