DYNAMIC INTERACTION BETWEEN FIBROBLASTS AND TUMOR CELLS AND THEIR EXOSOMES IN ADVANCED PROSTATE CANCER

Abstract

Prostate Cancer (PCa) is the most commonly diagnosed malignancy among men worldwide and remains a significant cause of cancer-related deaths in men. Despite advances in diagnostics and therapeutics, the disease often progresses to aggressive, therapy-resistant stages. Increasing evidence highlights the tumor microenvironment (TME) as a critical regulator of disease progression. Among its components, fibroblasts, particularly cancer-associated fibroblasts (CAFs) and their secreted exosomes, play an important role in modulating tumor growth, invasion and treatment response. However, the mechanisms by which patient-derived fibroblasts and their exosomes influence prostate cancer remain incompletely understood. This work investigated the dynamic interaction between primary patient-derived prostate fibroblasts, their exosomes, and PCa tumor cells, focusing on functional regulation and exosome-mediated signaling. Four pairs of CAFs and non-cancer-associated fibroblasts (NCAFs) were isolated from patients after radical prostatectomy and were comprehensively characterized. Exosomes were successfully isolated and confirmed using nanoparticle tracking analysis, transmission electron microscopy, and western blotting for canonical markers, establishing their identity as exosomes. Functional co-culture studies revealed patient-specific but distinct effects of fibroblasts on PCa cells. CAFs generally enhanced tumor cell viability and motility, whereas NCAFs showed a stronger influence on promoting proliferation. Exosome treatments partially mirrored these effects, demonstrating that fibroblast-derived exosomes can recapitulate key functional alterations in tumor cells. Importantly, context-specific responses were observed between the two PCa cell lines studied. The androgen-dependent LNCaP cells and the androgen-independent, more aggressive LNCaP C4-2 cells. While LNCaP cells remained largely non-migratory, CAFs and their exosomes significantly promoted migration in LNCaP C4-2 cells, suggesting that fibroblast-derived exosomes may contribute to metastatic progression. To further investigate reciprocal signaling, PCa cells-derived exosomes were applied to fibroblasts and hTERT-immortalized foreskin fibroblasts. While LNCaP C4-2-derived exosomes increased fibroblast viability, they failed to induce significant activation in patient fibroblasts. They unexpectedly reduced α-SMA expression in hTERT cells, indicating variable fibroblast responses to tumor-derived signals. Additionally, global microRNA profiling of fibroblasts and their exosomes revealed a distinct expression pattern differentiating CAFs from NCAFs. Several miRNAs associated with cancer-related pathways were enriched in CAFs and their exosomes, and validation experiments confirmed their selective packaging. These findings highlight a potential role of fibroblast-derived exosomal miRNAs in modulating PCa cell behavior and provide insight into their contribution to tumor progression. Overall, this work demonstrates that patient-derived fibroblasts and their exosomes are not passive bystanders but active modulators of PCa biology. The study provides new insights into the molecular and functional interplay between stromal and tumor cells, identifies potential biomarkers for disease stratification, and emphasizes the therapeutic relevance of targeting tumor-stroma communication. By advancing our understanding of the role of fibroblast-derived exosomes in PCa, these findings open avenues for developing novel diagnostic strategies and more personalized treatment approaches for advanced disease.