Extracellular vesicles (EVs) are of great interest to study the cellular mechanisms of cancer development and to diagnose and monitor cancer progression. EVs are a highly heterogeneous population of cell derived particles, which include microvesicles (MVs) and exosomes (EXOs). EVs deliver intercellular messages transferring proteins, lipids, nucleic acids, and metabolites with implications for tumour progression, invasiveness, and metastasis. Epidermal Growth Factor Receptor (EGFR) is a major driver of cancer. Tumour cells with activated EGFR could produce EVs disseminating EGFR itself or its ligands. This review provides an overview of EVs (mainly EXOs and MVs) and their cargo, with a subsequent focus on their production and effects related to EGFR activation. In particular, in vitro studies performed in EGFR-dependent solid tumours and/or cell cultures will be explored, thus shedding light on the interplay between EGFR and EVs production in promoting cancer progression, metastases, and resistance to therapies. Finally, an overview of liquid biopsy approaches involving EGFR and EVs in the blood/plasma of EGFR-dependent tumour patients will also be discussed to evaluate their possible application as candidate biomarkers.

This study aimed to standardize the generation of primary three-dimensional (3D) cell cultures derived from spontaneous canine tumors and to compare two culture systems: a ready-to-use bioreactor (VITVO®) and a miniaturized plate-based system (MPBS). A total of 27 tumors, including mainly hemangiosarcomas, oral melanomas, urothelial carcinomas, and osteosarcomas, were collected from three Italian veterinary hospitals. Fresh samples were used to establish 3D cell cultures and to perform histological and immunohistochemical analyses for phenotypic characterization and comparison with primary tumors. Tissue dissociation proved to be a critical step. A manual protocol combining mechanical and enzymatic methods significantly improved cell viability and cellularity compared to automated dissociation, which yielded fewer viable single cells and limited growth. Both culture systems successfully supported 3D cell cultures using the same scaffold material. The plate-based system was particularly suitable for small biopsy samples without compromising cellularity, whereas VITVO® was more appropriate for larger tissue specimens. An inverse correlation was observed between the extent of necrosis in primary tumors and the cellularity of the corresponding 3D cell cultures. Immunohistochemical analyses confirmed that most 3D cell cultures retained the phenotypic characteristics of the original tumors, while osteosarcoma models exhibited limited growth, likely due to stromal predominance. Overall, these findings highlight the importance of optimizing dissociation protocols, scaffold size, and culture conditions to preserve tumor heterogeneity and viability. This workflow provides a standardized and reproducible platform for generating representative 3D models for comparative oncology research.