Development of 3D breast cancer model to study extracellular vesicle crosstalk

Chabria, Yashna
3D model systems are crucial in establishing novel therapeutics and understanding cell interactions in the tumor microenvironment. Extracellular vesicles (EVs) are promising carriers of therapeutic cargo for tumor-targeted delivery, and biocompatible hydrogels offer controlled EV delivery. The three core elements of this study included generation and scale up of GFP-EVs in a FiberCell bioreactor, followed by assessment of EV encapsulation and release from hyaluronic acid hydrogels and finally establishing a dynamic 3D breast cancer model that mimics the tumor niche. MDA-MB-231 cells were genetically modified to produce CD63-GFP-labeled EVs. 1x109 of these cells were cultured in a 20kD bioreactor with the introduction of serum free media once stable culture was established. Over five weeks, multiple collections of GFP-labeled EVs were isolated by size exclusion chromatography and characterized by Nanoparticle Tracking Analysis (NTA), western blot, and Transmission Electron Microscopy (TEM). Furthermore, tyramine modified hyaluronic acid (HA-TA) hydrogels were established with hydrogen peroxide and horse radish peroxidase crosslinkers to investigate EV release patterns in static and dynamic conditions. To establish a dynamic 3D multicellular model of breast cancer, isolation and characterization of patient-derived tumor and lymph node stromal cells was performed. Mixed stromal/epithelial tumor spheroids and lymph node spheroids encapsulated in alginate hydrogels were formed. Spheroid viability was assessed and these were introduced into a dynamic multi in vitro organ (MIVO®) system with their respective endothelial barriers to establish crosstalk and support secretome analysis using angiogenesis arrays and ELISA. GFP expression was demonstrated in transduced cells with longitudinal expression confirmed in cells throughout bioreactor culture. NTA and TEM revealed both plasma-EVs and GFP-EVs in the size range of 30-200nm with an intact lipid bilayer were successfully isolated. Initial harvests of GFP-EVs contained subpopulations in a higher size range which disappeared within a few days of serum withdrawal, highlighting initial serum contamination. Western blot confirmed the expression of EV markers CD63, TSG101, CD81, CD 82. Successful incorporation and release of plasma-derived EVs from the hydrogels was demonstrated, with release patterns dependent on loaded EV concentrations and hydrogel formulations. Further investigation into EV release patterns using GFP-EVs under static and dynamic conditions highlighted a significant increase in EV release under fluidic flow conditions. Characterization of tumor and LN stromal populations confirmed presence of stromal markers and absence of hematopoietic markers. Spheroid growth within the alginate gel was monitored maintenance of an intact structure, cell viability and metastatic potential shown. Secretome analysis of the spheroid culture in the dynamic fluidic system supported the tumor-mimicking characteristics of the 3D breast cancer model system. These preliminary findings demonstrate the potential of the fibercell bioreactor system that supported efficient, serum free and reproducible scale up of GFP-EV production which will facilitate tracking EV transfer in the cancer setting. HA-TA hydrogels also showed promise for incorporation and sustained release of EVs. Finally, the 3D organoids in the dynamic system mimicked the tumor niche, demonstrating compelling potential for future study of intercellular EV trafficking and therapeutic potential prior to clinical translation.
NUI Galway
Publisher DOI
Attribution-NonCommercial-NoDerivs 3.0 Ireland