Development of 3D breast cancer model to study extracellular vesicle crosstalk
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Date
2024-01-31Embargo Date
2025-01-29
Author
Chabria, Yashna
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Abstract
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.