Towards the Development of an in Vitro 3D Co-Culture Cancer Model for the Design of Dendrimer-Anti-Cancer Drug Conjugates

Towards the Development of an in vitro 3D Co-Culture Cancer Model for the Design
of Dendrimer-Anti-cancer Drug Conjugates
 

Justin Moore, Elizabeth Bielski, and Sandro R.P. da Rocha*

School of Pharmacy & Chemical and Life Science Engineering

Virginia Commonwealth University Ð Richmond, VA 23298-0533

Two dimensional (2D) monolayer cells cultured on flat substrates have been for decades an important in vitro tool used to test and understand numerous biomedical problems. Traditionally, these cell culture assays have been a monolayer of cells cultured in vitro. However, these 2D cultures have recently come under scrutiny for their significant departure when compared to the in vivo tumor microenvironment they are designed to mimic. In 2D monolayer cell cultures cell-to-cell/cell-to-matrix interactions, biomedical gradients and signaling pathways are lost, tumor characteristics that significantly affect the drug transport. The long-range of this project is to establish a 3D in vitro model to test the ability of dendrimer-anti-cancer therapeutics to treat solid tumors, and the impact of their chemistry in terms of its ability to penetrate deep regions of the solid tumor environment. The first part of my project is establishing a more relevant model of the tumor microenvironment. This consisted of a transition from the traditional 2D cultures to a 3D model that better represents the architecture of solid tumors. Furthermore, the establishment of a co-culture system using cancer-associated stromal cells was an appropriate subsequent step in to achieve a more relevant in vitro system to test and understand the cytotoxicity profiles of the different systems. The stromal cell type used were cancer-associated fibroblast (CAF) because of their important role in creating an extracellular matrix which has been shown to impact drug penetration. Optimization of the ratios of cancer/stromal cell lines was required to see what initial ratio would result in a 70%/30% stromal/cancer cell ratio, a ratio representative of the in vivo microenvironment. Briefly, at a range of different ratios of 4T1 metastatic breast cancer cells and N1H 3T3 GFP fibroblast cells were seeded in flat-bottom 96 well plates, which were coated with agarose gel. The MCS were allowed to grow for 4 days then were dissociated. Then, using Flow Cytometry the co-cultured spheroids were characterized. It was found that the fibroblast and 4T1 cells are growing with similar growth kinetics within the co-culture. Previously, using an MTT assay, we tested the cytotoxicity of doxorubicin (DOX) on mono-culture MCS, and then compared these results to a 2D model system assay.  The MTT assay showed that much higher concentration of DOX are required to achieve 50% of cell death (IC50%) in the 3D spheroid (ca. 13 fold). Additionally, Dendrimer-Dox-conjugate had an increased toxicity profiles in both system which may suggest that the dendrimer conjugation has more ability to penetrate the spheroids and each cell in general. Next steps include establishing a cytotoxicity protocol for the 3D co-culture system and how manipulating (size, charge, and functional groups) impacts the Dendrimer-DOX penetration.   

Corresponding author: srdarocha@vcu.edu

Acknowledgements: VCU for a Start-up Grant; NSF-DMR Grant # 1643770 and VCU IMSD - NIHR25GM090084 for a scholarship for JM.