Abstract B23: Extravasation of breast cancer cells to a bone-cell conditioned microenvironment in functional 3D microvascular networks generated by vasculogenesis in a microfluidic system
Authors: Jeon Jessie S. , Bersini Simone , Dubini Gabriele , Charest Joseph , Moretti Matteo , Kamm Roger D. 
Published: May 2015

Abstract
Cancer cell metastases arise due to a series of biological processes starting from malignant tumor cells invading from primary to distal organs. Bone is one of the preferred sites of metastasis for various tumors including breast cancer. Understanding metastatic invasion to the secondary site requires studying the individual steps in metastasis, particularly those leading up to and immediately following extravasation of a circulating tumor cell from a blood vessel into the remote tissue. Microfluidic systems can provide cells with a controllable and reproducible 3D bone micro-environment to study cancer extravasation to a degree not possible by standard tissue culture methods while also simultaneously providing in situ imaging capabilities for visualization. We investigate the critical steps of cancer extravasation: tumor cells adhering to the endothelium and subsequently transmigrate across it into a hydrogel model for the bone extracellular space. Our model system consists of a functional microvascular network generated through vasculogenesis in a bone-mimicking microenvironment all within a microfluidic system.
The microfluidic system contains a central hydrogel region flanked by two lateral media channels. The hydrogel region is filled with cells dispersed inside a fibrin gel. The two channel system enables easy access to the microvascular network for the addition of cancer cells for extravasation. Cells of passage 6 or lower MSCs, harvested from patients undergoing hip arthroplasty, were differentiated to osteo lineage. GFP-human umbilical vein endothelial cells (HUVECs) were suspended at 2·107 cells/ml in EMG2 + thrombin and combined with osteo-differentiated and non-differentiated MSCs mixed at a 9:1 ratio, and inserted into the gel channel to complete the seeding. RFP-expressing BOKL, which is an MDA-MB-231 cell line specific to bone metastasis enabled live-cell imaging via fluorescent microscopy.
We created a functional microvascular network in a bone cell-conditioned microenvironment with the addition of osteo-differentiated and non-differentiated MSCs. Immunofluorescent imaging of α-smooth muscle actin in the system confirmed the presence of mural cells differentiated from MSCs wrapped around the generated vascular network. Visual confirmation of 3μm microspheres flowing through the vessels confirmed that the vascular network generated in this system was indeed perfusable. Finally, the osteo-cell conditioned microenvironment was confirmed by staining for osteocalcin. BOKL were then perfused into the microvasculature to model the extravasation process. The percentage of cancer cell extravasation in a device containing a microvascular network seeded with osteo-differentiated MSCs was 3.8 fold higher (56.5±4.8%) than the case with HUVECs alone (14.7±3.7%). Permeability values of the vasculature were analyzed by monitoring the leakage of fluorescent 70kDa dextran from the microvessels over time. Vasculature in the bone cell-conditioned microenvironment exhibited a higher permeability (4.12±0.75)·10-6 cm/s compared to the HUVEC only condition (0.89±0.31)·10-6 cm/s. While the increase in extravasation rates may due to a combination of factors, the higher permeability of the vasculature in osteo-cell conditioned environment may also be one of the contributing factors.
Microfluidics offers the capability to create organ-specific microenvironments that can capture extravasation events, leading to tumor metastasis. This system therefore captures certain aspects of cell-cell and cell-matrix interactions for preferential metastasis to bone, offering potential for drug screening.
Citation Format: Jessie S. Jeon, Simone Bersini, Gabriele Dubini, Joseph Charest, Matteo Moretti, Roger D. Kamm. Extravasation of breast cancer cells to a bone-cell conditioned microenvironment in functional 3D microvascular networks generated by vasculogenesis in a microfluidic system. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B23. doi:10.1158/1538-7445.CHTME14-B23