Metastasis is responsible for 90% of cancer-related deaths. It is particularly challenging to investigate because it is a complex process composed of several hidden steps that are difficult to resolve in vivo. Thus, a new generation of in vitro microfluidic models has recently been developed to study in greater detail individual metastatic steps, although few have been used to investigate the role of immune cells in cancer.
In this project, we aim to develop a new microfluidic model for studying the role of macrophages in extravasation and recolonization, and to find underlying signaling pathways that could be blocked to prevent them from facilitating metastasis. Recently, metastasis associated macrophages (MAMs) have been identified in mouse studies as a specific subpopulation that increase extravasation and metastasis; we propose to exploit the high imaging capabilities of our microfluidic models combined with mouse studies to further investigate the underlying mechanisms.
A previously characterized microfluidic device is used to generate microvascular networks (μVN) and replicate cancer cell extravasation in vitro. The device has 3 distinct but connected regions for containing hydrogels, all flanked by channels containing media. Briefly, endothelial cells (ECs; GFP-HUVECs) are seeded at 4×10^6/ml in fibrin (2.5 mg/ml) in the central gel region, while the two lateral gel regions are filled with fibroblast in fibrin (2.5 mg/ml) at 5×10^6/ml; following 4 days of culture in EGM media, a 3D perfusable μVN is formed. Fluorescently tagged MDA-MB-231 are perfused through the µVN and their transmigration through the vessel walls (i.e. extravasation) is imaged with a confocal microscope. To test whether macrophage increase extravasation and physically assist cancer cells during that process, immortalized bone marrow-derived macrophages (BMDM) isolated from C57BL/6 mice were directly mixed with GFP-HUVECs in the central gel region at a final concentration of 40,000/ml. BMDMs were found in the fibrin gel surrounding the microvessels, closely associated with endothelial cells. The diffusive permeability of the µVN was measured with fluorescent rhodamine dextran, and was found to be similar to values reported in vivo (2.21×10^-7 cm/s). 33.3± 20.4% of MDA-MB-231 extravasated within 6 hrs, as imaged on fixed devices stained with Phalloidin and DAPI. Extravasation was also quantified in real-time, and was shown to increase from 10% within 1 hr, to 75% after 12 hrs.
Another co-culture system was used for different imaging capabilities, consisting of a monolayer of endothelial cells formed on top of a 2.5 mg/ml collagen gel seeded with or without RAW macrophages (400,000/ml) in a Matek dish. This system was used to assess if macrophages increase adhesion of cancer cells prior to extravasation. MDA-MB-231 were placed on the monolayer; 2 hrs later they were counted in three regions marked on the plate, and imaged with the confocal microscope before and after three PBS washes. Adhesion was found to be little affected by the presence of macrophages: 56±7 % adhered without RAW vs. 62±20 % in their presence (p=0.25; unpaired t-test). We also performed RT-PCR on HUVECs cultured in a transwell plate, in presence (or not) of RAW macrophages placed in a collagen gel, to test if macrophages upregulated adhesion molecules of ECs such as P-selectin, E-selectin, VCAM-1 or ICAM-1, but did not detect any significant difference.
In conclusion, we successfully developed a 3D co-culture system with macrophages surrounding a network of perfusable microvessels for studying the role of macrophages in cancer cell extravasation. Our results suggest macrophages do not affect adhesion. Further studies will investigate their effect on extravasation and recolonization, and investigate pharmacological ways to block these processes in vitro and in vivo.
Citation Format: Alexandra Boussommier, Roger D. Kamm. Characterizing the role of macrophages in breast cancer cell extravasation in 3D microfluidic models. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B165.
- ©2016 American Association for Cancer Research.