Melanoma is the most aggressive form of skin cancer, which invades deeper layers of the skin and has a propensity to metastasise early. In an effort to provide insight into the mechanism by which melanoma cells metastasise, we examined differential cell rolling, adhesion and transmigration within an isogenic model of melanoma progression under physiological shear flow conditions. These experimental conditions can easily be studied using Cellix's microfluidic pumps and biochips. The dimensions of the biochips can mimic those of human capillaries thereby enabling researchers to simulate in vivo microenvironments. Cellix's 8-channel Mirus Evo Nanopump enables researchers to conduct up to 8 assays simultaneously in microfluidic biochips while single assays may be conducted using the ExiGo pump. The microcapillary walls of Cellix's biochips may be pre-coated with Selectins, adhesion molecules such as VCAM, ICAM, chemoattractants or other proteins of interest. It is also possible to culture endothelial cells (e.g. HUVECs, HMVECs, HLMECs) via the Kima pump, resulting in a set-up for studying cell-cell interaction. In this study, we determine if the steps in extravastion differed in an isogenic melanoma cell line model of progression.
- Cell rolling, adhesion & migration assays
- Cell observation with brightfield, phase contrast and immunostaining microscopy
- WM793, WM793-P1, WM793-P2, 1205Lu interaction to rhVCAM-1 studies
- 1205Lu-GFP cells to endothelial monolayer studies
- Transmigration studies of 1205Lu-GFP cells
- Adhesion profile of Melanoma cell lines on rhICAM-1, rhVCAM-1 and HUVEC
- Metastatic movement of Melanoma cell line using VenaT4 biochip
Aim: To analyze rolling, addition and migration profile of Melanoma cell line on rhICAM-1, rhVCAM-1 and Human Umbilical Vein Endothelial Cells.
Vena 8™ biochip micro-channels were coated with rhICAM-1 and rhVCAM-1 overnight in humid conditions at 40°C.
Melanoma Cell lines (WM793, WM793-P1, WM793-P2, 1205Lu) were infused in the channels coated with the adhesion molecules. Alternatively, 1205Lu were perfused over HUVECs which had been cultured in VenaEC™ biochips.
Perfusion was executed via Mirus Evo Nanopump for 8-assays in parallel or to the ExiGo pump for single assays at a shear stress of 0.5 dyne/cm2.
Results: WM793, WM793-P1and WM793-P2 didn’t adhere to the adhesion molecules at constant shear stress, whereas 1205Lu cells adhered to rhVCAM-1 under similar shear. To determine the threshold shear stress, a decreasing shear stress of 5, 2 and 0.5 dynes/cm2. 1205Lu GFP cells were subjected to shear stress of 0.5 dynes/cm2 over HUVEC.
Discussion: Results showed that shear stress plays an important role in the extravasation process. The ability of 1205-Lu cells to attach to V-CAM under higher shear stress may contribute to its extravasation abilities, thus contributing to its high metastatic potential.
VenaT4 biochip micro-channels were coated overnight in humid conditions at 4°C with rh VC AM- 1(20 µg /ml).
After incubation, the thin film was removed from the microwells and 30 µl of Type 1 Bovine Collagen gel with chemo-attractant (Fibronectin 5 µg/ml) was added in the well. Control wells were Fibronectin free.
1205-Lu GFP cells were infused though the channel at 0.5 dynes/cm2 for 30 mins.
Connect to Mirus Evo Nanopump for 8-assays in parallel or to the ExiGo pump for single assays.
Results: 1205-Lu GFP adhered to rhVCAM at constant shear stress of 0.5 dyne /cm2 . The cells transmigrated through the 8μm diameter pore size of the VenaT4 biochip after continuous perfusion at the same rate. Analysis was done on the basis of diameter measurement of the cells in the images. The rate of transmigration of cells in the control well (image not given) was low compared to the transmigration of cells into the well which contained the chemo-attractant.
Discussion: Cellix’s VenaT4 biochip for transmigration and invasion assays is an efficient model to analyze the ability of Melanocytes and leukocytes to migrate through the endothelium under flow conditions. In this study, we focused on cell diameter and fluorescent intensity for analysing the transmigration of cells through the microporous membrane embedded in the VenaT4 biochip and in so doing, proving the technology as a ground breaking and valuable new addition to microfluidic area particularly for cancer invasion assays.