Ovarian cancer has a high mortality rate among gynecological malignancies. Ovarian cancer stem cells and their vascular niche are able to remodel tumor microenvironment. However, previous ovarian cancer stem cell models have not permitted the in-detail analysis because of their limited reproducibility.
The advantage of our protocol include the reproducibility of its 3D co-culture system, which facilitate the study of the effects of ovarian cancer stem cells-derived tumor initiation capabilities in patient during angiogenesis. Demonstrating the procedure will be Yuko Miyagawa, a research fellow from our laboratory. To isolate cancer stem cells from human ovarian cancer ascites obtained via paracentesis, Collect 100 to 250 milliliters of patient ascites and centrifuge the sample within 24 hours of acquisition.
Resuspend the pellets in two milliliters of OCSC medium and add eight milliliters of 30%non-ionic density gradient medium in PBS to each sample. Transfer the cell suspensions to individual 15 milliliter tubes and carefully layer two milliliters of additional OCSC medium onto the cell solution in each tube. Separate the cells by density gradient centrifugation and carefully transfer the OCSC at the interphase into a new 15 milliliter tube.
Wash the collected cells with a full volume of PBS and resuspend the pellets in fresh OCSC medium. Seed the cells onto ultra low attachment culture dishes and incubate the cultures at 37 degrees Celsius and 5%carbon dioxide. After five days of culture, collect the cells in one 15 milliliter tube per plate by centrifugation.
After a second centrifugation in PBS, resuspend the cells in one milliliter of proteolytic and collagenolytic enzyme cell detachment solution for 10 minutes at 37 degrees Celsius. At the end of the incubation, mix well by pipetting and return the cells to 37 for another five minutes. At the end of the incubation, mix the cells again by pipetting and fill the tube with PBS for washing by centrifugation.
Then, resuspend the pellet in OCSC medium and seed the cells on new ultra low attachment culture dishes. To set up a HUEhT-1 endothelial cell culture, wash the HUEhT-1 cells with PBS and treat the cells with 0.025%trypsin for three minutes at room temperature. When the cells have detached, stop the reaction with five milliliters of endothelial cell growth medium two, and collect the cells in a 15 milliliter tube for centrifugation.
Then, resuspend the pellet in HUEhT-1 medium at a 1.5 by 10 to the fifth concentration and seed the cells on collagen coated culture dishes. To set up an interactive co-culture plate for a tube formation assay, assemble the NICO-1 device according to the manufacturer's instructions and place the system on ice. Wash the assembly with 300 microliters of cold PBS before adding 300 microliters of chilled extracellular matrix based hydrogel to the device for a 60 minute incubation at 37 degrees Celsius.
At the end of the incubation, immerse a sterile 13 millimeter filter in 100%ethanol followed by a one minute submersion in PBS. At the end of the incubation, place an O-ring and the equilibrated filter into the appropriate chambers in the body of the assembly. When the device has been assembled, resuspend the HUEhT-1 cells at 1.25 by 10 to the fifth cells per milliliter of endothelial cell growth medium supplemented with 2%fetal calf serum concentration and add 1.2 milliliters of cells to each extracellular matrix based hydrogel coated well.
Then, add 1.5 milliliters of five day cultured OCSC to one well and place the NICO-1 system in the cell culture incubator. Tube formation can be observed by light microscopy and the formation cell networks on the extracellular matrix based hydrogel can be measured by quantifying the number of branches that form within the co-culture over time. Here, a comparison of the tube formation activity induced by two different CSC can be observed.
In this analysis, the number of formed vascular tubes dramatically increased over time during co-culture with the second CSC population. In these images, the angiogenic property of HUEhT-1 after vascular endothelial growth factor treatment without CSC2 co-culture can be observed as a positive control. In this OSCS endothelial cell co-culture model using the NICO-1 system, HUEhT-1 cells were co-cultured with CSC2 for 20 hours.
As observed via time lapse video imaging, the HUEhT-1 cells formed vascular tubes over the course of the 20 hour CSC co-culture. Recent discovery have led to the clinical use of PARP inhibitors for ovarian cancer, defective in homologous recombination repair, as well as anti-angiogenic bevacizumab. Our protocol using 3D co-culture system can provide insight regarding the angiogenic property of ovarian cancer stem cells in tumor metastasis.
