This technique will allow researchers to isolate breast cancer stem cells at maximum purity and viability for the performance of both in vitro and in vivo functional characterization assays. Isolated breast cancer stem cells can also be used to help elucidate the molecular events that control tumorigenicity and metastasis, and to design therapeutic strategies to target these cells. With minor modifications specific to the cell and tissue types of interest, this method can also be applied to other systems.
To generate a single cell suspension from a cell culture, wash an 80%confluent breast cancer cell culture with PBS and treat the cells with an appropriate cell dissociation solution for five minutes at room temperature. Once the cells have detached, add five milliliters of culture medium to neutralize the cell dissociation solution and transfer the cells to a 50 milliliter conical tube for centrifugation. Resuspend the pellet in five milliliters of PBS for counting.
After counting, centrifuge the cells again, then resuspend the cells at one times 10 to six cells per milliliter of ALDH substrate buffer. To generate a single cell suspension from a tissue sample, use surgical blades in a crisscross technique to mince the tumor tissue into approximately one millimeter size pieces. Place the pieces in a 50 milliliter conical tube containing 10 milliliters of dissociation buffer.
Seal the tube with parafilm for a 40 minute incubation at 37 degrees Celsius in a shaker and sediment the digested tissue pieces by centrifugation. Resuspend the pellet in five milliliters of trypsin using a five milliliter pipette and incubate the tissue in a 37 degree water bath for five minutes. At the end of the incubation, vigorously pipette the tissue several times to release single cells and add enough DMEM/F-12 medium to bring the total volume in the tube to 25 milliliters.
Collect the cells by centrifugation, resuspend the pellet in one milliliter of Displace DNAse, and incubate for five minutes in a 37 degree Celsius water bath. At the end of the incubation, bring the total volume to 10 milliliters with PBS. Mix the cells by pipetting and passage them through a 40 micron cell strainer into a 50 milliliter tube.
After centrifugation, resuspend the cells in five milliliters of fresh PBS for counting. To perform a colony forming assay, after sorting, resuspend the cells in complete medium and add the appropriate number of breast cancer stem cells to each of the three tubes containing two milliliters of medium per tube, labeled one times 10 to the second, two times 10 to the second, or five times 10 to the second cells. Pipette up and down five times to thoroughly mix the cell suspension and plate the cells into individual wells of a six well plate.
Gently swirl the plate to obtain uniform cell distributions and place the plate in the cell culture incubator until colonies appear. At the end of the incubation, wash the cells with one milliliter of PBS per well and stain the cells with 500 microliters of 0.05%crystal violet solution per well for 30 seconds. At the end of the incubation, wash the wells two times with two milliliters of water per well to remove the excess dye, and count and record the total number of colonies per well by light microscopy at a four or 10 times magnification.
To perform a mammosphere assay, after sorting, resuspend the cells in complete mammosphere medium and plate the at a seating density of five times 10 of the second cells per square centimeter area per well in a 96 well ultra-low attachment cell culture plate. Before counting the number of mammospheres per well by light microscopy, place the plate in the cell culture incubator for five to 10 days. To set up a 3D culture model, carefully add 50 microliters of basement membrane extract to each well of a 96 well plate without bubbles and place the plate at 37 degrees Celsius for one hour to allow the solution to polymerize.
Add 100 microliters of PBS after 10 minutes to avoid drying of the gel layer. At the end of the incubation, suspend the sorted cells to five to 50 times 10 to the third cells per 200 microliters of 3D culture medium concentration and replace the PBS in each well of the 96 well plate with 200 microliters of media containing cells. Then place the plate in the cell culture incubator for 10 to 14 days before assessing the cultures for organoid formation.
Human breast cancer cells that exhibit enhanced ALDH enzymatic activity express high levels of CD44 and low to negative levels of expression of CD24, and can be classified as breast cancer stem cells. The proportion of breast cancer stem cells within the bulk population can vary between cell lines or patients and often depends on the disease stage, with more aggressive breast cancer typically displaying a higher proportion of breast cancer stem cells. The ability of a single breast cancer cell to self-renew and generate colonies of 50 cells can be assessed by colony forming assays.
The ability of breast cancer stem cells to self-renew under anchorage-independent experimental conditions can be assessed by mammosphere assays. Culturing breast cancer cells and basement membrane extract allows breast cancer stem cells to form 3D structures that recapitulate in vivo conditions. In addition, in vivo mouse xenograft models can be used to understand the differences in growth, self-renewal, differentiation, and/or the tumor initiating ability of breast cancer stem cells compared to non-breast cancer stem cells or bulk cell populations.
Following this procedure, the isolated breast cancer stem cells can be used in transcriptomic, kinomic, and metabolomic studies. This method provides researchers with an opportunity to explore the role of breast cancer stem cells in tumor initiation, metastasis, recurrence, and therapy resistance.
