This protocol allows the identification and isolation of cancer stem cells in a robust manner using functional assays and phenotypic characterization. Cancer stem cell isolation from a tumor sample could be a platform for guiding the clinical application of specific therapies to individual tumors, predicting resistance and consequent reoccurrence. To prepare non-adherent suspension cultures, first coat the cell culture containers with 50 microliters per centimeters squared of 15 milligrams per milliliter of polyHEMA, and place the containers in a 37 degrees Celsius drying oven for at least two days.
When the containers are fully dry, wash in 80 to 90 percent confluent cancer cell line culture with PBS, and detach the cells with one to two milliliters of trypsin EDTA. After five minutes at 37 degrees Celsius, arrest the reaction with two to four milliliters of fresh cell culture medium, and wash the dissociated cells by centrifugation. Re-suspend the pellet in fresh culture medium for counting, and dilute the cells in fresh sphere culturing medium containing two percent methyl cellulose at a 500 to 2000 cells per centimeter squared per culture dish concentration.
To ensure sphere monoconality, seed each cell line at a low density in an anchorage-free environment. Then seed the cells onto the polyHEMA coated plates for a five day incubation at 37 degrees Celsius and five percent carbon dioxide, adding ten nanograms per milliliter of epidermal growth factor and ten nanograms per milliliter of basic fibroblast growth factor to the cell culture medium every two days. Three to twelve days after plating, three-dimensional ball-shaped cell colonies should be observed by light microscopy.
To obtain derived adherent populations, transfer the spheres to a new culture dish under the appropriate culture conditions for the original cancer cell line. After one to two days of culture, a cell monolayer should be observed growing around the adherent spheres with a morphology similar to that of the cell line of origin. To determine the sphere-forming capacity of the cancer cell line of interest, after completion of sphere-forming protocol, collect the spheres by centrifugation.
Gently resuspend the sphere pellet in fresh medium, and use a hemocytometer to count the number of spheres with a greater than 40 micrometer diameter. Then calculate the percentage ratio of the sphere obtained versus the number of cells initially plated. To determine the self-renewal ability of the cell line of interest, collect the spheres by centrifugation and gently resuspend the sphere pellet in trypsin EDTA for a five minute incubation at 37 degrees Celsius.
At the end of the incubation, add enzyme and activation medium to the tube, and pipette the solution to obtain a single-cell suspension. Using a hemocytometer and the trypan blue exclusion method, count the viable cells in the suspension and plate the cells in polyHEMA coated plates for a sphere-forming assay as just demonstrated. After eight days, use a hemocytometer to count the number of spheres with a more than 40 micrometer diameter, and calculate the percentage ratio of spheres obtained versus the number of cells initially plated.
To evaluate the area occupied by the spheres, place the culture dish onto the stage of an inverted microscope equipped with an image acquisition module and select a 100X to 400X magnification. Obtain images of at least ten random fields per condition, and use an appropriate image analysis software program to draw regions of interest around the spheres. Then measure the area of each region of interest in pixels and calculate the sphere projection area as the mean area of the pixels measured.
The functional characterization of the sphere-forming capacity, self-renewal, and projection area of the cancer stem cells allows a comparison of their distant lineage and tissue of origin. The sphere-forming protocol allows spherical colonies to be obtained in suspension from several endometrial and breast cancer cell lines or after gentle enzymatic digestion of tissues from human tumor samples. Both endometrial and breast cancer spheres give rise to a cell monolayer with similar morphologies to their cell line of origin one to two days after plating.
In these representative experiments, the hormonal receptor positive breast cancer MCF-7 cells demonstrated a higher sphere-forming capacity, self-renewal ability, and projection area than the triple-negative HCC1806 breast cancer cells. Besides cancer stem cell enrichment via the sphere-forming protocol, we recommend the further assessment of their stemness using complementary methods such as flow cytometry. In this representative analysis of spheres obtained from endometrial cell lines, four populations of cells expressing cancer stem cell markers were identified in each cell line by flow cytometry.
Evaluation of the tumor spheres by other molecular biology techniques can be confirmatory of stemness, and plasticity of cancer stem cells and facilitated the development of targeted therapies. Western blot analysis after gentle sphere harvesting also revealed a cancer stem cell phenotype for the in vitro generated spheres. This isolation protocol contributes to our understanding of cancer stem cell significance which clinically translates to understanding relapse, metastases, and treatment resistance.
