The overall goal of this in vitro method is to generate the three dimensional cell culture spheroids that enable the testing of current standard or experimental therapy regimens for head and neck squamous cell carcinoma. This method helps us to understand three dimensional tumor growth of head and neck cancer cells when exposed to radiation or chemo radiation treatment. It remains to be said that handling primary tumor cells is difficult and does not always lead to reliable three dimensional spheroid formation.
Demonstrating the procedure will be Sabina Schwenk-Zieger, a technician from our laboratory. While using primary cells from a tumor specimen, place the specimen on a suitable and sterile surface and cut it thoroughly with a sterile, single-use scalpel into very small pieces. After sufficient mechanical separation of the primary tissue, transfer it into a vial containing collagenase 1 and 2 and incubate it for one hour at 37 degrees celsius.
Next, sieve the mixture through a 70 micrometer Falcon cell strainer and wash the suspension with HBSS. After successful separation and subsequent washing, transfer the suspension containing one to two million cells into a T75 cell culture flask to grow to subconfluency at 37 degrees celsius for up to ten days. Under a microscope, confirm tumor cell growth.
Count the cells in culture. Using an ultra-low adhesion 96-well plate with concave round bottoms, seed 5, 000 primary tumor cells, or 1, 000 to 2, 000 cells of intermediate cell line, in 200 to 300 microliters of media into the wells. Next, culture the cells at 37 degrees celsius.
Perform media changes every other day. Pay attention not to aspirate the spheroid with the pipette during media changes. Culture the spheroids until the three dimensional spheroid-shaped cell conglomerates are observed.
Keep in mind to exclude the wells with irregular or multiple spheroid formation from further investigation. Subsequently, change the media to media with chemo therapeutics and/or monoclonal antibodies at desired concentrations. Add Cisplatin at the concentrations of 2.5, five, or 10 micromolar or 5-Fluorouracil at 30 micromolar.
In this procedure, measure the spheroid size after digital photo documentation on day six, day 10 and day 16 with a graphic software. After centrifugation of the plate at 520g for 2.5 minutes, remove the supernatant. Next, wash the cells with 1x PBS and centrifuge the plate again, followed by removing the supernatant.
Next, add 100 microliters of enzymatic cell detachment solution to each vial to allow the spheroids to dissolve. Subsequently, incubate the plate for eight minutes at 37 degrees celsius. Check for successful dissolving of the spheroids under the microscope.
Add 100 microliters of DMEM. Next, centrifuge the plate at 520g for 2.5 minutes. Then remove the supernatant and suspend the cells in 100 microliters of DMEM.
Subsequently, if necessary, perform a commercially-available colorimetric proliferation assay and read the assay in an ELISA reader. All cell lines could generate reliable spheroids with differences in size and time of readout. Primary cells did form reproducible spheroids in ultra-low attachment plates.
The Ki-67 staining reveals the periphery of the culture showing much higher proliferation rates than central cells, mimicking nutrient distribution in solid mucosal tumors that often show necrotic cores. Here, the effect of several chemo therapeutics and radiation on spheroid size. Radiation with two gray prior to treatment with Cisplatin, lead to a significant decrease in spheroid size compared to Cisplatin alone.
With further establishment of generating spheroids from primary human cells, this is how the concept of therapy testing could be implemented. Spheroids would be generated after tumor biopsy, and then tested for molecular characteristics and therapy susceptibility. We were able to establish a protocol to generate reproducible spheroids from cell suspensions for both cell lines and primary human tumor cells.
This multimodal assay is sufficiently sensitive to identify small differences between groups. In future, the assay could be used to first, assess individual response to standard and experimental therapy protocols. Second, further characterize the tumor cells from fresh specimens.
And third, correlate therapy response to molecular patterns. The use of primary cells and the generation of spheroids would allow the preservation of as many characteristics of in vivo tumor growth as possible in combination with a cost-efficient assay for studying individual therapy response.
