3D Tumor Spheroid Assay Details
Assay procedure. Tumor cells autonomously assemble to form spheroids in round-bottom 96 well plates. Compounds incubate for three days before readout of cell quantity with either cell viability reagent CellTiter-Glo-3D or luciferase measurement.
Example of U87MG mono-spheroids. Left: Comparison of the size of U87MG spheroids at day 0 and 3. Right: Concentration-response curve of treatment with a small molecule MEK1/MEK2 inhibitor on U87MG mono-spheroids. Positive control (0 % viability) is staurosporine treatment; negative control (100 % viability) is vehicle control. ‘day 0’ shows the number of viable cells at the start of treatment.
Assay procedure for co-culture spheroids: Firefly-luciferase expressing tumor cells and renilla-expressing stroma cells are seeded in a round-bottom 96 well plate and left to assemble for spheroid formation for three days. Compounds incubate for three days before firefly-luciferase and renilla-luciferase quantification from the lysate of the spheroid. The cytotoxic and anti-proliferative effects of compounds can be determined for both tumor and stroma cells separately.
Example showing reduced sensitivity of DLD1 cells against a MEK kinase inhibitor in co-spheroids: DLD1 tumor cells were cultured in spheroids as mono-culture (black) or together with HS27A stroma cells (blue). Spheroids were treated for 72 h with the drug. Subsequently, the viability of tumor cells was analyzed by measurement of firefly luminescence.
- Co-spheroids with tumor & stroma cells, luciferase measurement only in tumor cells
- Co-spheroids with firefly luciferase-labeled tumor & renilla-luciferase labeled stroma cells
Setup: IC50 value determination and single concentration testing are possible.
Cell lines: Please inquire to receive a list of cell lines that are established in the 3D Tumor Spheroid Assay format. New assays based on additional cell lines can be established according to the client’s research needs.
Controls: DMSO only treatment serves as high control. Staurosporine treatment (at 1x10-5M) serves as low control.
Turnaround time: Results will be available after 3 to 4 weeks. Expedited scheduling and data delivery can be arranged prior to study commencement.
Report: A detailed report including assay conditions, methodology, and comprehensive evaluation of data as well as raw data for each analysis will be provided. Photographs are optional.
Screening Facility: The assay is performed in Freiburg, Germany.
Compound requirements: In brief: 30 µl of the stock solution with 1000x of the highest assay concentration or the equivalent of solid material.