3D Tumor Spheroid Assays for Anti-Cancer Therapeutics

The 3D Tumor Spheroid Assay is used to analyze the therapeutic effect and tumor-penetrating capacity of anti-cancer therapeutics. The viability assay performed on spheroids in a 3D setup has a high translational value making it a valuable tool for lead candidate selection for in vivo studies.

3D tumor spheroids recapitulate some of the complex processes that compounds face in an in vivo situation such as cellular barriers and cells adapted to gradients of oxygen and nutrient concentrations. The spheroids are composed of cells in various proliferative and metabolic states similar to tumors. Therefore, the 3D Tumor Spheroid Assay has a high predictive value for pre-clinical drug research.

  • Mono-spheroid assay with human tumor cell lines
  • Co-culture spheroid testing with quantification of both, stroma and tumor cells
  • High-throughput compatible setup

3D Tumor Spheroids

Using spheroids for efficacy testing allows the detection of both cytotoxic and anti-proliferative effects of drugs.

Tumor cells assemble in round-bottomed multi-well plates to form spheroids. Within three days they grow to a multiple of their original size allowing testing of compounds for proliferation, or killing, of cells in the original spheroid. The example shown on the right: U87 glioblastoma tumor spheroids at day 1 and day 3 after seeding.

3d Tumor spheroids grown for 3 days derived of U87 cell culture

Assay Details

Procedure: Mono-Culture Spheroids

assay procedure for 3D tumor spheroid assay with cell titer glo for testing of cell proliferation

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.


Mono-Culture Spheroids

Mono-Culture Spheroids

Example of U87MG mono-spheroids. Left: Comparison of the size of U87MG spheroids at day 0 and 3. Right: Concentration-response curve of trametinib treatment 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.

Co-Culture Spheroids

3d tumor spheroids with luciferase to quantify co-culture

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.

co-culture 3D tumor spheroids with tumor and stroma cells used for dose-response testing of a compound for anti-proliferative effects

Example showing reduced sensitivity of DLD1 cells against trametinib 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 MEK kinase inhibitor trametinib. Subsequently, the viability of tumor cells was analyzed by measurement of firefly luminescence.

Screening options:

  1. Co-spheroids with tumor & stroma cells, luciferase measurement only in tumor cells
  2. Co-spheroids with firefly luciferase-labeled tumor & renilla-luciferase labeled stroma cells


Screening Formats

Setup: IC50 value determination and single concentration testing are possible.

Custom-tailored assays can be performed.

Cell lines that are not part of the list 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.