Cancer Spheroid

In vitro Cancer Spheroid Assay
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Cancer spheroids and tumoroids are versatile, three-dimensional (3D) in vitro models generated from cancer cells and tumor tissues. Because these 3D cancer models more closely resemble complex tumor biology and cellular interactions observed in vivo, scientists are using them to evaluate candidate immune cell products and accelerate preclinical drug development for urgently needed cellular immunotherapies in solid tumor applications. The simple and sensitive assays of the Maestro Z accurately measure tumor growth and immune cell killing of 3D cancer spheroid models.

The Challenge of Solid Tumors


  • Tumor heterogeneity can lead to acquired resistance through antigen escape.


  • The tumor microenvironment (TME) can develop mechanisms to evade or suppress the immune response​
  • Tumor expression of PD-L1 can trigger the inhibitory PD-1 pathway​
  • Common immune-suppressive cytokines found in TME: IL-10, TGFB, IL-4, and IL35​


  • The composition and structure of tumors can prevent t-cell trafficking into the tumor.
Common in vitro cancer spheroid or solid tumor cell killing assay challenges infographic: heterogeneity, immunosuppression and infiltration

Cancer Spheroid Assays

Track in vitro tumor growth and immune cell killing in 3D spheroids

Accurately measuring cell potency is critical for developing and improving immunotherapies. Cancer spheroids placed in a CytoView-Z plate can be noninvasively monitored to track real-time growth and assess the potency of therapeutic candidates.


Cancer Spheroid imaged in a CytoView-Z impedance well bottom


Cancer Spheroid growth over time as measured by an increase in impedance


Change in cancer cell growth as measured by change in impedance after CAR T dosing


A cancer spheroid composed of HER2-expressing ovarian cancer cells in a CytoView-Z plate. Growth of spheroids of different starting sizes are monitored over 50 hours. The addition of HER2-targeted CAR T cells at different effector:target ratios at 24 hours shows a dose-dependent decrease in spheroid size.

Investigate immunotherapy potency on models that better reflect solid tumor biology

Solid tumors are characterized by a harsh tumor microenvironment, which can substantially impact interactions between tumors and immune therapies like chimeric antigen receptor (CAR) T-cell therapy. While two-dimensional (2D) cell cultures remain an indispensable platform for high-throughput drug screening and toxicity testing in oncology, 3D cancer spheroids offer an in vitro model closer to the complex tumor biology. A comparison of CAR T cell-mediated cytolysis in 3D in vitro models and 2D monolayers using the Maestro Z found a decreased potency of HER-2 specific CAR T cells in the 3D tumor model- much as solid tumors demonstrate increased resistance to treatment in vivo.


%Cytolysis of cancer spheroids with CAR T dosing


Cytolysis of Cancer Spheroids and monolayers of 72 hours after CAR T dosing


Cancer Spheroid Figure Legend


CAR T cell-mediated cytolysis of cancer spheroids is lower than the cytolysis of matching E:T ratios in monolayer cultures over time (A), resulting in a higher EC50 for the spheroid cultures (B).

Application Note: CAR-T Cell Potency Assessment with 3D Cancer Spheroid Models
Application Note: 3D cancer spheroids
Application Note:
CAR-T Cell Potency Assessment with 3D Cancer Spheroid Models

When it comes to measuring the effects of cell-based immunotherapies on solid tumors, 2D monolayers may not tell the full story. Download our new app note to discover how next-generation in vitro tumor models on Axion's Maestro Z platform can accelerate immunotherapy discovery—with no labels, dyes, or complicated steps.

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Assay Steps


Cancer Spheroid Assay Steps


Cancer spheroids are formed over four days in ulta-low attachment U-bottom plates. Individual spheroids are then transferred into each well of a CytoView-Z plate. One day post spheroid plating, CAR T cell suspension is added at desired effector to target (E:T) ratios. Changes in spheroid size are tracked label-free and in real time with the Impedance Module software.

Additional cancer spheroid culture and recording protocol is available.

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