Quantify T Cell Response in 3D Tumor Spheroids Using Advanced Flow Cytometry and Live-Cell Analysis


K McBain1; M Oliver1; L Kelsey1; C Szybut1; T Dale1
1 Sartorius, UK


Antigen recognition by T cells induces powerful effects, including infiltration and targeted killing, which aid in the elimination of cancer. Typically, therapeutic effects on T cell response have been evaluated using 2D cell models, which bear limited resemblance to the complex physiology of the in vivo tumor microenvironment. Here we provide two 3D spheroid-based solutions for monitoring immune-cancer cell interactions: immune cell killing (ICK) and tumor infiltrating lymphocytes (TILs).

Single spheroids were co-cultured with PBMCs and CD3/CD28 Dynabeads. To measure the rate of ICK, labeled tumor cells were imaged using the Incucyte® Live-Cell Analysis System and quantified for spheroid fluorescence over time. For the TILs assay, spheroids were washed to separate non-infiltrated immune cells from the TILs. Both assays required endpoint spheroid dissociation prior to analysis of cell subsets and cytokines using the iQue® Advanced Flow Cytometer and T cell characterization kits.

Separation of the TILs and non-infiltrated T cells enabled comparison of their activation status. Infiltrated cells maintained high CD69 expression (67.1 ± 0.6%) regardless of the number of Dynabeads present. Non-infiltrated cells were more sensitive to external stimuli with CD69 expression increasing from 2.3 ± 0.2% to 86.9 ± 0.7% with increasing Dynabead density. The effects of Dynabead activation were further profiled using the ICK assay. Incucyte data showed a 98.4% reduction in spheroid fluorescence in the presence of 50K Dynabeads compared to the non-activated control on day 4. Spheroid killing was accompanied by an increase in T cell activation and exhaustion marker expression (CD25 and PD-1) and cytokine production (IFNγ). These data demonstrate the study of complex 3D tumor models using advanced flow cytometry and live-cell analysis as a translational approach to in vitro profiling of immunotherapeutics.

Hosted By

The European Laboratory Research & Innovation Group Our Vision : To provide outstanding, leading edge knowledge to the life sciences community on an open access basis
Event Logo Find us on Facebook Follow us on Twitter

Get the App

Get this event information on your mobile by
going to the appstore or google play and search for 'elrig'
Eventflo Home
copyright ELRIG,, Labhoo Ltd 2003-2021