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Updated: Oct 3, 2025
A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells
Published on: January 22, 2021
Three-dimensional colon cancer organoids model the response to CEA-CD3 T-cell engagers
Alvaro Teijeira1,2,3, Itziar Migueliz1,4, Saray Garasa1,2,3
1Program of Immunology and Immunotherapy. Cima Universidad de Navarra. 31008. Pamplona, Spain.
Abstract:
Rationale: The CEA-CD3 T cell bispecific antibody cibisatamab (CEA-TCB) is currently undergoing clinical trials. Here we study its performance against three-dimensional tumor organoids in cocultures with T cells as compared to a higher affinity CEACAM5-CD3 (CEACAM5-TCB) bispecific antibody using time-lapse confocal microscopy. Methods: Pre-labelled spheroids derived from colon cancer cell lines and primary organoids derived from four colorectal cancer surgical specimens, which expressed different graded levels of CEA, were exposed in cocultures to T lymphocytes. Cocultures were treated with CEA-CD3 T-cell engagers and were followed by live confocal microscopy. Caspase 3 activation detected in real-time was used as an indicator of tumor cell death. Co-cultures were also set up with autologous tumor-associated fibroblasts to test the co-stimulatory effect of a fibroblast activated protein (FAP)- targeted 4-1BBL bispecific antibody fusion protein currently undergoing clinical trials. Results: Tumor-cell killing of 3D colon carcinoma cultures was dependent on the levels of surface CEA expression, in such a way that the lower affinity agent (CEA-TCB) did not mediate killing by human preactivated T cells below a certain CEA expression threshold, while the high affinity construct (CEACAM5-TCB) remained active on the low CEA expressing organoids. Modelling heterogeneity in the levels of CEA expression by coculturing CEA high and low organoids showed measurable but weak bystander killing. Cocultures of tumor organoids, autologous fibroblasts and T cells allowed to observe a costimulatory effect of anti-FAP-4-1BBL both to release IFNγ and to attain more efficacious tumor cell killing. Conclusion: Three-dimensional tumor cocultures with T cells using live confocal microscopy provide suitable models to test the requirements for colon-cancer redirected killing as elicited by CEA-targeted T-cell engagers undergoing clinical trials and treatment allow combinations to be tested in a relevant preclinical system.
Insights
This study shows that CEA-targeted T cell bispecific antibodies effectively kill colorectal cancer organoids, with efficacy dependent on CEA expression levels. Combining these with FAP-targeted 4-1BBL enhances tumor cell killing and cytokine release.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Carcinoembryonic antigen (CEA) is a target for colorectal cancer (CRC) therapies.
- CEA-CD3 T cell bispecific antibodies (CEA-TCBs) are in clinical trials for CRC.
- Three-dimensional (3D) tumor organoids offer a preclinical model for evaluating cancer therapies.
Purpose of the Study:
- To evaluate the efficacy of cibisatamab (CEA-CD3 TCB) against CEA-expressing colorectal cancer organoids.
- To compare CEA-TCB performance with a higher affinity CEACAM5-CD3 bispecific antibody.
- To assess the co-stimulatory effect of a fibroblast-activated protein (FAP)-targeted 4-1BBL bispecific antibody fusion protein.
Main Methods:
- Utilized time-lapse confocal microscopy to monitor T cell-mediated killing of 3D colon cancer organoids and primary CRC organoids.
- Assessed tumor cell death by real-time detection of Caspase 3 activation.
- Co-cultured organoids with T cells and autologous tumor-associated fibroblasts to test bispecific antibody efficacy and co-stimulatory effects.
Main Results:
- Tumor cell killing by CEA-TCB was dependent on surface CEA expression levels; lower affinity CEA-TCB required a CEA expression threshold.
- Higher affinity CEACAM5-TCB demonstrated activity even on low CEA-expressing organoids.
- Co-culture with anti-FAP-4-1BBL enhanced T cell-mediated killing and IFNγ release, particularly in the presence of fibroblasts.
Conclusions:
- 3D tumor organoid co-cultures with T cells provide a relevant preclinical model for evaluating CEA-targeted T cell engagers in colorectal cancer.
- Therapeutic efficacy of CEA-TCBs is influenced by CEA expression levels and antibody affinity.
- Combination therapies, such as CEA-TCB with FAP-targeted 4-1BBL, show promise for enhanced anti-tumor activity.
