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Tumor organoid–T-cell coculture systems


T cells are key players in cancer immunotherapy, but strategies to expand tumor-reactive cells and study their interactions with tumor cells at the level of an individual patient are limited. Here we describe the generation and functional assessment of tumor-reactive T cells based on cocultures of tumor organoids and autologous peripheral blood lymphocytes. The procedure consists of an initial coculture of 2 weeks, in which tumor-reactive T cells are first expanded in the presence of (IFNγ-stimulated) autologous tumor cells. Subsequently, T cells are evaluated for their capacity to carry out effector functions (IFNγ secretion and degranulation) after recognition of tumor cells, and their capacity to kill tumor organoids. This strategy is unique in its use of peripheral blood as a source of tumor-reactive T cells in an antigen-agnostic manner. In 2 weeks, tumor-reactive CD8+ T-cell populations can be obtained from ~33–50% of samples from patients with non-small-cell lung cancer (NSCLC) and microsatellite-instable colorectal cancer (CRC). This enables the establishment of ex vivo test systems for T-cell-based immunotherapy at the level of the individual patient.

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Fig. 1: Overview of the procedure.
Fig. 2: Examples of flow cytometry plots showing tumor reactivity of CD8+ T cells.
Fig. 3: Killing experiment overview.
Fig. 4: Quantification of the tumor organoid killing assay.
Fig. 5: Example of processing of live-cell imaging data.

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Data availability

The data that provide examples of the results that can be generated with this protocol are available from the corresponding author on reasonable request.


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We thank M. van Baalen, A. Pfauth and F. van Diepen for assistance with flow cytometry experiments; M. Mertz, L. Brocks and B. van den Broek for help with live-cell imaging experiments; M. van de Wetering, N. Sachs, H. Clevers and Hubrecht Organoid Technology (HUB) for advice on organoid cultures; and Merus for the provision of the anti-PD-1 antibody. This work was supported by the NWO gravitation program (NWO 2012-2022) (to E.E.V. on behalf of, KWF grant HUBR2014-7006 (to E.E.V.), the KWF Queen Wilhelmina Award (NKI 2013-6122, to T.N.S.) and ERC AdG SENSIT (to T.N.S.).

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Authors and Affiliations



C.M.C. and K.K.D. designed the protocol, performed the experiments and wrote the manuscript. L.F.F., S. Kelderman, S. Kaing and N.v.R. provided critical input in the design of the protocol. S.v.d.B. provided the protocol for Noggin-conditioned medium. T.N.S. and E.E.V. supervised the study.

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Correspondence to Emile E. Voest.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Marco Gerlinger, Michael James and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Dijkstra, K. K. et al. Cell 174, 1586–1598 (2018):

Integrated supplementary information

Supplementary Fig. 1 Quantification of the tumor organoid killing assay using fluorescent dyes.

Example of flow cytometry plots showing labeling of tumor cells with CellTrace Yellow and T cells with CellTrace Far Red.

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Cattaneo, C.M., Dijkstra, K.K., Fanchi, L.F. et al. Tumor organoid–T-cell coculture systems. Nat Protoc 15, 15–39 (2020).

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