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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The data that provide examples of the results that can be generated with this protocol are available from the corresponding author on reasonable request.
Blank, C. U., Haanen, J. B., Ribas, A. & Schumacher, T. N. The “cancer immunogram”. Science 352, 658–660 (2016).
Dijkstra, K. K., Voabil, P., Schumacher, T. N. & Voest, E. E. Genomics- and transcriptomics-based patient selection for cancer treatment with immune checkpoint inhibitors: a review. JAMA Oncol. 2, 1490–1495 (2016).
Pitt, J. M. et al. Resistance mechanisms to immune-checkpoint blockade in cancer: tumor-intrinsic and -extrinsic factors. Immunity 44, 1255–1269 (2016).
Sharma, P., Hu-Lieskovan, S., Wargo, J. A. & Ribas, A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell 168, 707–723 (2017).
Turcotte, S. et al. Phenotype and function of T cells infiltrating visceral metastases from gastrointestinal cancers and melanoma: implications for adoptive cell transfer therapy. J. Immunol. 191, 2217–2225 (2013).
Dijkstra, K. K. et al. Generation of tumor-reactive T cells by co-culture of peripheral blood lymphocytes and tumor organoids. Cell 174, 1586–1598 (2018).
Zaretsky, J. M. et al. Mutations associated with acquired resistance to PD-1 blockade in melanoma. N. Engl. J. Med. 375, 819–829 (2016).
Rosenberg, S. A. & Restifo, N. P. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348, 62–68 (2015).
Geukes Foppen, M. H., Donia, M., Svane, I. M. & Haanen, J. B. Tumor-infiltrating lymphocytes for the treatment of metastatic cancer. Mol. Oncol. 9, 1918–1935 (2015).
Scheper, W. et al. Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers. Nat. Med. 25, 89–94 (2018).
Simoni, Y. et al. Bystander CD8+ T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature 557, 575–579 (2018).
Gros, A. et al. PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors. J. Clin. Invest. 124, 2246–2259 (2014).
Strønen, E. et al. Targeting of cancer neoantigens with donor-derived T cell receptor repertoires. Science 352, 1337–1341 (2016).
Yossef, R. et al. Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy. JCI Insight 3, e122467 (2018).
Ye, Q. et al. CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor. Clin. Cancer Res. 20, 44–55 (2014).
Gros, A. et al. Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients. Nat. Med. 22, 433–438 (2016).
Cohen, C. J. et al. Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes. J. Clin. Invest. 125, 3981–3991 (2015).
Schumacher, T. N., Scheper, W. & Kvistborg, P. Cancer neoantigens. Annu. Rev. Immunol. 37, 173–200 (2019).
Verdegaal, E. M. et al. Successful treatment of metastatic melanoma by adoptive transfer of blood-derived polyclonal tumor-specific CD4+ and CD8+ T cells in combination with low-dose interferon-alpha. Cancer Immunol. Immunother. 60, 953–963 (2011).
Nozaki, K. et al. Co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes. J. Gastroenterol. 52, 206–213 (2016).
Tsai, S. et al. Development of primary human pancreatic cancer organoids, matched stromal and immune cells and 3D tumor microenvironment models. BMC Cancer 18, 335 (2018).
Jenkins, R. W. et al. Ex vivo profiling of PD-1 blockade using organotypic tumor spheroids. Cancer Discov. 8, 196–215 (2018).
Neal, J. T. et al. Organoid modeling of the tumor immune microenvironment. Cell 175, 1972–1988 (2018).
Weeber, F. et al. Preserved genetic diversity in organoids cultured from biopsies of human colorectal cancer metastases. Proc. Natl Acad. Sci. USA 112, 13308–13311 (2015).
van de Wetering, M. et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161, 933–945 (2015).
Sachs, N. et al. Long-term expanding human airway organoids for disease modeling. EMBO J. 38, e100300 (2019).
Schütte, M. et al. Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors. Nat. Commun. 8, 14262 (2017).
Drost, J. et al. Organoid culture systems for prostate epithelial and cancer tissue. Nat. Protoc. 11, 347–358 (2016).
Broutier, L. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat. Protoc. 11, 1724–1743 (2016).
Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat. Protoc. 10, 1474–1485 (2015).
Dudley, M. E., Wunderlich, J. R., Shelton, T. E., Even, J. & Rosenberg, S. A. Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients. J. Immunother. 26, 332–342 (2003).
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 CancerGenomics.nl), 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.).
The authors declare no competing interests.
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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Key reference using this protocol
Dijkstra, K. K. et al. Cell 174, 1586–1598 (2018): https://doi.org/10.1016/j.cell.2018.07.009
Integrated supplementary information
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). https://doi.org/10.1038/s41596-019-0232-9
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