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3D model for CAR-mediated cytotoxicity using patient-derived colorectal cancer organoids.

ABSTRACT: Immunotherapy using chimeric antigen receptor (CAR)-engineered lymphocytes has shown impressive results in leukemia. However, for solid tumors such as colorectal cancer (CRC), new preclinical models are needed that allow to test CAR-mediated cytotoxicity in a tissue-like environment. Here, we developed a platform to study CAR cell cytotoxicity against 3-dimensional (3D) patient-derived colon organoids. Luciferase-based measurement served as a quantitative read-out for target cell viability. Additionally, we set up a confocal live imaging protocol to monitor effector cell recruitment and cytolytic activity at a single organoid level. As proof of principle, we demonstrated efficient targeting in diverse organoid models using CAR-engineered NK-92 cells directed toward a ubiquitous epithelial antigen (EPCAM). Tumor antigen-specific cytotoxicity was studied with CAR-NK-92 cells targeting organoids expressing EGFRvIII, a neoantigen found in several cancers. Finally, we tested a novel CAR strategy targeting FRIZZLED receptors that show increased expression in a subgroup of CRC tumors. Here, comparative killing assays with normal organoids failed to show tumor-specific activity. Taken together, we report a sensitive in vitro platform to evaluate CAR efficacy and tumor specificity in a personalized manner.

SUBMITTER: Schnalzger TE

PROVIDER: S-EPMC6576164 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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3D model for CAR-mediated cytotoxicity using patient-derived colorectal cancer organoids.

Schnalzger Theresa E TE de Groot Marnix Hp MH Zhang Congcong C Mosa Mohammed H MH Michels Birgitta E BE Röder Jasmin J Darvishi Tahmineh T Wels Winfried S WS Farin Henner F HF

The EMBO journal 20190429 12

Immunotherapy using chimeric antigen receptor (CAR)-engineered lymphocytes has shown impressive results in leukemia. However, for solid tumors such as colorectal cancer (CRC), new preclinical models are needed that allow to test CAR-mediated cytotoxicity in a tissue-like environment. Here, we developed a platform to study CAR cell cytotoxicity against 3-dimensional (3D) patient-derived colon organoids. Luciferase-based measurement served as a quantitative read-out for target cell viability. Addi ...[more]

PMID: 31036555

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Project description:Cervical cancer is a leading cause of death among women globally, primarily driven by high-risk papillomaviruses. However, the effectiveness of chemotherapy is limited, underscoring the potential of personalized immunotherapies. Patient-derived organoids, which possess cellular heterogeneity, proper epithelial architecture and functionality, and long-term propagation capabilities offer a promising platform for developing viable strategies. In addition to αβ T cells and natural killer (NK) cells, γδ T cells represent an immune cell population with significant therapeutic potential against both hematologic and solid tumours. To evaluate the efficacy of γδ T cells in cervical cancer treatment, we generated patient-derived healthy and cancer ectocervical organoids. Furthermore, we examined transformed healthy organoids, expressing HPV16 oncogenes E6 and E7. We analysed the effector function of in vitro expanded γδ T cells upon co-culture with organoids. Our findings demonstrated that healthy cervical organoids were less susceptible to γδ T cell-mediated cytotoxicity compared to HPV-transformed organoids and cancerous organoids. To identify the underlying pathways involved in this observed cytotoxicity, we performed bulk-RNA sequencing on the organoid lines, revealing differences in DNA-damage and cell cycle checkpoint pathways, as well as transcription of potential γδ T cell ligands. We validated these results using immunoblotting and flow cytometry. We also demonstrated the involvement of BTN3A1 and BTN2A1, crucial molecules for γδ T cell activation, as well as differential expression of PDL1/CD274 in cancer, E6/E7+ and healthy organoids. Interestingly, we observed a significant reduction in cytotoxicity upon blocking MSH2, a protein involved in DNA mismatch-repair. In summary, we established a co-culture system of γδ T cells with cervical cancer organoids, providing a novel in vitro model to optimize innovative patient-specific immunotherapies for cervical cancer.

Dataset Information

3D model for CAR-mediated cytotoxicity using patient-derived colorectal cancer organoids.

Publications

3D model for CAR-mediated cytotoxicity using patient-derived colorectal cancer organoids.

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