Preclinical assessment of CAR-NK cell-mediated killing efficacy and pharmacokinetics in a rapid zebrafish xenograft model of metastatic breast cancer

Front Immunol. 2023 Oct 11:14:1254821. doi: 10.3389/fimmu.2023.1254821. eCollection 2023.

Abstract

Natural killer (NK) cells are attractive effectors for adoptive immunotherapy of cancer. Results from first-in-human studies using chimeric antigen receptor (CAR)-engineered primary NK cells and NK-92 cells are encouraging in terms of efficacy and safety. In order to further improve treatment strategies and to test the efficacy of CAR-NK cells in a personalized manner, preclinical screening assays using patient-derived tumor samples are needed. Zebrafish (Danio rerio) embryos and larvae represent an attractive xenograft model to study growth and dissemination of patient-derived tumor cells because of their superb live cell imaging properties. Injection into the organism's circulation allows investigation of metastasis, cancer cell-to-immune cell-interactions and studies of the tumor cell response to anti-cancer drugs. Here, we established a zebrafish larval xenograft model to test the efficacy of CAR-NK cells against metastatic breast cancer in vivo by injecting metastatic breast cancer cells followed by CAR-NK cell injection into the Duct of Cuvier (DoC). We validated the functionality of the system with two different CAR-NK cell lines specific for PD-L1 and ErbB2 (PD-L1.CAR NK-92 and ErbB2.CAR NK-92 cells) against the PD-L1-expressing MDA-MB-231 and ErbB2-expressing MDA-MB-453 breast cancer cell lines. Injected cancer cells were viable and populated peripheral regions of the larvae, including the caudal hematopoietic tissue (CHT), simulating homing of cancer cells to blood forming sites. CAR-NK cells injected 2.5 hours later migrated to the CHT and rapidly eliminated individual cancer cells throughout the organism. Unmodified NK-92 also demonstrated minor in vivo cytotoxicity. Confocal live-cell imaging demonstrated intravascular migration and real-time interaction of CAR-NK cells with MDA-MB-231 cells, explaining the rapid and effective in vivo cytotoxicity. Thus, our data suggest that zebrafish larvae can be used for rapid and cost-effective in vivo assessment of CAR-NK cell potency and to predict patient response to therapy.

Keywords: CAR-NK cells; ErbB2 (HER2); NK-92; PD-L1; breast cancer; cancer immunotherapy; in vivo imaging; zebrafish xenograft.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • B7-H1 Antigen / metabolism
  • Breast Neoplasms*
  • Cell Line, Tumor
  • Female
  • Heterografts
  • Humans
  • Killer Cells, Natural
  • Receptors, Chimeric Antigen*
  • Zebrafish

Substances

  • Receptors, Chimeric Antigen
  • B7-H1 Antigen

Grants and funding

This research was supported in part by the German Federal Ministry of Education (Clusters4Future SaxoCell, 03ZU1111DA) and German Red Cross Blood Donation Service internal grants to TT, the German Research Foundation (DFG) through EI1223/2-1 to JE, the DFG TRR67 (project 387653785), SPP2084 µBone (project KN 1102/2–1) and Transcampus (project tC2020_02_MED) to FK. The work at the TU Dresden is co-financed with tax revenues based on the budget agreed by the Saxonian Landtag. This work was supported by the Light Microscopy Facility of the TU Dresden CMCB Technology Platform.