Natural Killer Cell (NK-92MI)-Based Therapy for Pulmonary Metastasis of Anaplastic Thyroid Cancer in a Nude Mouse Model

Abstract

Objective: Natural killer (NK) cells represent the third largest population of lymphocytes, and they play an important role in immune surveillance against tumors. The lungs are a common metastatic site for anaplastic thyroid cancer (ATC), and metastasis is one of the most frequent causes of mortality in this type of cancer. In the current study, we evaluated the effects of NK cell-based immunotherapy for pulmonary metastasis of ATC and determined how it affects the effector molecules of NK cells.

Methods: Human NK cells (NK-92MI) were retrovirally transduced to express the effluc gene. Human ATC cells (CAL-62) were transduced with the effluc and Rluc genes. The cytotoxicity of NK cells against CAL-62 cells was assessed using the CytoTox 96^® Non-Radioactive Cytotoxicity Assay system. Pulmonary metastases of ATC were developed by i.v. injection of CAL-62, and metastasis growth was monitored using bioluminescence imaging (BLI). To treat the metastases, five million NK-92MI cells were injected twice into the caudal vein of nude mice. To assess the targetability of NK cells to ATC tumors, NK-92MI cells expressing the effluc gene (NK/F) were administered through the tail vein of nude mice with a pulmonary metastasis or tumor xenograft. BLI was subsequently performed at 1, 3, 24, and 48 h.

Results: NK/F and CAL-62 cells expressing the effluc or Rluc gene (CAL-62/F, CAL-62/R) were successfully established. Expression of the effluc and Rluc genes in NK/F, CAL-62/F, and CAL-62/R cells was verified by RT-polymerase chain reaction, western blotting, and luciferase assay. After coculture of NK-92MI and CAL-62/F cells for 24 h, the BLI signal intensity of CAL-62/F cells proportionally decreased with the number of cocultured NK cells. An ATC pulmonary metastasis mouse model was successfully generated, and NK cells significantly inhibited the growth of the metastasis (p < 0.01). The NK/F cells exhibited targetability to the pulmonary metastasis and tumor xenograft in the mouse model.

Conclusion: The results of present study suggest that NK cells are able to target ATC tumors and that NK cell-based immunotherapy may serve as an effective therapeutic approach for pulmonary metastases of ATC.

Keywords: anaplastic thyroid cancer; immunotherapy; mouse model; natural killer cells; pulmonary metastasis.

Figure 1

Establishment of NK cells expressing a reporter gene. (A) RT-polymerase chain reaction and (B) western blotting analyses showing effluc expression at the mRNA and protein levels, respectively, in NK/F cells. (C,D) Firefly luciferase activity was determined by bioluminescence imaging, and the intensity of the effluc was correlated with the cell number, R² = 0.91. effluc, enhanced firefly luciferase; Rluc, Renilla luciferase; NK/F, NK-92MI/effluc cells.

Figure 2

NK cell cytotoxicity to CAL-62/F (CAL-62) cells. Bioluminescence signals were monitored after NK cells were co-incubated with CAL-62/F at E:T ratios of 0.1:1, 0.2:1, 0.5:1, and 1:1 for 24 h. (A,B) The intensity of bioluminescence imaging signals in tumor cells was decreased a dose-dependent manner. (C) Cytotoxic activity of NK cells by using the CytoTox 96^® Non-Radioactive Cytotoxicity Assay system. (D) The antitumor effect of die NK cells to the CAL-62 was measured by the CCK-8 assay. Experiments were performed at least in triplicate, and values were plotted as mean ± SE. **p < 0.01, ***p < 0.001. effluc; enhanced firefly luciferase; NK, NK-92MI cells; CAL-62/F, CAL-62/effluc cells.

Figure 3

Generation of an anaplastic thyroid cancer pulmonary metastasis mouse model by intravenous injection of CAL-62/F cells. (A) Bioluminescence imaging (BLI) of mice after injection of CAL-62/F cells. (B) Average BLI signal intensities of the mice. (C) Bone metastases developed in some mice (almost 15%) 6 weeks after the injection. effluc, enhanced firefly luciferase; CAL-62/F, CAL-62/effluc cells.

Figure 4

The experimental schema of the in vivo NK cell-based immunotherapy for anaplastic thyroid cancer pulmonary metastases.

Figure 5

NK cell-based immunotherapy for anaplastic thyroid cancer (ATC) pulmonary metastasis. CAL-62/F cells were injected intravenously via the tail vein of nude mice, and after 7 days, NK cells were intravenously injected. After the in vivo experiment, the mice were sacrificed, and ex vivo experiments were performed. (A) In vivo bioluminescence imaging (BLI) of an ATC tumor. (B) Quantification of the BLI signals. (C) Hematoxylin and eosin staining of the ATC pulmonary metastases. (D) Ex vivo BLI of the ATC pulmonary metastases. Experiments were performed at least in triplicate, and the values represent the mean ± SE. *p < 0.05, **p < 0.01. effluc, enhanced firefly luciferase; CAL-62/F, CAL-62/effluc cells; NK, NK-92MI cells.

Figure 6

Targeting of NK cells to anaplastic thyroid cancer in animal models with pulmonary metastases or xenografts. CAL-62/R cells (1 × 10⁶ cells/100 μl PBS) were injected into mice via the tail vein or subcutaneously in the right thigh. After 21 or 42 days, NK/F cells were intravenously injected. Bioluminescence imaging was measured 1, 3, 24, and 48 h after injection of the NK/F cells. effluc, enhanced firefly luciferase; Rluc, Renilla luciferase; CAL-62/R, CAL-62/Rluc; NK/F, NK-92MI/effluc cells.

Figure 7

Apoptosis mechanism of anaplastic thyroid cancer induced by NK cell-based immunotherapy. (A) Western blotting of proteins related to the apoptosis signaling pathway. (B) Diagram of the apoptosis signaling pathway that was induced by NK cells.