Potential Therapeutic Effect of Natural Killer Cells on Doxorubicin-Resistant Breast Cancer Cells In Vitro

Abstract

Objective: The aim of this study was to explore the therapeutic effect of natural killer (NK) cells on human doxorubicin-sensitive and resistant breast adenocarcinoma.

Methods: Human doxorubicin-sensitive and resistant breast cancer cell lines (MCF-7 and MCF-7/ADR) were tagged with renilla luciferase (Rluc) (MCF-7/RC and MCF-7/ADR/RC). NK cells were tagged with enhanced firefly luciferase (effluc) using a recombinant retrovirus transfection (NKF). Expression of Rluc, effluc, and NK cell surface markers CD16, CD56 as well as death receptors, DR4 and DR5, were assessed by using flow cytometry. In vitro cytotoxic effect of NK to MCF-7 and MCF-7/ADR was measured and in vivo bioluminescence imaging was also performed to visualize MCF-7/RC, MCF-7/ADR, and NKF in an animal model.

Results: NK92-MI, MCF-7, and MCF-7/ADR cells were successfully labeled with Rluc or effluc. Both the target breast cancer cells (with Rluc) and therapeutic NK cells (with effluc) were noninvasively visualized in nude mice. Doxorubicin-resistant breast cancer cells (MCF-7/ADR) presented a higher expression of DR5 and were more sensitive to NK cells compared with doxorubicin-sensitive breast cancer cells (MCF-7).

Conclusion: The results of present study suggest that NK cell therapy has a therapeutic effect on doxorubicin-sensitive and resistant breast cancer cells.

Fig 1. Establishment of stable cell lines expressing reporter genes.

(A) Mcherry gene expression in DOX-sensitive and resistant breast cancer cells (MCF-7/RC, MCF-7/ADR/RC) was determined by using FACS.(B) effluc gene expression in NKF cell lines was determined by using FACS. PE = phycoerythrin. (C) Immunofluorescent staining of NKF cells using anti-Thy1.1-PE to assess effluc protein expression.(D) Mcherry expression under microscopy in MCF-7/RC and MCF-7/ADR/RC cells (10x).

Fig 2. Phenotype analysis of NK92-MI and NKF cells by flow cytometry.

NK92-MI and NKF cells did not express CD16. NK92-MI and NKF cells expressed CD56. Experiments were performed at least in triplicate.

Fig 3. In vivo bioluminescence imaging.

(A) Bioluminescence imaging (BLI) was performed after inoculation of different numbers of MCF-7/RC and MCF-7/ADR/RC cells into the left fore flank (Orange arrow, 1 × 10⁵ cells), left hind flank (Purple arrow, 3 × 10⁵ cells), and right hind flank (Green arrow, 9 × 10⁵ cells) of different nude mice (n = 3), respectively. (B) Average densities of BLI signals. There is a positive correlation between density and cell number of MCF-7/RC and MCF-7/ADR/RC. (C) BLI was acquired after inoculation of different number of NKF cells into the left fore flank (Orange arrow, 1 × 10⁵ cells), left hind flank (Purple arrow, 5 × 10⁵ cells), and right hind flank (Green arrow, 2.5 × 10⁶ cells) of nude mice (n = 3). (D) Average density of bioluminescence signals. There is a positive correlation between density and cell number of NKF. Experiments were performed at least in triplicate and mean values ± SD are plotted.

Fig 4. Cytolytic activity of NKF cells on MCF-7/RC and MCF-7/ADR/RC cells at different ratios.

Luciferase activity of MCF-7/RC and MCF-7/ADR/RC cells decreased in an effector-number dependent manner. Both breast cancer cell lines were significantly sensitive to NKF cells. Experiments were performed at least in triplicate and mean values ± SD are plotted.

Fig 5. Death receptor expression at the surface of MCF-7 and MCF-7/ADR cells.

Death receptor expression was determined by flow cytometry using anti-DR4-PE and anti-DR5-PE. Experiments were performed at least in triplicate and mean values ± SD are plotted.