Enhancement of Natural Killer Cell Cytotoxicity by Sodium/Iodide Symporter Gene-Mediated Radioiodine Pretreatment in Breast Cancer Cells

A phase II study of NK cell therapy in treatment of patients with recurrent breast cancer has recently been reported. However, because of the complexities of tumor microenvironments, effective therapeutic effects have not been achieved in NK cell therapy. Radioiodine (I-131) therapy inhibits cancer growth by inducing the apoptosis and necrosis of cancer cells. Furthermore, it can modify cancer cell phenotypes and enhance the effect of immunotherapy against cancer cells. The present study showed that I-131 therapy can modulate microenvironment of breast cancer and improve the therapeutic effect by enhancing NK cell cytotoxicity to the tumor cells. The susceptibility of breast cancer cells to NK cell was increased by precedent I-131 treatment in vitro. Tumor burden in mice treated with I-131 plus NK cell was significantly lower than that in mice treated with NK cell or I-131 alone. The up-regulation of Fas, DR5 and MIC A/B on irradiated tumor cells could be the explanation for the enhancement of NK cell cytotoxicity to tumor cells. It can be applied to breast cancer patients with iodine avid metastatic lesions that are non-responsive to conventional treatments.


Introduction
Breast cancer is the most common cancer and the second most common cause of cancer-related death in women, with more than one million cases and nearly 600,000 deaths occurring annually worldwide [1]. Breast cancer is characterized by a distinct pattern of metastasis involving regional lymph nodes, bone, lung, and liver, and the distant metastasis is closely associated with poor prognosis [2]. Once breast cancer has metastasized, it is usually not cured by current therapies, including high dose chemotherapy, likely due to subpopulations of slow-dividing chemoresistant cells present in metastatic cells [3]. In addition, the triple negative breast cancers lack a therapeutic target and have a poor prognosis [4]. Therefore, establishment of new therapeutic strategies is crucial to improving the prognosis of advanced breast cancer.
Tumor-specific immunotherapy offers considerable potential in management of patients with breast cancer; one of the effective immunotherapies is use of natural killer (NK) cells [5]. NK cells, a subset of lymphocytes capable of mediating cytotoxicity against tumor cells and virally infected cells, constitute a key component of the innate immune system [6]. NK cells have been shown to play an important role in controlling the growth of various tumor cell lines in mice [6,7]. A phase II study of NK cell therapy in treatment of patients with recurrent breast cancer has recently been reported [8]. However, because of the complexities of tumor microenvironments, effective therapeutic effects have not been achieved in NK cell therapy [9,10].
Radioiodine (I-131) therapy inhibits cancer growth through induction of apoptosis and necrosis of cancer cells [11]. In addition, it was shown that I-131 therapy can modify cancer cell phenotypes and enhance the effect of immunotherapy against cancer cells [12][13][14]. In particular, irradiated cancer cells show upregulated levels of Fas and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptor [15][16][17][18]. In breast cancer, I-131 therapy can be used, as the majority of breast cancer (70-80%) expresses human sodium/iodide symporter (hNIS), which is a specialized active iodide transporter [11,18,19]. hNIS expression and I-131 uptake by breast cancer cells has been suggested to provide supportive evidence for use of I-131 as an additional modality for treatment of breast cancer [20]. Therefore, it is expected that pretreatment with I-131 will result in modification of cancer cell phenotypes and enhance the susceptibility of breast cancer cells to NK cell cytotoxicity.
In this study, we attempted to determine whether Fas and TRAIL receptors of breast cancer cells are up-regulated by I-131 therapy and whether I-131 therapy can enhance NK cell cytotoxicity in vitro and in vivo.

Animals
Specific pathogen-free six-week-old female BALB/c nude mice (Hamamatsu, Shizuoka, Japan) were used in in vivo study. All animal experiment protocols were conducted in accordance with National Institutes of Health guidelines for the care and use of laboratory animals and approved by the Committee for the Handling and Use of Animals of Kyungpook National University.

I-125 Uptake Assay
The day before iodine uptake, 1610 5 , 2610 5 , 4610 5 , and 8610 5 MDA-231 and MDA-231/NF cells were plated in 24-well plates. The level of I-125 uptake was determined by incubation of the cells with 500 ml of Hank's balanced salt solution (HBSS) containing 0.5% bovine serum albumin (bHBSS), 3.7 kBq carrierfree I-125, and 10 mmol/L sodium iodide (specific activity of 740 MBq/mmol) at 37uC for 30 min. The blocking control study for hNIS was performed in an identical manner, with the exception of the addition of 1mM KClO 4 to the incubation buffer. After incubation, the cells were washed twice as quickly as possible with ice-cold bHBSS buffer and detached using 500 mL of trypsin. Radioactivity was measured using a gamma-counter (CobraII, Packard, Perkin Elmber, Waltham, MA, USA).
To evaluate the functional expression of the hNIS gene in vivo, MDA-231/NF cells (5610 5 ) in phosphate buffered saline (PBS) were implanted subcutaneously into the right flank of three mice. Fourteen days after tumor implantation, Tc-99m pertechnetate SPECT/CT scan was performed using the Inveon small animal imaging system (Siemens Medical Solutions, Knoxville, TN, USA). The mice were placed in a spread-prone position at 40 min after injection of Tc-99m pertechnetate (7.4 MBq/0.2 mL of 0.9% NaCl) into the tail vein, and scanned for 20 min. A 20% window was centered at the 140 keV photopeak of Tc-99m. The 3-D ordered subset expectation-maximization (OSEM) algorithm was used in reconstruction. The voxel size of the image matrix was 0.560.560.5 mm. All reconstructed images were normalized using a correction matrix derived from a uniform cylindrical phantom image prior to reconstruction.
To evaluate the functional expression of the effluc gene in vivo, MDA-231/NF cells in PBS were implanted subcutaneously into the right hind-flank (1610 5 ), left hind-flank (3610 5 ), and right fore-flank (9610 5 ) of three mice. After tumor implantation, bioluminescence imaging was performed using the IVIS lumina II imaging system (Caliper, Alameda, CA, USA), which included a highly sensitive CCD camera mounted on a light-tight specimen chamber.

Phenotype Marker Analysis
To determine the levels of

Cytotoxicity Assay
Cytotoxic activity of NK92-MI cells was assessed using the Calcein-AM release test. Calcein-AM was purchased from Invitrogen as a 1 mg/mL solution in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA). MDA-231 and MDA-231/NF cells were grown in 75 cm 2 flasks and incubated for 7 hr at 37uC in HBSS only or in HBSS containing 14.8 MBq of I-131. After 7 hr, I-131 containing medium was removed and cells were washed twice with HBSS. Cells were grown for three days. Then, irradiated or non-irradiated MDA-231/NF cells were re-suspended in the complete medium at a final concentration of 10 6 cells/ mL and were incubated with 5 mM Calcein-AM for 20 min at 37uC, allowing Calcein-AM to enter the target cells. The labeled target cells were then washed twice and dispensed at a concentration of 1610 4 target cells/well in a round-bottom, 96-well plate (Nunc, Roskilde, Denmark). The effectors were distributed in triplicate at effector : target (E : T) cell ratios from 2.5 : 1, 5 : 1, and 10 : 1 with at least three replicate wells for spontaneous (only target cells in complete medium) and maximum release (only target cells in medium plus 1% NP-40 cell lysis buffer). After incubation at 37uC in 5% CO 2 for 4 hr, each supernatant was harvested and transferred into new plates. Samples were measured using a multilabelcounter (VICTOR3, Perkin Elmer, San Diego, CA, USA) (excitation filter: 48569 nm; band-pass filter: 53069 nm). Data were expressed as arbitrary fluorescent units. NK cell cytotoxicity was calculated using the following equation: NK cell cytotoxicity (%) test release{spontaneous release maximum release{spontaneous release |100%

In vivo Animal Experiments
Twelve mice were divided into four groups for assessment of therapeutic effects (three mice per group); the experimental groups were referred to as the control, I-131, NK, and combined groups. In 12 mice, MDA-231/NF cells (5610 5 ) were implanted subcutaneously into the right flank.
In the control group, intravenous injection of PBS was administered at 14 days post-challenge. In the I-131 group, intraperitoneal injection of 29.6 MBq of I-131 was administered at 14 days post-challenge. In the NK group, NK92-MI cells (5610 6 ) were injected intravenously via tail vein at 17 and 18 days. A total of two doses were administered to each mouse with two days apart. The combined group received treatment with both I-131 at 14 days and NK92-MI cells at 17 and 18 days.
Bioluminescence imaging was performed using the IVIS lumina II imaging system (Caliper). From 14, 24, and 34 days postchallenge, mice received intraperitoneal injection with 100 mL of D-luciferin (30 mg/mL). After 5 min, mice were placed individually in the specimen chamber and images were then acquired. Grayscale photographic images and bioluminescent color images were superimposed using LIVING IMAGE, version 2.12 (Caliper, Alameda, CA, USA), and IGOR image analysis FX software (WaveMetrics, Lake Oswego, OR, USA). Bioluminescent signals were expressed in units of photons per cm 2 per second per steradian (P/cm 2 /sec/sr).

Statistical Analysis
All data are expressed as means 6 SDs and are representative of at least two separate experiments. The unpaired Student's t test and ANOVA analysis were used for determination of statistical significance. P values of ,0.05 were considered statistically significant.

Enhancement of NK Cell Cytotoxicity by I-131 Therapy in vivo
The mice were divided into four groups (control, I-131, NK, and combined groups) and PBS, I-131, NK92-MI or I-131 and NK92-MI were applied to each group. Tumor burden was monitored by bioluminescence imaging 14, 24, and 34 days after tumor inoculation.
Mice in the control group who received PBS showed a continuous increase in their tumor signal. The I-131 and NK groups showed moderate increases in tumor signals and the tumor signals of the I-131 and NK groups were consistently lower than that of the control group and significantly lower than that of the control group at 34 days, respectively (p = 0.017 and p = 0.001). The combined group showed a stationary tumor signal over time, which was consistently lower than that of the I-131 and NK groups, and significantly lower than that of the I-131 and NK groups at 34 days, respectively (p = 0.030 and p = 0.038) ( Figure 6). Tumor burdens measured by bioluminescence imaging did not differ significantly between the NK and I-131 groups (p = 0.155).

Discussion
Contemporary management of breast cancer with early detection, newer local control techniques, improved chemotherapy regimens, and targeted treatments has resulted in immense gains in survival in individuals with breast cancer [21]. Unfortunately, the triple negative breast cancers, a subset of breast cancers clinically defined by the absence of the estrogen receptor, progesterone receptor, and HER-2 over expression, have a higher propensity to metastasize to distant visceral organs, and have a worse outcome with a high rate of recurrence after adjuvant treatment [4]. Thus, the need for development of successful therapeutic options in an attempt to improve the outcome is urgent. An attractive approach to reducing the rate of recurrences in these individuals is use of immunotherapeutic strategies [6,22]. However, because of the complexities of tumor microenvironments, effective therapeutic effects may not be feasibly achieved in immunotherapy. Several researchers have reported obstacles to successful immunotherapy, such as tumor-derived suppression cytokines, the absence of danger signals, loss of MHC class molecules, and low antigen levels [22,23]. To overcome these impediments, modulation of the tumor microenvironment is essential. The current study showed that I-131 therapy can modulate microenvironment of breast cancer and improve the therapeutic effect through enhancement of NK cell cytotoxicity to tumor cells.
NK cells comprise 10-15% of all circulating lymphocytes and are also found in peripheral tissues, including the liver, peritoneal cavity, and placenta. Resting NK cells circulate in the blood; however, following activation by cytokines, they are capable of extravasation and infiltration into most tissues that contain pathogen infected or malignant cells [24]. NK92 is a human NK cell line first established in 1994 from a 50-year-old male patient with an aggressive NK cell lymphoma [25]. The NK92 cell line has been examined clinically as a treatment for advanced sarcoma and leukemia [25,26]. The parental NK92 cell line is highly dependent on the cytokine IL-2 and therapies involving these cells in vivo require superphysiological amounts of IL-2. However, an IL-2 independent cell line, NK92-MI, which has Enhancement of NK Cell by Radioiodine Therapy PLOS ONE | www.plosone.org been shown to be virtually identical to the parental cell line, may be a more appropriate choice for clinical therapies [26,27]. Nagashima et al. [28] reported that NK92-MI sustained proliferation in the absence of exogenously supplied IL-2 and showed greater in vivo anti-tumor activity in mice. The current study also showed that NK92-MI have anti-tumor activity to a breast cancer cell line in vitro and in vivo without IL-2 supplementation.
The cytotoxicity of NK cells are carried out by two main mechanisms. The first mechanism is granule-dependent cytotoxicity, where upon triggering by activating receptors [6]. Upon recognition of the ligands on the surface of the target cell surface by activating NK cell receptors, various intracellular signaling pathways drive NK cells toward cytotoxic action, which results in cytolysis of target cells [29]. When NK cells are activated by MIC A/B, which are ligands for the activating receptor NKG2D on the tumor surface, perforin and granzyme B are released to the tumor cell, resulting in mediation of apoptosis [30]. However, these processes are tightly controlled by a group of inhibitory receptors. These receptors act as negative regulators of NK cytotoxicity and inhibit the action of NK cells against 'self' targets. A main group of this type of receptors is NK cell immunoglobulin-like receptors (KIRs), which are mainly specific for self MHC Class-I molecules. Members of the KIR family recognize HLA-A, B and C alleles [31]. The second mechanism is the triggering of apoptosis pathways in the target cell via stimulation of death receptors by TRAIL or Fas ligand expressed on the surface of NK cells as well as secretion of TNF-a. NK cells express Fas ligand and TRAIL, which are both members of the TNF family and have been shown to induce target cell apoptosis when they bind their receptors on target cells [32]. In the current study, the levels of surface expression of Fas, DR5, and MIC A/B showed an increase on cells treated with I-131 in in vitro study. Up-regulation of Fas, DR5, and MIC A/B on the surface of irradiated tumor cells could explain the enhancement of NK cell cytotoxicity to tumor cells in both in vitro and in vivo studies.
Irradiation can alter immunogenecity of the tumor as well as the circumstances of the immunologic condition. Several groups have reported that radiation therapy concomitantly up-regulates the levels of Fas, DR5, and MIC A/B in several tumor cells. Ishikawa et al. [13] reported that external radiation therapy enhanced Fas and DR5 expression in glioma cell lines and cytotoxicity of NK cells was enhanced after radiation therapy. Zhou et al. [17] reported that DR5 expression was enhanced in melanoma cell lines by external radiation therapy and treatment with TRAIL resulted in significantly increased tumor cell apoptosis caused by radiation therapy. Xu et al. [33] also reported that radiation therapy up-regulated the level of MIC A/B and increased the sensitivity of NK cell killing in a pancreatic cancer cell line. However, external radiation therapy is limited in treatment of multiple metastatic lesions. Like external radiation therapy, I-131 therapy can also alter immunogenicity of tumor cells by providing radiation to the cells. In addition, I-131 therapy is applicable to treatment of multiple metastatic breast cancer, which can take up I-131 by hNIS expression [34]. Jeon et al. [15] reported that I-131 therapy can lead to up-regulated expression of Fas and enhance the killing activities of cytotoxic T cells in a colon cancer cell line. In accordance with previous studies, the present study demonstrated that I-131 therapy up-regulated the level of Fas, DR5 and MIC A/B expression in breast cancer cell line. Because the majority of breast cancers are known to express hNIS and take up iodide, it would be applicable in the clinical setting [19].
A limitation of the current study is the use of MDA-231/NF cells expressing hNIS, instead of parental MDA-231 cells, for I-131 therapy, and it is not straightforward in clinical practice. Expression of hNIS is known to be enhanced by certain agents such as a retinoic acid, and enhancement by hNIS inducible agents has been well investigated in breast cancer cells [35][36][37]. hNIS expression in breast cancer cells by the inducible agents (not