International Journal of

Radiation Oncology biology


Biology Contribution

Combination Effect of Regulatory T-Cell Depletion and Ionizing Radiation in Mouse Models of Lung and Colon Cancer Cheol-Hun Son, MS,*,y Jae-Ho Bae, MD, PhD,y Dong-Yeok Shin, PhD,* Hong-Rae Lee, MS,* Wol-Soon Jo, PhD,* Kwangmo Yang, MD, PhD,* and You-Soo Park, PhD* *Dongnam Institute of Radiological and Medical Sciences, Busan, Korea; and yDepartment of Biochemistry, Pusan National University School of Medicine, Yangsan, Korea Received Nov 21, 2014, and in revised form Dec 24, 2014. Accepted for publication Jan 12, 2015.

Summary Radiation therapy can induce and activate regulatory T cells (Tregs), which may promote tumor progression and recurrence. Our results showed that low-dose cyclophosphamide (LD-CTX) or anti-CD25 antibody combined with radiation therapy can trigger antitumor immune responses more strongly by suppressing Tregs. Specifically, LD-CTX was the most effective at inhibiting Tregs and augmenting systemic antitumor effect in radiation therapy. These Treg depletion strategies may further improve outcomes after radiation therapy.

Purpose: To investigate the potential of low-dose cyclophosphamide (LD-CTX) and anti-CD25 antibody to prevent activation of regulatory T cells (Tregs) during radiation therapy. Methods and Materials: We used LD-CTX and anti-CD25 monoclonal antibody as a means to inhibit Tregs and improve the therapeutic effect of radiation in a mouse model of lung and colon cancer. Mice were irradiated on the tumor mass of the right leg and treated with LD-CTX and anti-CD25 antibody once per week for 3 weeks. Results: Combined treatment of LD-CTX or anti-CD25 antibody with radiation significantly decreased Tregs in the spleen and tumor compared with control and irradiation only in both lung and colon cancer. Combinatorial treatments resulted in a significant increase in the effector T cells, longer survival rate, and suppressed irradiated and distal nonirradiated tumor growth. Specifically, the combinatorial treatment of LD-CTX with radiation resulted in outstanding regression of local and distant tumors in colon cancer, and almost all mice in this group survived until the end of the study. Conclusions: Our results suggest that Treg depletion strategies may enhance radiation-mediated antitumor immunity and further improve outcomes after radiation therapy. Ó 2015 Elsevier Inc. All rights reserved.

Reprint requests to: You-Soo Park, PhD, Department of Research Center, Dongnam Institute of Radiological and Medical Sciences, Jwadong-gil 40, Jangan-eup, Gijang-gun, Busan, 619-953 Korea. Tel: (þ82) 51-720-5841; E-mail: [email protected] This research was supported by the National R&D Program through the Dongnam Institute of Radiological & Medical Sciences (DIRAMS) Int J Radiation Oncol Biol Phys, Vol. -, No. -, pp. 1e9, 2015 0360-3016/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved.

funded by the Ministry of Science, ICT & Future Planning (505902014). C.-H.S. and J.-H.B. contributed equally to this work. Y.-S.P. and K.Y. are co-corresponding authors for this work. Conflict of interest: none.


Son et al.

International Journal of Radiation Oncology  Biology  Physics


Methods and Materials

Radiation therapy is widely used to treat cancer patients because it directly kills tumor cells and evokes tumorspecific immune responses (1-5). Obstacles to the success of this therapeutic approach are tumor recurrence and metastasis due to residual cancer cells. A variety of immunosuppressive factors, including transforming growth factor b (TGF-b), vascular endothelial growth factor, prostaglandin E2, and regulatory T cells (Tregs), are accumulated in the tumor microenvironment and may contribute to recurrence and metastasis (6, 7). Importantly, TGF-b is strongly induced by radiation (8) and is known to activate Tregs rather than effector T cells (9). Accordingly, Tregs attenuate local and systemic antitumor immune response and may contribute to the recurrence of tumors after radiation therapy (9, 10). Tregs constitute 5% to 10% of the circulating CD4þ T-cell population and have powerful immunosuppressive functions (7, 11), constitutively expressing the forkhead transcription factor Foxp3 (12, 13) and the high-affinity interleukin-2 (IL-2) receptor, CD25 (14). Tregs have been identified in multiple tumor types, including ovarian, non-small cell lung, pancreatic, and breast cancers, and high levels of Foxp3 expression among tumor-infiltrating lymphocytes correlate with poor prognosis and high risk of recurrence (15). Therefore, novel approaches to alter the pre-existing immunosuppressive and tolerogenic tumor microenvironment are required to sufficiently elicit antitumor effect after radiation therapy. Recently, various strategies to either reduce the number or inhibit the function of Tregs have been suggested to enhance the therapeutic effect of radiation (6, 12). A common strategy for Treg depletion is the injection of a monoclonal antibody (mAb) that binds to the CD25 molecule constitutively expressed by Tregs. Previous studies demonstrated that anti-CD25 mAb therapy significantly depleted Tregs and improved the regression of tumor (16-18). Other studies showed possible synergistic antitumor effects of combinatorial treatments including anti-CD25 mAb (6, 19). Cyclophosphamide (CTX) represents another strategy to inhibit Treg function. This DNA-alkylating agent is widely used in combination with other chemotherapy drugs to treat various types of cancers. At high doses CTX has side effects, including myelosuppression and cardiopulmonary toxicity. However, low-dose CTX (LD-CTX) treatment induces beneficial immunomodulatory responses, including selective Treg deletion and induction of effector T-cell stimulators, such as type I interferons, IL-7, IL-2, and IL-21 (20, 21). The combined effects of local irradiation and LD-CTX on the function of Tregs have not been reported, and previous studies mainly focused on local antitumor effects after combined treatments of ionizing radiation and Treg-depleting agents (9, 22). In this study we used anti-CD25 mAb and LD-CTX as means to deplete Tregs in combination with ionizing radiation and evaluated the systemic antitumor immunity in mouse models of lung and colon cancer.

Animals and cancer cell lines All animal experiments were approved (DI-2014-010) by the Dongnam Institute of Radiological & Medical Sciences Institutional Animal Care and Use Committee. C57BL/6 and BALB/C mice (male, 6 weeks old) were purchased from Central Lab. Animal (Seoul, Korea). C57BL/6derived Lewis lung carcinoma cells (LLCs; CRL-1642) and BALB/c-derived CT-26 colon carcinoma cells (CRL2638) were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in complete medium, which consisted of Dulbecco’s modified Eagle’s medium (LLC culture) or Roswell Park Memorial Institute 1640 medium (CT-26 culture) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/ mL streptomycin, and maintained at 37 C in a humidified atmosphere containing 5% CO2. Cell culture reagents were purchased from Life Technologies (Gaithersburg, MD).

Radiation treatment and regulatory T-cell depletion procedure C57BL/6 mice were subcutaneously inoculated with LLCs in the right leg (3  105 cells) and left flank (1.5  105 cells). BALB/c mice were subcutaneously inoculated with CT-26 cells in the right leg (1  106 cells) and left flank (0.5  106 cells). After tumors grew to approximately 8 to 10 mm in diameter, ionizing radiation (IR) was applied at a dose of 12 Gy (6-MV photon beam, dose rate of 6.1 Gy/ min) to the tumor on the right leg of mice using a linear accelerator (Elekta Infinity; Crawley, United Kingdom) (Fig. 1). The dosimetry was evaluated daily using an ionization chamber connected to an electrometer system according to the National Institute of Standards and Technology calibration guideline. Before irradiation, mice were anesthesized (intraperitoneal injection of 50 mg zoletile plus 5 mg rompun per kilogram body weight) and then placed in a customized restraining device and positioned. The irradiation field square was set up 20  20 (cm) and radiation focused on legs to minimize the whole-body exposure. Tumor sizes [tumor size (mm2) Z length (mm)  width (mm)] were measured twice per week. Low-dose CTX (50 mg/kg, C57BL/6 mice; 30 mg/kg, BALB/c mice; SigmaAldrich, St. Louis, MO) and anti-CD25 mAb (250 mg per mouse; clone PC61, BioXCell, West Lebanon, NH) were administrated intraperitoneally to the mice 3 days before radiation and then 2 times with 1-week intervals. Because drug sensitivities of cancer cells were not equal, the different dose of CTX was administrated (23, 24). The mice were killed using CO2 gas, and their organs were dissected for further analysis. The survival of mice was recorded as the percentage of surviving animals every day after tumor inoculation. The mice were allowed to die on their own in the

Volume -  Number -  2015

Enhanced systemic antitumor effect of RT


Tumor injection Right leg and left flank Right leg (Tumor: 8~10 mm)

CTX and anti-CD25 mAb treatment (Second)

CTX and anti-CD25 mAb treatment (First)

After 7 days

4 days

After 3 days Irradiation on right thigh

CTX and anti-CD25 mAb treatment (Third)

After 4 days

Survival rate

In vitro assay

Fig. 1. Schematic of the protocol for combination treatment of radiation and low-dose cyclophosphamide (CTX) or anti-CD25 monoclonal antibody (mAb). survival study. However, when body weight of mice seriously reduced over 20% or mice were immobile without food intake, the mice were killed. This study was performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Preparation of single-cell suspensions from spleen The spleens were isolated from the LLC and CT-26 tumorbearing mice 4 days after the second administration. Single-cell suspensions were obtained by homogenizing the spleens, followed by passing the homogenate through a 0.45-mm nylon mesh. Red blood cells were then lysed using 1  ACK (Ammonium-Chloride-Potassium) lysing buffer (Life Technologies) at room temperature for 1 to 2 minutes. Separated cells were washed 3 times with normal saline.

Preparation of single-cell suspensions from tumor tissue Tumor tissues were separated from the LLC- and CT-26ebearing mice at 4 days after the second Tregdepleting injection. Single-cell suspensions of tumor infiltrating leukocytes (TILs) were prepared from individual tumor tissues minced with scissors and dissociated using 0.04 mg/mL Liberase (Roche, Mannheim, Germany) in Roswell Park Memorial Institute medium 1640 at 37 C for 90 minutes. An equal volume of fetal bovine serum was added to the tubes to terminate digestion. Single cells were obtained by passage through 0.45-mm nylon mesh (BD Pharmingen, San Diego, CA). Separated cells were washed 3 times with normal saline.

Flow cytometric analysis The proportion or number of effector T cells and CD4þCD25þFoxP3þ cells within the total CD4þ cell population was evaluated after local irradiation combined with LD-CTX or anti-CD25 mAb treatment. Separated splenocytes and TILs were immunostained with anti-mouse CD4 phycoerythrin (PE), CD8 PE, and CD25 PE-cyanine 7 (PECy7) at 4 C for 30 minutes (all from BD Pharmingen). For

intranuclear staining with anti-mouse Foxp3 PE-Cy5 (eBioscience, San Diego, CA), the CD4/CD25 doublestained samples were fixed and permeabilized using the Transcription Factor Buffer Set (BD Pharmingen) according to the manufacturer’s instructions. Flow cytometry analysis was performed on a FC500 flow cytometer (Beckman Coulter, Brea, CA).

Statistical analysis For comparison among treatment groups, statistical analysis was performed using 1-way analysis of variance followed by Tukey’s multiple comparison test and logerank tests. Differences were considered statistically significant at P

Combination effect of regulatory T-cell depletion and ionizing radiation in mouse models of lung and colon cancer.

To investigate the potential of low-dose cyclophosphamide (LD-CTX) and anti-CD25 antibody to prevent activation of regulatory T cells (Tregs) during r...
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