Immunostimulatory Effects of Low-Dose Cyclophosphamide Are Controlled by Inducible Nitric Oxide Synthase

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Markus Loeffler, Jo¨rg A. Kru¨ger, and Ralph A. Reisfeld

Department of Immunology, The Scripps Research Institute, La Jolla, California

Abstract and various tumor cells. Besides its effects on pericytes, it has been Cyclophosphamide is a widely used chemotherapeutic drug implicated in autocrine growth stimulation, formation of tumor that was recently applied as either an antiangiogenic/ stroma, and control of the interstitial tumor pressure therefore antivasculogenic or an immunostimulatory agent in combi- making it a promising target for tumor vaccinations (7). nation with cancer immunotherapies. It has been previously We show here for the first time that the cyclophosphamide- shown that cyclophosphamide augments the efficacy of mediated inhibition of inducible nitric oxide synthase (iNOS) is antitumor immune responses by depleting CD4+CD25+ T directly linked to its immunostimulatory but not to its anti- regulatory cells and increasing both T-lymphocyte prolife- vasculogenic effects. Furthermore, we show that the newly ration and T memory cells. Furthermore, cyclophosphamide described mechanism applies to tumor bearing mice and can be was shown to mediate killing of circulating endothelial employed in different tumor models to greatly enhance the progenitors. However, the molecular basis for these obser- antitumor effect of an oral DNA vaccine targeting PDGF-B, delivered by attenuated Salmonella typhimurium to secondary vations has not yet been elucidated. We show here that the cyclophosphamide-mediated inhibition of inducible nitric lymphoid tissue. oxide synthase is directly linked to its immunostimulatory Materials and Methods but not to its antivasculogenic effects. Moreover, combined application of cyclophosphamide with a novel, oral DNA Animals, bacterial strains, cell lines, and plasmid vectors. Female À/À +/+ vaccine targeting platelet-derived growth factor B (PDGF-B), C57BL/6J iNOS and iNOS mice were purchased at 6 weeks of age from overexpressed by proliferating endothelial cells in the tumor The Jackson Laboratory (Bar Harbor, ME). Female BALB/c mice, 6 to vasculature, not only completely inhibited the growth of 8 weeks of age, were obtained from The Scripps Research Institute’s Rodent Breeding Facility. All animal experiments were done according to the NIH different tumor types but also led to tumor rejections in Guides for the Care and Use of Laboratory Animals and approved by our mice. These findings provide a new rationale at the Animal Care Committee. The attenuated S. typhimurium strain RE87 molecular level for the combination of chemotherapy and (AroAÀ) was provided by Remedyne Corp. (Santa Barbara, CA). Murine immunotherapy in cancer treatment. (Cancer Res 2005; D2F2 breast carcinoma cells were a gift from Dr. Wei-Zen Wei (Department 65(12): 5027-30) of Immunology, Karmanos Cancer Institute, Detroit, MI). MOPC-315 plasmacytoma cells and D121 Lewis lung carcinoma (LLC) cells were Introduction kindly provided by Dr. Lea Eisenbach (Weizmann Institute of Science, Rehovot, Israel). The cDNA encoding murine PDGF-B was purchased from Cyclophosphamide is a chemotherapeutic agent used to treat Invivogen (San Diego, CA) and the pcDNA3.1/Zeo control vector from various types of cancer. Although high doses of the drug lead to Invitrogen (San Diego, CA). immunosuppression, low-dose protocols have been shown to lead Oral immunization, tumor cell challenge, and treatment with to enhanced immune responses against various tumor antigens cyclophosphamide and antibodies. Mice were challenged either by s.c. (1). This immunostimulatory effect has been linked to the ability injection of 5 Â 105 MOPC-315 plasmacytoma cells in the left front flank, 5 of cyclophosphamide not only to decrease the number but also o.t. injection of 3 Â 10 D2F2 breast cancer cells in the left, second lowest 5 the functionality of CD4+CD25+ T regulatory cells (2, 3). Moreover, mammary fat pad, or by i.v. injection of 7.5 Â 10 LLCs in the lateral tail cyclophosphamide has been shown to trigger lymphocyte vein. Tumor volumes were measured in 2 dimensions and calculated as length / 2 Â width2. Treatment with cyclophosphamide was done in proliferation and increase the number of T lymphocytes with a hi + experimental groups of 8 mice with 175 mg/kg cyclophosphamide (Sigma- memory phenotype by up-regulation of CD44 CD4 and Aldrich, St. Louis, MO) as described previously (1), given i.p. every 6 days, hi + CD44 CD8 (4). Cyclophosphamide was also reported to have starting on days 3, 4, or 6 after tumor cell challenge with D2F2, MOPC-315, antivasculogenic effects (5), which at least in part can be or LLC cells, respectively. One hour after cyclophosphamide treatment, attributed to the suppression of circulating endothelial progen- antibodies (500 Ag) directed against either CD4 (clone GK 1.5) or CD8 (clone itors (CEP; ref. 6). The molecular mechanisms of both the 2.43), both from the National Cell Culture Center (Minneapolis, MN), were immunostimulatory and antivascular effects of cyclophosphamide injected i.p. every 6 days to deplete the respective CD4+ and CD8+ T cells. have yet to be defined. Mice were immunized 24 hours thereafter by gavage with 100 AL PBS 9 À Platelet-derived growth factor B (PDGF-B) is a 241–amino acid containing 10 S. typhimurium (AroA ) transformed with the plasmid protein that is mainly expressed in proliferating endothelial cells vectors indicated and as described previously (8). Determination of serum nitrite/nitrate concentration. After collec- tion of the respective blood samples, serum was obtained according to standard procedures and incubated for 15 minutes at 80jC. Thereafter, the Note: M. Loeffler and J.A. Kru¨ger contributed equally to this work. colorimetric assay was done according to the manufacturer’s protocol This is the Scripps Research Institute article no. 17026-IMM. (Roche Applied Science, Indianapolis, IN). Requests for reprints: Ralph A. Reisfeld, Department of Immunology, The Scripps Flow cytometry. For the determination of different T-cell subpopula- Research Institute, IMM13, R218, La Jolla, CA 92037. Phone: 858-784-8105; Fax: 858- 784-2708; E-mail: [email protected]. tions, splenocytes were stained for CD4, CD8, CD25, and Ly-6C I2005 American Association for Cancer Research. (BD PharMingen, San Diego, CA) by four-color flow cytometric analysis www.aacrjournals.org 5027 Cancer Res 2005; 65: (12). June 15, 2005

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Cancer Research and 105 cells were counted for each analysis. To enumerate CEPs, 200 AL NO (11). In contrast to these two immunostimulatory effects of heparinized peripheral blood were collected, RBC lysed with ACK lysis cyclophosphamide, there was no detectable iNOS dependence of buffer (Walkersville, MD), and cells stained for CD3, CD34, and flk-1 5 cyclophosphamide-mediated antivasculogenic effects as shown by (BD PharMingen) in three-color flow cytometric analyses with 2.5 Â 10 the lack of any significant difference in the decrease of CEPs, cells used for each analysis. indicated by CD3À/CD34+/flk-1+ cells (12), between iNOS+/+ and Statistical analysis. The statistical significance of differential findings À/À between experimental groups and controls was determined by Student’s iNOS mice after cyclophosphamide treatment. This finding can t test. Findings were regarded significant, if Ps < 0.05. be explained by recent reports that treatment with low-dose cyclophosphamide can lead to up-regulation of the antiangiogenic factor thrombospondin-1 (Tsp-1; ref. 5), which is known for its Results and Discussion ability to mediate apoptosis of endothelial cells in association with Based on the findings of Vig et al. (9), who established a link increased expression of proapoptotic Bax and decreased expression between the lack of iNOS and higher frequencies of memory T cells of antiapoptotic Bcl-2 molecules (13). Furthermore, Tsp-1 is in response to peptide immunizations, we hypothesized that nitric capable of sequestrating vascular endothelial growth factor (14), oxide could be a mediator of immunostimulatory and/or thereby preventing it from exerting such proangiogenic functions antivasculogenic effects of cyclophosphamide. To this end, as the induction of an increased expression of survivin, an inhibitor iNOSÀ/À mice were used to compare their generation of NO with of apoptosis protein in endothelial cells (15), and the expansion that of C57Bl/6J (iNOS+/+) control mice after either mock or low- (16) and mobilization of CEPs (17). dose cyclophosphamide treatment. As shown (Fig. 1A), treatment To determine, whether the immunomodulatory effects of low- with low-dose cyclophosphamide inhibited the generation of NO in dose cyclophosphamide therapy also apply to tumor-bearing mice, iNOS+/+ controls but not in iNOSÀ/À mice, showing a major role for cyclophosphamide therapy was combined with a DNA vaccine iNOS in the cyclophosphamide-mediated suppression of NO against PDGF-B, which is overexpressed by proliferating endothe- production (10). Moreover, cyclophosphamide was able to increase lial cells in the tumor microenvironment but not by any of the the percentage of CD8+ T cells with a memory phenotype by tumor cell lines used in our studies (data not shown). Three increasing the level of Ly-6C expression in iNOS+/+ but not in different preclinical mouse tumor models were applied. In the first iNOSÀ/À mice (Fig. 1B). We could also show that the cyclophos- experiment (Fig. 2A), we took advantage of the murine plasmacy- phamide-induced decrease of T cells with a regulatory phenotype toma model MOPC-315 and challenged syngeneic BALB/c mice s.c. (CD4+CD25+) is mainly mediated by iNOS as we observed a marked with MOPC-315 cells. Although mice were challenged with 50 times decrease of regulatory T cells in iNOS+/+ mice treated with the minimal lethal dose, a marked delay in the onset of tumor cyclophosphamide but not in iNOSÀ/À mice (Fig. 1C). The decrease growth in such mice was shown when treated with either observed in T cells with a regulatory phenotype can be explained by chemotherapy (cyclophosphamide) or immunotherapy. In our differences in the susceptibility of various T-cell subpopulations to approach, the immunotherapeutic entity consisted of an orally given DNA vaccine encoding PDGF-B, carried by attenuated S. typhimurium (AroÀ). Following gavage, these avirulent bacteria proved to be efficient in delivering plasmid DNA to such secondary lymphoid tissues as Peyer’s patches in the small intestine. As observed previously, this resulted in the subsequent induction of a robust CD8+ T cell–mediated immune response against target cells overexpressing the respective antigen (8). Although this inhibition of tumor growth was quite significant, the mice had to be sacrificed due to the huge tumor burden that ultimately reached the level of that in untreated or control-treated mice. In contrast to single therapy with the DNA vaccine, the combination of cyclophosphamide with this same vaccine could completely inhibit further growth progression of these highly aggressive tumors for 50 days (Fig. 2A). In a second experiment, BALB/c mice were challenged o.t. with D2F2 cells in a syngeneic murine breast cancer model (Fig. 2B). In this case, the treatment with cyclophosphamide was initiated at a time point when the tumors were still small (f10 mm3) compared with the previous experiment and thus more closely resembled clinical conditions. The group of mice treated with the combination therapy not only showed an inhibition of tumor growth for 80 days but more importantly revealed complete regression of tumors in three of eight mice. In view of the recently proposed three-stage model of cancer immunoediting (18), consisting of tumor cell elimination via cancer immunosurveillance, equilibrium, and escape, we conclude that our combination treatment was sufficient to revert tumors from phase 3 which Figure 1. Effects of cyclophosphamide on iNOSÀ/À. A, NO serum escaped immune recognition and destruction, to tumors in phase 1 concentrations from iNOS+/+ or iNOSÀ/À mice treated with cyclophosphamide as indicated by the complete remission in 37.5% of the mice (CTX) or PBS as a control. The respective percentages of CD8+Ly-6Chi T cells (B) and CD4+CD25+ T cells (C) as well as the number of CD3ÀCD34+flk-1+ cells challenged with D2F2 breast cancer cells, or to phase 2 of stable (D) were determined by flow cytometry. Columns, mean (n = 3); bars, +SD. disease as indicated by the majority of tumors not regrowing even

Cancer Res 2005; 65: (12). June 15, 2005 5028 www.aacrjournals.org

In a third approach, C57BL/6J mice were challenged i.v. with a lethal dose of syngeneic D121 LCC cells to induce experimental pulmonary metastases and groups of such animals were depleted in vivo of either CD8+ or CD4+ T cells with the corresponding antibodies (Fig. 2C). This combination treatment led to a significant increase in life span compared with control- or single therapy– treated mice. More importantly, the in vivo depletion experiments indicated that the increase in life span observed in the group of mice subjected to the combination therapy depended mainly on CD8+ T cells, suggesting that the improvement of the antitumor effect achieved by adding cyclophosphamide depends mainly on its immunostimulatory properties and not on any added antivasculogenic effect. We therefore asked ourselves whether cyclophosphamide was capable of exerting its immunomodulatory and antivasculogenic effects also in mice bearing tumors in the tumor escape phase of immunoediting. To this end, the different control and treatment groups of mice were analyzed for their percentages of T cells exhibiting either a memory or a suppressor phenotype as well as for the number of CEPs. As indicated (Fig. 3), cyclophosphamide, either given alone or in combination with the DNA vaccine against PDGF-B, was capable of lowering the serum concentration of NO (Fig. 3A), thereby augmenting the percentage of memory T cells (Fig. 3B) and decreasing both the percentage of suppressor T cells (Fig. 3C) and the number of CEPs (Fig. 3D). Taken together, we were able to show that the cyclophosphamide-mediated inhibition of iNOS is directly linked to its immunostimulatory effects but not to its antivasculogenic effects. Importantly, we showed that the mechanism described applies to tumor bearing mice in vivo by combining cyclophosphamide with

Figure 2. Reversal of tumor immune escape by combining immunotherapy and chemotherapy. A, BALB/c mice were first injected s.c. with 5 Â 105 MOPC-315 plasmacytoma cells and treated 4 days later, once palpable tumors were established, as described in Materials and Methods. Points, mean (n = 8); bars, FSE. B, 3 days after o.t. injection of 3 Â 105 D2F2 breast cancer cells, BALB/c mice bearing visible tumors were treated as indicated. Points, mean (n = 8); bars, FSE. C, survival curves represent results for C57BL/6J mice (n = 8) in each of the treatment and control groups after receiving 7.5 Â 105 D121 Lewis lung tumor cells i.v. In this setting, the treatment was started 6 days after tumor cell challenge, once pulmonary metastases were established (see Materials and Methods). Figure 3. Effects of cyclophosphamide (CTX) at the cellular level in tumor bearing mice. A, NO serum concentrations are depicted of tumor bearing mice after stopping the combination therapy. In fact, a decrease in the treated with either DNA vaccine control vector, control vector plus percentage of CD4+CD25+ T regulatory cells has been shown at cyclophosphamide, DNA vaccine PDGF-B vector, or the combination of DNA vaccine PDGF-B vector plus cyclophosphamide. Determinations of CD8+Ly-6Chi least in two cases to be sufficient for a conversion from tumor (B) and CD4+CD25+ T cells (C) as well as the number of CD3ÀCD34+flk-1+ cells escape to tumor rejection (19, 20). (D) was done by flow cytometry. Columns, mean (n = 3); bars, +SD. www.aacrjournals.org 5029 Cancer Res 2005; 65: (12). June 15, 2005

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Cancer Research a novel DNA vaccine against PDGF-B, which is expressed on Acknowledgments proliferating endothelial cells. This combinational therapy was Received 2/24/2005; revised 4/9/2005; accepted 4/14/2005. able to completely suppress tumor growth in two therapeutic Grant support: California Tobacco-Related Disease Research Program grant 12RT- tumor models and significantly enhance the life span in a 0002; EMD-Lexigen Research Center, Billerica, MA grant SFP1330 (R.A. Reisfeld); and DOD grant BC031079 (R.A. Reisfeld). therapeutic survival model. These findings provide a new The costs of publication of this article were defrayed in part by the payment of page rationale for combined treatments with agents, which, in addition charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. to cyclophosphamide, inhibit iNOS or the production of NO and We thank Dr. Wei-Zen Wei for kindly providing the D2F2 breast cancer cell line and therefore lead to active and effective cancer immunotherapy. Dr. Lea Eisenbach for providing the D121 LLC cell line.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Immunostimulatory Effects of Low-Dose Cyclophosphamide Are Controlled by Inducible Nitric Oxide Synthase

Markus Loeffler, Jörg A. Krüger and Ralph A. Reisfeld

Cancer Res 2005;65:5027-5030.

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