Eosinophil ablation and tumor development

Oral Oncology 35 (1999) 496±501

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Eosinophil ablation and tumor development D.T.W. Wong a,*, S.M. Bowen a, A. Elovic a, G.T. Gallagher a, P.F. Weller b a

Laboratory of Molecular Pathology, Division of Oral Pathology, Department of Oral Medicine and Diagnostic Sciences, Harvard School of Dental Medicine, Boston, MA 02115, USA b Department of Medicine, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, MA 02215, USA Received 30 October 1998; revised and accepted 29 December 1998

Abstract Tissue eosinophilia in squamous cell carcinoma has long been recognized; however, the role of eosinophils in tumor development remains unclear. Studies have reported both favorable and unfavorable prognoses for patients with tumors exhibiting tumorassociated tissue eosinophilia (TATE). This study seeks to elucidate the potential role of the eosinophil in squamous cell carcinoma development and provide an experimental model for future studies. The carcinogen-induced hamster oral cancer model was found to ful®ll these objectives. Eosinophils progressively in®ltrate into this carcinogen-induced oral cancer model. We now demonstrate that TATE is completely abolished by the use of an anti-interleukin-5 monoclonal antibody (mAb) preparation, TRFK-5. Clinical observations revealed that TRFK-5-treated hamsters exhibited smaller tumor burden and delayed onset of tumor development. The results suggest that anti-interleukin-5 antibody treatment may delay and/or inhibit tumor development, and that eosinophils may have a tumor-promoting role. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Tissue eosinophilia; Tumor development; Monoclonal antibody; Oral cancer

1. Introduction Tumor-associated tissue eosinophilia (TATE) in malignancies of epithelial origin has been reported since 1893 [1]. Despite this early observation, the role of eosinophils in carcinogenesis remained unclear. Studies have reported favorable prognoses in tumors demonstrating tissue eosinophilia [2±7], suggesting eosinophils may play a protective role against epithelial tumors. Other studies, however, suggest that eosinophils may play a role in promoting epithelial tumor growth [8±11]. Pretlow et al. [6] reported a signi®cant increase in survival, and decrease in metastases, in colonic carcinoma patients exhibiting TATE. Kapp and Livolsi [3] also suggested TATE to be a favorable prognostic indicator for tumors of the uterine cervix. Lowe and Fletcher [5,12] reported that massive tissue eosinophilia, in cases of squamous cell carcinoma of the oral cavity, external genitalia, anus, and bladder, was also associated with a favorable prognosis. Goldsmith et al. [2] reported that stromal eosinophilia in head and neck * Corresponding author. Tel.: +1-617-432-1834; fax: +1-617-4322449. E-mail address: [email protected] (D.T.W. Wong)

tumors is correlated with a favorable outcome. Tepper et al. [7] reported an eosinophil-dependent interleukin (IL)-4-mediated tumor rejection mechanism in rat. These studies jointly support the hypothesis that tumor eosinophilia is associated with a favorable prognosis. Though these studies suggest that TATE may have anti-tumor activity, a number of studies have suggested that eosinophils may play a role in tumor promotion. For example, Kruger-Krasagakes et al. [13] reported that eosinophils did not suppress growth in IL-5 transfected tumor cells. Horiuchi et al. [10] reported that heavy eosinophil in®ltration in well-di€erentiated squamous cell carcinomas of the oral cavity was a signi®cant factor for an unfavorable prognosis, and was not correlated with clinical stage or occurrence of metastasis. Another clue to the possible role of eosinophils in tumorigenesis occurred in 1990 when Wong et al. [11] found that eosinophils can produce a tumor promoting cytokine, transforming growth factor-a (TGF-a). The studies of eosinophils in experimental settings has been hampered by the lack of animal models that are de®cient in eosinophils. Yamaguchi et al. [14,15] showed IL-5 to be a highly speci®c factor for the maturation, terminal di€erentiation and ampli®cation of eosinophils in vitro. IL-5 is considered the most important cytokine

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D.T.W. Wong et al. / Oral Oncology 35 (1999) 496±501

involved in the regulation of functional eosinophil activity, as well as eosinophilia associated with allergic and parasitic diseases [16]. Recent studies have utilized an anti-IL-5 monoclonal antibody (mAb) preparation, TRFK-5, to successfully abolish eosinophil in®ltration in guinea pig [17±20], and monkey asthma models [19] for up to a 3-month period. We have recently used the TRFK-5 preparation to successfully ablate the in®ltration of eosinophils into hamster skin wounds [21]. Hence, the TRFK-5 anti-IL-5 mAb may be used to prevent the maturation, development and release of eosinophils, and prevent tissue eosinophilia. The hamster oral cancer model is an excellent animal model for experimental oral cancer studies [22]. Application of a 0.5% 7,12-dimethylbenz-(a)anthracene (DMBA) solution in mineral oil to the cheek pouch induces hyperplasia within 4 weeks, dysplasia in 4±6 weeks, carcinoma in situ in 6±10 weeks, and invasive carcinoma in 10±14 weeks. As in human squamous cell carcinomas [4], tumors in the hamster cheek pouch exhibit eosinophilia of the stromal connective tissues immediately adjacent to the invading front of malignant epithelium [9]. Importantly, we have demonstrated that eosinophils progressively in®ltrate into this oral cancer model and that TATE is a consistent histological feature of these tumors [23]. In this study we examined the e€ect of abolishing eosinophils on tumor development in the hamster oral cancer model. We now present data to demonstrate that the TRFK-5 anti-IL-5 mAb can successfully ablate the TATE. Abolishment of TATE in the hamster oral cancer model is associated with a delay in the onset of tumor development and a reduction in tumor burden. Our data suggest that eosinophils may serve a tumorpromoting role in the hamster oral cancer model. The stage is now set whereby an experimental strategy to block TATE and an experimental model of tumor development in place to de®ne the cellular and molecular contributions of eosinophils in epithelial carcinogenesis. 2. Materials and methods Twenty male Syrian golden hamsters, 60±90 days old, 81±90 g, were purchased from Charles River Laboratory (Wilmington, MA). All animals were treated according to the ``Guide for the Care and Use of Laboratory Animals'' (DHHS Publication No. (NIH) 85-23, Revised 1985). Animals were maintained in separate cages (one animal per cage) in an air-conditioned (24 C) animal room on a 12-h light, 12-h dark cycle, and fed with a commercial stock diet (Purina Formula Chow) and tap water ad libitum (protocol 48R95), over a 14-week period in the animal care facilities at the Harvard School of Public Health.

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Hamsters were divided into four groups. Group I consisted of ®ve hamsters which were treated thrice weekly with a 0.5% solution of DMBA (D-3254; Sigma Chemical Co., St. Louis, MO) dissolved in mineral oil (USP), painted in the left cheek pouch with a No. 4 soft sable brush. Group II consisted of ®ve hamsters, which were treated with mineral oil only. Group III consisted of ®ve hamsters which received 0.5% DMBA in mineral oil, and 5 mg of anti-IL-5 mAb (TRFK-5, 6.46 mg/ml, lot No.4), administered every second week via intraperitoneal injection. Group IV, with ®ve hamsters, received the 0.5% DMBA in mineral oil, in addition to 5 mg of control antibodies to bacterial b-galactosidase (GL-113 Control Ab, 6.0 mg/ml. lot No.V27f939-003) via intraperitoneal injection, every second week. The TRFK-5 mAb, as well as the control antibody to bacterial bgalactosidase, were kindly provided by Dr. Robert Egan at the Schering-Plough Research Institute. The TRFK-5 and control antibodies are of identical isotype, IgG1. Weekly observations included clinical examination of the cheek pouches, using a nasal speculum, and recording ®ndings of leukoplakia, erythroplakia, erythema, hyperkeratosis, tumor size, number, and morphology, and animal body weight. After the 14th week animals were sacri®ced by CO2 asphyxiation and their left cheek pouches harvested. Cheek pouches were photographed, and diagrammed to record ®nal clinical ®ndings. Dimensions of individual tumors were also measured and recorded. Tumors were then excised and ®xed in 10% formalin, processed and embedded in paran, slide mounted, and stained for microscopy. The femurs of representative animals from each of the four groups were also harvested. To facilitate the identi®cation of eosinophils in examined sections, all slides were stained with Fisher Giemsa (SG-28). Eosinophils stained in this manner exhibit bright orange ¯uorescence when viewed with a rhodamine ®lter at 552 nm [11]. Eosinophils were quanti®ed. Tumor invasiveness and di€erentiation were described. Tumor burden was calculated by using spherical volume, 0.75pr3, as most tumors were papillomatous and spherical. Those that were more plaque like, and elliptical, required the calculation LW2(0.52) [24]. Total volume was then divided by the number of tumors to obtain mean tumor burden. 3. Results 3.1. Anti-IL-5 (TRFK-5) mAb ablates TATE We have previously shown that progressive tissue eosinophilia is a consistent histological feature in the DMBA-induced hamster oral cancer model [23]. Normal hamster oral mucosa contains only an occasional eosinophil while DMBA-treated hamster cheek pouches

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the number of eosinophils observed, and whether the animal received TRFK-5 mAb, with p=0.001 and R (correlation coecient)=ÿ0.5480. Thus, animals receiving TRFK-5 mAb had signi®cantly less eosinophils than those who did not. Thus, the use of TRFK-5 mAb can decrease eosinophil in®ltration into hamster oral cancer development sites.

exhibit signi®cant increase in tissue eosinophilia after 6 weeks of carcinogen treatment. Thus, the hamster oral cancer model can be used to examine the role of eosinophils in tumor development. We have recently demonstrated that the anti-IL-5 mAb (TRFK-5) can ablate the in®ltration of eosinophils into hamster skin wounds [21]. We therefore ®rst examine whether the same TRFK-5 anti-IL-5 mAb can block TATE in the hamster oral cancer model. Hamster cheek pouches from all 20 animals were examined at the end of the tumor induction period (14 weeks). Since we have previously demonstrated that TATE is primarily associated with malignant oral epithelia, we randomly selected ten 200 ®elds containing malignant epithelium (Groups I, III, IV) and examined the lamina propria adjacent to the tumor epithelium. Eosinophils were quanti®ed using Giemsa-stained tissue sections and examined under rhodamine ¯uorescence microscopy [11]. We have previously demonstrated this histochemical staining coupled with rhodamine ¯uorescence will reveal all tissue eosinophils. The average number of eosinophils in tumor sections from Group I was 157‹20, Group II was 1‹1, Group III was 2‹4, and Group IV was 128‹15 (Fig. 1). Statistical analysis by Pearson's chi2 and Fisher's exact test revealed that there is a relationship between

With the successful depletion of TATE by the use of TRFK-5 mAb, we proceeded to examine if the ablation of eosinophil in®ltration altered the tumor development process. We ®rst compared the tumor burden of the four groups of animals at the end of the tumorinduction processes (Fig. 2). The animals in the positive control group (Group I) had a tumor burden of 0.13‹0.20 cm3. Mineral oil control group exhibited no tumor growth (Group II). Group III animals which received DMBA treatment plus the TRFK-5 anti-IL-5 mAb exhibited a smaller tumor burden than Group I animals (0.03‹0.04 cm3). Group IV animals, which received DMBA treatment and control mAb (b-galactosidase) injections, exhibited the highest tumor burden (0.46‹0.22 cm3). The tumor

Fig. 1. Quanti®cation of tumor-associated tissue eosinophilia. For each treatment group, 10 randomly selected tumor-epithelium associated ®elds were examined at 200. Group I=7,12-dimethylbenz(a)anthracene (DMBA) treatment, positive control; Group II=mineral oil, negative control; Group III=DMBA+TRFK-5 monoclonal antibody (mAb); Group IV=DMBA+b-galactosidase mAb.

Fig. 2. Comparison of tumor burden between treatment groups. Group I=7-12-dimethylbenz-(a)antracene (DMBA) treatment, positive control; Group II=mineral oil, negative control; Group III=DMBA+TRFK-5 monoclonal antibody (mAb); Group IV =DMBA+b-galactosidase mAb.

3.2. Tumor burden is lower in hamsters treated with the TRFK-5 anti-IL-5 mAb

D.T.W. Wong et al. / Oral Oncology 35 (1999) 496±501

burden in Group IV was greater than that in Groups I, II, and III. We also examined if any association existed between TATE, tumor numbers, and tumor burden. Regression analysis revealed that the number of eosinophils was related to the number of tumors developed, b=0.0249, 95% CI 0.0063±0.0434, p=0.012. However, no relation existed between TATE and average tumor volume in each animal, b=0.00002; 95% CI ÿ0.0004±0.0009, p=0.462. Neither was there a relationship between the number of eosinophils and the total tumor volume in each animal, b=0.0013, 95% CI ÿ0.0018±0.0044, p=0.379. 3.3. Tumor development is delayed in hamsters treated with TRFK-5 mAb The ®nal objective of this study was to examine the kinetics of tumor development in the di€erent treatment groups. We are particularly interested in the e€ect of the TRFK-5 anti-IL-5 mAb on the tumor induction process. Fig. 3 demonstrates that the ®rst example of tumor before week 6 was an animal in Group IV, which received DMBA treatment plus the control b-galactosidase mAb. By week 6 and thereafter the tumor induction pro®le was identical between Group I (DMBA positive control) and Group IV. All DMBA-treated

Fig. 3. Comparison of tumor incidence between treatment groups. Group I=7,12-dimethylbenz-(a)anthracene (DMBA) treatment, positive control; Group II=mineral oil, negative control; Group III=DMBA+TRFK-5 monoclonal antibody (mAb); Group IV =DMBA+b-galactosidase mAb.

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animals had clinically visible tumors by week 14. Interestingly, in Group III animals which received DMBA and the TRFK-5 anti-IL-5 mAb, the ®rst animal to develop clinically visible tumor was on week 8 (20%). At this time (week 8) three of the ®ve animals (60%) in Groups I and IV had tumors. By the end of the tumor induction period (week 14), only three of the ®ve animals (60%) in Group III had clinically visible tumors, while all animals in Groups I and IV had tumors. No clinical visible tumor was observed in any Group II animal. 4. Discussion In this report we have presented data that support the conclusion that eosinophils in®ltrated into oral cancer development sites in¯uence the tumor development process. Using the TRFK-5 anti-IL-5 mAb, we have shown that TATE can be ablated in tumor development sites. The depletion of TATE in tumor development sites is associated with a delayed onset of clinically visible tumors and a reduction in the tumor burden. Jointly, the delayed onset of tumor development and lower tumor burden suggest that eosinophils in®ltrated into tumor sites may play a tumor-promoting role. This study was prompted by our previous observation that eosinophils progressively in®ltrate into oral cancer development sites [23]. Tissue eosinophilia developed in speci®c association with malignant oral epithelium. The current literature suggests eosinophils could be either tumor protective or tumor promoting. Thus, there is a great need to have an animal model whereby the cellular and molecular contribution of the eosinophil to the tumor development process can be examined. Since there is no existing animal model that is de®cient in eosinophils, we attempted to experimentally deplete eosinophil in®ltration by the use of immunological reagents. The anti-IL-5 mAb, TRFK-5, which has been used successfully in a number of animal models to reduce eosinophilia, was also e€ective in ablating the TATE in the hamster oral cancer model. This was gratifying to us because we have previously shown eosinophils to progressively in®ltrate in this animal cancer model. The ability to ablate eosinophils in the hamster oral cancer model makes this a suitable animal model to examine the role of eosinophils in tumor development. We have also quanti®ed other in¯ammatory cells in the four experimental groups by the use of morphological criteria for neutrophils, lymphocytes, mast cells and macrophages. No signi®cant di€erences were observed between the di€erent treatment groups, suggesting that the e€ect of TRFK-5 mAb is selective for the depletion of eosinophils. This is similar to our recent data that TRFK-5 mAb selective depletes eosinophils in hamster skin wounds [21].

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It should be noted that it is currently unclear to us as to why Group IV animals developed the highest tumor burden. Perhaps it is the nature of the control mAb (bgalactosidase) that we used. Alternatively it could be the additional experimental manipulation to inject the mAb via intraperitoneal route that elicited the exaggerated response. We can control for these factors in future studies by the use of additional control mAb and the inclusion of an experimental arm which includes mock intraperitoneal injection (e.g. saline). The data that we are reporting in this study suggest that eosinophils in®ltrated into tumor development sites may promote carcinogenesis. One possible mechanism maybe via the eosinophil production of cytokines that may in¯uence the tumor development process. Eosinophils are now known to have the ability to elaborate a number of cytokines, some of which have been associated with tumor promotion. For example, we have shown that human eosinophils express TGF-a. TGF-a has tumor-promoting activity [25]. Transgenic mouse overexpressing TGF-a developed mammary and lacrimal tumors [26±28]. Transgenic mouse overexpressing TGF-a also had accelerated development of DMBAinduced mammary tumors [29]. While our data support a tumor-promoting role of eosinophils in carcinogenesis, we must be cautious in making generalizations based on our current data. Could this be a hamster-speci®c response? Are TATE observed in other tumor development models? It will be a challenge to be able to verify our ®ndings in human studies. However, it will be possible to validate our results in other experimental tumor development models. Should our studies be validated in other animal tumor models, TATE may serve as a negative prognostic indicator for human oral tumors. It will also be of interest to speculate on the potential use of the TRFK-5 anti-IL-5 mAb preparation to perhaps e€ectively reduce, delay, and maybe even prevent tumor development.

Acknowledgements Supported by grants from the National Institute of Health DE-08680 (D.T.W. Wong); AI20241, HL56386, AI41995 (P.F. Weller). A. Elovic is the recipient of a NIH Physician Scientist Award (DE-00323). We are particularly grateful to Dr. Robert Egan at the Schering-Plough Research Institute for providing us with the TRFK-5 anti-IL-5 mAb and the b-galactosidase mAb.

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