The Veterinary Journal 193 (2012) 222–227

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The Veterinary Journal

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Correlation of Foxp3 positive regulatory T cells with prognostic factorsin canine mammary carcinomas

J.H. Kim a,1, J.H. Hur b, S.M. Lee c, K.S. Im a, N.H. Kim a, J.H. Sur a,⇑a Department of Veterinary Pathology, Small Animal Tumor Diagnostic Center, College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong,Kwangjin-gu, Seoul 143-701, Republic of Koreab Korea Animal Clinic, Bupyeong-dong, Bupyeong-gu, Incheon 403-010, Republic of Koreac Cell Biology & Molecular Immunology Laboratory, Division of Biotechnology, College of Environmental & Bioresource Sciences, Jeonbuk National University, Ma-dong, Iksan-si,Jeonbuk 570-752, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Accepted 27 October 2011

Keywords:DogFoxp3Mammary carcinomaPrognostic factorRegulatory T cell

1090-0233/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.tvjl.2011.10.022

⇑ Corresponding author. Tel.: +82 2 450 4153.E-mail address: jsur@konkuk.ac.kr (J.H. Sur).

1 Present addresses: Department of Veterinary Clininary Medicine, University of Minnesota, St. Paul, MNCenter, University of Minnesota, Minneapolis, MN 554

Regulatory T cells (Treg) cells play a crucial role in tumor progression by suppressing anti-tumor immu-nity, but are not well-documented in veterinary oncology. To identify the characteristics of Treg cells intumor microenvironments, the numbers of Treg cells were analyzed and compared with histologicalprognostic factors and molecular biomarkers in canine mammary carcinoma (MC) tissues (n = 37).

Abundant Treg cells were associated with high histological grade and lymphatic invasion. The numbersof Treg cells infiltrating intratumoral areas markedly increased in tumors with poor prognostic factors,such as high histological grade, lymphatic invasion, and necrosis. These findings suggest that Treg cellsplay a role in canine MC progression. Furthermore, Treg cell numbers in intratumoral compartmentsmay provide a potential prognostic factor when assessing canine MCs, which may in turn lead to thedevelopment of new immunologic therapeutics.

� 2011 Elsevier Ltd. All rights reserved.

Introduction

Mammary gland tumors are the most common neoplasmdetected in female dogs, and approximately 50% are diagnosed asmalignant tumors (Sorenmo, 2003). Canine mammary carcinomas(MCs) are heterogeneous, having different biologic and pathologicfeatures and clinical behaviors (Nieto et al., 2000), which makes itdifficult to determine a prognosis and treatment for dogs withMCs.

In recent years, the immune response against tumors and themolecular aspects of oncology have been an area of particularinterest (Coussens and Werb, 2002; DeNardo and Coussens,2007). It has been suggested that cancer progression and/or regres-sion closely correlates with inflammation and host immune re-sponses (Coussens and Werb, 2002; DeNardo and Coussens,2007). There are a number of studies investigating the relationshipbetween the host immune system and tumors for development ofnovel immunotherapies against tumors (Lasalvia-Prisco et al.,2006).

ll rights reserved.

ical Science, College of Veter-55108, USA; Masonic Cancer

55, USA.

The lineage of CD3+ T cells includes CD8+ cytotoxic T lympho-cytes (CTL) and CD4+ helper T (Th) cells (Mason et al., 1992). CTLskill selectively target cells such as infected, damaged or dysfunc-tional cells (Kuppers and Henney, 1977), while Th cells that differ-entiate into Th1 or Th2 subsets with different cytokine profilesregulate the immune response with other immune cells (Mosmannet al., 1986). Th1 cells typically produce interferon-gamma (IFN-c),mediate protection against intracellular pathogens, and are in-volved in autoimmune diseases, whereas Th2 cells produce inter-leukin (IL)-1, IL-4, IL-5, and IL-25 and act in humoral immunityagainst extracellular pathogens (Abbas et al., 1996; Murphy andReiner, 2002).

In tumor-induced immune systems, tumor specific CD8+ CTLskill tumor cells by two main pathways: perforin and granzyme-mediated cell lysis mechanisms or cytokine-dependent tumor kill-ing (Kowalczyk et al., 2003; Cullen et al., 2010). Cytokines such asIFN-c, IL – 2 and tumor necrosis factor (TNF) –a are secreted fromCD4+ Th1 cells and can initiate an anti-tumor immune response(DeNardo and Coussens, 2007). However, tumors are rarely sup-pressed by the host immune response, since tumor cells acquireimmune tolerance.

The killing function of CTLs could be disrupted by Treg cellswith CD4+ Th2 cells and their associated cytokines, because Tregcells are a key player in peripheral immune tolerance and are knownto be essential for down-regulation of self-reactive lymphocytes

J.H. Kim et al. / The Veterinary Journal 193 (2012) 222–227 223

(Nishikawa and Sakaguchi, 2010). Therefore, suppression ofantitumor immune responses by Treg cells can promote tumorprogression and development (DeNardo and Coussens, 2007;Nishikawa and Sakaguchi, 2010). Because the tumor immuneresponse involves extremely complicated interactions betweenvarious factors such as cytokines, chemokines, angiogenic and lymph-angiogenic factors, and proteases (Yu and Rak, 2003; DeNardo andCoussens, 2007; Hojilla et al., 2008), the role of tumor-infiltratinglymphocytes in the tumor environment can be difficult to unravel.Nevertheless, determining the functional role of Treg cells isessential to understand tumor immunology.

Treg cells express the surface markers CD4 and CD25, althoughthe transcriptional factor forkhead (FHK) box P3 (Foxp3) is cur-rently considered the most reliable marker of Treg cells (Fontenotet al., 2005). Foxp3, a member of the FHK/winged helix familyof transcription factors, was identified in autoimmune diseases(Bennett et al., 2001) and is essential for the development andsuppressive function of Treg cells (Sakaguchi et al., 2008).

In humans, Treg cells have been shown to be higher in variouscancers and to correlate with poor prognostic factors and shortersurvival time in breast cancer (Clarke et al., 2006; Merlo et al.,2009). Inflammatory cells infiltrate canine MC more than benignmammary tumors (Estrela-Lima et al., 2010; Kim et al., 2010b).However, the characteristics and the function of infiltrating lym-phocytes including Treg cells are unclear. Although Treg cells areconsidered as significant modulators of the tumor immune system,Treg cells are still not well-described in the tumor microenviron-ment in veterinary oncology. Some studies have evaluated Tregcells in healthy dogs and dogs with cancer such as osteosarcoma,melanoma, mast cell tumor, and soft tissue sarcoma (Biller et al.,2007; Tominaga et al., 2010). However, in these studies, few sam-ples were evaluated and there was a lack of histopathologic data.

In the present study, the numbers of Foxp3+ Treg cells wereanalyzed in three different compartments of canine MCs and corre-lated with histological prognostic factors and expression of estro-gen receptor (ER) and epidermal growth factor (HER)-2.

Table 1Histological classification of tumors (WHO system).

Malignant mammary tumors (n = 37)

Without lymphatic invasion (n = 19) With lymphatic invasion (n = 18)

Simple carcinoma (n = 13) Simple carcinoma (n = 4)Tubular subtype (n = 5) Solid subtype (n = 2)Papillary subtype (n = 4) Anaplastic subtype (n = 2)Tubulopapillary subtype (n = 3) Carcinoma in mixed tumor (n = 4)Solid subtype (n = 1) Squamous cell carcinoma (n = 10)Complex carcinoma (n = 1)Malignant mixed tumor (n = 2)Squamous cell carcinoma (n = 3)

Materials and methods

Study population and samples

Archived formalin-fixed, paraffin embedded canine mammary gland tissueswere examined. Because lymphocytes infiltration is mostly observed in malignantmammary tumors compared to benign tumors, MC samples (n = 37) were used.Normal mammary tissue samples (n = 3) from clinically healthy dogs were usedas negative controls. All mammary tumor samples were acquired from dogs with-out any treatment such as chemotherapy and radiation therapy. Samples were fromcases of the Veterinary Medical Teaching Hospital of Konkuk University and privateanimal clinics and were submitted to the Department of Veterinary Pathology atKonkuk University between 2006 and 2008.

Histopathological examination

Sections (4 lm) were stained with hematoxylin and eosin (H&E) for evaluation.MCs were classified according to World Health Organization (WHO) criteria(Misdorp et al., 1999). Histological grades of canine MCs were well-differentiated(grade I), moderately differentiated (grade II), and poorly differentiated carcinoma(grade III) according to the Elston and Ellis grading system (Elston and Ellis, 2002).The presence of tumor necrosis and lymphatic invasion of tumor cells was recorded.

Immunohistochemistry

Immunohistochemistry (IHC) was performed as previously described(Tashbaeva et al., 2007). Briefly, slides were deparaffinized in xylene and hydratedin graded ethanol. Tissue sections (4 lm) were treated with 3% H2O2 solution for20 min at room temperature (RT), followed by three washes in phosphate bufferedsaline (PBS; pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4).Antigens were retrieved by boiling tissue sections in Tris–EDTA buffer (pH 9.0)for 20 min in a microwave oven (750 W, high power). Slides were cooled andwashed three times in PBS. Each section was subsequently incubated withanti-mouse/human Foxp3 antibody clone eBio7979 (diluted to 1:100; eBioscience)

overnight at 4 �C. The secondary polymer was applied to each slide for 40 min at RTusing the two-step Envision system horseradish peroxidase method (Dako REALEnvision kit). The slides were then washed four times in PBS and incubated withsubstrates. The color reaction was stopped by two washes in distilled water andcounterstaining with Harris hematoxylin was performed. A normal mouse lymphnode section was used as a Foxp3 positive control and mouse IgG1 (diluted to1:100; eBioscience) was used as a negative isotype control. Digital images wereacquired with an Olympus microscope (model BX41) and digital image transfersoftware (Leica Application Suite 2.7).

Foxp3+ cell determination

Foxp3+ cells were counted in the following three compartments: (1) intratu-moral; (2) within the adjacent stroma, and (3) in the distant stroma, according tomethods previously published (Mahmoud et al., 2011). The number of Foxp3+ cellswas analyzed in a 2.4 mm2 area per tumor section with automated image analysissoftware (Image Pro Plus 5.1). Images of the sections were acquired at 400� mag-nification (40� objective and 10� ocular) and 10 fields of Foxp3+ immunolabeledimages were taken for each compartment: intratumoral compartment (within tu-mor cell nests); adjacent stroma (one tumor cell size or less is the relative distancebetween a positive immunolabeled cell and its tumor nest); distant stroma (the dis-tance between positive cell and tumor nest is more than one tumor cell size). Thetotal number of Foxp3+ cells in each tumor was calculated by adding the countsof the three compartments.

Statistical analysis

Associations between Foxp3+ cell expression and categorical variables wereperformed using the Pearson’s v2-test. Associations of the mean number of Foxp3+cells infiltrating the three compartments according to histological type, histologicalgrade, lymphatic invasion of tumor cells, and necrosis were performed using a Stu-dent’s t test or analysis of variance (ANOVA) test. Statistical significance was estab-lished at P < 0.05. Statistical analysis was performed using Statistical Package forSocial Sciences (SPSS) 17.0.

Results

Clinical and histopathological data

MC samples (n = 37) from female dogs aged 2–19 years (mean10.8 ± 3.7 years) were examined. The dog breeds included Maltese(n = 11), Yorkshire terrier (n = 10), Shih Tzu (n = 4), Chihuahua(n = 1), Schnauzer (n = 1), mixed breed (n = 1), and unknown(n = 9). MC samples included simple carcinomas (n = 17), carci-noma in mixed tumors (n = 6), complex carcinomas (n = 1), andsquamous cell carcinomas (n = 13). MCs with evidence of lympha-tic invasion were described (n = 18). Histological grade was classi-fied as I (n = 8), II (n = 11), or III (n = 18). The histologicalclassification of tumors and evidence of lymphatic invasion areshown in Table 1.

Expression of Foxp3+ cells in canine MC

Foxp3 immunoreactivity was detected in the nuclei of infiltrat-ing lymphocytes (Fig. 1). Foxp3+ cells were observed within theadjacent, distant stroma, and intratumoral compartments in canineMCs (Figs. 2–4).

Fig. 1. Foxp3+ expression in the nuclei of lymphoid cells infiltrating caninemammary carcinoma. Immunohistochemical staining (Horseradish peroxidase andhematoxylin counterstain, bar = 36 lm).

Fig. 2. Foxp3+ cells within canine mammary carcinoma. Foxp3+ cells were locatedin the intratumor (T) and distant stroma (asterisk) areas. Immunohistochemicalstaining (Horseradish peroxidase and hematoxylin counterstain, bar = 70 lm).

Fig. 3. Foxp3+ cells within the intratumoral compartment (red arrows). Necrosiscan also be noted (asterisk). Immunohistochemical staining (Horseradish peroxi-dase and hematoxylin counterstain, bar = 36 lm).

Fig. 4. Foxp3+ cells in the adjacent stroma (red arrows). Immunohistochemicalstaining (Horseradish peroxidase and hematoxylin counterstain, bar = 36 lm).

224 J.H. Kim et al. / The Veterinary Journal 193 (2012) 222–227

Numbers of Foxp3+ cells

Foxp3+ cells were not found in normal mammary tissues andwere observed in 23/37 (54.1%) MCs. The mean number (±SD) oftotal Foxp3+ cells per 2.4 mm2 was 410.5 (±231.3). The mean num-bers of Foxp3+ cells in each compartment were as follows: adja-cent stroma 66.7 (±66.9), distant stroma 275.9 (±172.3), andintratumoral compartment 68.0 (±68.0). The numbers of Foxp3+cells are presented in Table 2.

Foxp3+ cells and histopathological features

Foxp3+ cells were present in tumors classified according to his-tological type as follows: simple carcinoma 7/17 (41.2%); complexcarcinoma 1/1 (100%); carcinoma in mixed tumor 4/6 (66.7%); andsquamous cell carcinoma 11/13 (84.6%) (P = 0.085). Foxp3+ cellswere found in histological grade I 1/8 (12.5%), II 8/11 (72.7%),and III 14/18 (77.8%) tumors (P = 0.005). Foxp3+ cells were ob-served in 15/18 (83.33%) MCs with and 8/19 (42.11%) without lym-phatic invasion (P = 0.010). Foxp3+ cells infiltrated 15/22 (68.2%)tumors with intratumoral necrosis and 8/15 (53.3%) withoutnecrosis (P = 0.361). A summary of Foxp3+ cells infiltration andhistopathological features is presented in Table 3.

The number of Foxp3+ cells was examined in the three definedtumor compartments per 2.4 mm2 of tumor tissue. The numbers ofFoxp3+ cells found in the adjacent, distant stroma, and intratumor-al compartments, and total numbers did not differ according to his-tological type. Foxp3+ cells in intratumoral compartments(P = 0.012) and total numbers (P = 0.037) were greatest in histolog-ical grade III tumor tissues. The numbers were higher in tumorswith lymphatic invasion than in those without, but only the num-ber of Foxp3+ cells in intratumoral compartments was statisticallysignificant (P = 0.016). Foxp3+ cells infiltrated more tumors withnecrosis than those without, mostly in the intratumoral compart-ment (P = 0.006). The correlation between histological variablesand Foxp3+ cells infiltration is presented in Table 4 and the num-bers of Foxp3+ cells within the three intratumoral compartments ispresented in Table 5.

Foxp3+ cells and immunohistochemical features

Foxp3+ cells were observed in 9/16 (56.3%) ER positive tumorsand in 13/19 (68.4%) ER negative tumors (P = 0.458), 11/14 (78.6%)

Table 2Number of Foxp3+ cells in mammary carcinomas with Foxp3+ cells.

Numbers of Foxp3+ cells (number/2.4 mm2)

Sample number Minimum Maximum Mean SDa

Adjacent stroma 23 0 323 66.7 66.9Distant stroma 23 55 677 275.9 172.3Intratumoral compartment 23 0 253 68.0 68.0Total number 23 88 865 410.5 231.3

a SD, standard deviation.

Table 3Comparison of Foxp3+ cell expression with histopathological and immunohistological features in canine mammary carcinomas.

Mammary carcinomas (n = 37) P⁄

With Foxp3+ cells (n = 23) Without Foxp3+ cells (n = 14)

Histological type n.s.Simple carcinoma 7/17 (41.2%) 10/17 (58.8%)Complex carcinoma 1/1 (100%) 0/1 (0%)Carcinoma in mixed tumor 4/6 (66.7%) 2/6 (33.3%)Squamous cell carcinoma 11/13 (84.6%) 2/13 (15.4%)

Histological grade 0.005I 1/8 (12.5%) 7/8 (87.5%)II 8/11 (72.7%) 3/11 (27.3%)III 14/18 (77.8%) 4/18 (22.2%)

Lymphatic invasion 0.010s 15/18 (83.3%) 3/18 (16.7%)� 8/19 (42.1%) 11/19 (57.9%)

Necrosis n.s.s 15/22 (68.2%) 7/22 (31.8%)� 8/15 (53.3%) 7/15 (46.7%)

Estrogen receptor status n.s.Positive 9/16 (56.3%) 7/16 (43.8%)Negative 13/19 (68.4%) 6/19 (31.6%)

HER-2 overexpression n.s.Positive 11/14 (78.6%) 3/14 (21.4%)Negative 12/23 (52.2%) 11/23 (47.8%)

s, presence; �, absence; �, Pearson’s v2-test; HER-2: Epidermal growth factor receptor-2; n.s., not significant.

Table 4Correlation of Foxp3+ cell numbers with histopathological and immunohistologicalvariables in different compartments (n = 23).

ADJ DIST INT Total numbers

Histological Typea n.s. n.s. n.s. n.s.Histological Gradea n.s. n.s. P = 0.012 P = 0.037Lymphatic invasionb n.s. n.s. P = 0.016 n.s.Necrosisb n.s. n.s. P = 0.006 n.s.Estrogen receptor statusb n.s. n.s. n.s. n.s.HER-2 overexpressionb n.s. n.s. n.s. n.s.

ADJ, adjacent stroma; DIST, distant stroma; INT, intratumoral compartment; HER-2,Epidermal growth factor receptor-2.

a ANOVA test.b Student t-test.

J.H. Kim et al. / The Veterinary Journal 193 (2012) 222–227 225

tumors with and 12/23 (52.2%) without HER-2 overexpression(P = 0.108) (Table 3). The numbers of Foxp3+ cells in the three com-partments did not correlate with ER status and HER-2 overexpres-sion in canine MCs (Table 4).

Discussion

The immune system protects the host against pathologicaldamage. The relationship between immune response and tumorhas been the subject of considerable debate and is still underextensive investigation. In veterinary oncology, although canineMCs are the most common neoplasm found in female dogs

(Sorenmo, 2003), the pathological aspects, prognosis estimation,and therapeutics are still not well described, because these tumorsare heterogeneous and involve a multitude of pathobiologicalfactors.

In the present study, the numbers of Treg cells were analyzedand compared with the malignant behavior of canine MCs. Tregcells were identified using Foxp3 as a marker, because Foxp3 isconsidered the best marker (Banham et al., 2006). Treg cells havebeen evaluated in human cancer, where a higher number of Tregcells has been associated with poor prognosis and reduced survival(Curiel et al., 2004; Hiraoka et al., 2006; Liu et al., 2011). It isknown that tumor progression and growth are promoted underthese circumstances, since Treg cells suppress the host immunesystem against the tumor (Nishikawa and Sakaguchi, 2010). Thereare a few reports in the veterinary literature which examine Tregcells in canine tumors (Biller et al., 2007; Tominaga et al., 2010).In these studies, the numbers of Treg cells in the peripheral bloodand tumor-draining lymph nodes were increased in dogs with can-cer compared to healthy dogs. However, these studies include asmall number of cases and do not describe histopathologicalfeatures.

In the current study, Treg cell infiltration was statistically corre-lated with high histological grade. Treg cells were frequently ob-served in tumors with evidence of lymphatic invasion. Inaddition, the numbers of Treg cells within the intratumoral com-partments were high in tumors with lymphatic invasion, high his-tological grade, and tumoral necrosis, which are all poor prognosticfactors (Hellmén et al., 1993; Misdorp, 2002). These results are

Table 5Numbers of Foxp3+ cells within the three intratumoral compartments (n = 23).

Foxp3+ cells in intratumoralcompartmenta

P

Histological grade I (n = 1) 41.0 0.012b

II (n = 8) 15.9 ± 18.1III (n = 14) 99.6 ± 69.5

Lymphatic invasion s (n = 15) 92.1 ± 71.7 0.016c

� (n = 8) 22.6 ± 25.2

Necrosis s (n = 15) 90.7 ± 73.0 0.006c

� (n = 8) 25.4 ± 26.0

a Numbers of Foxp3+ cells/2.4 mm2 (Mean ± SD).b ANOVA test.c Student’s t test.

226 J.H. Kim et al. / The Veterinary Journal 193 (2012) 222–227

consistent with previous human breast cancer (HBC) studies,which showed that intratumoral Foxp3 gene expression is higherin tumors than in normal tissue (Gupta et al., 2007) and that highernumbers of intratumoral Treg cells are associated with a worseprognosis (Liu et al., 2011). These results support the hypothesisthat recruitment of Treg cells enables tumor cells to evade the hostimmune response, but also that Treg cells in intratumoral areasmay facilitate canine MC progression.

Some authors have suggested that Treg cells can be an inde-pendent prognostic factor of ER positive HBC (Bates et al., 2006),while others have shown that Treg cells were not associatedwith ER status (Mahmoud et al., 2011). In dogs, lack of ERexpression is known to correlate with increasing tumor malig-nancy (de Las Mulas et al., 2005; Yang et al., 2006; Morriset al., 2009), but the correlation between ER and Treg cellswas not significant in our study. The relationship betweenHER-2 overexpression and clinicopathological features in canineMCs is also controversial with some authors reporting thatHER-2 overexpression correlates with malignant behavior (Dutraet al., 2004; Kim et al., 2010a) and others showing increased sur-vival rates for canine MCs overexpressing HER-2 (Hsu et al.,2009). The results of the present study indicated that the num-bers of Treg cells tended to increase in tumors with HER-2 over-expression, although this was not statistically significant. Otherstudies evaluating additional factors, such as cell proliferationor expression of cell adhesion molecules and cytokines, areneeded to determine the interaction of Treg cells with ER andHER-2 in tumor growth and progression. In particular, investigat-ing TGF-b and IL-10 produced by Treg cells (Hara et al., 2001;Nakamura et al., 2001; Chen et al., 2003) will help define thefunction of Treg cells in canine MCs.

Conclusions

Abundant Foxp3+ Treg cells were associated with high histolog-ical grade and lymphatic invasion. The numbers of Treg cells infil-trating intratumoral areas were higher in tumors with poorprognostic factors, such as high histological grade, lymphatic inva-sion, and necrosis. These results suggest that Treg cells may play acrucial role in tumor progression by suppressing the anti-tumorimmune system in canine MCs. Furthermore, Treg cell numbersin intratumoral compartments may represent a potential prognos-tic factor for canine MCs and may be the basis for the developmentof new immunological therapeutics.

Conflict of interest statement

None of the authors of this paper has a financial or personalrelationship with other people or organisations that could inappro-priately influence or bias the content of the paper.

Acknowledgements

We would like to thank Ms. R.H. Jang for her excellent technicalassistance and private animal clinics for providing canine mam-mary samples. J.H. Hur contributed equally to this paper with thefirst author. This report represents one part of a PhD thesis byJ.H. Hur. This research was supported by Basic Science ResearchProgram through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology(20110021337).

References

Abbas, A.K., Murphy, K.M., Sher, A., 1996. Functional diversity of helper Tlymphocytes. Nature 383, 787–793.

Banham, A.H., Powrie, F.M., Suri-Payer, E., 2006. FOXP3+ regulatory T cells: Currentcontroversies and future perspectives. European Journal of Immunology 36,2832–2836.

Bates, G.J., Fox, S.B., Han, C., Leek, R.D., Garcia, J.F., Harris, A.L., Banham, A.H., 2006.Quantification of regulatory T cells enables the identification of high-risk breastcancer patients and those at risk of late relapse. Journal of Clinical Oncology 24,5373–5380.

Bennett, C.L., Christie, J., Ramsdell, F., Brunkow, M.E., Ferguson, P.J., Whitesell, L.,Kelly, T.E., Saulsbury, F.T., Chance, P.F., Ochs, H.D., 2001. The immunedysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) iscaused by mutations of FOXP3. Nature Genetics 27, 20–21.

Biller, B.J., Elmslie, R.E., Burnett, R.C., Avery, A.C., Dow, S.W., 2007. Use of FoxP3expression to identify regulatory T cells in healthy dogs and dogs with cancer.Veterinary Immunology and Immunopathology 116, 69–78.

Chen, W., Jin, W., Hardegen, N., Lei, K.J., Li, L., Marinos, N., McGrady, G., Wahl, S.M.,2003. Conversion of peripheral CD4+CD25� naive T cells to CD4+CD25+regulatory T cells by TGF-beta induction of transcription factor Foxp3. TheJournal of Experimental Medicine 198, 1875–1886.

Clarke, S.L., Betts, G.J., Plant, A., Wright, K.L., El-Shanawany, T.M., Harrop, R.,Torkington, J., Rees, B.I., Williams, G.T., Gallimore, A.M., Godkin, A.J., 2006.CD4+CD25+FOXP3+ regulatory T cells suppress anti-tumor immune responsesin patients with colorectal cancer. PLoS ONE 1, e129.

Coussens, L.M., Werb, Z., 2002. Inflammation and cancer. Nature 420, 860–867.Cullen, S.P., Brunet, M., Martin, S.J., 2010. Granzymes in cancer and immunity. Cell

Death and Differentiation 17, 616–623.Curiel, T.J., Coukos, G., Zou, L., Alvarez, X., Cheng, P., Mottram, P., Evdemon-Hogan,

M., Conejo-Garcia, J.R., Zhang, L., Burow, M., Zhu, Y., Wei, S., Kryczek, I., Daniel,B., Gordon, A., Myers, L., Lackner, A., Disis, M.L., Knutson, K.L., Chen, L., Zou, W.,2004. Specific recruitment of regulatory T cells in ovarian carcinoma fostersimmune privilege and predicts reduced survival. Nature Medicine 10, 942–949.

de Las Mulas, J.M., Millan, Y., Dios, R., 2005. A prospective analysis of immuno-histochemically determined estrogen receptor alpha and progesterone receptorexpression and host and tumor factors as predictors of disease-free period inmammary tumors of the dog. Veterinary Pathology 42, 200–212.

DeNardo, D.G., Coussens, L.M., 2007. Inflammation and breast cancer. Balancingimmune response: Crosstalk between adaptive and innate immune cells duringbreast cancer progression. Breast Cancer Research 9, 212.

Dutra, A.P., Granja, N.V., Schmitt, F.C., Cassali, G.D., 2004. C-erbB-2 expression andnuclear pleomorphism in canine mammary tumors. Brazilian Journal of Medicaland Biological Research 37, 1673–1681.

Elston, C.W., Ellis, I.O., 2002. Pathological prognostic factors in breast cancer. I. Thevalue of histological grade in breast cancer: Experience from a large study withlong-term follow-up. Histopathology 41, 154–161.

Estrela-Lima, A., Araujo, M.S., Costa-Neto, J.M., Teixeira-Carvalho, A., Barrouin-Melo,S.M., Cardoso, S.V., Martins-Filho, O.A., Serakides, R., Cassali, G.D., 2010.Immunophenotypic features of tumor infiltrating lymphocytes frommammary carcinomas in female dogs associated with prognostic factors andsurvival rates. BMC Cancer 10, 256.

Fontenot, J.D., Rasmussen, J.P., Williams, L.M., Dooley, J.L., Farr, A.G., Rudensky, A.Y.,2005. Regulatory T cell lineage specification by the forkhead transcription factorfoxp3. Immunity 22, 329–341.

Gupta, S., Joshi, K., Wig, J.D., Arora, S.K., 2007. Intratumoral FOXP3 expression ininfiltrating breast carcinoma: Its association with clinicopathologic parametersand angiogenesis. Acta Oncologica 46, 792–797.

Hara, M., Kingsley, C.I., Niimi, M., Read, S., Turvey, S.E., Bushell, A.R., Morris, P.J.,Powrie, F., Wood, K.J., 2001. IL-10 is required for regulatory T cells to mediatetolerance to alloantigens in vivo. Journal of Immunology 166, 3789–3796.

Hellmén, E., Bergström, R., Holmberg, L., Spångberg, I.B., Hansson, K., Lindgren, A.,1993. Prognostic factors in canine mammary tumors: A multivariate study of202 consecutive cases. Veterinary Pathology 30, 20–27.

Hiraoka, N., Onozato, K., Kosuge, T., Hirohashi, S., 2006. Prevalence of FOXP3+regulatory T cells increases during the progression of pancreatic ductaladenocarcinoma and its premalignant lesions. Clinical Cancer Research 12,5423–5434.

J.H. Kim et al. / The Veterinary Journal 193 (2012) 222–227 227

Hojilla, C.V., Wood, G.A., Khokha, R., 2008. Inflammation and breast cancer:Metalloproteinases as common effectors of inflammation and extracellularmatrix breakdown in breast cancer. Breast Cancer Research 10, 205.

Hsu, W.L., Huang, H.M., Liao, J.W., Wong, M.L., Chang, S.C., 2009. Increased survivalin dogs with malignant mammary tumours overexpressing HER-2 protein anddetection of a silent single nucleotide polymorphism in the canine HER-2 gene.Veterinary Journal 180, 116–123.

Kim, J.H., Im, K.S., Kim, N.H., Yhee, J.Y., Nho, W.G., Sur, J.H., 2010a. Expression ofHER-2 and nuclear localization of HER-3 protein in canine mammary tumors:Histopathological and immunohistochemical study. Veterinary Journal 189,318–322.

Kim, J.H., Yu, C.H., Yhee, J.Y., Im, K.S., Sur, J.H., 2010b. Lymphocyte infiltration,expression of interleukin (IL)-1, IL-6 and expression of mutated breast cancersusceptibility gene-1 correlate with malignancy of canine mammary tumours.Journal of Comparative Pathology 142, 177–186.

Kowalczyk, D.W., Wlazlo, A.P., Giles-Davis, W., Kammer, A.R., Mukhopadhyay, S.,Ertl, H.C., 2003. Vaccine-induced CD8+ T cells eliminate tumors by a two-stagedattack. Cancer Gene Therapy 10, 870–878.

Kuppers, R.C., Henney, C.S., 1977. Studies on the mechanism of lymphocyte-mediated cytolysis. IX. Relationships between antigen recognition and lyticexpression in killer T cells. Journal of Immunology 118, 71–76.

Lasalvia-Prisco, E., Garcia-Giralt, E., Cucchi, S., Vazquez, J., Lasalvia-Galante, E.,Golomar, W., Larranaga, J., 2006. Advanced breast cancer: Anti-progressiveimmunotherapy using a thermostable autologous hemoderivative. BreastCancer Research and Treatment 100, 149–160.

Liu, F., Lang, R., Zhao, J., Zhang, X., Pringle, G.A., Fan, Y., Yin, D., Gu, F., Yao, Z., Fu, L.,2011. CD8(+) cytotoxic T cell and FOXP3(+) regulatory T cell infiltration inrelation to breast cancer survival and molecular subtypes. Breast CancerResearch and Treatment [Epub ahead of print].

Mahmoud, S.M., Paish, E.C., Powe, D.G., Macmillan, R.D., Lee, A.H., Ellis, I.O., Green,A.R., 2011. An evaluation of the clinical significance of FOXP3(+) infiltrating cellsin human breast cancer. Breast Cancer Research and Treatment 127, 99–108.

Mason, D.Y., Cordell, J.L., Gaulard, P., Tse, A.G., Brown, M.H., 1992.Immunohistological detection of human cytotoxic/suppressor T cells usingantibodies to a CD8 peptide sequence. Journal of Clinical Pathology 45, 1084–1088.

Merlo, A., Casalini, P., Carcangiu, M.L., Malventano, C., Triulzi, T., Menard, S.,Tagliabue, E., Balsari, A., 2009. FOXP3 expression and overall survival in breastcancer. Journal of Clinical Oncology 27, 1746–1752.

Misdorp, W., Else, R.W., Hellmén, E., Lipscomb, T.P., 1999. Histological Classificationof Mammary Tumors of the Dog and the Cat. Armed Forces Institute ofPathology, Washington, DC, second series, vol. VII.

Misdorp, W., 2002. Tumors of the mammary gland. In: Meuten, D.J. (Ed.), Tumors inDomestic Animals, Fourth Ed. Iowa State Press, Iowa, pp. 575–606.

Morris, J.S., Nixon, C., King, O.J., Morgan, I.M., Philbey, A.W., 2009. Expression ofTopBP1 in canine mammary neoplasia in relation to histological type, Ki67,ERalpha and p53. Veterinary Journal 179, 422–429.

Mosmann, T.R., Cherwinski, H., Bond, M.W., Giedlin, M.A., Coffman, R.L., 1986. Twotypes of murine helper T cell clone. I. Definition according to profiles oflymphokine activities and secreted proteins. Journal of Immunology 136, 2348–2357.

Murphy, K.M., Reiner, S.L., 2002. The lineage decisions of helper T cells. NatureReviews Immunology 2, 933–944.

Nakamura, K., Kitani, A., Strober, W., 2001. Cell contact-dependent immuno-suppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface-bound transforming growth factor beta. The Journal of Experimental Medicine194, 629–644.

Nieto, A., Pena, L., Perez-Alenza, M.D., Sanchez, M.A., Flores, J.M., Castano, M., 2000.Immunohistologic detection of estrogen receptor alpha in canine mammarytumors: Clinical and pathologic associations and prognostic significance.Veterinary Pathology 37, 239–247.

Nishikawa, H., Sakaguchi, S., 2010. Regulatory T cells in tumor immunity.International Journal of Cancer 127, 759–767.

Sakaguchi, S., Yamaguchi, T., Nomura, T., Ono, M., 2008. Regulatory T cells andimmune tolerance. Cell 133, 775–787.

Sorenmo, K., 2003. Canine mammary gland tumors. The Veterinary Clinics of NorthAmerica. Small Animal Practice 33, 573–596.

Tashbaeva, R.E., Hwang, D.N., Song, G.S., Choi, N.H., Lee, J.H., Lyoo, Y.S., Lee, S.J., Jung,D.I., Kim, H.Y., Sur, J.H., 2007. Cellular characterization of multidrug resistanceP-glycoprotein, alpha fetoprotein, and neovascular endothelium-associatedantigens in canine hepatocellular carcinoma and cirrhotic liver. VeterinaryPathology 44, 600–606.

Tominaga, M., Horiuchi, Y., Ichikawa, M., Yamashita, M., Okano, K., Jikumaru, Y.,Nariai, Y., Kadosawa, T., 2010. Flow cytometric analysis of peripheral blood andtumor-infiltrating regulatory T cells in dogs with oral malignant melanoma.Journal of Veterinary Diagnostic Investigation 22, 438–441.

Yang, W.Y., Liu, C.H., Chang, C.J., Lee, C.C., Chang, K.J., Lin, C.T., 2006. Proliferativeactivity, apoptosis and expression of oestrogen receptor and Bcl-2 oncoproteinin canine mammary gland tumours. Journal of Comparative Pathology 134, 70–79.

Yu, J.L., Rak, J.W., 2003. Host microenvironment in breast cancer development:Inflammatory and immune cells in tumour angiogenesis and arteriogenesis.Breast Cancer Research 5, 83–88.