Presence of Tumour-infiltrating FOXP 3 + Lymphocytes Correlates with Immature Tumour Angiogenesis in Renal Cell Carcinomas

Regulatory T cells (Tregs), characterised by a CD4+ CD25+ phenotype, play an essential role in immune regulation by facilitating peripheral tolerance and precluding autoimmunity (Thornton and Shevach, 1998). CD4+ CD25+ Tregs express the transcriptional repressor forkhead box P3 (FOXP3) which is essential for their development (Hori and Sakaguchi, 2004; Fontenot and Rudensky, 2005). The capacity of Tregs to suppress effector T-cell proliferation and cytokine production can enable tumour cells to elude immunological surveillance, as evidenced by data showing increased levels of Tregs in the peripheral blood of some cancer patients (Griffiths et al., 2007). A study of previously untreated renal cell carcinoma (RCC) patient demonstrated an association between increased levels of Tregs and an adverse overall survival (Griffiths et al., 2007). Interestingly, another study of RCC patients who had undergone radical or partial nephrectomy showed that increased levels of CD4+ CD25+ FOXP3− T cells were associated with an increased risk of death from RCC (Siddiqui et al., 2007).


Introduction
Regulatory T cells (Tregs), characterised by a CD4 + CD25 + phenotype, play an essential role in immune regulation by facilitating peripheral tolerance and precluding autoimmunity (Thornton and Shevach, 1998).CD4 + CD25 + Tregs express the transcriptional repressor forkhead box P3 (FOXP3) which is essential for their development (Hori and Sakaguchi, 2004;Fontenot and Rudensky, 2005).The capacity of Tregs to suppress effector T-cell proliferation and cytokine production can enable tumour cells to elude immunological surveillance, as evidenced by data showing increased levels of Tregs in the peripheral blood of some cancer patients (Griffiths et al., 2007).A study of previously untreated renal cell carcinoma (RCC) patient demonstrated an association between increased levels of Tregs and an adverse overall survival (Griffiths et al., 2007).Interestingly, another study

Presence of Tumour-infiltrating FOXP3 + Lymphocytes Correlates with Immature Tumour Angiogenesis in Renal Cell Carcinomas
Hai-Lun Zhan, Xin Gao*, Xiang-Fu Zhou, Xiao-Yong Pu, De-Juan Wang Vasculature is a critical process in the tumour growth and metastasis.For most of solid tumours, microvessel density (MVD) correlates with poor prognosis.However, MVD remains a controversial prognostic predictor for RCC (Kirkali et al., 2001;Sabo et al., 2001a;Suzuki et al., 2001;Dekel et al., 2002;Kinouchi et al., 2003;Yildiz et al., 2008;Qian et al., 2009), a significant contributory factor being that blood vessels in RCC may be regarded as being monolithic in structure and function, which might not accurately reflect the functional diversity based on microvessel differentiation and location.Mature blood vessels are usually covered by pericytes, and pericyte coverage is regarded as an indicator of vascular maturation (Gerhardt and Semb, 2008).
Since Tregs are associated with tumour progression and MVD is critical for tumour prognosis, it was hypothesized that the level of FOXP3 and MVD correlate with RCC pathology and be associated with disease prognosis.To this end, we determined the level of MVD and FOXP3 + Treg in tissues resected from RCC patients by immunohistochemistry and analysis their correlation with the clinicopathological parameters and survival data.

Patients and tissue specimens
The study protocol was approved by the Institutional Review Board of The Third Affiliated Hospital, Sun-Yat Sen Universtity (Guangzhou, China).Patients were informed of the investigative nature of the study, and written consent in accordance with institutional regulations was obtained prior to study entry.Sixty two patients with unilateral or sporadic RCC treated with radical nephrectomy or nephron-sparing surgery were enrolled between January 1998 and January 2009.None of the enrolled patients had received preoperative immunotherapy or renal arterial embolization therapy.Paraffin blocks were selected based on the availability of complete clinicopathological and follow-up data for the patients.The selected tissue sections (4μm thickness) were taken from areas away from necrotic and haemorrhagic tissue.For each patient, three tissue samples, obtained from different areas with a 5 mm diameter scope in the centre of tumour nests, the nearest cancerous margin, and normal kidney tissue (designated as interior, rim and normal, respectively) were studied.Analysed pathological characteristics for each patient included tumour size, tumour TNM stage, nuclear grade and pathological type.Tumours were staged according to the 2002 WHO TNM staging system.Nuclear grade was assigned according to the criteria of Fuhrman et al. (1982).Specimens were evaluated by a pathologist blinded to the outcome.
Overall survival was evaluated.Patient follow-up was completed on 1 st March 2009.The duration of followup was calculated from the date of nephrectomy to the date of cancer progression (i.e., distant metastases after nephrectomy for the primary tumour), death or last followup.All patients were prospectively monitored by serum creatinine, abdomen ultrasonography and chest X-ray every 1-6 months according to the postoperative time and individual factors.For suspicious cases, computed tomography and/or magnetic resonance imaging were used to verify the development of metastases.

FOXP3 immunohistochemistry
Immunohistochemistry was performed as previously described (Bates et al., 2006).Briefly, paraffin sections were deparaffinized and hydrated.After microwave antigen retrieval, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes.Immunohistochemistry to label FOXP3 + Treg was performed using diluted rabbit anti-human FOXP3 monoclonal antibody (Sigma, England, dilution 1:700) for 120 min in 37 ℃.After incubation with primary antibodies, secondary antibody serially, the sections were developed in 3, 3'-diaminobenzidine solution under the microscope and counterstained with haematoxylin.The control tissues used for immunostaining of FOXP3 were lymph nodes.Negative control slides omitting the primary antibodies were included in all assays.
Each tissue sample in the tumour interior, tumour rim and normal tissue was respectively examined using × 100 optical fields (10 fields per location) and the number of FOXP3 + lymphocytes was recorded in each field.The mean value of these measurements was assigned as the score for each location.

Double immunohistochemistry for microvessels
Paraffin sections (4 μm) of tumour tissue samples were deparaffinized and rehydrated, prior to heating in a microwave in 0.01mol/L citrate buffer (pH 7.8) for antigen retrieval.Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol.The sections were incubated over night at 4℃ with a combination of mouse anti-human CD31 monoclonal antibody diluted 1:10 (M0823; Dako, Copenhagen, Denmark) to stain endothelial cells and a rabbit antihuman a-SMA (smooth muscle actin) monoclonal antibody directly conjugated to alkaline phosphatase diluted 1:400 (ab32575; Abcam, Cambridge, UK) to detect pericytes.The immunoreaction for CD31 was detected by a horseradish peroxidase labelling goat anti-mouse IgG (DS-0002; Zhongshan Goldenbridge, Beijing, China), and the α-SMA immunoreaction was detected with the alkaline phosphatase goat anti-rabbit IgG (DS-0002; Zhongshan Goldenbridge, Beijing, China) at 37℃ for 25min.Staining was carried out using the alkaline phosphatase substrate Vector Red and the chromogen 3, 3'-diaminobenzidine.The sections were counterstained with haematoxylin, dehydrated and mounted.Normal mouse IgG1 was used as a substitute for the primary antibody in the negative controls.
MVD was assessed according to previously described criteria (Yao et al., 2007).Briefly, eight most vascularized areas (hotspots) were selected from the centre of tumour nests or in the peripheral areas or in the normal kidney, images were taken at a magnification of ×400 with a Nikon microscope.All CD31-positive entities associated with a-SMA-positive cells were counted as mature, and CD31positive entities associated with a-SMA-negative cells were counted as immature vessels.Stromal and necrotic areas were excluded from the analysis.The mean number of mature vessels or immature vessels of tumour rim or tumour interior was assigned as the score for each case.

Statistical analysis
Recorded data were independently processed by a biostatistician.The cut-off value for definition of the immunohistochemical marker subgroups was the median value.Cox proportional hazard regression models were fitted for univariate analysis.Comparisons of pathological features by patient subgroups were analysed using Fisher's exact test.Continuous data were compared by Mann-Whitney test.Overall survival was determined using the Kaplan-Meier method and the difference between groups assessed using the log-rank test.The correlation between FOXP3 + Tregs and MVD were evaluated by Spearman's correlation.Data were analyzed using SPSS 15.0 (SPSS, Inc., Chicago, IL, USA).A two-tailed P<0.05 was considered to indicate statistical significance.

Patient population
A total of 62 patients with RCC were enrolled into the DOI:http://dx.doi.org/10.7314/APJCP.2012.13.3.867 FOXP3 and Renal Cell Carcinoma Immature Angiogenesis study and followed up for a median (range) duration of 57.2 (5.5-102) months.The patients' clinicopathological characteristics are summarised in Table 1.At the last follow-up 12 patients had died.Higher overall survival was significantly associated with early tumor stage, lower nuclear grade, and pathological type (clear cell RCC) (Log rank test; P<0.001, P=0.033, and P=0.007, respectively).

FOXP3 + Tregs in normal kidney tissue and RCC microenvironment.
In normal kidney tissue and RCC, FOXP3 was expressed exclusively by stroma infiltrating lymphocytes; tumour cells were negative for FOXP3 in all cases (Figure 1).The number of FOXP3 + cells in the tumour interior (5.06 (1.0-15)/high power field [HP]) or the rim (9.2 (2.0-25.0)/HP)was higher than in normal kidney tissue (0.8 (0-2.4)/HP), with statistically significant differences (P<0.05).Using a cut-off point of the median value, FOXP3 + cells in the tumour interior or rim were divided into 'high' and 'low' FOXP3 + infiltration groups.The association of FOXP3 + cells with pathological features of the tumour are summarised in Table 2.The presence of FOXP3 + cells in the tumour interior or the rim was not significantly associated with overall survival, as determined by univariate analysis (RR=0.957,P=0.138 and RR=0.970,P=0.068, respectively; Figure 3A and B).

MVD and pathological characteristics in RCC.
The number of mature microvessel in the tumour interior (4.0 (2-7.75)/HP) or the rim (4.7 (3.0-12.0)/HP)was higher than normal kidney tissue (1.0 (1.0-2.0)/HP), with a statistically significant difference (P<0.05).Furthermore, the number of immature microvessels in the tumour interior (25 (12.9-36.11)/HP)or the rim (23.4 (14.3-36.0)/HP)was also higher than normal tissue (1.0 (1.0-2.0)/HP), as illustrated in Figure 2 (P<0.05) The relationship between immature MVD in the tumour interior or rim and clinical pathological characteristics is summarised in Table 3.A negative      3) and overall survival (P=0.002,P=0.007, as shown in Figure 3 C and D).However, there was no correlation with the nuclear grade or pathological type.There was a negative correlation between immature MVD with tumor stage and with overall survival (P=0.005;P=0.001, respectively) (Table 3).There were no correlation between the mature MVD and tumour size, tumour stage, nuclear DOI:http://dx.doi.org/10.7314/APJCP.2012.13.3.867 FOXP3 and Renal Cell Carcinoma Immature Angiogenesis grade, pathological types (Table 3) and overall survival in the tumour interior or the rim (P>0.05,respectively; Figure 3E and F).

Discussion
To our knowledge, this is the first report describing the relationship between tumour-infiltrating FOXP3 + Tregs and MVD, and its effect on overall survival in patients with RCC.
CD4 + CD25 + Foxp3 + Tregs have been documented to be increased in the of RCC patients compared with normal subjects, and may be associated with RCC pathology and prognosis (Cesana et al., 2006).Furthermore, the clinical significance of tumour-infiltrating FOXP3 + Tregs in the cancer microenvironment, as a prognostic factor for poor survival, has been demonstrated in many studies with a variety of malignancies (Seo et la., 2001;Liyanage et al., 2002).As such, we expected the proportion of FOXP3 + Tregs to increase in tumour infiltrating lymphocytes among patients with more aggressive forms of RCC.On the contrary, FOXP3 + cells were not found to be significantly associated with RCC pathology or patient outcome, which is consistent with Siddiqui et al. (2007).In human malignancies, the role of infiltrating FOXP3+Tregs in disease biology remains a subject of debate.Alvaro et al. (2005) found that increased FOXP3 expression within reactive lymphocytes in Hodgkin's lymphoma patients was associated with improved survival.Siddiqui et al. (2007) further noted that CD4 + CD25 + Foxp3 -T cells, but not CD4 + CD25 + FOXP3 + T cells, were associated with higher TNM staging, larger tumour size, presence of coagulative tumour necrosis, and poorer cancer-specific survival.
Increased MVD has been associated with early progression in a number of tumours, including breast, colon and prostate.However, the correlations between MVD and pathological factors in RCC remains controversial.In our study, we recorded the mature MVD and immature MVD in the different locations of the tumour.Our results showed negative correlations between the immature MVD in the tumour interior or rim and tumour size, tumour stage and overall survival, but no correlations with nuclear grade or pathological type.In addition, we found no correlation between the mature MVD and tumour size, tumour stage, nuclear grade, pathological type or overall survival.The data showed that distinct types of vasculature in RCC correlated with a distinct prognosis, and the immature MVD was more important to the prognosis of RCC than mature MVD.
Tumour angiogenesis can lead to immunosuppression, as a result of defective lymphocyte recruitment through a complex process involving impairment of immune cell adhesion to endothelial cells in vessels.Vascular endothelial growth factor (VEGF) is secreted at high levels in many tumours, including RCC, and it is a major regulator of tumour growth and metastasis with a key role in tumour angiogenesis (Folkman, 1995).VEGF has also been linked to immunosuppression and shown to inhibit the development of dendritic cells (Gabrilovich et al., 1998) and enhance the number of FOXP3 + Tregs within the tumour microenvironment (Giatromanolaki et al., 2008).Recently, the correlation between the presence of FOXP3 + Tregs and high vessel density were confirmed in endometrial adenocarcinomas (Gupta et al., 2007) and breast carcinoma (Li et al., 2006).The increased Treg cells in metastasis in RCC patients were reduced after treatment with angiogenesis inhibitors such as sunitinib (Finke et al., 2008;Ko et al., 2009) In RCC, the presence of FOXP3 + lymphocytes was significantly associated with high immature angiogenesis in the tumour interior, scored as vessels expressing the endothelial marker CD31, but not expressing the pericyte marker α-SMA.The numbers of Tregs in RCC may be an indirect affect due to their promotion of tumour vasculature.As previously studied, Tregs play the immunosuppressive function through the secretion of some cytokines such as IL-10, IL-4, TGF-β, while the TGF-β may also act on tumour vascular pericytes and promote tumour angiogenesis (Gaengel et al., 2009).Recently, Dace et al. (2008) found that IL -10 also promote pathological angiogenesis by regulating macrophage responses to hypoxia.
For many cancers, abundant tumor vascularity is associated with a greater risk for tumor progression (Sabo et al., 2001a).It is generally accepted that increased vascularization is associated with tumor growth and greater invasive potential.Multiple studies have found that in contrast to other neoplasms, lower MVD in RCC is associated with poor prognosis (Yoshino et al., 1995;Köhler et al., 1996;Delahunt et al., 1997;Herbst et al., 1998;Sabo et al., 2001aSabo et al., , 2001b)).It is unclear why poorly vascularized RCC exhibits a worse prognosis.It is possible that the decreased MVD in high grade RCC reflects an inability of the vascularization to keep pace with tumor growth and increased tumor vasculature permeability may compensate for the lower MVC (Sabo et al., 2001b).Another possibility is that tumor progression may results in larger vascular channels within a RCC tumor and compensates for the lower MVD in providing sufficient blood flow (Delahunt et al., 1997).This difference in vascularisation activities between RCC and many other cancers may also result in reduced metabolism and extensive necrosis in late stage RCC.As the tumor becomes necrotic, viable tumor cells may gain access to the vascular system resulting in tumor metastasis (Sabo et al., 2001b).
In conclusion, immune suppression and angiogenesis in the tumour microenvironment simultaneously exist in major tumours, including RCC.In our study, the correlation between the presence of FOXP3 + Tregs and immature MVD in RCC were confirmed, which may be one factor contributing to poor treatment outcome and survival after metastasis of RCC.The mechanism responsible for the interaction FOXP3 + Tregs and immature MVD warrants further study.The association of MVD and FOXP3 with overall survival may also suggest that they be useful prognosis Combined treatment with immunotherapy and anti-angiogenic therapies would be of benefit to patients with RCC.

Figure 1 .
Figure 1.The Immunohistochemistry for the FOXP3 + Cells at 200× Magnification.The number of FOXP3 + cells in the tumor interior (A) or the rim (B) was more than in the normal kidney tissue (C).Quantification of immunohistochemistry result detecting FOXP3 + Treg (D) in normal tissue, tumor interior or rim

Figure 3 .Figure 4 .
Figure 3. Kaplan-Meier Curves for Patients with RCC.A) FOXP3 in the tumour interior (log-rank test, P=0.138); B) FOXP3 in the tumour rim (log-rank test, P=0.068); C) the immature MVD in the tumour interior (log-rank test, P=0.007); D) the immature MVD in the tumour rim (log-rank test, P=0.012); E) the mature MVD in the tumour interior (log-rank test, P=0.126); F) the mature MVD in the tumour rim (log-rank test, P=0.086).The dotted line represents the negative group (below the cut-off point) and the solid line the positive group (above the cut-off).The cut-off point was the median value of FOXP3, immature MVD and mature MVD