Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 in canine simple mammary gland adenocarcinomas

Abstract

The expression of 5 markers associated with angiogenesis, proliferation, and apoptosis was studied in 26 canine simple mammary gland adenocarcinomas (SMGAs). The adenocarcinomas were graded histologically, and tissue sections were immunohistochemically stained for the expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), intra-tumor microvessel density, and tumor proliferation (PI) using antibodies against VEGF, VEGFR-2, von Willebrand factor, and Ki-67 antigen, respectively. Apoptotic indices (AI) were determined by an apoptosis assay. Markers VEGF and VEGFR-2 were detected in 96% and 100% of SMGAs, respectively. A high correlation between histologic grade and PI (r = 0.73), a moderate correlation between VEGF and histologic grade (r = 0.33), and between VEGF and PI (r = 0.42) were found. There was a significant difference in median PI among the 3 histologic grade groups (r < 0.05). Vascular endothelial growth factor may stimulate tumor cell proliferation through an autocrine loop, since VEGF and VEGFR-2 were expressed in most tumors.

Introduction

The formation of new microvasculature in a process known as angiogenesis is critical for the growth and metastasis of malignant tumors (1). Vascular endothelial growth factor (VEGF) is a protein that plays a key role in tumor angiogenesis (2). Two VEGF receptors are present on endothelial cells, VEGF Receptor-1 (VEGFR-1) and VEGF-2 (VEGFR-2) (3). Both receptors bind to VEGF with high affinity (4,5); however, only VEGFR-2 is capable of mediating angiogenesis (6). Receptor VEGFR-1 is thought to play a regulatory role by decreasing the availability of VEGF to VEGFR-2 (6,7). Vascular endothelial growth factor and VEGFR-2 are associated with tumor cell proliferation (8–10). The simultaneous expression of both molecules on tumor cells may indicate that VEGF has an autocrine function, stimulating tumor cell growth (11); it may also decrease tumor apoptosis (12). In many human malignant tumors the increased expression of VEGF has been correlated with the histologic grade of malignancy (13–15). Moreover, the relationship between the degree of angiogenesis, often expressed as intra-tumor microvessel density (iMVD), and many parameters including tumor proliferation index (PI), an expression of tumor cell proliferation, and tumor apoptotic index (AI), an expression of tumor cell death by apoptosis, have been extensively studied in human cancer (8,10,16,17,18–24).

Mammary tumors comprise 52% of all neoplasms affecting female dogs (25), of which, 41% to 53% are malignant (26). Based on their histologic features, canine mammary tumors can be classified into several types including simple carcinoma, complex carcinoma, and carcinoma arising in a benign tumor (27).

In the veterinary literature, VEGF, VEGFR-2, iMVD, and PI have been evaluated in few canine tumors (16,28–35). However, the relationship between these parameters in canine mammary neoplasia is still unclear, and studying this relationship may aid in understanding their role in the progression of mammary neoplasia in dogs.

We previously evaluated the expression of these markers in canine cutaneous squamous cell carcinomas, trichoepitheliomas (16), and fibrosarcomas (36). The purpose of this study was to evaluate the expression of these markers in canine simple mammary gland adenocarcinomas.

Materials and methods

Tissue selection and histologic grading

Histologic sections previously diagnosed as canine simple mammary gland adenocarcinoma (SMGA), and submitted between 2002 to 2004 to the Prairie Diagnostic Services at the Western College of Veterinary Medicine, Saskatoon, Saskatchewan were evaluated independently by 2 of the authors (ANA and BAK). Twenty-six cases receiving consensus diagnosis were selected and each given a histologic grade from 1 to 3 based on the degree of tubular formation, the presence of mitotic figures, and nuclear pleomorphism according to established criteria (27). Histologic grading was performed independently by the same authors and tumors receiving different histologic grades were reevaluated and a consensus was established.

Immunohistochemical staining for VEGF, VEGFR-2, vWf, and Ki-67

An avidin-biotin-complex (ABC) immunohistochemical staining method was used on formalin-fixed paraffin-embedded tissues (37). Consecutive 5-μm tissue sections were cut from each paraffin block, and then mounted and dried on glass slides. Tissues were deparaffinized in Citrosolv (Fisher Scientific, Ottawa, Ontario), then dehydrated in graded alcohol solutions. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in absolute methanol for 12 min at room temperature. Antigen was retrieved by steaming the tissues in a vegetable steamer for 30 min using 0.01 M sodium citrate solution at pH 6.8. Non-specific protein binding was saturated using 4% horse serum (Invitrogen, Burlington, Ontario) in phosphate buffered saline (PBS) (Invitrogen) for 20 min at 42°C, except when staining for von Willebrand factor where 4% goat serum was used. Primary antibodies consisted of: monoclonal mouse anti-human VEGF121 isoform, 1/20 (NeoMarkers, Fremont, California, USA); monoclonal mouse anti-human VEGFR-2, 1/1000 (Santa Cruz Biotechnology, Santa Cruz, California, USA); polyclonal rabbit anti-human vWF, 1/2000 (Dakocytomation, Mississauga, Ontario); and prediluted monoclonal mouse anti-human Ki-67 (Zymed Laboratories, San Francisco, California, USA). The diluted primary antibodies were applied to tissue sections, and the slides incubated 14 to 18 h at 4°C. A biotinylated polyclonal secondary horse anti-mouse antiserum (Vector Laboratories, Burlington, Ontario) was applied to tissues for VEGF, VEGFR-2, and Ki-67 staining at a dilution of 1/400 for 20 min at 42°C. A 1/400 dilution of biotinylated polyclonal sheep anti-rabbit secondary antiserum was used to stain tissues for vWf. Reagent (ABC) was prepared according to the manufacturer’s directions (Vecastain Elite ABC kit; Vector Laboratories) and applied for 35 min at 42°C. Color development was achieved by applying a 3-3′-diaminobenzine-tetrahydrochloride (DAB) reagent (Sigma, Oakville, Ontario) for 5 min. Tissues were counterstained with hematoxylin (Sigma), rehydrated in graded alcohol, and mounted with coverslips.

When staining tissues for VEGF and VEGFR-2, serial sections of tissues were also stained with omission of the primary antibody and with an isotype matched irrelevant antibody (Neo Markers). Tissue from a canine splenic hemangiosarcoma was used as a positive control for VEGF. Tissue from a canine squamous cell carcinoma, previously known to express VEGFR-2 (16), was used as a positive control for VEGFR-2. Tissue from a normal canine lymph node was used as a positive control for Ki-67. Normal vessels in the surrounding tissue of each tumor were used as a positive control for vWf. Five normal canine skin tissues containing mammary glands were also stained for VEGF, VEGFR-2, and vWf.

Terminal deoxynucleotidyl transferase-mediated 2-deoxyuridine 5-triphosphate end-labeling assay for apoptosis

Apoptosis was detected using the ApopTag Peroxidase In Situ detection kit (Chemicon International, Temecula, California, USA). Briefly, 5-μm tissue sections were deparaffinized and rehydrated as described. Protein was digested with 20 μg/mL proteinase K solution (Invitrogen/VWR, Saskatoon, Saskatchewan) for 15 min at room temperature. Endogenous peroxidase activity was blocked with 3.0% hydrogen peroxide. An equilibration buffer supplied with the kit was applied for 15 s, followed by working strength terminal deoxynucleotidyl transferase enzyme for 1 h at 37°C. The slides were covered with an anti-digoxigenin conjugate and incubated in a humidified chamber for 30 min at room temperature. Color development and counterstaining were as described for immunohistochemical stains. Tissue from a canine lymphosarcoma was used as positive control for the TUNEL assay.

Immunohistochemical scoring for VEGF and VEGFR-2

A score ranging from 0 to 2 was assigned to each tumor section. Tumors were given a score of 0 if there was no staining, a score of 1 if there was pale brown staining and a score of 2 if there was dark brown staining. All tumors were evaluated independently by 2 of the authors (ANA and BAK). In instances of discordant scores, a score was established by consensus.

Evaluation of microvessel density, Ki-67, and apoptosis

Intra-tumor microvessel density was evaluated with antibodies detecting vWf (Figure 1), using the “hot-spot method” (38). Slides were examined at low power (40×) to identify 10 areas with high vessel density followed by re-examination of each area at high-power (400×) and counting of all positively staining structures irrespective of whether or not a vessel lumen was identified. Continuous vessels were counted as one vessel. The average of the 10 fields was determined and was expressed as count per high-power field in each tumor.

Figure 1.

Immunohistochemical staining of endothelial cells for von Willebrand factor antigen in simple mammary gland adenocarcinoma. Note the density of labeled vessels (arrows). Avidin-Biotin-complex diaminobenzidine method, hematoxylin counterstain. Bar = 50 μm.

A similar method was used to evaluate sections stained with Ki-67 monoclonal antibody to demonstrate dividing tumor cells. The tissue sections were first examined at low power (40×) to identify 5 areas with high numbers of positive cells. Within each area, a high power field (400×) was selected and 200 cells were counted. A total of 1000 cells were counted in the 5 high power fields. The average number of the positive cells in the 5 fields represented the PI. The apoptotic index was similarly determined by counting 200 cells in 5 selected areas and averaging numbers of positive cells using the TUNEL assay in five 400× fields.

Statistical analysis

Statistical analysis was performed using Statistix 7 for Windows©2000 (Analytical Software, Tallahassee, Florida, USA). The strength of the association between the 6 variables evaluated (histologic grade, VEGF expression, VEGFR-2 expression, iMVD, PI, and AI) was assessed by Spearman Rank correlation coefficient. Correlation coefficients were interpreted as follows: r = 0, no correlation; 0 < r < 0.1, trivial correlation; 0.1 ≤ r < 0.3, slight correlation; 0.3 ≤ r < 0.5, moderate correlation; 0.5 ≤ r < 0.7, substantial or high correlation; and r ≥ 0.7 very high correlation (39).

The Kruskal-Wallis test was used to test for differences in the median iMVD, AI, and PI among the VEGF expression, VEGFR-2 expression, or histologic grade groups of SMGAs. This test was also used to evaluate the differences in VEGF or VEGFR-2 expression levels among the different histologic grades of SMGAs.

Results

Of the 26 SMGAs, 11 (42.3%) were assigned a histologic grade of 1; 12 (46.2%), grade 2; and 3 (11.5%), grade 3. Vascular endothelial growth factor and VEGFR-2 antigens were detected in 96% and in 100% of the tumors, respectively (Figures 2 and 3). The results for each of the parameters evaluated are shown in Table 1. A high correlation between histologic grade and proliferation index (PI) (r = 0.73), a moderate correlation between VEGF expression and PI (r = 0.42), and a moderate correlation between VEGF and histologic grade (r = 0.33) were found (Spearman rank correlation test). Correlation coefficients between other variables were < 0.3.

Figure 2.

Immunohistochemical staining of tumor cells for VEGF antigen in simple mammary gland adenocarcinoma. Note the dark staining in the cytoplasm of tumor cells (arrows). Avidin-Biotin-complex diaminobenzidine method, hematoxylin counterstain. Bar = 50 μm.

Figure 3.

Immunohistochemical staining of tumor cells for VEGFR-2 antigen in simple mammary gland adenocarcinoma. Note the dark staining in the cytoplasm of tumor cells (arrows). Avidin-Biotin-complex diaminobenzidine method, hematoxylin counterstain. Bar = 50 μm.

Table 1.

Simple mammary gland adenocarcinoma VEGF, VEGFR-2, iMVD, PI, AI, and histological grade scores^a

Tumor identification	Histologic grade	VEGF score	VEGFR-2 score	iMVD count (s)b	PI (s)	AI (s)
1	1	1	1	19.9 (6.5)	15 (4.6)	2.8 (1.5)
2	1	1	2	8.3 (2.3)	3.8 (1.6)	2.6 (1.1)
3	1	2	1	12.2 (3.7)	20.2 (3.7)	17.2 (5.9)
4	1	1	2	6.4 (2.8)	5.8 (3.3)	4 (1)
5	1	1	1	11.9 (6.7)	32.4 (6)	10.2 (2.6)
6	1	1	1	10 (7)	13.4 (5.3)	22.2 (5.8)
7	1	1	2	6.8 (2.8)	18.8 (5.3)	7 (1.2)
8	1	1	2	15.6 (3.7)	26.6 (5.1)	7.4 (2.1)
9	1	1	2	12.1 (5.7)	7 (2.9)	0 (0)
10	1	1	2	11.2 (6.1)	42.8 (8.6)	0 (0)
11	1	2	2	17.2 (7.6)	38 (9.7)	0 (0)
12	2	1	2	16.2 (6.4)	44.4 (8.8)	11.8 (2.6)
13	2	1	2	12.2 (6.4)	16 (5.8)	11.2 (4.4)
14	2	1	2	9 (2.7)	42 (10.4)	3.8 (0.8)
15	2	2	2	16.3 (12.1)	23.4 (4)	23.6 (5.4)
16	2	1	1	10.5 (4.7)	41.8 (6.1)	6.4 (3.5)
17	2	2	1	4.7 (1.7)	54.4 (2.7)	8.6 (2.9)
18	2	1	1	7.5 (3.1)	42 (4.1)	4.8 (1.5)
19	2	2	2	13.8 (5.3)	46.4 (4.3)	11.6 (1.9)
20	2	1	1	16.2 (5.4)	58.4 (9.1)	12.6 (3)
21	2	2	2	13 (5.9)	44.8 (5.5)	5.2 (1.3)
22	2	1	2	16.2 (4.3)	25 (4.9)	2 (0.7)
23	2	2	2	9.4 (3.3)	59.6 (8.5)	3.6 (1.1)
24	3	2	1	28.5 (6.5)	47.2 (5.2)	1.8 (0.8)
25	3	1	1	11.9 (2.6)	66.6 (10.1)	10.2 (1.9)
26	3	2	1	8.8 (3.7)	49.2 (8.4)	0 (0)

^aAI — apoptotic index, PI — proliferation index, VEGF — vascular endothelial growth factor, VEGFR-2 — VEGF receptor-2, iMVD — intra-tumor microvessel density.

^bs — standard deviation.

There was a significant difference (P = 0.0013) in median PI among the different tumor histologic grades (Kruskal-Wallis test). The median PI increased as the histologic grade increased; however, the difference in median PI was statistically significant only when grade 3 or 2 tumors were compared to grade 1 tumors. The median PI of grade 3 tumors was higher than the median PI of grade 2 tumors but the difference was not significant (see Table 2 for details).

Table 2.

Comparison of median PI, AI, and iMVD between the 3 histological grade groups in canine SMGA^a

Histologic grade
Parameter	I	II	III	P-value
Median PI	18.8	43.2	49.2	0.0013b
Median AI	4.0	7.5	1.8	0.22
Median iMVD	11.9	12.6	11.9	0.89

^aSMGA — simple mammary gland adenocarcinoma, PI — roliferation index, AI — apoptotic index, iMVD — intra-tumor microvessel density.

^bSignificant P-value. Grades III and II had a significantly higher median PI than grade I. The difference in median PI between grades II and III was not statistically significant.

There were no significant differences in median iMVD, PI, or AI among the different expression levels of VEGF, VEGFR-2, and no significant difference in median iMVD, or AI among the histologic grade groups (Kruskal-Wallis test). In normal skin tissues, VEGFR-2 expression was detected in normal mammary epithelium as well as dermal fibroblasts, while VEGF expression was weak or absent. Normal vessels within mammary tissue stained positive for VEGFR-2 and vWF.

Discussion

In this study, 26 canine simple mammary gland adenocarcinomas were histologicaly graded and immunohistochemically evaluated for the expression of VEGF and VEGFR-2, iMVD, PI, and AI.

We found a moderate correlation between VEGF and histologic grade. In agreement with this, a previous study found a significant difference in the number of VEGF positive cells among the different histologic grade groups of malignant canine mammary tumors (28). Only one study previously examined VEGFR-2 expression in canine mammary tumors (34). Several histologic subtypes were evaluated including malignant mixed tumors and tumors with squamous differentiation. Contrary to the present study, a significant difference in VEGFR-2 expression was found among the different histologic grades, with highest levels being found in less differentiated tumors (34). The absence of correlation between VEGFR-2 and histologic grade in SMGAs in this study may be related to sample size, or may indicate that VEGFR-2 is not related to histologic grade. Additional studies are needed to clarify the role of VEGF in mediating angiogenesis, and the relationship between VEGF and VEGFR-2, and histologic grade in canine SMGAs.

Ki-67 is a nuclear protein expressed in proliferating cells, which has been evaluated in many canine tumors (35,40–45). High index values of Ki-67 were positively correlated with metastasis, death from neoplasia, low disease-free survival rate and low overall survival rates in canine mammary tumors (45). Ki-67 expression is also a useful prognostic indicator in canine mast cell tumors (40). The finding, in the present study, of a significant difference in PI among the different histologic grade groups is in agreement with previous studies (40,45). The lack of a significant difference in PI between grade 3 and 2 tumors could be due to the small sample size.

The detection of VEGF and VEGFR-2 in most canine SMGAs, and the presence of a moderate correlation between VEGF expression and PI may indicate that VEGF promotes tumor growth through an autocrine loop. Similar to our finding in dogs, human breast cancer cells have been shown to have an autocrine VEGF activity (46). Such activity may provide a survival signal for tumor cells through the activation of the PI3/Akt pathway (47). Using a breast carcinoma cell line, the reduction of VEGF expression resulted in a significant decrease in the basal activity of PI3-kinase and, eventually, induced apoptosis (47). Vascular endothelial growth factor autocrine signaling may also result in the increased expression of the chemokine receptor CXCR4 on the surface of tumor cells (48). The binding of this receptor to its ligand, stromal-derived factor-1, stimulates tumor cell migration (49).

The development of angiogenesis in breast cancer is complex and depends on several angiogenic growth factors (50). The assessment of iMVD in breast cancer may relate to histologic grade and prognosis (51). Only 3 studies evaluated iMVD in canine mammary tumors (35,52,53), 2 of which found a correlation between iMVD and histologic grade (35,53). In the third study, iMVD was not correlated with patient age, sex, reproductive status, clinical tumor stage, or histologic type (52). The absence of correlation in this study may indicate that other angiogenic factors are more important than VEGF in promoting angiogenesis in this particular histologic subtype. The disparity between this study and other canine and human studies, however, may also relate to differences in markers used to evaluate iMVD (54), or could be due to the small sample size used herein.

Normal rodent mammary epithelial cells may express VEGF and VEGFR-2 (55). The expression of both molecules is important for the growth of mammary gland during pregnancy and lactation (55). Mammary stromal cells may also express VEGF (56). The expression of VEGFR-2 in normal mammary stromal cells is unknown. The normal mammary tissue used in this study belonged to a nonpregnant and nonlactating bitch. While VEGFR-2 expression was found in normal mammary epithelial and stromal cells, the expression of VEGF in these 2 cell populations was minimal or absent. This expression pattern may suggest that VEGF signaling may be important only during mammary development throughout pregnancy and lactation.

In conclusion, it was hypothesized that VEGF, VEGFR-2, iMVD, and PI would correlate with histologic grade and hence with malignancy of canine SMGAs. However, only VEGF expression and PI correlated with histologic grade. It was also concluded that VEGF may promote tumor growth through an autocrine loop. Since most tumors were positive for VEGF and VEGFR-2, these tumors may respond to therapy targeting these molecules.