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Year : 2020  |  Volume : 63  |  Issue : 2  |  Page : 205-209
Microvessel density and vascular endothelial growth factor receptors in breast carcinoma under the influence of rapamycin and platelet factor 4

1 Department of Pathology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, Kubang Kerian, Kelantan, Malaysia
2 Department of Chemical Pathology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, Kubang Kerian, Kelantan, Malaysia
3 Department of Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, Kubang Kerian, Kelantan, Malaysia

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Date of Web Publication18-Apr-2020


Background: Vascular endothelial growth factor receptors (VEGFRs) are major endothelial growth factor receptors that influence the growth of a tumor. Microvessel density ( MVD) is the quantification method of various aspects of tumor vasculature that indicates angiogenic activity. This study aims to analyze the correlation between MVD to the expression of VEGFRs on breast cancer tissue. Materials and Method: A total of 60 N-methyl-N-nitrosourea (MNU)-induced breast carcinomas in rats were suppressed by using antiangiogenic drugs. The rats were then sacrificed, and the tumor was fixed in 10% formalin, paraffin embedded, and immunohistochemistry stained using VEGFRs and CD34. Result: One-way ANOVA test showed a significant difference in all markers that have been used (P < 0.05) on MNU-breast tumor treated with rapamycin (M= 90.1664, SD= 7.4487), PF4 (M= 93.7946, SD= 7.1303) and rapamycin + PF4 (M= 93.6990, SD= 1.8432). We obtained a significant reduction of MVD count on breast carcinoma for rapamycin group (M= 25.6786, SD= 9.7075) and rapamycin + PF4 group (M= 30.5250, SD= 13.6928) while PF4 group (M=47.7985, SD=4.8892) showed slightly increase compared to control (M= 45.1875, SD= 4.4786). There was a moderately strong, positive correlation between angiogenic markers; Flt-1 (r= 0.544, n=60, P < 0.005) and Flt-4 (r= 0.555, n= 60, P < 0.005) while Flk-1 (r= 0.797, n= 60, P < 0.005) showed a strong, positive correlation with MVD. Conclusion: MVD was strongly correlated to the VEGFRs expression on breast carcinoma.

Keywords: Breast, MNU-induced breast carcinoma, microvessel density, vascular endothelial growth factor receptors

How to cite this article:
Muhammad Sakri MS, Abdul Rahman WF, Tengku Din TA, Idris FM, Jaafar H. Microvessel density and vascular endothelial growth factor receptors in breast carcinoma under the influence of rapamycin and platelet factor 4. Indian J Pathol Microbiol 2020;63:205-9

How to cite this URL:
Muhammad Sakri MS, Abdul Rahman WF, Tengku Din TA, Idris FM, Jaafar H. Microvessel density and vascular endothelial growth factor receptors in breast carcinoma under the influence of rapamycin and platelet factor 4. Indian J Pathol Microbiol [serial online] 2020 [cited 2021 Dec 1];63:205-9. Available from: https://www.ijpmonline.org/text.asp?2020/63/2/205/282697

   Introduction Top

Rapid development and proliferation of a tumor have been associated with a broad range of factors of neovascular.[1],[2] Several studies supported an important role played by angiogenic activities which correspond with the aggressiveness of a tumor[3] and was reported to have an association with the types of a tumor.[4] Interestingly, the recent study had found that tumor progression and metastases are highly dependent on angiogenesis as well.[5] It has been well known that angiogenesis played an essential role in supporting the growth of a tumor with gene and proteins involvement.[6] The absence of angiogenesis is severely restricting the tumor to grow beyond 1–2 mm.[7]

Quantification of various aspects of tumor vasculature might provide an indication of angiogenic activity. The often-quantified aspect of tumor vasculature is MVD.[8] CD34 is the marker for endothelial cell and was found to be the most sensitive marker to demonstrate microvessel.[9] The previous studies have demonstrated MVD as a prognostic indicator for a wide range of cancers. MVD provides information about the degree of angiogenic activity thus help to guide decision for treatment.[10],[11] Based on this assumption, quantification of microvessel density is thought to constitute a surrogate marker for the efficacy of antiangiogenic agents as well as a means by which to assess which patients are good candidates for antiangiogenic therapy prior to treatment.

Most of the studies on MVD had reported positive correlations between MVD and tumor recurrence, although some reports have no or even negative correlations between these endpoints. Some of the discrepancies may be explained by the fact that the details of the methodology had been used to assay MVD can influence its value as a prognostic indicator. Despite these technical issues, MVD as measured by the hotspot method of Weidner is a valuable prognostic indicator for a wide range of tumor types.[12]

Vascular endothelial growth factor (VEGF) exists in multiple isoforms which have distinct physical and biological properties.[13] They elicit their angiogenic responses via three specific tyrosine kinase receptors known as: VEGFR-1 [also referred as fms-like tyrosine kinase 1 (Flt-1)], VEGFR-2 [also known as kinase insert domain-containing receptor (KDR) or its murine homolog, fetal liver kinase 1 (Flk-1)] and VEGFR-3 [also known as fms-like tyrosine kinase 4 (Flt-4)].[13],[14] The receptors share 45% homology sequences and possess similar organizational structure composed of seven immunoglobulin-like domains in the extracellular domain, a single hydrophobic transmembrane domain, and an intracellular cytoplasmic domain with tyrosine-kinase activity essential for signal transduction.[15]

Flt-1 is a positive regulator of monocyte and macrophage migration and has been described as a positive and negative regulator of Flk-1 signaling capacity. Negative regulation is exerted, at least in part, by an alternatively spliced soluble Flt-1 variant that binds to VEGF and thereby prevents VEGF from binding to Flk-1.[16] Flk-1 is implicated in all aspects of normal and pathological vascular-endothelial-cell biology, whereas Flt-4 is important for lymphatic endothelial cell development and function. Recently, tumor therapies that are based on neutralizing anti-VEGF antibodies and small-molecular-weight tyrosine-kinase inhibitors that target the VEGFRs have been developed. These new strategies for tumor treatment show the clinical relevance of inhibiting VEGF signal-transduction pathways that are exaggerated in pathological angiogenesis.

   Materials and Method Top

Animal procedures

Sixty females Sprague Dawley (SD) rats were obtained from the Animal Research and Services Center and the ethical clearance was obtained from the Animal Ethics Committee. The rats were kept at the Animal House Unit. Caging and rat's handling was performed in accordance with good laboratory practice which had been outlined by the Animal House. The rats were kept in groups of six and were fed with a standard laboratory diet.[15],[17] The rats were divided into 4 groups and each group was given different interventions drug(s); a control (untreated) group 1 (n = 15), rapamycin-treated group 2 (n = 15), platelet factor 4-treated group 3 (PF4) (n = 15), and drug combination of rapamycin and PF4-treated group (rapamycin+PF4) (n = 15).

MNU, rapamycin, and PF4 preparation and tumor induction

Sigma Aldrich provided N-methyl-N-nitrosourea (MNU) in the form of crystallizing. MNU was dissolved in freshly-prepared 0.9% normal saline prior induction process. The MNU was injected intraperitoneally at a dose of 70 mg/kg body weight into 21-day-old rats[15] and were weighed and palpated for mammary tumor lesions weekly. The daily inspection and palpation were done to monitor the onset of tumors at the mammary regions. Rapamycin was mixed with absolute ethanol and had been diluted in mixtures of 10% polyethene glycol (PEG)-400, 8% ethanol: 10% Tween-80 to a final concentration of 20 μg/0.2 ml, while PF4 was dissolved in normal saline with a final concentration of 20 μg/0.2 ml.

Study design

The study was conducted viain vivo which involved the induction of MNU carcinogen in 60 female Sprague Dawley rats to induce breast carcinoma. After a breast tumor developed within 40 days of induction of MNU carcinogen, the tumor was then suppressed using angiogenic inhibitor either single treatment using platelet factor 4 (PF4) or rapamycin or drug combination in which taking both PF4 and rapamycin by intratumoral injection. After 5 days of intervention, the rats were terminated, while for the control group, the rats were terminated after 45 days of tumor induction and immunohistochemistry stained using CD34. All results were then statistically analyzed using SPSS version 21 software.

Immunohistochemistry staining and scoring

Tissue sections were dewaxed and antigen retrieval process were performed in citrate buffer by using a pressure cooker. The sections were incubated with primary antibody for 1 hour at room temperature. UltraVision ONE Large Volume Detection system HRP Polymer kit were applied to the tissue sections with added DAB Plus substrate system (Cat. No. TL-125-PHJ, LabVision, USA) and counterstained with Hematoxylin to visualise the reactivity.

Expression of all antibodies was assessed using a semi-quantitative scoring system developed by (Klein et al. 2001).[18]

This scoring system is the multiplication of a proportion score and an intensity score. Each sample was scored for the percentage of stained tumor cells (0, absence of staining; 1, <30% of tumor cells stained; 2, 30%–60% of tumor cells stained; 3, >% of tumor cells stained) and the intensity score estimates the average staining intensity of positive tumor cells: 0 = negative; 1 = weak; 2 = moderate; and 3 = intense. Both scores were multiplied together to give a final numerical score ranging from 0 to 9. The scoring was determined according to no staining (0), weak (1, 2, or 3 points), mild (4 points), and strong (6, 7, or 9 points). Scoring was performed in a double-blind manner by three independent investigators. Any disagreements were resolved by discussion to obtain a final score.

Statistical analysis

The expressions of CD34 between experimental groups were determined by Pearson correlation test. The result was determined using the Pearson correlation coefficient (r) ranges from −1 to +1.

   Result Top

The analysis of VEGFRs expression on breast carcinoma

One-way ANOVA test shows a significant difference (P < 0.05) in all experimental groups. Rapamycin (M = 90.1664, SD = 7.4487) on Flt-1 marker shows better effect compared to PF4 (M = 93.7946, SD = 7.1303) and rapamycin + PF4 (M = 93.6990, SD = 1.8432) treatment [Figure 1]. Rapamycin (M = 89.9043, SD = 7.2542) also showed better antiangiogenic effects than PF4 (M = 98.9175, SD = 2.0487) and rapamycin + PF4 (M = 91.2330, SD = 4.0934) treatment on Flk-1 which also known as true VEGFR stimulant. On the other hand, PF4 treatment (M = 98.9175, SD = 0.6061) seems ineffective against VEGFR markers even with the combination with rapamycin. The rapamycin + PF4 group was determined to give the best effects on Flt-4 marker compared to rapamycin and PF4 groups.
Figure 1: Expression of VEGFRs signaling protein receptor on rat's mammary carcinoma. The VEGFRs expressions are highly reflected in the efficacy of treatment given to suppress angiogenesis via VEGFRs signaling blockage. All treatment groups showed reduced VEGFRs expressions withP value is <0.05

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The analysis of microvessel density (MVD) expression

The blood vessel is very crucial for tumor survival. To further investigate the dependence of tumor cells to the blood vessel, we do an investigation on MVD by counting blood vessel present [Figure 2] under high objective magnification of the microscope. There were 3 hotspots selected based on the highest number of blood vessels present and observed by 3 persons. From the findings, rapamycin found to be a potent angiogenic inhibitor. The tissues appeared to have low blood vessels number [Figure 3]. The result obtained also indicated that rapamycin is preventing the formation of a new blood vessel through the mTOR pathway. Rapamycin + PF4 group was also found to act as a good inhibitor of angiogenesis which blocked nearly 30% of new vessel formation compared to control. However, PF4 did not show much effect on suppressing new blood vessel formation as expected. Some of the samples found to have as much blood vessel as found in the control group.
Figure 2: Expression of CD34 immunostaining on 100× (a) and 400× (b) power magnification. Each hotspot has chosen based on an accumulation of vessel stained. MVD was counted via high power magnification (400×). The arrow shows stained vessels

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Figure 3: MVD count on MNU-induced mammary carcinoma under the influence of rapamycin, PF4, and combination of rapamycin and PF4. The result showed significant vascular suppressed on rapamycin and drug combination groups

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The correlation between angiogenic marker expression and MVD

To find the correlation between VEGFRs and MVD, the expression for each angiogenic marker: Flt-1, Flk-1, and Flt-4 was compared to the count of blood vessels stained by CD34. We found that all angiogenic markers were significantly correlated to the blood vessels obtained [Table 1]. There was a moderately, positive correlation between angiogenic markers with MVD in all treatment groups: Flt-1 (r = 0.544, n = 60, P < 0.005) and Flt-4 (r = 0.555, n = 60, P < 0.005), while Flk-1 (r = 0.797, n = 60, P < 0.005) showed a strong, positive correlation with MVD. These findings explained that the tumor growth is highly dependent on angiogenesis. This finding also suggests that MVD count can provide valuable information regarding the prognostic indicator of a tumor.
Table 1: Correlation test between VEGFRs and the CD34 expression on MNU-induced rat's mammary carcinoma. All VEGFRs' markers strongly correlated to CD34 expression

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   Discussion Top

Since tumor angiogenesis demonstrated a crucial role in the invasiveness of mammary tumor progression, the antitumor and antiangiogenic effects of rapamycin and PF4 as single agents were determined and their nature of interactions while being combined was evaluated. It is exciting to know that many predecessor studies often focus on the mTOR signaling pathway in elaborating the antitumor effects of rapamycin. However, we had found that antitumor and antiangiogenic effects of rapamycin were elicited via VEGF signaling cascade. Rapamycin was determined to react well on all angiogenic receptors: Flt-1, Flk-1, and Flk-4 which resulted of markers' suppression. These were proved at both gene and protein levels. The treatment with rapamycin also gave us neo-angiogenesis blockage and resulted in significant inhibition of mammary tumor progression. With the suppressions of angiogenic markers and down-regulation of the genes, the tumor was forced to reduce its capacity due to the lack of nutrient and oxygen. This will lead to reduce the size of a tumor and reduce the aggressiveness of a tumor. This finding had driven us to a conclusion that rapamycin is an excellent angiogenic inhibitor which is useful to block neo-vasculation and good to control the aggressiveness of a tumor.

Meanwhile, decades of studies have shown the effectiveness of PF4 as a potential endogenous angiogenesis inhibitor in murine melanoma, human colon carcinoma, lung metastasis, brain carcinoma, head and neck squamous carcinoma as well as HUVEC cell line.[19],[20],[21],[22],[23] Nonetheless, the effect of PF4 in a breast tumor notably in the MNU-induced mammary tumor was determined to cause only slight effects in vivo. These findings disagree with the previous findings of PF4in vitro andin vivo which showed that it is a potent antiangiogenic drug.[24] Hence, the effect of the PF4 drug was investigated, and findings showed that the drug did not inhibiting neovascularization but promoted and exacerbated the mammary tumor progression. In fact, the tumor only showed transient regression and re-grows rapidly after 2 days post-treatment, then transformed to a more aggressive phenotype which reflected by a marked increase in VEGF expression at gene and protein levels. Unlike earlier reports which implicated that PF4 elicited its antiangiogenic effect via direct interaction with VEGF receptors, we found that PF4 treatment did not effectively inhibit all VEGF receptors expression both at gene and protein levels. From a rather mechanistic perspective, a possible explanation for this phenomenon could be the fact that PF4 selectively binds to VEGF-dependent heparin binding such as VEGF165 consequently restricting receptor dimerization and activation.[25],[26] Moreover, studies also showed that PF4 was unable to inhibit the VEGF isoforms which lack heparin-binding capacity such as VEGF121, to the VEGF receptor of the cells or notably to the Flk-1.[27] However, further investigations will be necessary to elucidate the mechanism by which PF4 exert its effect in this tumor model.

The interaction of two or more drugs for the same purposes will increase in the effects of one or both drugs to create synergism effects. A recent study found that there is synergism exists between VEGF and PLGF in vivo, especially during the pathological situations as evidenced by impaired tumorigenesis and vascular leakage in Plgf -/- mice.[28] Based on our study, the expression of VEGFRs treated by rapamycin group found to produce a better reduction of expression compared to the rapamycin + PF4 group. In contrast, rapamycin + PF4 group demonstrated a significant tumor regression. The regression process was thought to be the effect of rapamycin drug.

On the other hand, our study had found that rapamycin + PF4 treatment had led to down-regulation of all VEGFRs expression. Based on the evidence gathered, we suggested that rapamycin is neither synergistic nor additive with PF4. Indeed, PF4 might be an antagonist toward the rapamycin activity as an antitumor instead of an antiangiogenic agent.

The analysis of MVD is associated with the onset of tumor angiogenesis; a hallmark of neoplastic transformation1 and a valuable prognostic indicator for a wide range of tumor types.[12],[28] Based on the experiment, we found that rapamycin and rapamycin + PF4 were proven effective against angiogenic activity in the cancer environment. In contrast, PF4 did not prove effective, but some of a tumor in the group became more aggressive and more vessels detected.

In additional, microvessel density total mean count reflects inter-capillary distance, and thus it is influenced by both angiogenic and non-angiogenic factors such as VEGF, fibroblast growth factor (FGF), transforming growth factor (TGF), and oxygen supply.[29] In our study, we found that mTOR not only plays a pivotal role in tumor cell proliferation but also stimulates the increase of angiogenic factors' secretion such as VEGF and FGF. Interestingly, blocking mTOR resulted in significant reduction in the number of blood vessel together with the reduction of the aggressiveness of a tumor.

From our study, we found the outstanding newly formed blood vessel in the untreated control group. This finding coincides with the previous study which stated aggressive ductal carcinoma lesions with increased VEGF expression; MVD count had been determined to be high.[8] MVD had been determined to be high with aggressive ductal carcinoma in situ lesions where there was an increment of vascular endothelial growth factor expression.[30] Similarly, our findings have seen a significant increment of MVD in greater expression of VEGF and aggressive tumor. On the other hand, antiangiogenic drug that has been used for treatment gave effects on MVD which were found to be less in the count with less of VEGF expression obtained. This finding supported the previous finding by Schneider and Sledge (2007), who found the association between MVD and angiogenesis. In the present finding, we found that there were significant correlations between the VEGF receptors: Flt-1, Flk-1, and Flt-4 to the total MVD count thus explained the correlation between MVD and angiogenesis. These findings had driven us to the conclusion that the aggressiveness of a tumor is highly dependent on blood vessel formation.

   Conclusion Top

In conclusion, we found that MVD was positively correlated to the VEGFRs expression and suppression on mTOR leads to a significantly reduced VEGF production thus suppressing angiogenesis. This finding helps to improve the understanding of cancer therapy by targeting angiogenesis.

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Conflicts of interest

There are no conflicts of interest.

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Correspondence Address:
Wan Faiziah Wan Abdul Rahman
Department of Pathology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJPM.IJPM_496_19

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