Silencing of S100A4, a metastasis-associated protein, in endothelial cells inhibits tumor angiogenesis and growth

Endothelial cells express S100A4, a metastasis-associated protein, but its role in angiogenesis remains to be elucidated. Here we show that knockdown of S100A4 in mouse endothelial MSS31 cells by murine specific small interference RNA (mS100A4 siRNA) markedly suppressed capillary-like tube formation in vitro, in early stage after the treatment, along with down- and up-regulation of some of the pro-angiogenic and anti-angiogenic gene expression, respectively. Of particular note is that intra-tumor administration of the mS100A4 siRNA in a human prostate cancer xenograft significantly reduced tumor vascularity and resulted in the inhibition of tumor growth. These findings show that S100A4 in endothelial cells is involved in tube formation, and suggest its potential as a molecular target for inhibiting tumor angiogenesis, which warrants further development of endothelial S100A4-based strategies for cancer treatment.

Introduction S100A4, a member of the S100 family of EF-hand calcium-binding proteins, is involved in the regulation of a variety of biological processes, including tumor progression and metastasis [1]. It is highly expressed in various metastatic tumor cells and its elevated expression is associated with the poor prognosis of different types of human cancers [2][3][4][5][6][7]. The involvement of S100A4 in the promotion of tumor metastasis has been demonstrated by several approaches [8][9][10][11]. Furthermore, recent studies on mice that lack the S100A4 gene [12], and on mice carrying tumor cells subjected to siRNA-mediated S100A4-downregulation [13][14][15][16][17][18], have shown suppression of tumor development and metastasis formation. Although the precise mechanism by which S100A4 stimulates invasion and metastasis remains unknown, an extracellular role for S100A4 suggested by studies showing the secretion of S100A4 by cancer cells and the presence of S100A4 in the serum of cancer patients is intriguing [19]. In fact, the exogenous addition of S100A4 stimulates endothelial cell motility in vitro [20] and induces corneal neovascularization [19] and metastasis formation in vivo [21]. On the other hand, little is known about the effect of endothelial S100A4 on angiogenesis. We previously reported that S100A4 is expressed in both tumor cells, and endothelial cells [22,23]. Although endothelial cells have not been shown to secrete S100A4, and its biological function in these cells is unknown, the endothelial S100A4 may be pertinent for the regulation of tumor angiogenesis and metastasis, and thus an attractive target for cancer therapy.
In this study, we used siRNA-mediated depletion and DNA microarray analysis to investigate the function of S100A4 in endothelial cells. Furthermore, the effect of S100A4 knockdown on angiogenesis and tumor growth was examined in a xenograft cancer model. The results suggest that S100A4 in endothelial cells is involved in tube formation and thus siRNA-mediated inhibition of endothelial S100A4 could provide an effective anti-tumor RNAi medicine.

Transfection of siRNA in vitro
After 48 h of siRNA transfection using DharmaFECT No.1 (GE Healthcare, Waukesha, WI, USA), MSS31 cells were seeded on ECM-gel (SIGMA-Aldrich, St. Louis, MO, USA) thin-coated center-well dishes (60 mm, BD Falcon, Franklin Lakes, NJ, USA) at a cell density of 4 9 10 4 cells/cm 2 and cultured in the presence of 50 ng/ml HGF (PeproTech, Rocky Hill, NJ, USA). Capillary formation was assessed after 16 h of Matrigel culture [31]. RNA samples were extracted from transfected cells before seeding for realtime PCR analysis of S100A4 mRNA, using ISOGEN and platinum SYBR green qPCR superMix-UDG (Invitrogen/ Life Technologies).
Cell adhesion and wound healing assay Migration ability was determined using a cell adhesion and a wound healing assay. After 48 h of S100A4 siRNA or negative control siRNA transfection, MSS31 cells was plated on 24-muti-well plate (Nunc, Thermo Scientific, Japan) at a cell density of 4 9 10 4 cells/cm 2 and then counted the number of adhered cells at 30 min, 1 and 3 h. siRNA-transfected MSS31 cells were grown on 60 mm plates. After the cells reached sub-confluence, the cells were pretreated with mitomycin C (10 lg/ml) for 30 min and then the cells were wounded by scraping the monolayer and grown in medium for 16 h. The width of the wound was measured. Three different locations were visualized and photographed under a phase-contrast inverted microscope (ECLIPSE1000, Nikon, NY, USA). Data was represented as a percent of wound healing.

Real-time PCR analysis
Total RNA was extracted from cells by ISOGEN (Nippon Gene, Tokyo, Japan) and treated with DNase I (Takara, Otsu, Japan). Five lg of total RNA was used to produce cDNAs with oligo (dT) 12 primer by superscript III RNA polymerase (Invitrogen/Life Technologies). cDNA was diluted five-fold and used for quantitative PCR. For quantitation, aliquots of 5 ll of cDNA samples were subjected to quantitative PCR in 50 ll reactions using Platinum Quantitative PCR SuperMix-UDG (Invitrogen/Life Technologies) and Assays-on-Demand TaqMan primers/ probe sets (Applied Biosystems, Foster City, CA, USA) specific for target genes (Supplementary Table S1). Reactions were carried out using the ABI PRISM 7700 sequence detection System (Applied biosystems). The reactions were incubated at 50°C for 2 min, then heated to 95°C for 2 min followed by 45 cycles of 30 s at 95°C, 15 s at 60°C, and 20 s at 72°C. The GAPDH gene and b-actin gene were used as an internal control.

Microarray analysis
RNA quantity and quality were determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), as per manufacturer's instructions. Total RNA was amplified and labeled with Cyanine 3 (Cy3) using Agilent Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies) according to the manufacturer's instructions. Briefly, 100 ng of total RNA was reversed transcribed to doublestrand cDNA, using a poly dT-T7 promoter primer. Primer, template RNA and quality-control transcripts of known concentration and quality were first denatured at 65°C for 10 min, then incubated for 2 h at 40°C with 5X first strand Buffer, 0.1 M DTT, 10 mM dNTP mix, and AffinityScript RNase Block Mix. The AffinityScript enzyme was then inactivated at 70°C for 15 min. cDNA products were used as templates for in vitro transcription to generate fluorescent cRNA. cDNA products were mixed with a transcription master mix in the presence of T7 RNA polymerase and Cy3 labeled-CTP and incubated at 40°C for 2 h. Labeled cRNAs were purified using QIAGEN's RNeasy mini-spin columns and eluted in 30 ll of nuclease-free water. After amplification and labeling, cRNA quantity and cyanine incorporation were determined using a Nanodrop ND-1000 spectrophotometer and an Agilent Bioanalyzer. For each hybridization, 1.65 lg of Cy3 labeled cRNA was fragmented, and hybridized at 65°C for 17 h on an Agilent Mouse GE 4x44K v2 Microarray (Design ID: 026655). After washing, microarrays were scanned using an Agilent DNA microarray scanner. Intensity values of each scanned feature were quantified using Agilent feature extraction software version 10.7.3.1, which performs background subtractions. Features that were flagged as having no errors (present flags) were used and features that were not positive, not significant, not uniform, or not above the background level were excluded. Features that were saturated and any population outliers (marginal and absent flags) were also excluded. Normalization was performed using Agilent GeneSpring GX version 11.0.2. (per chip: normalization to 75 percentile shift; per gene: normalization to median of all samples). There were 39,429 probes on the Agilent Mouse GE 4x44K v2 Microarray (Design ID: 026655), excluding control probes. The altered transcripts were quantified using the comparative method. A C twofold change in signal intensity was designated as a significant difference in gene expression.

Animals
Animal experiments were performed in compliance with the guidelines of the Institute for Laboratory Animal Research at the National Cancer Center Research Institute.
The mice were maintained under specific pathogen-free conditions with a 12-h light-dark cycle.

Study of xenografted tumors
Eight-to 11-week-old male athymic nude mice (CLEA Japan, Osaka, Japan) were used for all experiments. Anesthetized animals were subcutaneously injected with 3 9 10 6 PC-3M-Luc C6 cells in 100 ll sterile Dulbecco's PBS [30]. Nine days after subcutaneous inoculation of PC-3M-Luc C6 cells into mice (hereinafter referred to as day 1), tumors were treated twice with 50 lg siRNA/atelocollagen complex (day 1 and 3). After the treatment, tumor volume was measured at day 9, 12, and 15. Tumor size was measured with calipers and tumor volume was calculated by ab 2 /2, where a and b are the lengths of the long and short axes, respectively. Number of animals was 4 in each group and the experiment was performed twice.

Imaging of tumor angiogenesis
Day 9 after the siRNA treatment, signals of AngioSense-IVM-750 were measured using fluorescence molecular tomography (FMT, VisEn Medical/PerkinElmer Inc., Waltham, MA, USA). AngioSense-IVM-750 is a 250 kDa macromolecule that freely circulates while staying confined to the intravascular space. Relative value of angiogenesis of mS100A4 siRNA-treated tumor was measured when the negative siRNA control was set to 1.0 and the data was represented as relative angiogenesis. Blood vessels in tumors monitored by Angiosence day 9 after the siRNA treatment, tumor vessels were also visualized using AngioSense-IVM-680 (ViSen medical) by intravenous administration of 50 ll per mouse. AngioSense-IVM-680 is a near-infrared labeled fluorescent macromolecule that remains localized in the vasculature for extended periods of time and enables imaging of blood vessels and angiogenesis. Fifteen min after the dye administration, the vessel Fig. 2 Anti-angiogenesis activity of S100A4 siRNA in vitro. MSS31 cells were transfected with murine S100A4 siRNA (mS100A4 siRNA) or control siRNA. Two days after transfection, 9.9 9 10 4 MSS31 cells were seeded on ECM-gel thin-coated center-well dishes. RNA samples were extracted from transfected cells before seeding for real-time PCR analysis of S100A4 mRNA. HGF-induced capillary formation was assessed 16 h after Matrigel culture. a Relative expression of mS100A4 by real-time PCR analysis. *P = 0.01. b Western blot analysis of the expression of S100A4 in cells 24 h after siRNA transfection. Rabbit polyclonal anti-S100A4 antibody (Abcam, ab27957) and rabbit monoclonal anti-b actin antibody (Millipore, clone C4/MAB1501) were used.

Measurement of caspase-3/7 activity in vitro
Caspase-3/7, which plays key effector roles in apoptosis, was determined with the Apo-ONE Homogeneous caspase-3/7 assay (Promega) according to the manufacturer's instructions. Cells were incubated with the Apo-ONE caspase-3/7 assay reagent for 1.5 h at room temperature, and the fluorescence was then measured at 485Ex/535Em with a Wallac multi-label counter.
Double-immunostaining of CD31 and S100A4 B16-BL6 cells (1 9 10 6 cells) were injected subcutaneously into C57BL/6 mice (6-7 weeks of age, Nippon SLC, Shizuoka, Japan). Fourteen days after the injection, tumor tissues were removed, embedded in a tissue-freezing medium, and then cut into 5 lm-thick sections. For doubleimmunostaining of CD31 and S100A4, tissue sections were fixed in acetone for 10 min. The samples were blocked with PBS containing 0.1 % bovine serum albumin and 2 % goat serum and then incubated with a mixture of rat antimouse CD31 antibody (Pharmingen, San Diego, CA, USA) and rabbit anti-S100A4 antibody [23]. The sections were washed three times with PBS, and incubated with a mixture of TRITC-labeled goat anti-mouse IgG and FITC-labeled goat anti-rabbit IgG. After rinsing with PBS, the sections were mounted in 50 % glycerol in PBS containing 1 mg/ml p-phenylenediamine to inhibit photobleaching, and were observed under a confocal laser scanning microscope (Fluoview, Olympus, Tokyo, Japan). Using NIH Image J 1.42q software (http://rsb.info.nih.gov/ij), a ratio of S100A4 pixel values to CD31 pixel values was calculated for each image (n = 6) for determination of S100A4/CD31 double-positive areas.

Statistical analyses
Statistical analyses were conducted using Student's t test for in vitro screening of cell capillary morphogenesis and proliferation and evaluation of in vivo angiogenesis. A P value of 0.05 or less was considered significant.

Inhibition of capillary formation in endothelial cells by S100A4 siRNA
We first examined whether endothelial cells of tumor microvessels express S100A4. For this, we immunostained the microvessels in tumor tissues formed by B16-BL6 melanoma cells that express little S100A4 with anti-CD31 and anti-S100A4 antibody (Fig. 1, Supplementary Fig. S1). The results showed that there were S100A4-positive and -negative CD31 ? endothelial cells (arrows and arrowheads in Fig. 1, panels c and f). Quantification of each S100A4 ? and CD31 ? area in double-stained tissue sections showed that approximately half (49.3 ± 29.5 %, n = 6) of CD31 ? endothelial cells was S100A4-positive. These results suggest that there exist subpopulations of endothelial cells in tumors that might, or might not, be primed for angiogenesis. This prompted us to examine the role of S100A4 in angiogenesis and, to this end, we tested the effect of siRNA-mediated depletion of S100A4 on capillary formation in mouse endothelial MSS31 cells. Specifically, murine S100A4 siRNA (mS100A4 siRNA) completely blocked S100A4 expression in MSS31 cells at both the mRNA and protein levels (Fig. 2a, b). Hepatocyte growth factor (HGF)-induced capillary formation was assessed 16 h after Matrigel culture [2]. siRNA-induced knockdown of mS100A4 resulted in the inhibition of HGF- Fig. 5 mS100A4 siRNA specifically inhibited expression of mS100A4, but not human S100A4 (hS100A4). MSS31 (murine) and PC-3M-Luc C6 (human) cells were transfected with murine S100A4 siRNA (mS100A4 siRNA) and negative control siRNA. Two days after transfection, RNA samples were extracted from transfected cells and subjected to real-time PCR analysis of S100A4 mRNA. *P = 0.05 induced capillary formation in MSS31 cells in vitro, while control siRNA showed no inhibitory effect when compared to untreated controls (Fig. 2c). Additionally, suppression of cell growth of MSS31 cells was not detectable within 16 h of mS100A4 siRNA treatment (Fig. 2d) and the analysis of caspase 3/7 activity did not show caspase-dependent apoptotic cell death (Fig. 2e), excluding a possibility that the inhibition of tube formation by the siRNA is non-specific effect. These results indicate that S100A4 is important for tube formation of endothelial cells. In addition, cell adhesion and cell migration assay was performed. As shown in Fig. 3a, cell adhesion was significantly enhanced by inhibition of S100A4 by S100A4 siRNA as compared to N.C. siRNA (P = 0.0017). Wound healing assay revealed that knockdown of S100A4 inhibited cell migration significantly (P = 0.0003) in Fig. 3b. Taken together, these results of multiple angiogenesis assay clearly indicate that S100A4 is significantly involved in angiogenesis.

Inhibition of tumor angiogenesis by S100A4 siRNA
To examine the effect of mS100A4 siRNA on tumor angiogenesis, PC-3M-Luc C6 cells were inoculated into athymic mice. Nine days after the injection (hereinafter referred to as day 1), tumors were treated twice with 50 lg siRNA/atelocollagen complex intratumorally. After the treatment, tumor angiogenesis was measured at day 9 (Fig. 4a). Administration of a fluorescent imaging agent revealed a strong inhibition of tumor angiogenesis by mS100A4 siRNA (Fig. 4b), without a discernible reduction in tumor volume (Supplementary Fig. S3). The mS100A4 siRNA was designed to inhibit specifically murine S100A4 expression, and not human S100A4 in PC-3M-Luc C6 cells (Fig. 5). Thus, these findings showed that endothelial cells were the primary targets of mS100A4 siRNA, not human tumor cells. Additionally, AngioSense analysis demonstrated that vessel formation in tumors was clearly inhibited by mS100A4 siRNA (Fig. 4c, Supplementary Fig. S2, Table 1). Thus, the results indicate that mS100A4 siRNA acts directly upon and inhibits host angiogenesis. We also found that mS100A4 siRNA inhibits tumor growth, with a significant reduction in tumor volume by day 15 (Table 2).

Discussion
Using B16BL6 tumor tissues little expressing S100A4, we stained tumor microvessels for CD31 and S100A4 and found that there are subpopulations of endothelial cells in tumors, S100A4-positive and -negative ones. This observation motivated us to examine a possible role of endothelial S100A4 by silencing it. The multiple angiogenesis assay including tube formation, adhesion, and migration analysis of endothelial cells clearly indicated that endothelial S100A4 plays a crucial role in angiogenesis. S100A4-positive endothelial cells in tumors may represent the ones primed for neoangiogenesis. A comparison of the gene expression profiles of siRNA-treated cells with those of untreated cells showed that endothelial S100A4 acts upstream of a variety of angiogenesis-related genes. These findings were confirmed in a xenograft tumor model, where intratumor administration of siRNA distinctly reduced tumor angiogenesis and growth.
In the present study, mouse siRNA was delivered in vivo using atelocollagen, a highly purified type I collagen with low immunogenicity. Atelocollagen forms nano-sized particles when mixed with oligonucleotides such as double stranded RNAs and DNAs via electrostatic binding, and is incorporated into cells by endocytosis [43,44]. In xenografted tumor tissues, many cell types can take up the  . 6 Modulation of the expression of angiogenesis-related genes by S100A4 siRNA. Verification of microarray analysis via RT-PCR. RT-PCR analysis was performed under linear amplification conditions. The GAPDH gene was used as an internal control. a Genes in mS100A4 knockdown cells with lower expression levels included angiogenesis-associated genes. b Genes in mS100A4 knockdown cells with higher expression levels included anti-angiogenesis genes complex, including human prostate cancer cells, endothelial cells and stromal cells. However, the specificity of the siRNA for mouse S100A4 suggests that the primary target of the S100A4 siRNA was the mouse vasculature. Microarray analysis further confirmed the molecular mechanism of S100A4-mediated angiogenesis in endothelial cells. Significant changes in angiogenesis-promoting gene expression occurred in S100A4 siRNA-treated endothelial cells. Among the genes exhibiting altered expression levels, aqp1, map3k5, and fgf18 are highly expressed in tumor-associated blood vessels in several human tumors [45][46][47][48]. Furthermore, our results indicate that S100A4 may negatively regulate anti-angiogenic genes, such as cdkn1a, thbs1, and spry4. This means that S100A4 has a dual effect on angiogenesis-related gene expression: it up-regulates angiogenic genes and down-regulates anti-angiogenic ones, thereby determining the fate of endothelial cells in association with tumorigenesis. The biological significance of all these genes has been proven in vitro and in vivo analysis of angiogenesis in previous reports.
Our sequential observations revealed that mS100A4 knockdown induced apparent cell growth inhibition of MSS31 cells in a later stage after siRNA treatment. It suggests a possible role of S100A4 in regulating endothelial cell growth, which may, in part, account for a drastic inhibition of neoangiogenesis in mS100A4 siRNA-administered tumor. Further analysis is required to know whether S100A4 promotes growth and survival of neovascular endothelial cells.
In the human cancer xenograft model used in this study, only host S100A4 was targeted to explore the role of S100A4 in angiogenesis. However, S100A4 in tumor cells is also involved in invasion and metastasis. Therefore, it would be interesting to investigate whether administration of an agent that inhibits both mouse and human S100A4 could suppress the metastasis of xenografted human tumor cells in immuno-compromised mice. If suppression occurs, it would suggest that inhibition of S100A4 could reduce tumor angiogenesis and by inference, tumor metastatic ability, which would have important implications for the development of cancer therapeutics. Furthermore, it would underscore the advantages of S100A4 inhibition over conventional angiogenesis factor/receptor inhibition. Studies are currently under way to develop such agents.
In conclusion, the present results show the importance of endothelial S100A4 expression in tumor angiogenesis. As the generation of a new vascular supply is causally involved in the progression of the majority of solid tumors, siRNA-mediated inhibition of endothelial S100A4 could provide an effective anti-tumor RNAi medicine. Thus, the development of inhibitors targeting endothelial S100A4 could represent a promising approach for effective antiangiogenesis and anti-metastasis therapy.