VEGF-A acts via neuropilin-1 to enhance epidermal cancer stem cell survival and formation of aggressive and highly vascularized tumors.
Journal: 2017/August - Oncogene
ISSN: 1476-5594
Abstract:
We identify a limited subpopulation of epidermal cancer stem cells (ECS cells), in squamous cell carcinoma, that form rapidly growing, invasive and highly vascularized tumors, as compared with non-stem cancer cells. These ECS cells grow as non-attached spheroids, and display enhanced migration and invasion. We show that ECS cell-produced vascular endothelial growth factor (VEGF)-A is required for the maintenance of this phenotype, as knockdown of VEGF-A gene expression or treatment with VEGF-A-inactivating antibody reduces these responses. In addition, treatment with bevacizumab reduces tumor vascularity and growth. Surprisingly, the classical mechanism of VEGF-A action via interaction with VEGF receptors does not mediate these events, as these cells lack VEGFR1 and VEGFR2. Instead, VEGF-A acts via the neuropilin-1 (NRP-1) co-receptor. Knockdown of NRP-1 inhibits ECS cell spheroid formation, invasion and migration, and attenuates tumor formation. These studies suggest that VEGF-A acts via interaction with NRP-1 to trigger intracellular events leading to ECS cell survival and formation of aggressive, invasive and highly vascularized tumors.
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Oncogene 35(33): 4379-4387

VEGF-A acts via neuropilin-1 to enhance epidermal cancer stem cell survival and formation of aggressive and highly vascularized tumors

INTRODUCTION

Non-melanoma skin cancer is the most commonly diagnosed cancer in the United States with over two million patients being treated each year.13 This disease is associated with exposure to ultraviolet light, chemicals, chronic wounding and viral infection.1,4 Squamous cell carcinoma tumors are aggressive, have a high risk of metastasis,3 and comprise 16% of these cancers.3 Tumors cannot grow beyond 1–2 mm in diameter in the absence of a vascular network5 and so tumor survival requires that these cells trigger angiogenesis.6 Vascular endothelial growth factor (VEGF) is a well-characterized inducer of angiogenesis that stimulates endothelial cell survival and proliferation, and blood vessel formation.7,8 VEGF has an important role in skin cancer development.6,9 Transgenic and knockout mouse studies indicate that VEGF is required for tumor formation,10,11 and that VEGF directly modulates cancer cell behavior.6,1216 VEGF receptors (VEGFR1, 2 and 3) are expressed in keratinocytes although the data on VEGFR2 is controversial.6,12,13,1618 VEGF has been shown to be important in cancer stem cell survival in several systems,12,1921 and VEGF stimulates endothelial cell-mediated construction of vasculature around the stem cell niche.6,22,23 Limited information is available regarding the role of VEGF-A signaling and angiogenesis in epidermal cancer stem (ECS) cells.12

We recently identified a limited subpopulation (0.15%) of highly aggressive cells in squamous cell carcinoma.24 These cells express stem cell markers and display characteristics of ECS cells, including growth as spheroids in non-attached conditions, and enhanced migration and invasion. Enriched populations of these cells form highly vascularized and aggressive tumors as compared with non-stem cancer cells. Aggressive tumor formation can be observed following injection of as few as 100 cells in immunocompromised mice.24 In the present study we show that ECS cells produce enhanced levels of angiogenic factors that maintain ECS cell survival and also induce vessel formation in a human umbilical vein endothelial cells (HUVEC) cell tube-formation assay and drive formation of highly aggressive and highly vascularized tumors. In ECS cell culture models, reducing VEGF-A level by treatment with small interfering RNA (siRNA) or anti-VEGF-A, reduces ECS cell spheroid formation, proliferation, migration and invasion. In addition, treatment with bevacizumab, a clinically used anti-VEGF therapy, markedly reduces xenograft tumor size and vascularization. These findings suggest that ECS cell-derived angiogenic factors act in an autocrine/paracrine manner to maintain ECS cell function, and also stimulate endothelial cell-mediated vascularization. Surprisingly, ECS cells lack VEGFR1 and VEGFR2 and so the VEGF-A action is not mediated via these receptors. Instead, our studies suggest a novel mechanism whereby VEGF-A acts via neuropilin-1 (NRP-1) to stimulate ECS cell survival.

RESULTS

ECS cells form large and highly vascularized tumors

Our recent studies demonstrate that human epidermal squamous cell carcinoma tumors contain a small subpopulation (0.15%) of cells that that are highly efficient at migration, invasion and tumor formation.24 These cells can be enriched to comprise ~ 12% of the culture24 when grown as non-adherent spheroids as shown in Figure 1a. These ECS cells produce enhanced levels of a cadre of key stem cell marker proteins, including Suz12, Bmi-1 and Ezh2 (Figure 1b). Moreover, we observe enhanced formation of H3K27me3, a marker of Ezh2 action. An important finding is that the ECS cells form large, aggressive and highly vascularized tumors as compared with the non-stem cancer cells (Figure 1c). To quantify the increase in vascularization, we measured CD31 (PECAM-1), an endothelial cell marker, specifically associated with vascular structures.19 SCC-13 monolayer and spheroid tumors were grown in NSG mice for 4 week and then harvested and stained with anti-CD31. Figure 1d shows hematoxylin/eosin, anti-CD31 and anti-K5 staining. K5 is a keratin that is specifically expressed in epithelial cells.25 This staining reveals highly elevated anti-CD31 staining in the ECS cell-derived (spheroid) tumors, which is localized in vascular structures as shown by the arrows (Figure 1e). These vascular features are surrounded by mesenchymal tissue, whereas the adjacent tumor cells are K5 positive. The graph summarizes a semiquantitative image analysis of the CD31 staining using ImageJ software. Analysis reveals a threefold increase in CD31 immunoreactivity in the ECS cell-derived tumors. This was further confirmed by detection of increased CD31 levels by immunoblot (Figure 1f). An antibody that detects human nuclear antigen, a specific marker of human cells, stains the K5-positive tumor cells, but does not stain the surrounding mesenchymal tissue (Figure 1g), indicating that the tumor localized mesenchymal tissue and vasculature is derived from the host tissue. The arrow shows the location of corresponding human nuclear antigen and K5 identical epithelial cells.

An external file that holds a picture, illustration, etc.
Object name is nihms766539f1.jpg

Epidermal cancer stem (ECS) cells form large and highly vascularized tumors. (a) Images of SCC-13 cells grown as monolayer (attached) or spheroid (unattached) cultures. (b) Stem marker expression in monolayer cells versus spheroids. Cells were grown for 10 days and total extracts were prepared for immunoblot detection of the indicated proteins. Similar results were observed in each of three independent experiments. (c, d) Tumor morphology and histology. Non-stem cancer cells and ECS cells (0.1 million/injection site) were injected in each front flank in NSG mice. After 4 weeks, the tumors were harvested and photographed. Primary tumors were paraffin-embedded, sectioned and stained with H&amp;E, or antibodies detecting CD31 (endothelial cells) and K5 (epithelial cells). The arrows indicate blood vessels, and the bars =125 microns. (e) ImageJ quantification of CD31 distribution. CD31 distribution was quantified using ImageJ and expressed as a percentage of the total area. The values are mean ± s.e.m., n =6, and the asterisks indicate a significant difference, P<0.05. The arrows indicate CD31 staining. (f) CD31 level. Tumor extracts from non-stem cancer (monolayer) and ECS cell (spheroid)-derived tumors were electrophoresed for immunoblot detection of CD31. (g) K5 and human nuclear antigen (HNA) distribution. The arrow shows HNA- and K5-positive human epithelial tumor cells. Note that the mesenchymal cells, which include the blood vessels, are K5 and HNA negative.

Evidence for enhanced angiogenic activity in ECS cell-derived tumors

A major goal of the present study is to identify factors that drive development of the vascularized phenotype. Biological evidence for enhanced angiogenic activity in ECS cell-derived tumors was obtained by incubating tumor extracts with HUVEC cells and monitoring ability of these extracts to stimulate HUVEC cell tube (vessel) formation.19Figure 2a shows tube-formation (left panels) and ImageJ analysis of tube formation, which is typically characterized by counting junctions, segments and nodes that are the elements of the vessel network (right panels). Formation of these structures was not detected in cells treated with no extract (not shown). Figure 2b plots the increase in formation of junctions, segments and nodes in cells treated with ECS cell tumor-derived extract as compared with non-stem cancer cell tumor extract and no extract. These findings indicate that extracts derived from ECS cell tumors are enriched in agents that stimulate angiogenesis.

An external file that holds a picture, illustration, etc.
Object name is nihms766539f2.jpg

VEGF-A angiogenic activity in ECS cell-derived tumors. (a) ECS cell tumor extracts stimulate vessel formation. Extracts were prepared from non-stem cancer cell- or ECS cell-derived tumors and 300 μg of lysate was incubated with HUVEC cultures for 18 h. Images were collected (left panels) and analyzed for detection of junction, segment and node formation (right panels). (b) Plot showing frequency of junctions, segments and nodes detected by ImageJ analysis of images in panel a. (c) Anti-VEGF-A inhibits vessel formation. ECS cell tumor lysate (300 μg) was treated with 0 or 10 μg/ml anti-VEGF-A and then tested in a HUVEC assay for ability to stimulate vessel formation. The values are mean ± s.e.m., n =3, and the asterisk indicates a significant difference, P<0.05. (d) VEGF signaling protein expression in tumors. Tumor extracts were prepared for immunoblot detection of the indicated proteins. (e) Level of mRNA-encoding VEGF signaling protein in tumors. RNA was isolated from non-stem cancer- and ECS cell-derived tumors for qRT-PCR measurement of mRNA-encoding angiogenic signaling proteins. The values are mean ± s.e.m., n = 3, and the asterisks indicate a significant difference, P<0.05. (f, g) Bevacizumab suppresses tumor formation. ECS cells (0.1 million) were injected into the two front flanks of NSG mice, treatment was initiated with 0 or 10 mg/kg bevacizumab and tumor size and morphology was monitored. The values are mean ± s.e.m., n = 6, and the asterisks indicate a significant difference, P<0.05. The tumor images are from 4 week tumors. (h) Immunoblot detection of CD31 in ECS cell tumors. ECS cells (0.1 million) were injected into each front flank of NSG mice, and bevacizumab was delivered by intraperitoneal injection three times per week at a level of 10 mg/kg body weight. After 4 weeks the tumors were harvested and extracts were prepared for detection of CD31.

VEGF-A is an important cancer-related angiogenesis factor.8,12,26Figure 2c shows that junction, segment and node formation is reduced by treating the ECS cell-derived extracts with anti-VEGF-A, suggesting that VEGF-A is a key angiogenic factor in ECS cell-derived tumors. In the classical model of VEGF-A signaling, VEGF-A interacts with VEGFR2 to stimulate angiogenesis and expression of angiogenesis-related target genes.8,12,26 Hypoxia inducible factor (HIF-1α) transcription factor is known to increase VEGF-A level and is a marker of angiogenesis.27,28 We therefore monitored the level of VEGF-A, VEGFR2 and HIF-1α in tumor extracts. Figure 2d shows that VEGF-A and HIF-1α levels are elevated in extracts prepared from ECS cell-derived tumors. However, VEGFR2 is not detected in these tumors, which suggests that the classical VEGF-A/VEGFR2 signaling pathway is not active. In addition, we show that VEGFR1 is also absent. VEGFR1 also interacts with VEGF-A, but demonstrates much weaker VEGF-dependent tyrosine phosphorylation upon VEGF-A binding.29 However, NRP-1, a VEGFR2 co-factor that enhances VEGF-A/VEGFR2 interaction and activation,29 is elevated in ECS cell-derived tumors. The lower panel in Figure 2d uses HUVEC cell extract as a positive control to assure that the antibodies to VEGFR1, VEGFR2 and NRP-1 are working.17 Moreover, Figure 2e confirms that VEGF-A, NRP-1 and HIF-1α mRNA levels are elevated in ECS cell-derived tumors, but VEGFR1 and VEGFR2 are expressed at vanishingly low levels.

We next examined the impact of inhibiting VEGF-A action on growth and vascularization of ECS cell-derived tumors. At 2 days after subcutaneous injection of 100 000 ECS cells, NSG mice were treated by intraperitoneal injection of bevacizumab, an inhibitor antibody that binds VEGF-A, at 10 mg/kg body weight on 3 alternate days each week. Figure 2f shows that bevacizumab treatment substantially reduces tumor growth and markedly reduces visible vascularization (Figure 2g). This reduced vascularization is confirmed by showing reduced CD31 levels in the bevacizumab-treated tumors (Figure 2h).

VEGF-A and NRP-1 have an important role in cultured ECS cells

These studies suggest that VEGF-A does not act via interaction with the traditional VEGF-A receptors, VEGFR2 or VEGFR1. To understand the mechanism of VEGF-A action, we examined VEGF-A action in cultured ECS cells. We have previously shown that spheroid formation, in non-attached culture conditions, is a measure of ECS cell survival.24Figure 3a shows that spheroid formation is reduced in anti-VEGF-A-treated ECS cells, and this is associated with a shift in spheroid diameter toward smaller sizes (Figure 3b). Moreover, anti-VEGF-A treatment reduces ECS cell ability to migrate on plastic (Figure 3c) and to invade matrigel (Figure 3d). We next assessed the role of VEGFR1, VEGFR2 and NRP-1 as mediators of VEGF-A action. Figure 3e is an immunoblot showing that VEGFR1 and VEGFR2 levels are not detectable in ECS cell spheroids, derived from SCC-13 cells, or from another surface epithelial cell lines (HaCaT). In contrast, NRP-1 is present in SCC-13 and HaCaT cell-derived ECS cells. HUVEC cells extracts were electrophoresed as a control, as these cells express VEGFR1, VEGFR2 and NRP-1.17 We next assessed the role of VEGF-A, VEGFR1, VEGFR2 and NRP-1 in mediating biological responses. Figures 3f and g show that treatment with VEGF-A- or NRP-1-siRNA attenuates ECS cell spheroid formation and migration, but that VEGFR1- or VEGFR2-siRNA have no impact. Figure 3h confirms the extent of siRNA-dependent knockdown of VEGF-A and NRP-1. We typically see nearly complete knockdown of VEGF-A and a 50% reduction in NRP-1.

An external file that holds a picture, illustration, etc.
Object name is nihms766539f3.jpg

VEGF-A signaling proteins and control of ECS cell function. (a, b) Anti-VEGF-A impact on spheroid formation. SCC-13 cells were seeded at 40 000 cells/well in ultra-low attachment plates in spheroid medium containing 10 μg/ml anti-VEGF or control IgG. After 3 days, spheroid number, and size distribution, were analyzed and expressed as mean ± s.e.m., n = 4, and the asterisks indicate a significant difference, P<0.05. (c, d) Anti-VEGF-A treatment suppresses ECS cell migration and invasion. ECS cells were plated at confluent density on conventional culture plates and then scratched to create uniform wounds. Anti-VEGF-A (0 or 10 μg/ml) was added at the time of wounding and wound width was monitored at 0, 8 and 18 h. ECS cells were seeded into a transwell chamber atop a matrigel layer in the presence of 0 or 10 μg/ml anti-VEGF-A and cell invasion was monitored at 18 h. The asterisks indicate a significant difference, n = 3, P<0.05. (e) VEGF-A signaling protein expression. Extracts were prepared from monolayer cultures of the indicated cell lines for immunoblot detection of the indicated proteins. (f, g) VEGF-A and NRP-1 are required for ECS cell spheroid formation and migration. SCC-13 monolayer cells were electroporated with 3 μg of control-, VEGF-A-, VEGFR1-, VEGFR2- or NRP-1-siRNA and plated in triplicate in ultra-low attachment dishes at 40 000 cells per well and spheroids were photographed after 5 days. In parallel, the electroporated cells were seeded at confluence in conventional plates, permitted to attach and uniformly wounded using a 10 μl pipet tip. Spheroid medium was added and wound closure was monitored at 0, 16 and 24 h. (h) Immunoblot confirmation of VEGF-A and NRP-1 knockdown in siRNA-treated ECS cells used in panels f and g. We routinely achieve a near-complete reduction in VEGF-A level and a 50% reduction in NRP-1 level.

NRP-1 is required for tumor formation

The cell culture studies suggest that NRP-1 is required for VEGF-A action in ECS cells, and so we next examined the impact of inhibiting NRP-1 on tumor growth and vascularization. For this purpose, we have used an inhibitor of NRP-1 called EG00229.2932 We first assessed the impact of this agent on spheroid growth and ECS cell invasion through matrigel. To assess the impact on ECS cell spheroid formation, cells (40 000) were plated in ultra-low attachment plates in the presence of increasing concentrations of EG00229 and spheroid size was monitored after 3 days. Figure 4a shows that treatment with EG00229 reduces spheroid size. We also assessed the ability of EG00229 to suppress ECS cell matrigel invasion. Figure 4b shows that EG00229 reduces invasion by 25%. Having confirmed this agent is active in cultured cells, we assessed its impact on tumor formation. At 2 days after subcutaneous injection of 100 000 ECS cells in each of the front flanks in NSG mice, treatment was initiated by intraperitoneal injection of EG00229 at 10 mg/kg body weight on 3 alternate days each week. EG00229 treatment substantially reduces tumor growth (Figure 4c), and dose–response studies indicate that the compound is active over a wide range of concentrations (Figure 4d). EG00229 treatment reduces visible vascularization (Figure 4e) and reduced vascularization was confirmed by showing a reduction in CD31 staining in EG00229-treated tumors (Figure 4f) and a reduction in CD31 level as assessed by immunoblot (Figure 4g). We also stained tumor sections to detect CD31 and K5. Figure 4h shows that CD31 staining is reduced in EG00229-treated cultures. K5 staining confirms that the tumor cells are of epithelial origin. The unstained areas show mouse connective tissue and vasculature that is interpolated into the tumor.

An external file that holds a picture, illustration, etc.
Object name is nihms766539f4.jpg

EG00229 suppresses ECS cell function and tumor formation. (a) EG00229 impact on ECS cell spheroid formation. SCC-13 cells were seeded at 40 000 cells per well in ultra-low attachment plates in spheroid medium containing 0–100 μM EG00229. After 3 days, spheroid number was analyzed and expressed as mean ± s.e.m., n = 3, P<0.05. (b) EG00229 impact on ECS cell matrigel invasion. ECS cells were seeded into a transwell chambers atop a matrigel layer in the presence of 0 or 100 μM EG00229 and invasion was monitored at 18 h. The values are mean ±s.e.m. and the asterisk indicates a significant difference, n = 3, P<0.05. (ch) EG00229 suppresses tumor formation and vascularization. ECS cells were enriched by growth for 10 days as spheroids, and 0.1 million cells were injected into NSG mice in each front flank. The mice were treated with 0 or 10 mg EG00229/kg body weight for various times (c) or various doses of EG00229 for 4 week (d) given three times per week by IP injection. Tumor formation was monitored using calipers and tumors were photographed on week 4 at the time of harvest (e, 10 mg/kg body weight). The values are mean ± s.e.m., n =6, and the asterisks indicates a significant difference, P<0.05. EG00229 treatment did not impact animal weight. Sections were stained with anti-CD31 and processed by ImageJ and staining intensity was expressed as % area (f). The values are mean ± s.e.m., n =6, and the asterisk indicates a significant difference, P<0.05. The reduction in CD31 level was confirmed by immunoblot of tumor extracts (g). Similar results were observed for each of three tumor comparisons. Tumor sections were prepared from tumors treated with 0 or 10 mg/kg body weight EG00229 and then stained to detect CD31 (vascularization marker) and keratin 5 (K5, epithelial cell marker) (h). The arrows indicate CD31-positive blood vessels. The bar =125 μm. (i) Evidence for NRP-1/VEGF-A interaction. Extracts were prepared from SCC-13-derived ECS cells in lysis buffer and 200 μg of extract was immunoprecipitated with anti-NRP-1 or anti-VEGF-A followed by immunoblot to detect the indicated epitopes. Total extract (25 μg) was electrophoresed as a control. Similar results were observed in three experiments.

VEGF-A and NRP-1 interaction

The above studies suggest that VEGF-A and NRP-1 may physically interact. To test this, we prepared extracts from ECS cells for immunoprecipitation. Figure 4i shows that VEGF-A co-precipitates with NRP-1 (top panel) and NRP-1 co-precipitates with VEGF-A (bottom panel). The interactions are specific as no co-precipitation is observed when non-specific immunoglobulin is utilized.

ECS cells form large and highly vascularized tumors

Our recent studies demonstrate that human epidermal squamous cell carcinoma tumors contain a small subpopulation (0.15%) of cells that that are highly efficient at migration, invasion and tumor formation.24 These cells can be enriched to comprise ~ 12% of the culture24 when grown as non-adherent spheroids as shown in Figure 1a. These ECS cells produce enhanced levels of a cadre of key stem cell marker proteins, including Suz12, Bmi-1 and Ezh2 (Figure 1b). Moreover, we observe enhanced formation of H3K27me3, a marker of Ezh2 action. An important finding is that the ECS cells form large, aggressive and highly vascularized tumors as compared with the non-stem cancer cells (Figure 1c). To quantify the increase in vascularization, we measured CD31 (PECAM-1), an endothelial cell marker, specifically associated with vascular structures.19 SCC-13 monolayer and spheroid tumors were grown in NSG mice for 4 week and then harvested and stained with anti-CD31. Figure 1d shows hematoxylin/eosin, anti-CD31 and anti-K5 staining. K5 is a keratin that is specifically expressed in epithelial cells.25 This staining reveals highly elevated anti-CD31 staining in the ECS cell-derived (spheroid) tumors, which is localized in vascular structures as shown by the arrows (Figure 1e). These vascular features are surrounded by mesenchymal tissue, whereas the adjacent tumor cells are K5 positive. The graph summarizes a semiquantitative image analysis of the CD31 staining using ImageJ software. Analysis reveals a threefold increase in CD31 immunoreactivity in the ECS cell-derived tumors. This was further confirmed by detection of increased CD31 levels by immunoblot (Figure 1f). An antibody that detects human nuclear antigen, a specific marker of human cells, stains the K5-positive tumor cells, but does not stain the surrounding mesenchymal tissue (Figure 1g), indicating that the tumor localized mesenchymal tissue and vasculature is derived from the host tissue. The arrow shows the location of corresponding human nuclear antigen and K5 identical epithelial cells.

An external file that holds a picture, illustration, etc.
Object name is nihms766539f1.jpg

Epidermal cancer stem (ECS) cells form large and highly vascularized tumors. (a) Images of SCC-13 cells grown as monolayer (attached) or spheroid (unattached) cultures. (b) Stem marker expression in monolayer cells versus spheroids. Cells were grown for 10 days and total extracts were prepared for immunoblot detection of the indicated proteins. Similar results were observed in each of three independent experiments. (c, d) Tumor morphology and histology. Non-stem cancer cells and ECS cells (0.1 million/injection site) were injected in each front flank in NSG mice. After 4 weeks, the tumors were harvested and photographed. Primary tumors were paraffin-embedded, sectioned and stained with H&amp;E, or antibodies detecting CD31 (endothelial cells) and K5 (epithelial cells). The arrows indicate blood vessels, and the bars =125 microns. (e) ImageJ quantification of CD31 distribution. CD31 distribution was quantified using ImageJ and expressed as a percentage of the total area. The values are mean ± s.e.m., n =6, and the asterisks indicate a significant difference, P<0.05. The arrows indicate CD31 staining. (f) CD31 level. Tumor extracts from non-stem cancer (monolayer) and ECS cell (spheroid)-derived tumors were electrophoresed for immunoblot detection of CD31. (g) K5 and human nuclear antigen (HNA) distribution. The arrow shows HNA- and K5-positive human epithelial tumor cells. Note that the mesenchymal cells, which include the blood vessels, are K5 and HNA negative.

Evidence for enhanced angiogenic activity in ECS cell-derived tumors

A major goal of the present study is to identify factors that drive development of the vascularized phenotype. Biological evidence for enhanced angiogenic activity in ECS cell-derived tumors was obtained by incubating tumor extracts with HUVEC cells and monitoring ability of these extracts to stimulate HUVEC cell tube (vessel) formation.19Figure 2a shows tube-formation (left panels) and ImageJ analysis of tube formation, which is typically characterized by counting junctions, segments and nodes that are the elements of the vessel network (right panels). Formation of these structures was not detected in cells treated with no extract (not shown). Figure 2b plots the increase in formation of junctions, segments and nodes in cells treated with ECS cell tumor-derived extract as compared with non-stem cancer cell tumor extract and no extract. These findings indicate that extracts derived from ECS cell tumors are enriched in agents that stimulate angiogenesis.

An external file that holds a picture, illustration, etc.
Object name is nihms766539f2.jpg

VEGF-A angiogenic activity in ECS cell-derived tumors. (a) ECS cell tumor extracts stimulate vessel formation. Extracts were prepared from non-stem cancer cell- or ECS cell-derived tumors and 300 μg of lysate was incubated with HUVEC cultures for 18 h. Images were collected (left panels) and analyzed for detection of junction, segment and node formation (right panels). (b) Plot showing frequency of junctions, segments and nodes detected by ImageJ analysis of images in panel a. (c) Anti-VEGF-A inhibits vessel formation. ECS cell tumor lysate (300 μg) was treated with 0 or 10 μg/ml anti-VEGF-A and then tested in a HUVEC assay for ability to stimulate vessel formation. The values are mean ± s.e.m., n =3, and the asterisk indicates a significant difference, P<0.05. (d) VEGF signaling protein expression in tumors. Tumor extracts were prepared for immunoblot detection of the indicated proteins. (e) Level of mRNA-encoding VEGF signaling protein in tumors. RNA was isolated from non-stem cancer- and ECS cell-derived tumors for qRT-PCR measurement of mRNA-encoding angiogenic signaling proteins. The values are mean ± s.e.m., n = 3, and the asterisks indicate a significant difference, P<0.05. (f, g) Bevacizumab suppresses tumor formation. ECS cells (0.1 million) were injected into the two front flanks of NSG mice, treatment was initiated with 0 or 10 mg/kg bevacizumab and tumor size and morphology was monitored. The values are mean ± s.e.m., n = 6, and the asterisks indicate a significant difference, P<0.05. The tumor images are from 4 week tumors. (h) Immunoblot detection of CD31 in ECS cell tumors. ECS cells (0.1 million) were injected into each front flank of NSG mice, and bevacizumab was delivered by intraperitoneal injection three times per week at a level of 10 mg/kg body weight. After 4 weeks the tumors were harvested and extracts were prepared for detection of CD31.

VEGF-A is an important cancer-related angiogenesis factor.8,12,26Figure 2c shows that junction, segment and node formation is reduced by treating the ECS cell-derived extracts with anti-VEGF-A, suggesting that VEGF-A is a key angiogenic factor in ECS cell-derived tumors. In the classical model of VEGF-A signaling, VEGF-A interacts with VEGFR2 to stimulate angiogenesis and expression of angiogenesis-related target genes.8,12,26 Hypoxia inducible factor (HIF-1α) transcription factor is known to increase VEGF-A level and is a marker of angiogenesis.27,28 We therefore monitored the level of VEGF-A, VEGFR2 and HIF-1α in tumor extracts. Figure 2d shows that VEGF-A and HIF-1α levels are elevated in extracts prepared from ECS cell-derived tumors. However, VEGFR2 is not detected in these tumors, which suggests that the classical VEGF-A/VEGFR2 signaling pathway is not active. In addition, we show that VEGFR1 is also absent. VEGFR1 also interacts with VEGF-A, but demonstrates much weaker VEGF-dependent tyrosine phosphorylation upon VEGF-A binding.29 However, NRP-1, a VEGFR2 co-factor that enhances VEGF-A/VEGFR2 interaction and activation,29 is elevated in ECS cell-derived tumors. The lower panel in Figure 2d uses HUVEC cell extract as a positive control to assure that the antibodies to VEGFR1, VEGFR2 and NRP-1 are working.17 Moreover, Figure 2e confirms that VEGF-A, NRP-1 and HIF-1α mRNA levels are elevated in ECS cell-derived tumors, but VEGFR1 and VEGFR2 are expressed at vanishingly low levels.

We next examined the impact of inhibiting VEGF-A action on growth and vascularization of ECS cell-derived tumors. At 2 days after subcutaneous injection of 100 000 ECS cells, NSG mice were treated by intraperitoneal injection of bevacizumab, an inhibitor antibody that binds VEGF-A, at 10 mg/kg body weight on 3 alternate days each week. Figure 2f shows that bevacizumab treatment substantially reduces tumor growth and markedly reduces visible vascularization (Figure 2g). This reduced vascularization is confirmed by showing reduced CD31 levels in the bevacizumab-treated tumors (Figure 2h).

VEGF-A and NRP-1 have an important role in cultured ECS cells

These studies suggest that VEGF-A does not act via interaction with the traditional VEGF-A receptors, VEGFR2 or VEGFR1. To understand the mechanism of VEGF-A action, we examined VEGF-A action in cultured ECS cells. We have previously shown that spheroid formation, in non-attached culture conditions, is a measure of ECS cell survival.24Figure 3a shows that spheroid formation is reduced in anti-VEGF-A-treated ECS cells, and this is associated with a shift in spheroid diameter toward smaller sizes (Figure 3b). Moreover, anti-VEGF-A treatment reduces ECS cell ability to migrate on plastic (Figure 3c) and to invade matrigel (Figure 3d). We next assessed the role of VEGFR1, VEGFR2 and NRP-1 as mediators of VEGF-A action. Figure 3e is an immunoblot showing that VEGFR1 and VEGFR2 levels are not detectable in ECS cell spheroids, derived from SCC-13 cells, or from another surface epithelial cell lines (HaCaT). In contrast, NRP-1 is present in SCC-13 and HaCaT cell-derived ECS cells. HUVEC cells extracts were electrophoresed as a control, as these cells express VEGFR1, VEGFR2 and NRP-1.17 We next assessed the role of VEGF-A, VEGFR1, VEGFR2 and NRP-1 in mediating biological responses. Figures 3f and g show that treatment with VEGF-A- or NRP-1-siRNA attenuates ECS cell spheroid formation and migration, but that VEGFR1- or VEGFR2-siRNA have no impact. Figure 3h confirms the extent of siRNA-dependent knockdown of VEGF-A and NRP-1. We typically see nearly complete knockdown of VEGF-A and a 50% reduction in NRP-1.

An external file that holds a picture, illustration, etc.
Object name is nihms766539f3.jpg

VEGF-A signaling proteins and control of ECS cell function. (a, b) Anti-VEGF-A impact on spheroid formation. SCC-13 cells were seeded at 40 000 cells/well in ultra-low attachment plates in spheroid medium containing 10 μg/ml anti-VEGF or control IgG. After 3 days, spheroid number, and size distribution, were analyzed and expressed as mean ± s.e.m., n = 4, and the asterisks indicate a significant difference, P<0.05. (c, d) Anti-VEGF-A treatment suppresses ECS cell migration and invasion. ECS cells were plated at confluent density on conventional culture plates and then scratched to create uniform wounds. Anti-VEGF-A (0 or 10 μg/ml) was added at the time of wounding and wound width was monitored at 0, 8 and 18 h. ECS cells were seeded into a transwell chamber atop a matrigel layer in the presence of 0 or 10 μg/ml anti-VEGF-A and cell invasion was monitored at 18 h. The asterisks indicate a significant difference, n = 3, P<0.05. (e) VEGF-A signaling protein expression. Extracts were prepared from monolayer cultures of the indicated cell lines for immunoblot detection of the indicated proteins. (f, g) VEGF-A and NRP-1 are required for ECS cell spheroid formation and migration. SCC-13 monolayer cells were electroporated with 3 μg of control-, VEGF-A-, VEGFR1-, VEGFR2- or NRP-1-siRNA and plated in triplicate in ultra-low attachment dishes at 40 000 cells per well and spheroids were photographed after 5 days. In parallel, the electroporated cells were seeded at confluence in conventional plates, permitted to attach and uniformly wounded using a 10 μl pipet tip. Spheroid medium was added and wound closure was monitored at 0, 16 and 24 h. (h) Immunoblot confirmation of VEGF-A and NRP-1 knockdown in siRNA-treated ECS cells used in panels f and g. We routinely achieve a near-complete reduction in VEGF-A level and a 50% reduction in NRP-1 level.

NRP-1 is required for tumor formation

The cell culture studies suggest that NRP-1 is required for VEGF-A action in ECS cells, and so we next examined the impact of inhibiting NRP-1 on tumor growth and vascularization. For this purpose, we have used an inhibitor of NRP-1 called EG00229.2932 We first assessed the impact of this agent on spheroid growth and ECS cell invasion through matrigel. To assess the impact on ECS cell spheroid formation, cells (40 000) were plated in ultra-low attachment plates in the presence of increasing concentrations of EG00229 and spheroid size was monitored after 3 days. Figure 4a shows that treatment with EG00229 reduces spheroid size. We also assessed the ability of EG00229 to suppress ECS cell matrigel invasion. Figure 4b shows that EG00229 reduces invasion by 25%. Having confirmed this agent is active in cultured cells, we assessed its impact on tumor formation. At 2 days after subcutaneous injection of 100 000 ECS cells in each of the front flanks in NSG mice, treatment was initiated by intraperitoneal injection of EG00229 at 10 mg/kg body weight on 3 alternate days each week. EG00229 treatment substantially reduces tumor growth (Figure 4c), and dose–response studies indicate that the compound is active over a wide range of concentrations (Figure 4d). EG00229 treatment reduces visible vascularization (Figure 4e) and reduced vascularization was confirmed by showing a reduction in CD31 staining in EG00229-treated tumors (Figure 4f) and a reduction in CD31 level as assessed by immunoblot (Figure 4g). We also stained tumor sections to detect CD31 and K5. Figure 4h shows that CD31 staining is reduced in EG00229-treated cultures. K5 staining confirms that the tumor cells are of epithelial origin. The unstained areas show mouse connective tissue and vasculature that is interpolated into the tumor.

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EG00229 suppresses ECS cell function and tumor formation. (a) EG00229 impact on ECS cell spheroid formation. SCC-13 cells were seeded at 40 000 cells per well in ultra-low attachment plates in spheroid medium containing 0–100 μM EG00229. After 3 days, spheroid number was analyzed and expressed as mean ± s.e.m., n = 3, P<0.05. (b) EG00229 impact on ECS cell matrigel invasion. ECS cells were seeded into a transwell chambers atop a matrigel layer in the presence of 0 or 100 μM EG00229 and invasion was monitored at 18 h. The values are mean ±s.e.m. and the asterisk indicates a significant difference, n = 3, P<0.05. (ch) EG00229 suppresses tumor formation and vascularization. ECS cells were enriched by growth for 10 days as spheroids, and 0.1 million cells were injected into NSG mice in each front flank. The mice were treated with 0 or 10 mg EG00229/kg body weight for various times (c) or various doses of EG00229 for 4 week (d) given three times per week by IP injection. Tumor formation was monitored using calipers and tumors were photographed on week 4 at the time of harvest (e, 10 mg/kg body weight). The values are mean ± s.e.m., n =6, and the asterisks indicates a significant difference, P<0.05. EG00229 treatment did not impact animal weight. Sections were stained with anti-CD31 and processed by ImageJ and staining intensity was expressed as % area (f). The values are mean ± s.e.m., n =6, and the asterisk indicates a significant difference, P<0.05. The reduction in CD31 level was confirmed by immunoblot of tumor extracts (g). Similar results were observed for each of three tumor comparisons. Tumor sections were prepared from tumors treated with 0 or 10 mg/kg body weight EG00229 and then stained to detect CD31 (vascularization marker) and keratin 5 (K5, epithelial cell marker) (h). The arrows indicate CD31-positive blood vessels. The bar =125 μm. (i) Evidence for NRP-1/VEGF-A interaction. Extracts were prepared from SCC-13-derived ECS cells in lysis buffer and 200 μg of extract was immunoprecipitated with anti-NRP-1 or anti-VEGF-A followed by immunoblot to detect the indicated epitopes. Total extract (25 μg) was electrophoresed as a control. Similar results were observed in three experiments.

VEGF-A and NRP-1 interaction

The above studies suggest that VEGF-A and NRP-1 may physically interact. To test this, we prepared extracts from ECS cells for immunoprecipitation. Figure 4i shows that VEGF-A co-precipitates with NRP-1 (top panel) and NRP-1 co-precipitates with VEGF-A (bottom panel). The interactions are specific as no co-precipitation is observed when non-specific immunoglobulin is utilized.

DISCUSSION

Epidermal squamous cell carcinoma is among the most common cancers in humans, and so devising strategies that control this disease is an important goal.33 Recent studies suggest that tumors contain a small subpopulation of cancer stem cells, which exhibit self-renewal capacity, proliferate infrequently and are responsible for tumor maintenance and metastasis.34 Moreover, it has been proposed that these ‘slow cycling’ cells are not impacted by anticancer agents that kill rapidly growing tumor cells.35 As the cancer stem cells display enhanced migratory potential, invasiveness, tumor-forming potential and resistance to therapeutic intervention, eliminating the stem cell population is an important goal to halt tumor formation.35

We recently characterized a subpopulation of cells from squamous cell carcinoma that display properties of ECS cells.24 Detailed analysis reveals that ECS cell cultures are highly enriched for expression of epidermal and embryonic stem cell markers, including aldehyde dehydrogenase 1, K15, CD200, K19, Oct4, Bmi-1, Ezh2 and trimethylated histone H3. These cells are important, as injection of as few as 100 cells in immunocompromised mice results in the formation of large and rapidly growing highly invasive and aggressive tumors.24 This is in contrast to tumors derived from non-stem cancer cells which form small, circumscribed and non-vascularized tumors.

It is accepted that tumors cannot exceed 1 mm in size in an avascular state and so tumors must induce vascularization to remain viable.36 In the course of our studies, we observed that ECS cell-derived tumors are hypervascularized compared with tumor formed by non-stem cancer cells (Figure 1).24,37 As vascularization is key contributor to an aggressive tumor phenotype, it is important to understand why ECS cells drive angiogenesis more efficiently than non-stem cancer cells.

VEGF-A regulation of tumor ECS cell survival and vasculature

VEGF is a well characterized and important regulator of angiogenesis.7,20 Cancer cells produce VEGF-A, which produces autocrine and paracrine effects on cancer cells and on the surrounding endothelial cells.29 Our studies show that more VEGF-A is produced by ECS cell-derived tumors as compared with non-stem cancer cell-derived tumors. Consistent with a role for VEGF-A in maintaining the ECS cell phenotype, treatment with anti-VEGF-A reduces ECS cell spheroid formation and spheroid size, and also reduces ECS cell migration and invasion. We further show that treatment with VEGF-A-siRNA produces a similar change. Thus, VEGF-A directly interacts with ECS cells to maintain and enhance the stem cell phenotype (that is, enhance spheroid formation, migration and invasion).24,37 We further demonstrate that ECS cell tumor cell extracts stimulate HUVEC cell vessel formation more efficienty than non-stem cancer cell tumor extract. Again, VEGF-A appears to be a key stimulator of this process, as treating the extracts with anti-VEGF-A redues HUVEC cell vessel formation. We also examined the role of VEGF-A in driving tumor formation. Treatment of mice with bevacizumab, a clinically used form of anti-VEGF-A, which sequesters and prevents VEGF-A action, reduces ECS cell xenograft tumor vasculariztion and growth. Loss of vascularization is evidenced by reduced redness of the tumors and reduced presence of the CD31 endothelial cell marker.

NRP-1 mediates the action of VEGF-A

The classical mechanism of VEGF-A action is via VEGF-A interaction with VEGFR2 to drive intracellular signal transduction.8,12,26 However, this mechanism is not active in ECS cells, as these cells lack both VEGFR1 and VEGFR2. Thus, VEGFR1 and VEGFR2 do not mediate VEGF-A action in our system. These findings suggested that VEGF-A enhances ECS cell survival and angiogenesis via a novel mechanism. Therefore, we attempted to identify the molecular entity that mediates VEGF-A action in ECS cells.

NRP-1 is a 922 amino-acid protein that includes a large (860 amino acid) extracellular glycoprotein domain, a 22 amino acid transmembrane segment and a 40 amino acid intracellular region that does not possess intrinsic enzymatic activity. VEGF-A interacts with NRP-1 and this leads to enhanced VEGF-A interaction with VEGFR, leading to enhanced angiogenesis.3840 Consistent with this role, NRP-1 overexpression increases blood vessel formation,41 and inactivation causes abnormal vasculature formation leading to embryonic lethality.42 NRP-1 is expressed in various human tumors, including prostate cancer, breast cancer, melanoma and pancreatic adenocarcinoma.4347 In some model systems, NRP-1 expression increases angiogenesis and tumor formation;48,49 however, how NRP-1 regulates tumor formation is not well understood. As noted above, the prevailing dogma has been that NRP-1 functions as a co-receptor with VEGFR2 and other receptors. However, NRP-1 has been reported to mediate VEGF-A action, independent of VEGFR1 and VEGFR2, in a limited number of systems.31,5052 For example, Panc-1 pancreatic carcinoma cells express VEGF and NRP-1, but not VEGF receptors. In this cell type, VEGF-mediated stimulation of proliferation requires NRP-1,52 In addition, restoration of NRP-1 in NRP-1-negative pancreatic cancer cells altered cell survival,50 and VEGF can operate via NRP-1 to stimulate malignant progression in metastatic renal cell carcinoma.53,54

Our present studies suggest that VEGF-A interacts with NRP-1, via a novel signaling pathway that does not require VEGF receptors, to activate cellular processes in ECS cells that enhance ECS cell survival and tumor formation.

VEGF-A regulation of tumor ECS cell survival and vasculature

VEGF is a well characterized and important regulator of angiogenesis.7,20 Cancer cells produce VEGF-A, which produces autocrine and paracrine effects on cancer cells and on the surrounding endothelial cells.29 Our studies show that more VEGF-A is produced by ECS cell-derived tumors as compared with non-stem cancer cell-derived tumors. Consistent with a role for VEGF-A in maintaining the ECS cell phenotype, treatment with anti-VEGF-A reduces ECS cell spheroid formation and spheroid size, and also reduces ECS cell migration and invasion. We further show that treatment with VEGF-A-siRNA produces a similar change. Thus, VEGF-A directly interacts with ECS cells to maintain and enhance the stem cell phenotype (that is, enhance spheroid formation, migration and invasion).24,37 We further demonstrate that ECS cell tumor cell extracts stimulate HUVEC cell vessel formation more efficienty than non-stem cancer cell tumor extract. Again, VEGF-A appears to be a key stimulator of this process, as treating the extracts with anti-VEGF-A redues HUVEC cell vessel formation. We also examined the role of VEGF-A in driving tumor formation. Treatment of mice with bevacizumab, a clinically used form of anti-VEGF-A, which sequesters and prevents VEGF-A action, reduces ECS cell xenograft tumor vasculariztion and growth. Loss of vascularization is evidenced by reduced redness of the tumors and reduced presence of the CD31 endothelial cell marker.

NRP-1 mediates the action of VEGF-A

The classical mechanism of VEGF-A action is via VEGF-A interaction with VEGFR2 to drive intracellular signal transduction.8,12,26 However, this mechanism is not active in ECS cells, as these cells lack both VEGFR1 and VEGFR2. Thus, VEGFR1 and VEGFR2 do not mediate VEGF-A action in our system. These findings suggested that VEGF-A enhances ECS cell survival and angiogenesis via a novel mechanism. Therefore, we attempted to identify the molecular entity that mediates VEGF-A action in ECS cells.

NRP-1 is a 922 amino-acid protein that includes a large (860 amino acid) extracellular glycoprotein domain, a 22 amino acid transmembrane segment and a 40 amino acid intracellular region that does not possess intrinsic enzymatic activity. VEGF-A interacts with NRP-1 and this leads to enhanced VEGF-A interaction with VEGFR, leading to enhanced angiogenesis.3840 Consistent with this role, NRP-1 overexpression increases blood vessel formation,41 and inactivation causes abnormal vasculature formation leading to embryonic lethality.42 NRP-1 is expressed in various human tumors, including prostate cancer, breast cancer, melanoma and pancreatic adenocarcinoma.4347 In some model systems, NRP-1 expression increases angiogenesis and tumor formation;48,49 however, how NRP-1 regulates tumor formation is not well understood. As noted above, the prevailing dogma has been that NRP-1 functions as a co-receptor with VEGFR2 and other receptors. However, NRP-1 has been reported to mediate VEGF-A action, independent of VEGFR1 and VEGFR2, in a limited number of systems.31,5052 For example, Panc-1 pancreatic carcinoma cells express VEGF and NRP-1, but not VEGF receptors. In this cell type, VEGF-mediated stimulation of proliferation requires NRP-1,52 In addition, restoration of NRP-1 in NRP-1-negative pancreatic cancer cells altered cell survival,50 and VEGF can operate via NRP-1 to stimulate malignant progression in metastatic renal cell carcinoma.53,54

Our present studies suggest that VEGF-A interacts with NRP-1, via a novel signaling pathway that does not require VEGF receptors, to activate cellular processes in ECS cells that enhance ECS cell survival and tumor formation.

MATERIALS AND METHODS

Chemicals and reagents

Trypsin and Dulbecco’s modified Eagle’s medium were purchased from Invitrogen (Frederick, MD, USA). β-actin antibody (A5441) was obtained from Sigma (St Louis, MO, USA). Anti-H3K27me3 (07-449) was purchased from Millipore (Bedford, MA, USA). Ezh2 (612667) antibody was obtained from BD Transduction Labs (San Jose, CA, USA). Mouse monoclonal antibody to VEGF-A (MAB293) was purchased from R&amp;D Systems (Minneapolis, MN, USA). Antibodies to CD31 (ab28364), Bmi-1 (ab14389), NRP-1 (ab81321), VEGFR2 (ab2349) were from Abcam (Cambridge, MA, USA). Human recombinant VEGF-A (ab9571) was from Abcam. Anti-VEGFR1 (2893) was from Cell Signaling (Danvers, MA, USA). Anti-keratin 5 (AF 138) was obtained from Covance (Princeton, NJ, USA). Horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin (NXA931) and donkey anti-rabbit immunoglobulin (NA934V) were obtained from GE Healthcare (Buckinghamshire, UK) and used at a 1:5000 dilution. Mouse monoclonal anti-human nuclear antibody (MAB1281, clone 235-1) was from Milllipore. Avastin (Roche, 4 ml at 25 mg bevacizumab per ml, CAS216974-75-3) was purchased from the University of Maryland Cancer Center Pharmacy.

Cell culture

Monolayer cultures of SCC-13 squamous cell carcinoma cells were maintained in a Dulbecco’s modified Eagle’s medium containing 5% fetal bovine serum, 200 mM L-glutamine and 100 mM sodium pyruvate and appropriate antibiotics.24 ECS cell spheroids were grown by plating 40 000 cells/well in ultra-low attachment six-well cluster dishes and growing for 0–10 days in spheroid medium (DMEM/F12 (1:1) (DMT-10-090-CV, Mediatech Inc, Manassa, VA, USA) containing 2% B27 serum-free supplement (17504-044, Invitrogen), 20 ng/ml EGF (E4269, Sigma), 0.4% bovine serum albumin (B4287, Sigma) and 4 μg/ml insulin (#19278, Sigma Chemical).24

Electroporation

For electroporation, 1 × 10 cells were electroporated with 3 μg of control-(sc-37007, Santa Cruz, Dallas, TX, USA), VEGF-A- (sc-29520, Santa Cruz), or HIF-1α-(THEHC-00001, Dharmacon, Lafayette, CO, USA) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany). The cells were plated, permitted to recover overnight and then harvested, and the electroporation was repeated using the same siRNA.55 After a 24-h recovery, the cells were utilized for experiments. Reduced level of the siRNA targeted protein was confirmed by immunoblot.

Tumor xenograft growth assays

Monolayer or spheroid-derived cancer cells were prepared as a single-cell suspension by trypsin treatment, resuspended in phosphate-buffered saline containing 30% Matrigel and 100 μl containing 100 000 cells was injected subcutaneously at two sites in the front ventral flanks of five 6-week old female NOD scid IL2 receptor gamma chain knockout mice (NSG mice) using a 26.5-gauge needle. Tumor growth was monitored by measuring tumor diameter and calculating tumor volume using the formula, volume = 4/3π × (diameter/2)24,56 Tumors were photographed and sectioned for histology, and samples were harvested to prepare extracts for immunoblot, RNA detection and immunostaining.24 These experiments were reviewed and approved by the University of Maryland Baltimore Institutional Animal Care and Use Committee. Differences in tumor number were assessed using the t-test.

Bevacizumab treatment

Patient-grade bevacizumab was purchased as 100 mg Bevacizumab in 4-ml sterile phosphate buffer (NDC50242-060-01, Genentech, Inc, San Francisco, CA, USA). The solution was diluted in sterile phosphate buffer to 1 mg/ml for intraperitoneal injection of 0.2 ml three times per week (M, W, F) for a final level of 10 mg/kg body weight. Control mice were injected with phosphate buffer.

EG00229 treatment

EG00229 is a small molecular inhibitor of VEGF-A interaction with NRP-1.2931 For cell culture studies, EG00229 (R&amp;D Systems, #4931) was dissolved in DMSO to produce a 100 mM (50 mg/ml) stock. For in vivo studies, EG00229 was diluted to 2 mM (1 mg/ml) in 20% captisol, a non-toxic delivery vehicle.57 In brief, 40% captisol (RC-0C7-020, CyDex Pharmaceuticals, Lawrence, KS, USA) was prepared in sterile water by stirring overnight at 25°C followed by filter sterilization. The 40% captisol solution (50 ml) was diluted 1:1 with sterile water and supplemented with 0.5 ml of 1N acetic acid to create 20% captisol solution. The 50 mg/ml EG00229 stock was diluted 1:50 into 20% captisol solution and 0.2 ml was injected intraperitoneally three times per week (M, W, F) to achieve a final level of 10 mg/kg body weight.

Immunostaining

Primary tumors were paraffin-embedded and sectioned. The sections were deparaffinized by treatment with xylene followed by alcohol rehydration. Antigens were recovered by heating the slides in Na-Citrate buffer (10 mM, pH 6.5) for 20 min. Endogenous peroxidase activity was blocked by treatment with 3% H2O2 in phosphate-buffered saline for 20 min. The sections were rinsed in phosphate-buffered saline and non-specific sites were blocked overnight at 4°C in phosphate-buffered saline containing 1% bovine serum albumin and 5% normal goat or horse serum. Antigen was detected using the Vectastain ABC kit (Vector Labs, Burlingame, CA, USA). Sections were incubated with primary antibody for 2 h at room temperature followed by biotin-conjugated secondary antibody for 1 h. Antibody binding was visualized using 3,3-diaminobenzidine peroxidase substrate.

Immunoblot analysis

Tumor tissue was pulverized in liquid nitrogen and placed in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin).24,37 The resulting lysates were sonicated, centrifuged and supernatants were collected. Equivalent amounts of protein were electrophoresed on denaturing and reducing 12% poly-acrylamide gels and transferred to nitrocellulose. The membrane was blocked by 5% non-fat dry milk and then incubated with appropriate primary (1:1000) and secondary antibody (1:5000). Secondary antibody binding was visualized using chemiluminescence detection technology.24 For co-precipitation, ECS cells (10-day spheroids) were collected and extracts were prepared in lysis buffer. Human VEGF-A (100 ng, ab9571) was added to 200 μg of cell lystate followed by addition of mouse immunoglobulin or mouse anti-VEGF-A (1 μg, MAB293, R&amp;D Systems) or rabbit anti-NRP-1 (1 μg, ab81321) followed by incubation overnight at 4°C. Protein A agarose beads (20 μl of 50% slurry, pre-washed in lysis buffer) was added to 200 μg of lysate and incubated for 3 h at 4°C with shaking followed by two washes with lysis buffer and resuspension in 40 μl of Laemmli loading buffer with reducing agent. Lysates were electrophoresed on denaturing and reducing 10% polyacrylamide gels and transferred to nitrocellulose for subsequent immunoblot analysis. Immunoprecipitations were run adjacent a lane containing 25 μg of total extract. For immunoblot primary antibodies were diluted 1:1000 and secondary antibodies 1:5000, and antibody binding was visualized by chemiluminescence.

HUVEC cell angiogenesis assay

Twenty-four well cluster dishes were coated with a 10 mg/ml solution of BD Matrigel (354234, BD Biosciences, Franklin Lakes, NJ, USA) and incubated at 37°C for 30 min. Early passage HUVEC cells (1.2 × 10 cells) (Lonza, Basel, Switzerland; CC-2519) are plated in endothelial basal medium basal medium (Lonza, CC-3121) supplemented with human epidermal growth factor, hydrocortisone, bovine brain extract, ascorbic acid, fetal bovine serum and gentamicin/amphotericin-B (Lonza, CC-4133). For tube-formation assay, this medium was supplemented with 0 or 300 μg of tumor extract. The plates were the incubated for 18 h at 37°C and microscopic images were collected for analysis of junction, segment and node formation using ImageJ angiogenesis analyzer software (http://imagej.nih.gov/ij/macros/toolsets/).19

Quantitative reverse transcriptase PCR

RNA was isolated according to the Illustra RNAspin Mini RNA Isolation Kit protocol and 1 μg of RNA was used for complementary DNA synthesis. PCR was performed on the Roche 480 Lightcycler. Primers included: VEGF-A (forward 5′-CCTGGTGGACATCTTCCAGGAGTACC, reverse 5′-GAAGC TCATCTCTCCTATGTGCTGGC), VEGFR1 (forward 5′-CTAGGATCCGTGACTTAT TTTTTCTCAACAAGG, reverse 5′-CTCGAATTCAGATCTTCCATAGTGATGGGC TC), VEGFR2 (forward 5′-CTGGCATGGTCTTCTGTGAAGCA, reverse 5′-AATAC CAGTGGATGTGATGCGG), NRP-1 (forward 5′-AGGACAGAGACTGCAAGTA TGAC, reverse 5′-AACATTCAGGACCTCTCTTGA), and HIF-1α (forward 5′-CC TGAGCCTAATAGTCCCAGTG, reverse 5′-GGTGACAACTGATCGAAGGAACG).

Chemicals and reagents

Trypsin and Dulbecco’s modified Eagle’s medium were purchased from Invitrogen (Frederick, MD, USA). β-actin antibody (A5441) was obtained from Sigma (St Louis, MO, USA). Anti-H3K27me3 (07-449) was purchased from Millipore (Bedford, MA, USA). Ezh2 (612667) antibody was obtained from BD Transduction Labs (San Jose, CA, USA). Mouse monoclonal antibody to VEGF-A (MAB293) was purchased from R&amp;D Systems (Minneapolis, MN, USA). Antibodies to CD31 (ab28364), Bmi-1 (ab14389), NRP-1 (ab81321), VEGFR2 (ab2349) were from Abcam (Cambridge, MA, USA). Human recombinant VEGF-A (ab9571) was from Abcam. Anti-VEGFR1 (2893) was from Cell Signaling (Danvers, MA, USA). Anti-keratin 5 (AF 138) was obtained from Covance (Princeton, NJ, USA). Horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin (NXA931) and donkey anti-rabbit immunoglobulin (NA934V) were obtained from GE Healthcare (Buckinghamshire, UK) and used at a 1:5000 dilution. Mouse monoclonal anti-human nuclear antibody (MAB1281, clone 235-1) was from Milllipore. Avastin (Roche, 4 ml at 25 mg bevacizumab per ml, CAS216974-75-3) was purchased from the University of Maryland Cancer Center Pharmacy.

Cell culture

Monolayer cultures of SCC-13 squamous cell carcinoma cells were maintained in a Dulbecco’s modified Eagle’s medium containing 5% fetal bovine serum, 200 mM L-glutamine and 100 mM sodium pyruvate and appropriate antibiotics.24 ECS cell spheroids were grown by plating 40 000 cells/well in ultra-low attachment six-well cluster dishes and growing for 0–10 days in spheroid medium (DMEM/F12 (1:1) (DMT-10-090-CV, Mediatech Inc, Manassa, VA, USA) containing 2% B27 serum-free supplement (17504-044, Invitrogen), 20 ng/ml EGF (E4269, Sigma), 0.4% bovine serum albumin (B4287, Sigma) and 4 μg/ml insulin (#19278, Sigma Chemical).24

Electroporation

For electroporation, 1 × 10 cells were electroporated with 3 μg of control-(sc-37007, Santa Cruz, Dallas, TX, USA), VEGF-A- (sc-29520, Santa Cruz), or HIF-1α-(THEHC-00001, Dharmacon, Lafayette, CO, USA) siRNA using the Amaxa electroporator and the VPD-1002 nucleofection kit (Germany). The cells were plated, permitted to recover overnight and then harvested, and the electroporation was repeated using the same siRNA.55 After a 24-h recovery, the cells were utilized for experiments. Reduced level of the siRNA targeted protein was confirmed by immunoblot.

Tumor xenograft growth assays

Monolayer or spheroid-derived cancer cells were prepared as a single-cell suspension by trypsin treatment, resuspended in phosphate-buffered saline containing 30% Matrigel and 100 μl containing 100 000 cells was injected subcutaneously at two sites in the front ventral flanks of five 6-week old female NOD scid IL2 receptor gamma chain knockout mice (NSG mice) using a 26.5-gauge needle. Tumor growth was monitored by measuring tumor diameter and calculating tumor volume using the formula, volume = 4/3π × (diameter/2)24,56 Tumors were photographed and sectioned for histology, and samples were harvested to prepare extracts for immunoblot, RNA detection and immunostaining.24 These experiments were reviewed and approved by the University of Maryland Baltimore Institutional Animal Care and Use Committee. Differences in tumor number were assessed using the t-test.

Bevacizumab treatment

Patient-grade bevacizumab was purchased as 100 mg Bevacizumab in 4-ml sterile phosphate buffer (NDC50242-060-01, Genentech, Inc, San Francisco, CA, USA). The solution was diluted in sterile phosphate buffer to 1 mg/ml for intraperitoneal injection of 0.2 ml three times per week (M, W, F) for a final level of 10 mg/kg body weight. Control mice were injected with phosphate buffer.

EG00229 treatment

EG00229 is a small molecular inhibitor of VEGF-A interaction with NRP-1.2931 For cell culture studies, EG00229 (R&amp;D Systems, #4931) was dissolved in DMSO to produce a 100 mM (50 mg/ml) stock. For in vivo studies, EG00229 was diluted to 2 mM (1 mg/ml) in 20% captisol, a non-toxic delivery vehicle.57 In brief, 40% captisol (RC-0C7-020, CyDex Pharmaceuticals, Lawrence, KS, USA) was prepared in sterile water by stirring overnight at 25°C followed by filter sterilization. The 40% captisol solution (50 ml) was diluted 1:1 with sterile water and supplemented with 0.5 ml of 1N acetic acid to create 20% captisol solution. The 50 mg/ml EG00229 stock was diluted 1:50 into 20% captisol solution and 0.2 ml was injected intraperitoneally three times per week (M, W, F) to achieve a final level of 10 mg/kg body weight.

Immunostaining

Primary tumors were paraffin-embedded and sectioned. The sections were deparaffinized by treatment with xylene followed by alcohol rehydration. Antigens were recovered by heating the slides in Na-Citrate buffer (10 mM, pH 6.5) for 20 min. Endogenous peroxidase activity was blocked by treatment with 3% H2O2 in phosphate-buffered saline for 20 min. The sections were rinsed in phosphate-buffered saline and non-specific sites were blocked overnight at 4°C in phosphate-buffered saline containing 1% bovine serum albumin and 5% normal goat or horse serum. Antigen was detected using the Vectastain ABC kit (Vector Labs, Burlingame, CA, USA). Sections were incubated with primary antibody for 2 h at room temperature followed by biotin-conjugated secondary antibody for 1 h. Antibody binding was visualized using 3,3-diaminobenzidine peroxidase substrate.

Immunoblot analysis

Tumor tissue was pulverized in liquid nitrogen and placed in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin).24,37 The resulting lysates were sonicated, centrifuged and supernatants were collected. Equivalent amounts of protein were electrophoresed on denaturing and reducing 12% poly-acrylamide gels and transferred to nitrocellulose. The membrane was blocked by 5% non-fat dry milk and then incubated with appropriate primary (1:1000) and secondary antibody (1:5000). Secondary antibody binding was visualized using chemiluminescence detection technology.24 For co-precipitation, ECS cells (10-day spheroids) were collected and extracts were prepared in lysis buffer. Human VEGF-A (100 ng, ab9571) was added to 200 μg of cell lystate followed by addition of mouse immunoglobulin or mouse anti-VEGF-A (1 μg, MAB293, R&amp;D Systems) or rabbit anti-NRP-1 (1 μg, ab81321) followed by incubation overnight at 4°C. Protein A agarose beads (20 μl of 50% slurry, pre-washed in lysis buffer) was added to 200 μg of lysate and incubated for 3 h at 4°C with shaking followed by two washes with lysis buffer and resuspension in 40 μl of Laemmli loading buffer with reducing agent. Lysates were electrophoresed on denaturing and reducing 10% polyacrylamide gels and transferred to nitrocellulose for subsequent immunoblot analysis. Immunoprecipitations were run adjacent a lane containing 25 μg of total extract. For immunoblot primary antibodies were diluted 1:1000 and secondary antibodies 1:5000, and antibody binding was visualized by chemiluminescence.

HUVEC cell angiogenesis assay

Twenty-four well cluster dishes were coated with a 10 mg/ml solution of BD Matrigel (354234, BD Biosciences, Franklin Lakes, NJ, USA) and incubated at 37°C for 30 min. Early passage HUVEC cells (1.2 × 10 cells) (Lonza, Basel, Switzerland; CC-2519) are plated in endothelial basal medium basal medium (Lonza, CC-3121) supplemented with human epidermal growth factor, hydrocortisone, bovine brain extract, ascorbic acid, fetal bovine serum and gentamicin/amphotericin-B (Lonza, CC-4133). For tube-formation assay, this medium was supplemented with 0 or 300 μg of tumor extract. The plates were the incubated for 18 h at 37°C and microscopic images were collected for analysis of junction, segment and node formation using ImageJ angiogenesis analyzer software (http://imagej.nih.gov/ij/macros/toolsets/).19

Quantitative reverse transcriptase PCR

RNA was isolated according to the Illustra RNAspin Mini RNA Isolation Kit protocol and 1 μg of RNA was used for complementary DNA synthesis. PCR was performed on the Roche 480 Lightcycler. Primers included: VEGF-A (forward 5′-CCTGGTGGACATCTTCCAGGAGTACC, reverse 5′-GAAGC TCATCTCTCCTATGTGCTGGC), VEGFR1 (forward 5′-CTAGGATCCGTGACTTAT TTTTTCTCAACAAGG, reverse 5′-CTCGAATTCAGATCTTCCATAGTGATGGGC TC), VEGFR2 (forward 5′-CTGGCATGGTCTTCTGTGAAGCA, reverse 5′-AATAC CAGTGGATGTGATGCGG), NRP-1 (forward 5′-AGGACAGAGACTGCAAGTA TGAC, reverse 5′-AACATTCAGGACCTCTCTTGA), and HIF-1α (forward 5′-CC TGAGCCTAATAGTCCCAGTG, reverse 5′-GGTGACAACTGATCGAAGGAACG).

Acknowledgments

This work was supported by grants from the Maryland Stem Cell Research Foundation (RLE) and the National Institutes of Health (RLE - CA131074, CA184027) and a pilot grant from the Greenebaum Cancer Center (P30 CA134274).

Departments of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MA, USA
Departments of Dermatology, University of Maryland School of Medicine, Baltimore, MA, USA
Departments of Reproductive Biology, University of Maryland School of Medicine, Baltimore, MA, USA
Departments of Marlene and Stewart Greenebaum Cancer, University of Maryland School of Medicine, Baltimore, MA, USA
Correspondence: Dr RL Eckert, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Rm 103, Baltimore, MA 21201, USA. ude.dnalyramu@trekcer

Abstract

We identify a limited subpopulation of epidermal cancer stem cells (ECS cells), in squamous cell carcinoma, that form rapidly growing, invasive and highly vascularized tumors, as compared with non-stem cancer cells. These ECS cells grow as non-attached spheroids, and display enhanced migration and invasion. We show that ECS cell-produced vascular endothelial growth factor (VEGF)-A is required for the maintenance of this phenotype, as knockdown of VEGF-A gene expression or treatment with VEGF-A-inactivating antibody reduces these responses. In addition, treatment with bevacizumab reduces tumor vascularity and growth. Surprisingly, the classical mechanism of VEGF-A action via interaction with VEGF receptors does not mediate these events, as these cells lack VEGFR1 and VEGFR2. Instead, VEGF-A acts via the neuropilin-1 (NRP-1) co-receptor. Knockdown of NRP-1 inhibits ECS cell spheroid formation, invasion and migration, and attenuates tumor formation. These studies suggest that VEGF-A acts via interaction with NRP-1 to trigger intracellular events leading to ECS cell survival and formation of aggressive, invasive and highly vascularized tumors.

Abstract

Footnotes

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Footnotes

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