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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cell. Author manuscript; available in PMC 2014 February 28.
Published in final edited form as:
PMCID: PMC3587980
NIHMSID: NIHMS441266

Targeting placental growth factor/neuropilin 1 pathway inhibits growth and spread of medulloblastoma

SUMMARY

Medulloblastoma is the most common pediatric malignant brain tumor. Although current therapies improve survival, these regimens are highly toxic and associated with significant morbidity. Here, we report that placental growth factor (PlGF) is expressed in the majority of medulloblastomas independent of their subtype. Moreover, high expression of PlGF receptor neuropilin 1 (Nrp1) correlates with poor overall survival in patients. We demonstrate that PlGF and Nrp1 are required for the growth and spread of medulloblastoma: PlGF/Nrp1 blockade results in direct antitumor effects in vivo, resulting in medulloblastoma regression, decreased metastases, and increased mouse survival. We reveal that PlGF is produced in the cerebellar stroma via tumor-derived Sonic hedgehog (Shh) and show that PlGF acts through Nrp1—and not vascular endothelial growth factor receptor 1 (VEGFR1)—to promote tumor cell survival. This critical tumor-stroma interaction—mediated by Shh, PlGF, and Nrp1 across medulloblastoma subtypes—supports the development of therapies targeting PlGF/Nrp1 pathway.

INTRODUCTION

Medulloblastoma is the most common malignant pediatric brain tumor and constitutes 20% of all brain tumors in children (Louis, 2007). The standard of care includes initial surgery followed by radiation and chemotherapy. Despite high survival rates with current regimens, patients encounter devastating morbidity, including decline in cognition and intellect (Fouladi et al., 2005), and secondary malignancies (Goldstein et al., 1997). Therefore, there is an urgent need for new therapies that reduce the significant morbidity associated with current treatments.

Medulloblastomas can be classified into at least 4 distinct molecular groups, suggesting the possibility of developing targeted therapies directed against key drivers (Northcott et al., 2012). For example, therapy targeting Sonic hedgehog (Shh) signaling, a driver of one genetic subset of medulloblastomas, showed promising results initially (Rudin et al., 2009). However, tumors rapidly developed resistance mutations (Yauch et al., 2009), thus emphasizing the importance of alternative targeted treatment strategies. Targeting tumor-stroma interactions could provide an alternative targeting strategy, given the critical role of the tumor microenvironment in cancer progression and greater genetic stability of the stromal compartment (Carmeliet and Jain, 2011; Fukumura et al., 2010; Goel et al., 2011).

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family and is involved in bone marrow-derived cell activation, endothelial stimulation, pathologic angiogenesis and wound healing (Carmeliet et al., 2001; Fischer et al., 2008, Dewerchin and Carmeliet, 2012). PlGF is overexpressed in breast and gastric carcinomas but downregulated in colon and lung carcinomas (Fischer et al., 2007; Xu and Jain, 2007). While in some models PlGF promotes tumor growth (Schmidt et al., 2011), in other models PlGF inhibits tumor growth (Xu et al., 2006). PlGF is mitogenic and proangiogenic (Ziche et al., 1997), and blockade of PlGF has resulted in antiangiogenic and antitumor effects in some models (Fischer et al., 2007; Van de Veire et al., 2010) but not in others (Bais et al., 2010). VEGFR1, the only known tyrosine kinase receptor for PlGF, was shown to be required for certain tumors that respond to anti-PlGF therapy (Yao et al., 2011). However, direct inhibition of VEGFR1 activity did not explain anti-PlGF effects in other models (Casanovas et al., 2005; Dawson et al., 2009), suggesting an alternative signaling mechanism for PlGF independent of VEGFR1.

Since PlGF is dispensable during development, we proposed anti-PlGF as a therapeutic approach for pediatric tumors (Jain and Xu, 2007). Here, we demonstrate that PlGF is necessary for medulloblastoma growth irrespective of genetic background. We found that PlGF is produced by the cerebellar stroma in response to Shh ligand secreted by tumor cells. We also found that in medulloblastoma PlGF signaling via Nrp1 and not VEGFR1 directly conveys pro-survival signals and that Nrp1 expression correlated with poor overall survival. Together with the reported minimal side effects of anti-PlGF blockade (Fischer et al., 2007, Lassen et al., 2012), these findings identify PlGF and Nrp1 as potential targets for therapy of pediatric medulloblastoma.

RESULTS

PlGF is expressed in medulloblastomas regardless of their genetic background

By IHC we found that PlGF is expressed in a variety of brain tumors of children and adults including medulloblastoma, ependymoma, atypical teratoid and rhabdoid tumor and GBM (Figure 1A, C). We compared expression of angiogenic and vascular remodeling factors in pediatric medulloblastomas (n = 5) against normal pediatric cerebella (n = 2; 3 and 5 years of age) and normal adult cerebella (n = 4; all > 60 years of age). We found a marked overexpression of PlGF (14-fold) and Nrp1 (11-fold) in tumor tissues. VEGF receptors were either slightly upregulated (VEGFR2/KDR; 2-fold) or modestly downregulated (VEGFR1/FLT1; 2.6-fold) (Figure 1B and Supplemental Table ST1). The patterns of gene expression in medulloblastoma and immature brain were quite similar, suggesting that factors active during cerebellar development may also be activated in pediatric brain tumors.

Figure 1
Expression of PlGF and its receptors in brain tumors

We analyzed 32 clinical samples of different medulloblastoma subtypes by immunohistochemistry (IHC). Of these tumors, ~90% (29 in 32) diffusely expressed PlGF (Figure 1C, D) across all World Health Organization (WHO) subtypes (Louis, 2007) and all main molecular subtypes of medulloblastoma with the lowest expression in the Wnt group (Figure 1D). While both PlGF receptors were expressed, Nrp1 expression was more striking than VEGFR1 (Figure 1C and Supplemental Figure S1A). Analysis of publicly available copy number data (Tumorscape, Broad Institute) showed ~13% of medulloblastomas with gains of the chromosomal region containing PlGF (Supplemental Figure 1B), suggesting one direct genetic mechanism of PlGF upregulation. Taken together, our data show that PlGF is present in the majority of human medulloblastomas and is not associated with any particular subtype.

PlGF blockade leads to medulloblastoma regression

Mice with D283-MED and D341-MED human medulloblastoma xenografts exhibited clinical symptoms (CS) in a period of 3 to 4-weeks from implantation, and showed a similar profile to clinical samples (Figure 1C and Supplemental Figure S1C). Using the same classification methods (Taylor et al., 2011), D341-MED were classified as Group 3 and D283-MED as Group 4 medulloblastoma, respectively (Figure 1C). These tumors grew rapidly and spread along the spinal cord via cerebrospinal fluid (Figure 2A), closely resembling the clinical behavior of human medulloblastoma.

Figure 2
Inhibition of PlGF leads to regression of medulloblastoma

We treated established tumors with TB403, a dual blocking antibody for human and murine PlGF from Roche (Nielsen and Sengelov, 2012). TB403 (αPlGF [R]) treatment led to inhibition of primary tumor growth and spinal metastasis (Figure 2A–B, and Supplemental Figure S1D). Anti-PlGF treated mice survived significantly longer both in the D283-MED (p < 0.0001, 60% no CS) and D341-MED (p < 0.0001, 70% no CS) models (Figure 2C).

We also tested the effects of another dual anti-human/murine anti-PlGF Ab, C9.V2 from Genentech (Bais et al., 2010). C9.V2 (αPlGF [G]) treated D283-MED bearing mice showed a significant survival increase (Figure 2C) and a delay in tumor growth, although not tumor regression (Supplemental Figure S1E).

We next evaluated the effect of anti-PlGF blockade in Smo/Smo transgenic mice (Hatton et al., 2008). Immunohistochemical analysis of medulloblastomas from Smo/Smo mice – representative of the Shh subgroup of tumors – showed expression of PlGF and Nrp1 (Figure 1C). When Smo/Smo mice reached 6–8 weeks of age, we treated with the murine specific anti-PlGF antibody 5D11D4 (Fischer et al., 2007) or C9.V2 for 3 weeks. Anti-PlGF treated mice had significantly smaller tumors and less body weight loss (body weight 29.4 g vs. 23.1 g, p = 0.0044), and did not show a hunched posture seen in control-treated mice (Figure 2D, E).

Furthermore, PlGF levels in Smo/Smo mice with established medulloblastoma were dramatically increased compared to wild-type mice (Supplemental Figure S1F). Together, these data indicate that PlGF contributes to the progression of medulloblastoma and can be targeted by a number of different anti-PlGF antibodies.

Medulloblastoma recruits host-derived cerebellar stromal cells that secrete PlGF and sustain tumor growth

We found that circulating murine (m)PlGF levels were highest in younger mice and that the medulloblastoma growth was enhanced in younger mice (Supplemental Figure S1G, H). Further, growth of untreated D283-MED tumors was accompanied by a significant increase in tumor-derived human (h)PlGF in the cerebellum and in the blood (Figure 3A). The levels of stromal mPlGF also increased with time and by 5-weeks contributed to about 80% of the total blood PlGF concentration (mPlGF = 15 pg/ml; hPlGF = 3 pg/mL).

Figure 3
Role of stromal PlGF in growth of medulloblastoma

Implantation of D283-MED with hPlGF silenced (Supplemental Figure S1I–J) resulted in significant delays in tumor growth and spinal cord spread, and increased survival (Figure 3B, D). These tumors also induced substantially lower levels of host stromal mPlGF secretion (Figure 3A). Thus, initial production of hPlGF by the tumor cells appeared critical for the initial growth of the tumors and stimulation of stromal mPlGF production. Re-expression of PlGF2 in a PlGF shRNA-3′UTR construct in D283-MED cells (Supplemental Figure S1I) rescued rapid tumor growth and short survival phenotype (Figure 3B, D).

To determine the importance of stromal PlGF, we blocked mPlGF with 5D11D4 either starting at day 1 after tumor implantation (prevention), or starting at day 21 when tumors produced CS (intervention). Mice treated from day 1 had significantly lower tumor burden, no spinal spread and increased average overall survival (p < 0.0001) (Figure 3C) similar to mice with tumors that had genetically silenced PlGF (62.5% no CS in both PlGF inhibited groups, Figure 3D). Mice treated from day 21 showed significant reduction in the size of primary tumors and in the average number of spinal metastasis per mouse (2.0 vs. 5.0, p < 0.001) (Figure 3E) and prolonged survival (p < 0.0001, 60% no CS) (Figure 3C, D). There was a small additional effect of TB403 (which blocks both hPlGF and mPlGF) compared to 5D11D4 (which only blocks mPlGF) (Supplemental Figure S1K).

We then implanted Smo/Smo medulloblastoma tumors in the cerebellum of syngeneic plgf−/− mice. We found a significantly lower tumor take rate, delayed growth, and increased survival in the plgf−/− mice compared to the wild-type controls (2/10 vs. 11/11 and Figure 4A, Supplemental Figure S2A). Overall, our results indicate that medulloblastoma tumor cells secrete PlGF but that a substantial portion of PlGF is derived from the stroma and medulloblastoma growth requires stromal PlGF.

Figure 4
Role of Shh in stromal PlGF secretion

Tumor-secreted Shh ligands stimulate paracrine stromal production of PlGF

We investigated stromal production of PlGF and examined the role of Shh, a key driver of tumorigenesis in medulloblastoma. Tumor-derived Shh ligands were shown to sustain tumorigenesis by signaling activation in the stromal compartment (Walter et al., 2010). D283-MED and D341-MED medulloblastoma models secreted Shh ligands (Supplemental Figure S2B), despite absence of genetic changes in Shh pathway. For stroma, we isolated cerebellar glial and external granule layer cells, neither of which produced significant amount of basal Shh or PlGF (Figure 4B–C and Supplemental Figure S2C). Stimulation with Shh induced a dose-dependent secretion of PlGF by granule cells but had no detectable effect on glial cells (Figure 4B). Stimulation of the Wnt pathway which is also involved in normal cerebellum development (Pei et al, 2012), by Wnt-3a or Wnt-7a proteins, did not induce PlGF secretion (Supplemental Figure S2D). Medulloblastoma cells were unresponsive to Shh stimuli, perhaps due to high basal levels of endogenous PlGF in vitro (Supplemental Figure S2E).

In co-cultures of isolated mouse granule cells and D341-MED cells, the secreted levels of Shh increased in a time-dependent manner and correlated with increased secretion of mPlGF (Figure 4C). Treatment with the Shh inhibitor cyclopamine led to a significant decrease in circulating mPlGF levels in mice bearing D283-MED (Figure 4D). In addition, we found that medulloblastoma-bearing mice treated with anti-mPlGF therapy had increased circulating hPlGF (Figure 4E) while tumors decreased in size (Figure 3C). These tumors also showed upregulated expression of Shh and its effector Gli1 (Figure 4F). Collectively, these data suggest that medulloblastoma co-opts the stroma to produce PlGF through paracrine Shh signaling, possibly through activation of Gli1. Furthermore, the tumor responds to a dramatic drop in stromal PlGF levels by secreting Shh, upregulating Gli1 and increasing tumor cell production of PlGF as a compensatory mechanism (Figures 3A and and4E).4E). We down-regulated Shh with shRNA in tumor cells (D283-shSHH tumors) and showed significant growth delay and significant increase in survival (p < 0.001, Figure 4G–H).

Blockade of PlGF has modest effects on angiogenesis and no effect on tumor-associated macrophages (TAMs) in medulloblastoma

PlGF regulates the angiogenic switch and anti-PlGF therapy can inhibit angiogenesis in some tumor models (Fischer et al., 2007; Rolny et al., 2011). We analyzed the number and function of vessels in anti-PlGF Ab treated or shPLGF medulloblastomas. We found a significant but modest decrease in tumor microvascular density (~30% decrease, p < 0.0001, Figure 5A–B) but no change in tumor vessel diameter, vascular permeability or hypoxic fraction (Supplemental Figure S3A and data not shown).

Figure 5
Inhibition of PlGF has moderate anti-angiogenic effect on medulloblastoma

Inhibition of PlGF may also affect tumors by reducing TAM infiltration (Fischer et al., 2007) or by inducing TAM polarization towards an anti-tumor phenotype (Rolny et al., 2011). We analyzed changes in TAMs in medulloblastoma after PlGF blockade. Medulloblastomas had a very small number of TAMs at baseline and anti-PlGF therapy did not significantly change the TAM recruitment or polarization (Supplemental Figure S3B). Thus, the effects of anti-PlGF therapy on angiogenesis and TAMs are unlikely to mediate the dramatic tumor reduction measured by optical frequency domain imaging (OFDI) (Figure 5C–D).

PlGF directly activates pro-survival pathways in medulloblastoma

We next sought to determine if PlGF has direct effects on medulloblastoma cells. We stimulated D283-MED and D341-MED cells with rhPlGF, and Smo/Smo medulloblastoma cells isolated from transgenic mice with rmPlGF, and evaluated the activation of canonical survival pathways. Stimulation with PlGF induced marked phosphorylation of pro-survival/pro-growth pERK1/2 in all medulloblastoma lines as well as activation of PI3K/Akt in the Smo/Smo cells (Figure 6A and Supplemental Figure S4A–B). We used the MEK inhibitor U0126 to test if maintenance of MAPK signaling is essential for medulloblastoma viability. Treatment with non-toxic doses of U0126 induced a rapid decrease in medulloblastoma cell viability (Supplemental Figure S4C). We compared the levels of ERK1/2 activation and apoptosis in control versus treated tumor tissues. Blockade of stromal PlGF by 5D11D4 induced a decrease in pERK1/2 and increase in apoptosis of tumor cells in both the xenograft and transgenic models but no difference in cell proliferation (Supplemental Figure S5). Overall, these data suggest that stromal PlGF actively sustains the survival of tumor cells through activation of the MAPK cascade.

Figure 6
PlGF signaling in medulloblastoma is dependent on Nrp1

Survival signaling is mediated via Nrp1 but not VEGFR1

To determine the transduction of PlGF survival signaling, we investigated the role of Nrp1 and VEGFR1. We established a stable D283-MED shNRP1-GFP tagged cell line, and tested the response of high versus low Nrp1-expressing cell populations to rhPlGF. Cells with relatively low level of Nrp1 knockdown (selected as 5% GFP-dimmest cells) showed activation of ERK1/2, while cells with high level of Nrp1 knockdown (lowest Nrp1 expression, selected as top 5% GFP-brightest cells) failed to activate ERK in the presence of PlGF (Figure 6B). D283-MED shNRP1 tumors exhibited a significant growth delay in mice (Supplemental Figure S6A) and did not exhibit spinal cord metastasis (Figure 6C–D), which significantly extended survival (p < 0.0001, 43% no CS) (Figure 6E). D283-MED shNRP1 tumors had preserved proliferation activity measured by Ki-67 implying that genetic inhibition of Nrp1 does not affect medulloblastoma cell proliferation (Supplemental Figure S6D). Interestingly, genetic silencing of Nrp2 did not affect growth of tumor cells in vitro or in vivo (data not shown).

We treated human medulloblastoma cell lines and murine Smo/Smo cells with species-specific Nrp1-blocking antibodies and analyzed PlGF-mediated activation of MAPK and Akt signaling. We detected increased pERK1/2 and pAkt after PlGF stimulation. Blockade of Nrp1 prevented the activation of MEK1/2 and Akt by PlGF to a similar extent compared to anti-PlGF antibody (Figure 6A).

A recent report suggested that response to anti-PlGF therapy is restricted to a subset of VEGFR1 expressing tumors (Yao et al., 2011). VEGFRs are classically necessary partners of Nrp1 for effective signal transduction (Bielenberg et al., 2006). Thus, we next examined the relative role of VEGFR1 in PlGF signaling in medulloblastoma. When we co-immunoprecipitated VEGFR1 and Nrp1 in unstimulated and PlGF-stimulated D283-MED and Smo/Smo cells, VEGFR1 and Nrp1 were pulled-down together. However, we found very small differences in the amount of complex between control and stimulated cells and minor differences in tyrosine phosphorylation of VEGFR1 before and after PlGF stimulation (Supplemental Figure S6B and data not shown). Moreover, when we genetically inhibited VEGFR1 in D283-MED cells (D283-MED shVEGFR1), we found no effect on pERK1/2 following PlGF stimulation (Supplemental Figure S6C). Furthermore, when we implanted stable D283-MED shVEGFR1 cells in vivo, we found that the tumors with VEGFR1 down-regulation exhibited no difference in growth (Figure 6G and Supplemental Figure S6E). Finally, when we crossbred Smo/Smo transgenic mice (Hatton et al., 2008) with VEGFR1/Flt1-tyrosine kinase knockout (flt1TK−/−) mice (Hiratsuka et al., 1998), we found that double homozygote (Smo/Smo/flt1TK−/−) mice developed tumors similar to Smo/Smo transgenic mice, further showing that VEGFR1 activity is not necessary for development of medulloblastoma (Figure 6H).

Intracellular domain of Nrp1 is essential for effect of PlGF

Nrp1 is a receptor with short cytoplasmic tail, and is widely regarded as a non-signaling receptor. To determine whether Nrp1 is involved in transduction of downstream PlGF signaling, we overexpressed a mutant form of Nrp1 in D283-MED cells (Nrp1-ΔSEA) that retains its functional binding sites for PlGF but lacks the three intracellular C-terminal amino acids (SEA-COOH) that bind PDZ-adaptor signaling proteins and is unable to transduce signaling. Mice bearing medulloblastomas with overexpressed Nrp1-ΔSEA showed significant tumor growth delay and increased survival (p < 0.01, 100% no CS) (Figure 6C, F). These results demonstrate that PlGF activation of intracellular Nrp1-mediated signaling is a requirement for cell growth and survival in medulloblastoma.

In the therapeutic setting, mice treated with anti-Nrp1 antibody (Pan et al., 2007) showed significant tumor growth delay and improved survival (p < 0.01, 100% no CS) (Figure 7A–B). Moreover, we found that high expression of Nrp1 in tumors significantly correlates with a decreased overall survival in an independent cohort of medulloblastoma patients (p = 0.0058) (Figure 7C). Collectively these results support evaluation of the available PlGF and Nrp1 antibodies in medulloblastoma patients (Lassen et al., 2012, Martinsson-Niskanen et al., 2011, Weekes et al. 2010).

Figure 7
Growth of medulloblastoma is dependent on Shh stimulated PlGF secretion via Nrp-1 signaling

DISCUSSION

Role of PlGF in cancer remains unclear, perhaps due to context dependent effects of this growth factor in tumor progression. We show here that 1) PlGF is expressed in the majority of human pediatric medulloblastomas regardless of their molecular/genetic subtype and is required for the growth and spread of three subtypes of medulloblastoma models in mice; 2) the majority of PlGF protein is secreted by cerebellar stromal cells in vivo; 3) the stromal PlGF production is stimulated via paracrine Shh secretion by the cancer cells; and 4) the PlGF/Nrp1/MAPK signaling axis is critical for survival of medulloblastoma in vitro and in vivo (Figure 7D). These findings provide insights into the role of Shh, PlGF and Nrp1 in medulloblastoma and offer a different approach to targeted therapy.

There may be several mechanisms of PlGF upregulation in medulloblastoma, including copy number gain or mutations of PlGF or its regulators. In addition, medulloblastoma cells also secrete Shh, which stimulates production of stromal PlGF. Since we observed a strong tumor dependence on stromal-derived PlGF, blockade of PlGF presents targeted therapy that bypasses the tumor cell and disrupts a tumor-stroma interaction that is vital for disease progression. Our study provides insight into a role for Shh in medulloblastoma that seems to be unrelated to the tumor’s genetic origin and provides a new potential for use of Shh pathway inhibitors. If stimulation of Shh pathway in stromal cells is crucial for production of survival factor PlGF, inhibition of Shh signaling in stromal cells might prove to be as important as in the tumor cells without facing a challenge of acquired genetic resistance (Metcalfe and de Sauvage, 2011). Combined Shh and PlGF inhibition might therefore offer the maximum benefit in disrupting tumor-stroma interactions.

The exact role of PlGF in tumor growth and the potential benefit of its inhibition has been a matter of intense debate (Bais et al., 2010; Coenegrachts et al., 2010; Fischer et al., 2007; Fischer et al., 2008; Rolny et al., 2011; Schmidt et al., 2011; Van de Veire et al., 2010; Xu et al., 2006; Yao et al., 2011). What has emerged from our work is that the utility of PlGF inhibition in the clinic will be tumor-context dependent. We propose that tumor type, stage, microenvironment, and maybe even age of the patient will be major contributors to the outcome of therapy. While in some tumors PlGF may be important as an angiogenic factor, in other tumors – such as medulloblastoma – it might represent a vital growth factor for tumor survival. Although PlGF binding receptors have been extensively studied (Fischer et al., 2008), the exact downstream signaling and role in cancer cell survival is not well understood. Yao et al have shown that the effect of anti-PlGF inhibition depends on the presence of functional VEGFR1 in tumor cells (Yao et al., 2011). However, another report showed that genetic ablation of VEGFR1 tyrosine kinase domain in the host had no effect on tumor growth (Bais et al., 2010). Our study shows the critical importance of Nrp1 in medulloblastoma and reveals PlGF/Nrp1 axis as a necessary survival mechanism. The cytoplasmic protein docking (SEA) domain of Nrp1 is necessary for PlGF pro-survival signal transduction. Taken together, these data indicate that either PlGF or Nrp1 inhibition can provide therapeutic benefit.

Intriguingly, despite the initial tumor stabilization and regression, none of our treatments led to complete eradication of medulloblastomas. After initial regression tumors reached a plateau and persisted without clinical symptoms. When investigated by IHC, these remaining tumors showed reduced Nrp1 expression (Supplemental Figure S5A). The mechanisms of resistance after anti-PlGF therapy in medulloblastoma remain to be deciphered. Although we did not observe tumor recurrence in our models, it is conceivable that cells that do not depend on PlGF/Nrp1 axis for survival could lead to medulloblastoma recurrence. Therefore, anti-PlGF or anti-Nrp1 therapy in medulloblastoma would likely be used in concert with conventional regimens. Recent clinical trials of humanized anti-PlGF antibody TB-403 showed that anti-PlGF therapy was well tolerated with minimal side effects (Lassen et al., 2012; Martinsson-Niskanen et al., 2011).

Both TB403 and 5D11D4 therapy led to regression of medulloblastoma, which was dependent on Nrp1 signaling. C9.V2 was also active but induced a growth delay rather than tumor regression. This antibody has been reported to have the best effects in VEGFR1 dependent tumors (Yao et al., 2011), presumably by blocking PlGF-VEGFR1 interaction, but may be less potent in blocking PlGF-Nrp1 interaction (Bais et al., 2010). However, 5D11D4 was shown to potently block binding of PlGF to both Nrp1 and VEGFR1 (Fischer et al., 2007). This raises an important issue for development of therapeutic antibodies. In medulloblastoma PlGF signals via Nrp1 and therapy with an antibody blocking predominantly the interaction with VEGFR1 that might be efficacious in VEGFR1 overexpressing tumors (Yao et al., 2011) would likely be less effective than an antibody that potently blocks PlGF interaction with Nrp1.

In summary, we show that both tumor and stromal-derived PlGF signals through Nrp1 to directly sustain growth and spread of medulloblastoma; and inhibition of this pathway leads to regression and improved mouse survival. In conjunction with the low adverse effects, the PlGF/Nrp1 axis represents a promising target for therapy of medulloblastoma.

EXPERIMENTAL PROCEDURES

Clinical samples, histology and molecular classification

De-identified samples of medulloblastoma, ependymoma, glioblastoma, AT&RT, retinoblastoma and normal cerebella were obtained from the Department of Pathology, Massachusetts General Hospital, after IRB approval (protocol#: 2008-P-000079/3) and 42 clinically annotated samples treated according to the German HIT ‘91 protocol (Rutkowski et al. 2007) and the subsequent HIT 2000 protocol (ClinicalTrials.gov Identifier: NCT00303810) at the University Children’s Hospital Münster, Germany, between years 1998–2010 were collected following institutional approval.

Cell lines and animal models

Human medulloblastoma cell lines included D283-MED (ATCC® Number: HTB-185) and D341-MED (ATCC® Number: HTB-187) (Friedman et al., 1988; Friedman et al., 1985). Smo/Smo medulloblastoma mice (Hatton et al., 2008) were obtained from the Jackson Laboratory. flt1TK−/− mice (Hiratsuka et al. 1998) were generously provided by Dr. Shibuya, Tokyo University, Japan. Plgf−/− mice were obtained from Dr. Anne Croy, Queen’s University, Kingston, Canada.

Anti-PlGF and anti-Nrp1 antibodies

5D11D4, a specific anti-murine PIGF antibody, and TB403, a dual anti-human/murine PlGF antibody previously used in translational studies and phase I clinical trials (Lassen et al., 2012; Martinsson-Niskanen et al., 2011, Fischer et al., 2007) were provided by Hoffmann-La Roche Inc. Both anti-PlGF Abs were administered by intraperitoneally (i.p.), 3x/week, 25 mg/kg. C9.V2 dual anti-human/murine anti-PlGF antibody (Bais et al., 2010, Yao et al., 2011) was kindly provided by Genentech and applied i.p. twice a week, 30 mg/kg. Specific human anti-Nrp1 antibody (Pan et al., 2007) was provided by Genentech and applied i.p. twice a week, 10 mg/kg.

Cyclopamine treatment

SCID mice bearing D283-MED were treated with a single dose of cyclopamine (Sigma-Aldrich, MO), 25 mg/kg, s.c. Samples were collected 48 hours after injection.

Tumor growth and response to therapy

Tumor growth, progression and response to therapy were followed by 1) clinical evaluation of posterior fossa symptoms and body weight, 2) whole body bioluminescence and blood GLuc levels, 3) small animal MRI, 4) Optical Frequency Domain Imaging (OFDI). Tumors were evaluated by IHC after harvesting.

Isolation of granular cells and tumor-stroma co-culture

Cerebellar granule cells were purified as previously described (Hatten et al., 1985) with slight modifications. Co-cultures of granule cells and D341-MED were performed at a 1/1 or 1/2 ratio.

ELISA

ELISA studies were performed on plasma using MS6000 Human Growth Factor I Kit (PlGF, VEGF, sFlt1, bFGF; Meso-Scale Discovery, Gaithersburg, MD) measured on SI2400 (Meso-Scale Discovery). ELISA measurements for mPlGF were performed using Quantikine MP200 kit (R&D Systems, Minneapolis, MN) measured on Benchmark Plus microplate spectrophotometer (Bio-Rad Laboratories, Hercules, CA). The levels of hShh in conditioned medium from granule cells/D341-MED co-cultures were measured using the hShh ELISA kit (ab100639; Abcam, Cambridge, MA).

Cell viability

Medulloblastoma cells were cultured with indicated concentrations of MEK inhibitor U0126 or control vehicle for 48hrs. Cell viability was assessed by quantifying metabolic active cells through measurements of cellular contents of ATP using the CellTiter-Glo luminescence assay (Promega, WI).

Immunoblotting and co-Immunoprecipitation

The following antibodies and reagents were used: PlGF, hVEGFR1, mVEGFR1 (Abcam); Nrp1, VEGFR2, Shh, phospho-ERK1/2(Thr202/Tyr204), ERK1/2; phospho-Akt(Ser473), Akt, β-Actin, c-Caspase3, anti-rabbit IgG-HRP (Cell Signaling, MA); Tubulin, anti-mouse IgG-HRP (Sigma-Aldrich); U0126 (Calbiochem, MA).

PCR arrays, qRT-PCR, and shRNA studies

Gene expression was performed using commercially available human angiogenesis PCR Array (PAHS-024; SABiosciences, CA) according to the manufacturer’s guidelines. Mission® human-shPLGF, human shNRP1 and non-targeting shRNA control lentiviral transduction particles were purchased from Sigma. Retroviral shVEGFR1 and shSHH was obtained from OriGene. Cells were infected according to manufacturer’s protocol.

Statistical analyses

All data are expressed as mean ± standard error of the mean. Survival curves are plotted with the Kaplan-Meier method. For statistical analysis JMP Statistical analysis software was used (SAS Institute Inc, NC). 2-tailed T-tests were used between data comparing only two groups. Least squares means contrasts were used to assess significance of multiple data points such at tumor growth curves. For survival data, a Log-Rank test was employed. We considered a p-value less than 0.05 to be statistically significant.

Highlights

  • PlGF is expressed in medulloblastomas regardless of their genetic subtype
  • Majority of PlGF protein is secreted by cerebellar stromal cells
  • Stromal PlGF production is stimulated via paracrine Shh secretion by the cancer cells
  • Targeting PlGF/Nrp1 pathway suppresses growth and spread of medulloblastoma

Supplementary Material

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Acknowledgments

We would like to thank Dr. James M. Olson for his help with the Smo/Smo medulloblastoma; M. Dewerchin for advice and experimental assistance; Carolyn J. Smith, Anna Khachatryan, Kathryn Kinzel and Barbara Riesmeier for technical assistance; Dr. A. John Iafrate for assistance with aCGH; the MGH Vector Core (NIH/NINDS P30 NS045776 interdepartmental Neuroscience Center (Core C) grant) for reagents; Dr. Marek Ancukiewicz for statistical advice; Drs. Emmanuelle diTomaso, Shom Goel, David Kodack, Kate Munro, Brian Seed, Robert Wechsler-Reya, and Michael Weidner for invaluable helpful suggestions. This study was partially supported by grants from NCI (P01-CA080124, R01-CA163815), NCI Proton Beam Federal Share, National Foundation for Cancer Research Grants and Hoffmann-La Roche Inc (to R.K.J.). P.C. is supported by grant IUAP06/30 from the Federal Government Belgium, Long term structural funding Methusalem by the Flemish Government, grant GOA2006/11 from the Concerted Research Activities, Stichting Emmanuel van der Schueren (Belgium) and is inventor on patent applications, claiming subject matter related to the results described in this paper. The aforementioned patent application has been licensed, which may result in a royalty payment. M.S. is the recipient of a Paul Calabresi Career Development Award in Clinical Oncology (2K12CA090354-11) at the Massachusetts General Hospital. Analysis of clinical samples from University of Muenster was funded by Rolf Dierichs Foundation and Deutsche Krebshilfe fellowship to L.R. R.K.J is a consultant to Noxxon Pharma and holds equity and serves on the Board of Directors of Xtuit Pharmaceuticals, Inc.

Footnotes

Accession numbers

Array CGH data have been deposited in Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible under accession number GSE 37412.

Supplemental Data

The Supplemental Data include six Supplemental Figures and one Supplemental Table, Supplemental Experimental Procedures and Supplemental References.

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References

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