WO2023223022A1 - Method and compositions for tumorigenesis inhibition - Google Patents

Abstract

Methods and compositions for substantially preventing or inhibiting tumour growth and/or angiogenesis, said method including the step of co-targeting neuropilin-1 (NRP1) and neuropilin-2 (NRP2) in endothelial cells.

Description

Method and Compositions forTumorigenesis Inhibition

Angiogenesis constitutes a critical driver of tumour growth and metastatic dissemination. W ithout the expansion of a vascular network to supply oxygen and nutrients to the tumour,growth cannot proceed past a few millimetres in size [1]—[3]. Vascular endothelial growth factor (VEGF)-dependent stimulation of vascular endothelial growth factor receptor-2 (VEGFR-2) represents one of major and most widely acknowledged signalling pathways which promotes angiogenesis,yet,counterintuitively,the clinicalbenefits of targeting the VEGF/ VEGFR-2 axis have remained modest. Only minimal increases in progression-free survivalrates for various tumour types, including lung,breast,kidney,and colon cancers,have been reported following treatment [3]. Only when combined with chemotherapy have such therapies become recognised asan effective strategy against cancer growth,anti-angiogenics acting to selectively prune leaky,and immature tumour-associated vessels to facilitate more efficient delivery of chemotherapeutic agents [4]—[6]. The identification of novel combinations of angiogenic targets to enhance the therapeutic index of anti-VEGF/VEGFR-2 strategies remains paramount however, on account of cancers developing numerous adaptive mechanisms to escape tumourtherapy [7].

Neuropilin 1 (NRP1) and 2 (NRP2) are type-1 transmembrane glycoprotein co-receptors for VEGFs and VEGFRs [8], [9].Notably, NRP1 is known to form complexes with VEGF-A,the principle pro- angiogenic factor, and VEGFR-2 to promote angiogenic signalling, whilstNRP2 preferentially bindsVEGF-C and VEGFR-3 to propagate lymphangiogenic signalling [3], [10], [11]. Unsurprisingly, neuropilin (NRP) over-expression is often considered synonymous with an enhanced rate of tumour growth,invasiveness and angiogenesis in a number of differentcancer types,including carcinoma [12],colorectal [13], melanoma [14], myeloid leukaemia [15], breast [16] and lung cancer [17]. This is facilitated, at least in part, by their ability to interactwith integrin receptors,for example integrin α5β1,to enhance tumour cellspreading and extravasation [18],[19].

Owing to their ability to associate with a diverse range of receptors, in turn forming holoreceptors to propagate a plethora of downstream pro-angiogenic signalling cascades, NRPs are promising targets for tumour therapies [3].For example,the NRPl-specific smallmolecule inhibitors EG00229 and AT WLPPR have been demonstrated to inhibit NRP1-VEGFR-2 signalling, and impair both tumour angiogenesis and tumour growth in vivo [20]—[22] Tandem-virtual screening and cell-based screening were since utilised by Borriello et al., to identify a series of non-peptide VEGF-NRP antagonists, notably NPRa-47 and NRPa-308, which display anti-angiogenic and anti-proliferative capabilities in vitro, in addition to anti-tumorigenic effects on experimentalbreast cancer in vivo [23],[24].More recently, the anti-tumour potential of NRPa-308 was employed against experimental clear cell Renal Cell Carcinoma (ccRCC), a highly vascularised cancer arising from the overexpression of VEGF-A. Compared to the tyrosine kinase inhibitor sunitinib, the current reference treatment for ccRCC, NRPa-308 was found to suppress ccRCC cell proliferation, migration and invasiveness to a greater extent.Supporting gene invalidation studies proceeded to ratify these findings,and alluded to the factthatboth NRP1 and NRP2 should be completely inhibited to obtain maximaltherapeutic effect [25]. Itis therefore an aim ofthe presentinvention to provide a method of substantially preventing, supressing or inhibiting tumour growth and/or substantially preventing, supressing or inhibiting tumour angiogenesis.

It is a further aim of the present invention to provide a method of supressing metastasis.

In a first aspect of the invention there is provided a method of substantially preventing or inhibiting tumour growth and/or angiogenesis, said method including the step of co-targeting neuropilin-1 (NRP1)and neuropilin-2 (NRP2)in endothelialcells.

Typically co-targeting the expression of both NRP1 and NRP2 severely inhibits primary and secondary tumour growth and angiogenesis, to a greater extent than when either NRP receptor is targeted alone.

Typically the growth and/orangiogenesisisprevented in solid tumour cells or cancers. Further typically co-targeting NRP-1 and NRP-2 provides synergistic impairment of tumour development and/or vascularisation. The co-targeting, co-supression or co-inhibition provides a synergistic effect over that to be expected from targeting orinhibition ofNRP-1 orNRP-2 individually.

In one embodiment the co-targeting of NRP-1 and NRP-2 includes the substantially simultaneous inhibition of endothelial NRP-1 and In one embodimentthe co-targeting includesthe suppression ofNRP- 1 and/or NRP-2 genes and/or gene expression. Typically the co- targeting of NRP-1 and NRP-2 reduces the expression of the NRP1 and NRP2 genes.

In one embodiment the depletion of both NRP1 and NRP2 severely impairs EDA-fibronectin (EDA-FN) secretion. Typically the expression ofendothelialNRPsis essential.

In one embodiment dual-targeting of endothelialNRPs is effective at supressing metastasis. Typically the metastasis is hematogenous metastasis.

In one embodiment dual-targeting of endothelial NRP1 and NRP2 provides a means to retard primary tumorigenesis and significantly reduce secondary site angiogenesis.

In a second aspect of the invention there is provided a method to reduce metastatic load and/or angiogenic potential in secondary metastatic sites said method including dual-targeting of endothelial NRP1 and NRP2.

In a third aspect ofthe invention there is provided a composition for the dual-targeting or co-targeting ofendothelialNRP1 and NRP2 said composition provided in a pharmaceutically acceptable carrier. Typically the composition includes any one of peptide, protein, monoclonal antibody, small molecule, oligonucleotide or antisense oligonucleotide.

In one embodiment the composition includes any one or any combination ofNRPa-308,EG00229 orSunitinib.

In one embodimentthe composition includes CEND-1 cyclic peptide.

In one embodiment the composition includes anti-NRP antibody ASP1948.

In one embodiment the composition includes humanized monoclonal antibody ATYR2810.

In a further aspect of the invention there is provided a method and/or composition to reduce metastatic load and/or angiogenic potential in primary and/or secondary tumour sites, said method including dual-targeting ofNRP1 and NRP2.

Specific embodiments of the invention are now described with reference to the following figureswherein:

Figure 1 shows the dual targeting of endothelial expressed NRPs effectively inhibits primary tumour growth. Inducible, endothelial specific deletion of NRPs,either individually,or in combination was achieved by crossing mice expressing the PDGFb.iCreER promoter of Cre-recombinase to those floxed for NRP1, NRP2 or NRP1/NRP2. A) Experimental schematic: tamoxifen-induced activation of Cre- recombinase and thus deletion of targets was employed via the following regime. Cre-positive and Cre-negative littermate control mice received intraperitoneal (IP) injections of tamoxifen (75 mg/kg bodyweight, 2mg/ml stock) thrice weekly (Monday, W ednesday, Friday) for the duration ofthe experimentfrom D-4 to DI7 to induce Cre-recombinase activity.CMT19T lung carcinoma cells (1X10⁶) were implanted subcutaneously (SC) into the flank of mice at DO and allowed to grow untilD18.

B) Representative images of CMT19T tumours harvested on D18 removed from Cre-negative and positive mice.Scale bar shows 5 mm.

C) Raw tumour volume growth kinetics from 10 days post CMT19T injection to harvest. Tumour volume calculated using the formula: length x width² x 0.52.Errorbars show mean ± SEM;N =3 (n>12).

D) Quantification ofrelative tumour volumes measured on D18.Data presented as a percentage of the average tumour volume (mm³) observed in their Cre-negative littermate controls. Error bars show mean ± SEM;N =3 (n>12).

E) Quantification of tumour weight (g) measured on DI8. Data presented aspercentages ofthe average tumourweight(g) observed in respective littermate controls. Error bars show mean ± SEM; N =3 (n>12).

F) (Leftpanels) Representative tumour sections from Cre-negative and Cre-positive tumours showing endomucin⁺ blood vessels.Scale bar = 100 pm. (Kight panels') Confirmation of endothelial-specific target depletion in tumour sections from Cre-negative and Cre-positive tumours.

G) Quantification of % blood vessel density per mm². Mean quantification performed on 3x ROIs per tumour section, from 1-3 sections per tumour.Data presented as a percentage ofthe average % vessel density observed in their Cre-negative littermate controls. Errorbars show mean ± SEM;N =3 (n>12).

H) Representative tumour sections from Cre-negative and Cre- positive tumours showing EDA-FN coverage around endomucin⁺ blood vessels.Scale bar = 100 μm.

I) Quantification of mean EDA-FN area (pm²) surrounding vessels, performed on >10 vessels/tumour. Error bars show mean ± SEM; n≥3.

J) Delayed experimental schematic: tamoxifen-induced activation of Cre-recombinase and thus deletion of targets was employed via the following regime. Cre-positive and Cre-negative littermate control mice received intraperitoneal (IP) injections of tamoxifen (75 mg/kg bodyweight, 2mg/ml stock) thrice weekly (Monday, W ednesday, Friday) from D7 to induce Cre-recombinase activity. CMT19T lung carcinoma cells (1X10⁶) were implanted subcutaneously (SC) into the flank ofmice atD0 and allowed to grow untilD18.

K) Representative images of CMT19T tumours harvested on D18 removed from Cre-negative and positive mice.Scale bar shows 5 mm.

L) Raw tumour volume growth kinetics from 10 days post CMT19T injectionsto harvest.Errorbars show mean ± SEM;n>6.

M) Raw tumour volume growth change measured between Dll and D18, n>6.

N) Quantification of tumour volume (mm³) (Leftaxis) and weight (g) (Kight axis') measured on D18.Data presented as percentages of the average tumour volume and weight observed in respective littermate controls.Errorbars show mean ± SEM;n>6.

O) Representative tumour sections from Cre-negative and Cre- positive tumours showing endomucin⁺ blood vessels and Ki-67⁺ proliferating cells.Scale bar = 100 pm. P) Quantification of% blood vesseldensity per mm² (Leftaxis)and % number of Ki-67⁺ proliferating cells per mm² (Light axif from CMT19T tumours. Mean quantification performed on 3x ROIs per tumour section, from 1-3 sections per tumour. Data presented as a percentage of the average % vessel density/ % number of Ki-67⁺ proliferating cells observed in their Cre-negative littermate controls. Errorbars show mean ± SEM;n>6. Asterixisindicate significance.

Figure 2: Shows primary tumour development and angiogenesis are susceptible to the effects ofco-targeting endothelialNRP1 and NRP2 in multiple cancer models.Inducible,endothelial specific deletion of NRPs, either individually,or in combination was achieved by crossing mice expressing the PDGFb.iCreER promoter of Cre-recombinase to those floxed forNRP1/NRP2.

A) Experimental schematic: tamoxifen-induced activation of Cre- recombinase and thus deletion of targets was employed via the following regime. Cre-positive and Cre-negative littermate control mice received intraperitoneal (IP) injections of tamoxifen (75 mg/kg bodyweight, 2mg/ml stock) thrice weekly (Monday, W ednesday, Friday) from D7 to induce Cre-recombinase activity. B16F10 melanoma cells (4xl0⁵) were implanted subcutaneously (SC) into the flank ofmice atDO and allowed to grow untilD18.

B) B16F10 tumours harvested on D18 removed from Cre-negative and positive mice.Scale bar shows 5 mm.

C) Raw tumour volume growth kinetics from 10 days post B16F10 injection to harvest. Tumour volume calculated using the formula: length x width² x 0.52.Errorbars show mean ± SEM;N =2 (n>10).

D) Quantification of tumour volume (mm³) (Leftaxis) and weight (g) (Right axis) measured on D18.Data presented as percentages of the average tumour volume and weight observed in respective littermate controls.Errorbars show mean ± SEM;N =2 (n>10).

E) Quantification of mean animal weight measured at point of harvest.Data presented as a percentage of the average animalweight observed in respective littermate controls.Error bars show mean ± SEM, N =2 (n>10).

F) Experimental schematic: Cre-positive and Cre-negative littermate controlmice received intraperitoneal (IP) injections of tamoxifen (75 mg/kg bodyweight, 2mg/ml stock) thrice weekly (Monday, W ednesday, Friday) from D7 to induce Cre-recombinase activity. PyMT-BO1 breast cancer cells (1X10⁵) were implanted orthotopically into the flank ofmice atDO and allowed to grow untilDI5.

G) PyMT-BO1 tumours harvested on D15 removed from Cre-negative and positive mice.Scale bar shows 5 mm.

H) Raw tumourvolume growth kinetics from 10 dayspostPyMT-BO1 injection to harvest. Tumour volume calculated using the formula: length x width² x 0.52.Errorbars show mean ± SEM;n>10.

I) Quantification of tumour volume (mm³) (Left axis') and weight (g) (Kight axis') measured on D15.Data presented as percentages of the average tumour volume and weight observed in respective littermate controls.Errorbars show mean ± SEM;n>10.

J) Raw tumourvolume growth change measured between D7 and DI5, n>10.

K) Quantification of mean animal weight measured at point of harvest.Data presented as a percentage of the average animalweight observed in respective littermate controls.Error bars show mean ± SEM, n>10.

L) Representative tumour sections from Cre-negativeand Cre-positive tumours showing endomucin⁺ blood vessels.Scale bar = 100 pm. M ) Quantification of % blood vessel density per mm² from PyMT- BO1 tumours.Mean quantification performed on 3x ROIsper tumour section,from 1-3 sectionsper tumour.Data presented as a percentage of the average % vessel density observed in their Cre-negative littermate controls. Error bars show mean ± SEM; n>10. Asterixis indicate significance.

Figure 3: Shows targeting endothelial NRPs reduces metastatic load and angiogenic potential in secondary metastatic sites. Inducible, endothelial specific deletion of NRPs, either individually, or in combination was achieved by crossing mice expressing the PDGFb.iCreER promoter of Cre-recombinase to those floxed for NRP1/NRP2.

A) Experimental metastasis schematic: tamoxifen-induced activation of Cre-recombinase and thus deletion oftargetswas employed via the following regime. Cre-positive and Cre-negative littermate control mice received intraperitoneal (IP) injections of tamoxifen (75 mg/kg bodyweight, 2mg/ml stock) thrice weekly (Monday, W ednesday, Friday) from D3 to induce Cre-recombinase activity. B16F10 luciferase⁺ melanoma cells (1X10⁶) were intravenously (IV) injected into the tailvein ofmice atDO and allowed to disseminate untilD14.

B) (Leftpanels)Representative images oflungsharvested on D14 from Cre-negative and positive mice showing metastatic lung nodules. (Right panels) Corresponding representative bioluminescence (photons/sec/mm²)imaging of3 lungsdetected using Brukerimager.

C) Quantification of number of metastatic nodules per lung at D14. Data presented as percentages of the average number of nodules observed in respective littermate controls.Error bars show mean ± SEM; N =2 (n>8). D) Representative images of B16F10 metastatic nodules from Cre- negative and Cre-positive lungs showing NRP1,NRP2 and endomucin expression,and targetknockdown.Scale bar = 250 pm.Boxed images show highlighted magnified regions.

E) Representative images of B16F10 metastatic nodules from Cre- negative and Cre-positive lungs showing endomucin⁺ blood vessels. Scale bar = 250 pm.

F) Quantification of nodule vascular density (Left axis) and nodule area (pm²) (Kight axis').Data presented as percentages of the average vascular density and nodule area observed in respective littermate controls. Error bars show mean ± SEM; N =2 (n>18). Asterixis indicate significance.

Figure 4: Shows endothelial NRPs regulate VEGFR-2 turnover to sustain pro-angiogenic signalling responses.

A) Representative tumour sections from Cre-negative and Cre- positive CMT19T tumours showing colocalisation between VEGFR-2 and endomucin⁺ blood vessels.Scale bar = 100 pm.

B) Quantification of % VEGFR-2⁺ vessels per mm², performed on lOx ROIspertumour.Error bars show mean ± SEM;n>3.

C) Quantification of % y>-VEGFR-2^Y1175+ vessels per mm²,performed on 10x ROIspertumour.Errorbars show mean ± SEM;n>3.

D) (Top panel) Schematic showing method for surface protein labelling and stripping. (Bottom panel) Ctrl siRNA-treated ECs were seeded onto 10 pg/ml FN for 48 hours before being incubated in serum-free OptiMEM for 3 hours.ECs were then stimulated with 30 ng/ml VEGF-A for the indicated timepoints before being labelled with 0.3 mg/mlbiotin.Unreacted biotin was quenched with 100 mM glycine. ECs were then either lysed or incubated with 100 mM MESNA to strip off biotin labelled proteins.Unreacted MESNA was quenched with 100 mM iodoacetamide before lysis.EC lysates were immunoprecipitated with Protein G Dynabeads™ coupled to anti- biotin primary antibody.Immunoprecipitated biotin-labelled proteins were separated by SDS-PAGE and subjected to W estern blotanalysis. Membranes were incubated in anti-VEGFR-2 primary antibody.

E) siRNA-treated ECs were seeded onto 10 pg/mlFN for 48 hours, then incubated in serum-free OptiMEM for 3 hours.ECswere subject to 5 minutes of stimulation with 30 ng/ml VEGF-A before being washed twice on ice with PBS and lysed.Lysateswere quantified using the DC protein assay, separated by SDS-PAGE and subjected to W estern blot analysis.Membranes were incubated in anti-VEGFR-2, anti-y>-VEGFR-2^Y1175 and anti-HSC70 primary antibodies.

F) Confirmation oftargetdepletion by siRNA transfection.

G) Quantification of total VEGFR-2 expression following VEGF-A stimulation relative to respective unstimulated lysates.Quantification shows mean densitometric analysis obtained using ImageJ™ . Error bars show mean ± SEM;N>3.

H) siRNA-treated ECs were seeded onto acid-washed,oven-sterilised coverslips pre-coated with 10 pg/ml FN for 3 hours. ECs were incubated in serum-free OptiMEM for 3 hours,before being subject to 5 minutes of stimulation with 30 ng/mlVEGF-A.Coverslips were fixed in 4% PEA, blocked and permeabilised. ECs were incubated with anti-VEGFR-2 and anti-Rab7 primary antibodies overnight at 4°C before incubation with appropriate Alexa fluor secondary antibodies at RT for 1 hour. Coverslips were mounted with flouromount G with DAPI™ .Panels show representative images of unstimulated and VEGF-A stimulated siRNA-treated ECs.Error bars show 10 pm. I) Quantification of VEGFR-2⁺ Rab7 vesicles/cell.Error bars show mean ± SEM; n=30.Asterixis indicate significance.J) siRNA-treated ECs were seeded onto 10 pg/mlFN for 48 hours,then incubated in serum-free OptiMEM ± 10 LIM M G-132 for 3 hours.ECs were then stimulated, lysed and prepped for W estern blot analysis in the same manner asE).

Figure 5:Supplementary figureswherein

A) Determination of prognostic value of NRP1 and NRP2 receptor mRNA expression in lung carcinoma patients (n = 719) using www.kmplot.com. Kaplan-Meier survival plot of lung carcinoma patients with high NRP1 mRNA expression (Affymetrix ID: 210615_at).

B) Kaplan-Meier survival plot of lung carcinoma patients with high NRP2 mRNA expression {Affymetrix ID: 214632_ai). Respective logrankp valuesare shown.

C) Quantification of mean animal weight measured at point of harvest.Data presented as a percentage of the average animalweight observed in respective littermate controls.Error bars show mean ± SEM, N =3 (n>12).

D) siRNA-treated ECs were seeded onto 10 pg/mlFN for 48 hours, then incubated in serum-free OptiMEM for 3 hours.ECswere subject to 5 minutes of stimulation with 30 ng/ml VEGF-A before being washed twice on ice with PBS and lysed. Lysates were cleared by centrifugation for 30 minutes at 4 °C, allowing for the isolation of soluble and insoluble fractions.Soluble and insoluble fractions were quantified using the DC protein assay, separated by SDS-PAGE and subjected to W estern blotanalysis.Membraneswere incubated in anti- EDA-FN primary antibody. Panels show representative levels of EDA-FN expression quantified from soluble and insoluble fractions, in the presence orabsence ofVEGF-A stimulation.

E) Quantification of relative EDA-FN expression. Quantification shows mean densitometric analysis obtained using ImageJ™ . Error bars show mean ± SEM;N =3.

F) Quantification of mean animal weight measured at point of harvest.Data presented as a percentage of the average animalweight observed in respective littermate controls.Error bars show mean ± SEM, n>6.

G) Delayed experimental schematic:tamoxifen-induced activation of Cre-recombinase and thus deletion of targets was employed via the following regime. Cre-positive and Cre-negative littermate control mice received intraperitoneal (IP) injections of tamoxifen (75 mg/kg bodyweight, 2mg/ml stock) on D12 and D14 to induce Cre- recombinase activity. CMT19T lung carcinoma cells (1x10⁶) were implanted subcutaneously (SC) into the flank of mice at DO and allowed to grow untilD16.

H) Representative images of CMT19T tumours harvested on D16 removed from Cre-negative and positive mice.Scale bar shows 5 mm.

I) Raw tumour volume growth kinetics from 10 days post CMT19T injectionsto harvest.Errorbars show mean ± SEM;n>9.

J) Representative tumour sections from Cre-negative and Cre-positive tumours showing endomucin⁺ blood vessels.Scale bar = 100 pm.

K) Quantification of % blood vessel density per mm² from CMT19T tumours. Mean quantification performed on 3x ROIs per tumour section,from 1-3 sectionsper tumour.Data presented as a percentage of the average % vessel density observed in their Cre-negative littermate controls. Error bars show mean ± SEM; n>9. Asterixis indicate significance. Figure 6 shows that endothelialNRP1/NRP2 knockdown results in a synergistic effectwith the chemotherapeutic cyclophosphamide.

The present invention discloses the reciprocal co-targeting of endothelial neuropilins to provide effective inhibition against tumorigenesis. This is provided by abrogating extra-domain A fibronectin (EDA-FN)-dependentvesselstimulation and growth.

Presently, there have been no in depth assessments comparing the anti-angiogenic effects of depleting either NRP receptor individually versus when they are targeted together. To this end, we generated genetically modified mouse models that enabled us to perform temporal endothelial-specific deletions of either NRP gene individually, or in combination. Utilising these models, we demonstrate that in multiple paradigms of cancer, co-targeting the expression of both NRP1 and NRP2 severely inhibits primary and secondary tumour growth and angiogenesis, to a greater extent than when either NRP receptor is targeted alone.W e go on to revealthat the depletion of both NRP1 and NRP2 severely impairs EDA- fibronectin (EDA-FN) secretion in vivo and in vitro, which likely impedes pathologicalvesselstability and growth.W e also demonstrate that NRP depletion stimulates the rapid degradation of VEGFR-2, metering surface receptor availability for VEGF-Aus-induced pro- angiogenic responses.

Results

Dual targeting of endothelial expressed NRPs inhibits prim ary tumour growth and angiogenesis by impairing vesselstability. As co-receptors for VEGF family receptors, endothelial neuropilins (NRPs) are becoming increasingly recognised as candidate targets for supressing pathologies typified by uncontrolled vascular expansion, such as cancer and retinopathy. Investigations have, however, persisted in elucidating their function separately from one another, rather than in conjunction. For example, by crossing NRP2-floxed (NRP2^flfl) mice [27] with mice expressing a tamoxifen-inducible Pdgfb-iCreER^T2 promoter [28],we previously showed that endothelial NRP2 (NRP2^EC) promotes pathological angiogenesis to support the progression of primary tumours in a lung-carcinoma model. Acute, endothelial-specific depletion ofNRP2 impaired tumour development and vascularisation approximately 2-fold,revealing a novel,effective therapeutic strategy against cancerous growth [29]. Indeed, Kaplan- Meier plots indicate a significantly reduced overall patient survival following diagnosis with lung carcinoma when either NRP1 or NRP2 mRNA expression iselevated [30] (Suppl.Figure 5A-B).

To ascertain whether endothelial NRP1 and NRP2 contribute in a non-redundant, synergistic manner during angiogenesis-dependent tumour development, we crossed NRP2^flfl.Pdgfb-iCreER^T2 (NRP2^flfl.EC^KO ) mice with NRPl^flfl.Pdgfb-iCreER^T2 (NRPl^flfl.EC^KO ) mice to generate NRPl^flfl;NRP2^flfl.Pdgfb-iCreER^T2 (NRP1^flflNRP2^flfl.EC^KO ) animals and compared the effects ofan acute endothelial-specific depletion of NRP1,NRP2 or NRP1;NRP2 during subcutaneous allograft tumour growth using CMT19T lung carcinoma cells. Tamoxifen administrations were performed thrice weekly starting 4 days prior to CMT19T cell implantation, continuing until day 18 (D18),atwhich pointprimary tumourswere harvested (Figure 1A).NRP1 ^flfl.EC^KO and NRP2^flfl.EC^KO animals developed significantly smaller tumours (—50%) compared to their respective Pdgfb- iCreER^T2-negative (Pdgfb-iCreER^T2 ) controls,as observed previously [29]. In comparison, tumours harvested from NRPl^flflNRP2^flfl.EC^KO mice grew significantly smaller than either NRPl^flfl.EC^KO or NRP2^flfl.EC^KO tumours,and only to —20% the size ofthose harvested from controlmice (Figure 1B-E).No changes to gross animalweight were observed (Suppl. Figure 5C). Immunofluorescence imaging of endomucin⁺ blood vessels verified that our tamoxifen regimen effectively silenced target expression.Notably,NRPl^flflNRP2^flfl.EC^KO tumours also exhibited significantly less vasculature than either NRPl^flfl.EC^KO or NRP2^flfl.EC^KO tumours (Figure 1F-G), suggesting thatthe dualtargeting ofboth NRP1 and NRP2 elicits a compounded anti-angiogenic response to inhibittumour developmentand growth.

The extracellular matrix (ECM) component fibronectin containing extra domain-A (EDA-FN) is a known marker of tumour vasculature, and is essentialforthe developmentofa metastatic microenvironment [20], [31]—[33]. As both NRP1 and NRP2 have been reported to regulate FN fibrillogenesisin ECsin the past [29],[34],we considered whether the deposition of EDA-FN around tumour vessels would be perturbed in our knockout models. Compared to respective Cre- negative control tumours, only those depleted for both NRP1 and NRP2 saw a significant reduction in EDA-FN coverage around tumour vasculature (Figure 1H-I), suggesting that both endothelial NRPs facilitate tumour angiogenesis by promoting vessel stability. Deoxycholate studies employing siRNA-transfected wild-type (WT) mouse-lung ECs validated these findings,combined depletion ofboth NRP1 and NRP2 resulted in a significant reduction in EDA-FN expression from both unstimulated and VEGF-A₁₆₅-stimulated insoluble fractions (Suppl.Figure 5D-E).

To determine if co-targeting endothelial NRP1 and NRP2 impedes tumour growth in already established tumours, we next performed intervention CMT19T allograft studies in our NRPl^flflNRP2^flfl.EC^KO animals whereby we delayed tamoxifen administrations until 7 days after cellimplantation (Figure 1J).By doing so,we aimed to provide a more clinically relevant study design, where treatment is initiated once a cancer has become vascularised. Following this regimen, we observed severe impediments to tumour growth and angiogenesis following the combined loss of endothelial NRP1 and NRP2. Tumours in NRPl^flflNRP2^flfl.EC^KO mice were significantly smaller than controls or NRPl^flfl.EC^KO or NRP2^flfl.EC^KO animals (Figure 1K- N). Again,no changes in mean animalweightwere observed between groups (Suppl. Figure 5F).In addition to a significant reduction in tumour vascularity, NRPl^flflNRP2^flfl.EC^KO tumours also exhibited significantly fewer Ki67⁺ proliferating cells compared to control tumours (Figure 1O-P).

Finally, to exclude tumour size as a statistical confounder, and therefore assess whether the loss of endothelial NRP1 and NRP2 directly influences pathological angiogenesis, tamoxifen administration was suspended further until day 12 (Suppl. Figure 5G). Tumour growth was tracked from day 7, and tumours were harvested from allanimals on day 16. W hilstno significantreduction in tumour volume was detected between NRPl^flflNRP2^flfl.EC^KO mice and Cre-negative control mice, (Suppl. Figure 5H-I), immuno- labelling of endomucin-positive vessels revealed a —50% reduction in tumour vascularity (Suppl. Figure 5J-K). These results strongly suggest that co-depletion of endothelial NRP1 and NRP2 influences tumour angiogenesis regardless of tumour size,and in already highly vascularised tumours.

Primary tumour development and angiogenesis is susceptible to the effects of co-targeting endothelial NRP1 and NRP2 in multiple cancermodels.

As the endothelial co-depletion of NRP1 and NRP2 was found to effectively impair primary lung carcinoma growth and angiogenesis, we proceeded to assess the efficacy of their co-depletion in other paradigms of cancer. To investigate whether the loss of NRP expression influences melanoma development, B16-F10 melanoma cellswere subcutaneously implanted and allowed to grow for a period of 18 days, following our intervention-based tamoxifen regime as previously described in Figure II (Figure 2A). From Dll, NRPl^flflNRP2^flfl.EC^KO tumours grew significantly smaller than their controlcounterparts,and when excised were found to have developed to only —10% the size of controltumours.Indeed,a smallnumber of tumours were found to have regressed entirely (Figure 2B-D). No changes in mean animal weight were observed between control and NRPl^flflNRP2^flfl.EC^KO mice (Figure 2E).

In a similar manner, we assessed the impact of co-depleting endothelialNRP1 and NRP2 on a luminalB model of breast cancer. PyMT-BO1 cancer cells [35] were orthotopically implanted into the fourth inguinal mammary gland of NRPl^flflNRP2^flfl and NRPl^flflNRP2^flfl.EC^KO mice,and allowed to grow over a period of 15 days.Again,tamoxifen administration was delayed untilday 7 to allow for palpable tumours to develop prior to the onset oftargetdepletion (Figure 2F). Compared to NRPl^flflNRP2^flfl control tumours, those grown in NRPl^flflNRP2^flfl.EC^KO animals developed —65% smaller by day 15 (Figure 2G-J), alongside no significant alterations in mean animalweight (Figure 2K).Unlike the B16-F10 tumours,which failed to grow more than ~2 mm in size in our NRPl^flflNRP2^flfl.EC^KO mice, we were able to process our PyMT-BO1 tumours for immunofluorescence imaging analysis. NRPl^flflNRP2^flfl.EC^KO PyMT- BOl tumours were —70% less vascularised than respective Cre- negative controltumours (Figure 2L-M),corroborating our CMT19T studies, and confirming that the expression of NRP1 and NRP2 is essentialfortumourangiogenesisin multiple cancermodels.

Targeting endothelial NRPs reduces metastatic load and angiogenic potentialin secondary metastatic sites.

Not only are murine B16-F10 cells a well-established, aggressive tumour model for preclinical investigations into melanoma progression,but they are also known to preferentially metastasise to the lungs ofC57/BL6 mice [36].To investigate whether dual-targeting of endothelial NRPs is effective at supressing hematogenous metastasis,we measured pulmonary seeding 14 days post intravenous injection ofluciferase⁺-tagged B16-F10 cells (Figure 3A).

NRPl^flflNRP2^flfl.EC^KO mice were found to develop significantly fewer metastatic lung nodules than controlmice,subsequently confirmed by bioluminescence imaging (Figure 3B-C). Immunofluorescence staining of lung metastases revealed a robust expression of both NRP1 and NRP2 colocalising to endomucin⁺ vasculature in control nodules, but not in lung nodules of NRPl^flflNRP2^flfl.EC^KO mice

(Figure 3D).Furthermore,lung metastases of NRPl^flflNRP2^flfl.EC^KO mice were observed to be significantly smaller and less vascularised than their control counterparts (Figure 3E-F).These results clearly demonstrate that the dual-targeting of endothelial NRP1 and NRP2 can be implemented not only as a means to retard primary tumorigenesis, but also to significantly reduce secondary site angiogenesisand growth.

Endothelial NRPs regulate VEGFR-2 turnover to sustain pro- angiogenic signalling responses.

Sustained hyperactivation of VEGFR-2 is largely considered one of the most critical aspects of pathological angiogenesis during tumour growth. Both NRP1 and NRP2 are also known co-receptors of VEGFRs and their respective VEGF signalling moieties [8], [9].W e therefore examined whether VEGFR-2 signalling is perturbed in tumour vasculature depleted for NRP1 and NRP2 by measuring VEGFR-2 and phosphorylated-VEGFR-2^Y1175 localisation to endomucin⁺ vessels.W hilstNRPl^flfl.EC^KO and NRP2^flfl.EC^KO CMT19T tumours saw reductions in VEGFR-2 localisation of approximately 30% and 10% respectively,we observed a compounded reduction of over 50% in NRPl^flflNRP2^flfl.EC^KO tumours (Figure 4A-B).Likewise, simultaneous depletion of both endothelialNRP1 and NRP2 resulted in an equivalent loss of phosphorylated-VEGFR-2^Y1175 expression from tumourvessels (Figure 4C).

To further elucidate how endothelial NRPs co-operate to influence VEGFR-2 activity,we examined VEGFR-2 dynamics in vitro.First,we established total surface expression of VEGFR-2 in Ctrl siRNA- treated ECs remained intact up to 5 minutes after stimulation with VEGF-A165 (Figure 4D). Lysates from Ctrl, NRP1, NRP2 and NRP1/2 siRNA-treated ECs were subsequently analysed by W estern blotting to assess changes in VEGFR-2 expression following an acute 5 minute period of VEGF-A₁₆₅ stimulation. Interestingly, total VEGFR-2 expression was significantly diminished in stimulated ECs depleted for both NRP1 and NRP2 compared to unstimulated knockdown ECs (Figure 4E-G).

Following internalisation, VEGFR-2 is shuttled from Rab5⁺ early endosomes to either Rab4/Rabll⁺ recycling endosomes or rapidly degraded via Rab7⁺ late endosomes. W e therefore proceeded to determine any changes in the fraction of VEGFR-2 localising to Rab7⁺ punctae following 5 minutes of VEGF-A165 stimulation. siNRPl/2 depleted ECs displayed a significantly greaterproportion of VEGFR-2 present in Rab7⁺ vesicles compared to siCtrlECs (Figure 4H-I), suggesting that NRP1 and NRP2 promote VEGFR-2-induced pro-angiogenic responses by moderating receptor turnover. To test this hypothesis,we treated VCtrland siNRP1/2 ECswith 10 LIM M G- 132, a well characterised proteosome inhibitor [37]—[39]. MG-132 treatment effectively rescued total VEGFR-2 expression in VEGF- Ai65-stimulated siNRPl/2 depleted ECs (Figure 4J),confirming that NRP co-depletion stimulates the rapid translocation of VEGFR-2 from Rab7⁺ late endosomesto the proteosome fordegradation.

Turning to figure 6 where it is shown that the knockdown of endothelial NRP1/NRP2 responds in a synergistic manner with a clinically relevant chemotherapeutic (cyclophosphamide). PyMT-BO1 cancer cellswere orthotopically implanted on DO and allowed to grow until day seven (D7). On D7, 100mg/kg cyclophosphamide was administered alongside 75 mg/kg tamoxifen to induce target deletion. Tamoxifen injections were maintained thrice weekly until harvest on D15. Tables C and D clearly show the synergistic effect of NRP1, NRP2 and cyclophosphamide in the presence and absence of endothelial-specific NRP1 and NRP2 deletion

Discussion

Pathologicalangiogenesis is a core driver ofaggressive tumorigenesis, yet the clinical benefits of targeting principle regulators of pro- angiogenic cascades have thus far shown limited efficacy [3]. W e demonstrate that the endothelial-specific co-targeting of both NRP receptors, NRP1 and NRP2, provides effective inhibition against tumour growth and secondary site metastasis in multiple cancer models,likely by potentiating the rapid delivery ofVEGFR-2 to late- endosomes for degradation.Importantly,we highlight the importance of targeting the expression of both NRPs simultaneously for maximum therapeutic effect.

Previous investigations have indeed demonstrated that targeting the expression of either NRP1 or NRP2 individually confers some anti- tumorigenic response. For example, inhibiting NRP1 binding to VEGF-A165 enhances the anti-tumour efficacy ofVEGF-Awsblocking antibodies such as bevacizumab to modulate tumour cellproliferation and angiogenesis [40], [41]. The NRP1 inhibitor EG00229 has also been demonstrated to exert significant tumour-suppressive effects in gliomas and squamous cell carcinomas [42]—[44]. Equally, treatment with the NRP2-specific monoclonal antibody N2E4 inhibited pancreatic ductal adenocarcinoma (PDAC) cell tumour growth and metastasis by blocking interactions with β1 integrin to inhibit FAK/Erk/HIF-1α/ VEGF-A₁₆₅ signalling [45]. In addition, blocking NRP2 binding to VEGF-C was shown to reduce tumoral lymphangiogenesis and metastasis of breast adenocarcinoma and glioblastoma cells [46].Naturally,ithas since been elucidated thatco- targeting the functions of both NRP1 and NRP2 may provide enhanced anti-tumorigenic responses [11].

Consistent with the above investigations, we confirm that an endothelial-specific deletion of either NRP gene significantly impairs tumour development and tumour angiogenesis. Critically however, dual loss of both NRP1 and NRP2 was found to reduce primary tumour growth and primary tumour angiogenesis by a greater extent than when either molecule was targeted individually.Furthermore,co- targeting NRP1 and NRP2 expression effectively inhibited secondary site metastasis compared to control animals. Given both NRP1 and NRP2 are known to modulate primary and secondary tumour microenvironments by interacting with integrins to remodel the tumoral ECM [20], [45], [47], of which FN is known as a major component [48],it follows thatthe impaired tumour growth exhibited following NRP co-depletion likely arises as a result of perturbations in EDA-FN fibrilassembly and deposition.Indeed,EDA-FN hasbeen demonstrated to facilitate tumour growth and invasiveness by promoting matrix stiffness, sustaining tumour-induced angiogenesis and lymphangiogenesis via VEGF-A₁₆₅[49] and VEGF-C respectively [50]. For example, Su et al., revealed that EDA-FN secretion promoted VEGFR-2 recruitment to [31 integrin sites, upregulating VEGFR-2 phosphorylation and pathological angiogenesis during hepatic fibrosisin a CD63-dependentmanner [49]. As they are canonicalco-receptors for VEGFR-2 in endothelialcells, we hypothesised that NRP co-depletion would provide effective inhibition of VEGFR-2-induced responses. NRP1/NRP2 knockout tumour vasculature was found to express significantly less VEGFR-2 than either NRP1 or NRP2 knockouttumours,in addition to reduced phosphorylated VEGFR-2 expression. Mechanistic studies utilising siRNA transfected W T mouse-lung ECs subsequently revealed that dual loss of NRP1 and NRP2 promotes the rapid translocation of VEGFR-2 complexes to Rab7+ late endosomes for proteosomal degradation upon acute VEGF-A165 stimulation, likely resulting in a severely moderated VEGFR-2 response.This work supports that of Ballmer-Hofer et al.,who delineated that in the absence of NRP1,or in ECs stimulated with a non-NRPl-binding VEGF-A isoform, VEGFR-2 is re-routed to the degradative pathway specified by Rab7 vesicles. Importantly, this was found to occur only following 30 minutes VEGF-A stimulation [51], suggesting that the rate of VEGFR-2 degradation is accelerated when NRP1 and NRP2 are lost in tandem,aswe observed changesafter 5 minutesofstimulation.

In conclusion,our findings show thatthe activity ofendothelialNRPs together is required for sustained tumour angiogenesis,and support a hypothesis that maximum anti-angiogenic efficacy can be achieved by co-targeting both NRP1 and NRP2 rather than targeting either receptor individually.Dualloss of both NRPs was found to severely abrogate tumour development and tumour angiogenesis in multiple models of cancer,in addition to secondary tumour development.This work provides strong evidence for the need to develop noveltargeted therapeutics specific for both endothelialNRP1 and NRP2 receptors, againstpathologies characterised by uncontrolled vascularexpansion. M aterials and M ethods

Anim algeneration

All experiments were performed in accordance with UK home office regulations and the European LegalFramework for the Protection of Animals used for Scientific Purposes (European Directive 86/609/EEC), prior to the start of this project.NRP1 (NRPl^flfl) [52] and NRP2 floxed (NRP2^flfl) [27] mice were purchased from Jackson Laboratories (Bar Harbour,Maine,USA),and were generated by gene targetinsertion ofembryonic stem cells (ESCs),enabling the insertion of loxP sites flanking exon 2 of the NRP1 gene,and exon 1 of the NRP2 gene. LoxP-tau-GFP FRT-flanked neo cassettes were inserted via homologous recombination. Neo cassettes were removed by crossing heterozygous animals to an flp recombinase transgenic line. Allanimalswere bred on a pure C57/BL6 background.

The PGR analysis to confirm floxing was carried out using the following oligonucleotide primers: Forward NRP1 primer: 5’ - A GGTTA GGCTTCA GGCCAA T- 3’, Reverse NRP1 primer: 5’- GGTA CCCTGGGTTTTCGA TT-3’. Forward NRP2 (W T reaction) primer (Reaction A): 5 ’-CA GGTGA CTGGGGA TA GGGTA -3 common NRP2 primer (Reaction A + B): 3’-

A GCTTTTGCCTCA GGA CCCA -3 forward NRP2 primer (^flfl reaction) (Reaction B):5’-CCTGA CTA CTCCCA GTCA TA G -3’.

Transgenic mice expressing a tamoxifen-inducible PDGFb-iCreER^T2 allele in vascular ECs were provided by Marcus Fruttiger (UCL, London, UK), and were generated by substituting the exon 1 of the PDGFb gene by the iCreER^T2-IRES-EGFP-pA sequence. PGR confirmation of Cre-recombinase status was performed using the following oligonucleotide primers: Forward primer: 5’-

GCCGCCGGGA TCA CTCTC-3 Reverse primer: 5’-

CCA GCCGCCGTCGCAA CT-3 NRPl^flfl and NRP2^flfl mice were bred with PDGFb.iCreER⁷² mice to generate NRPl^flfl.Pdgfb-iCreER^T2 and NRP2^flfl.PDGFb.iCreER animals. NRPl^flfl.Pdgfb-iCreER^T2 and NRP2^flfl.PDGFb.iCreER mice were subsequently bred to generate NRPl^flfl;NRP2^flfl.Pdgfb-iCreER^T2 animals. PDGFβ-iCreER^T2 expression was maintained exclusively on breeding males to ensure the production of both Cre-negative and positive offspring, and therefore the use oflittermate controls.

CM T19T tumour growth assays

Mice received intraperitoneal (IP) injections of tamoxifen (75 mg/kg bodyweight,2mg/ml stock in corn oil) thrice weekly for the duration of the experiment from D-4 to D17 to induce target deletion. CMT19T lung carcinoma cells (CR-UK CellProduction) (1x10⁶) were implanted subcutaneously (SC) into the flank of mice at DO and allowed to develop untilD18.On D18,mice were killed,and tumour volumes and weights measured. Tumour volume was calculated according to the formula:length x width² x 0.52.

Intervention tumour growth assays

CMT19T lung carcinoma cells (1x10⁶) or B16-F10 melanoma cells (4x10⁵) were implanted subcutaneously (SC) into the flank of mice at DO and allowed to develop until D18. PyMT-BO1 cells (1x10⁵ in matrigel)were implanted orthotopically into the inguinalmammary fat pad under anaesthesia, and allowed to develop until D15. Mice received intraperitoneal (IP) injections of tamoxifen (75 mg/kg bodyweight, 2 mg/ml stock) thrice weekly for the duration of the experiment from D7 to induce target deletion. On D18/D15, mice were killed,and tumourvolumesand weightsmeasured.

Tissue immunofluorescence analysis

Cryopreserved tumour sectionswere fixed in 4% PFA for 10 minutes, then washed in PBS 0.3% triton-X100, and in PBLEC (lx PBS, 1% Tween 20,0.1 mM CaCh,0.1 mM MgCh,0.1 mM MnCE),before being incubated in Dako protein block serum free (Agilent).Sections were then incubated overnight at 4°C in primary antibodies against NRP1 (clone AF566; R&D), NRP2 (clone Sc-13117; Santa-Cruz

Biotechnology (SCB)), endomucin (clone Sc-65495; SCB), Ki-67 (Abl5580;Abeam), EDA-FN (clone F6140;Sigma),VEGFR-2 (clone 2479; Cell signalling technologies (CST)), y>-VEGFR-2^Y1175 (clone 2478; CST). Following primary antibody incubation, sections were washed again in PBS 0.3% triton-XlOO before being incubated in the appropriate Alexa fluor secondary antibody for 2 hours at RT. Sectionswere blocked in Sudan black before mounting.Sectionswere imaged at 20 X magnification using a Zeiss Axiolmager M2 microscope (AxioCam MRm camera).

Tumour vascular density was assessed by counting the number of endomucin-positive vessels per mm² from 3 representative ROIs averaged per section,subsequently averaged over 3 sections/tumour. Vascular density of lung nodules was measured using a previously described ImageJ™ macro application [53].

Cellisolation,immortalisation,and cellculture Primary mouse lung microvascular endothelial cells (mLMECs) were isolated from W T C57/BL6 adult mice.mLMECs were twice subject to magnetic activated cell sorting (MACS) to positively select for endomucin⁺ ECs as previously described by Reynolds & Hodivala- Dilke [54]. ECs were immortalised using polyoma-middle-T-antigen (PyMT) by retroviraltransfection aspreviously described by Robinson etal[55].Immortalised mLMECs were cultured in media composed of a 1:1 mix ofHam’s F-12:DMEM (low glucose) medium supplemented with 10% FBS, 100 units/mL penicillin/streptomycin (P/S), 2 mM glutamax, 50 μg/mL heparin (Sigma).ECs were grown at 37°C in a humidified incubator with 5% CO₂ unless otherwise stated. For experimentalanalyses,plasticware was coated using 10 pg/mlhuman plasma fibronectin (FN) (Millipore).

EC stimulation was achieved using 30 ng/ml VEGF-A164 (VEGF-A) (mouse equivalent of VEGF-A₁₆₅) post 3 hours incubation in serum- free medium (OptiMEM®; Invitrogen).VEGF-A wasmade in-house as previously described by Krilleke etal[56]. siRNA transfection

Immortalised ECs were transfected with non-targeting controlsiRNA (VCtrl) or mouse-specific siRNA constructs against NRP1 or NRP2 (Dharmacon),suspended in nucleofection buffer (200 mM Hepes,137 mM NaCl, 5 mM KC1, 6 mM D-glucose, and 7 mM Na₂HPO₄ in nuclease-free water; filter sterilised). Nucleofection was performed using the Amaxa 4D-nucleofector system (Lonza) using program EO- 100 according to manufacturer’sinstructions.

VEGF-A 165stimulation ECs were incubated in serum-free OptiMEM® for 3 hours prior to VEGF-A165 stimulation (30 ng/ml) for the indicated timepoints.ECs were then immediately placed on ice,washed twice with ice-cold PBS, then lysed in electrophoresis sample buffer (ESB) (Tris-HCL:65 mM pH 7.4,sucrose:60 mM,3 % SDS).

Deoxycholate (DOC) Buffer-extraction

Following VEGF-A165 stimulation, ECs were lysed in DOC lysis buffer (20 mM Tris, pH 8.5, 1% sodium deoxycholate, 2 mM iodoacetamide,2 mM EDTA) in the presence of 100X Halt protease inhibitor cocktail,cleared by centrifugation,and the insoluble fraction isolated. Soluble and insoluble fractions were separated by SDS- PAGE and subjected to W estern blotanalysis.

W estern blotting

Equivalent protein concentrations were loaded onto 8% polyacrylamide gels and subjected to SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (Sigma) before being incubated in 5 % milk powder. Membranes were then incubated overnightin primary antibody diluted 1:1000 at4 °C.M embraneswere washed with 0.1% Tween-20 in PBS (PBST) and incubated in an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Dako) diluted 1:2000 for 2 hours at RT. Bands were visualised by incubation with a 1:1 solution of Pierce ECL W estern Blotting Substrate (Thermo). Chemiluminescence was detected on a ChemiDoc™ MP Imaging System (BioRad).Densitometric readings of band intensities were obtained using ImageJ™ . Primary antibodies were allpurchased from CST unless otherwise stated:y>-VEGFR-2^Y1175 (clone 2478; CST), VEGFR-2 (2479), NRP2 (3326), NRP1 (3725), HSC70 (clone B-6;SCB),EDA-FN (F6140;Sigma).

M etastasis experiments luciferase⁺-tagged B16-F10 melanoma cells (1X10⁶)were intravenously (IV) injected into the tail vein of mice at DO and allowed to disseminate untilD14.Mice received intraperitoneal(IP)injections of tamoxifen (75 mg/kg bodyweight, 2mg/ml stock) thrice weekly for the duration ofthe experiment from D3 to induce targetdeletion.On D14, mice were killed, and lungs removed for bioluminescence imaging and subsequentimmunofluorescence analysis ofsections.

Biotin-surface protein labelling

ECs were washed twice on ice with Soerensen buffer (SBS) pH 7.8 (14.7mM KH₂PO₄, 2mM Na₂HPO₄, and 120mM Sorbitol pH 7.8). Surface proteins were labelled with 0.3 mg/ml biotin (Thermo Scientific) in SBS for 30 minutes at 4 °C. Unreacted biotin was quenched in 100 mM glycine for 10 minutes. Biotin stripping was achieved by incubation with 100 mM MESNA (Sigma) for 75 minutes at 4 °C. Unreacted MESNA was quenched with 100 mM iodoacetamide (Sigma) for 10 minutes.ECs were lysed in lysis buffer (25 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM MgCl₂, 1 mM Na₃VO₄, 0.5 mM EGTA, 1% Triton X-100,5% glycerol,and protease inhibitors), and placed on ice as described previously [34]. Lysates were cleared by centrifugation at 12,000g for 20 minutes at 4°C,then quantified using the DC BioRad protein assay. Equivalent protein concentrationswere immunoprecipitated with Protein G Dynabeads™ (Invitrogen) coupled to a mouse anti-biotin primary antibody. Immunoprecipitated biotin-labelled proteins were separated by SDS- PAGE and subjected to W estern blotanalysis.

Immunocytochemistry

ECs were seeded onto acid-washed,oven sterilised glasscoverslips for 3 hours. Following VEGF-A stimulation, ECs were fixed in 4 % paraformaldehyde, washed in PBS, blocked and permeabilised with 10% goat serum in PBS 0.3% triton X-100. ECs were incubated in primary antibody diluted 1:100 overnight at 4 °C. Coverslips were then PBS washed and incubated in an appropriate Alexa fluor secondary antibody diluted 1:200 in PBS for 1 hour atRT.Coverslips were mounted using flouromount G with DAPI™ . Images were captured using a Zeiss Axiolmager M2 microscope (AxioCam MRm camera) at 63x magnification. Primary antibodies: VEGFR-2 (CST; 2479),Rab7 (CST;17286).

Statisticalanalysis

The graphic illustrations and analyses to determine statistical significance were generated using GraphPad Prism 9 software and Student’s t-tests unless otherwise stated. Statistical analysis between Cre-positive groups was performed using one-way ANOVA tests.Bar charts show mean values and the standard error ofthe mean (+SEM). Asterisks indicate the statistical significance of p values:p > 0.05 = NS (not significant),*p < 0.05,**p < 0.01,***p < 0.001 and ****p < 0.0001. References

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Claims

Claims

1. A method of substantially preventing or inhibiting tumour growth and/or angiogenesis, said method including the step of co-targeting neuropilin-1 (NRP1)and neuropilin-2 (NRP2)in endothelialcells.

2. A method according to claim 1 wherein co-targeting the expression of both NRP1 and NRP2 severely inhibits primary or secondary tumour growth and/or angiogenesis, to a greater extent than when eitherNRP receptoristargeted alone.

3. A method according to claim 1 wherein the growth and/or angiogenesisisprevented in solid tumour cells or cancers.

4. A method according to claim 1 wherein the co-targeting ofNRP-1 and NRP-2 includes the substantially simultaneous inhibition of endothelialNRP-1 and NRP-2.

5. A method according to claim 1 wherein the co-targeting includes the suppression of NRP-1 and/or NRP-2 genes and/or gene expression.

6. A method according to claim 5 wherein the depletion of both NRP1 and NRP2 severely impairs EDA-fibronectin (EDA-FN) secretion.

7. A method according to claim 1 wherein dual-targeting of endothelialNRPsiseffective atsupressing metastasis.

8. A method according to claim 7 wherein the metastasis is hematogenous metastasis.

9. A method according to claim 1 wherein dual-targeting of endothelial NRP1 and NRP2 provides a means to retard primary tumorigenesisand significantly reduce secondary site angiogenesis.

10. A method to reduce metastatic load and/or angiogenic potential in secondary metastatic sites said method including dual-targeting of endothelialNRP1 and NRP2.

11. A composition for the dual-targeting or co-targeting of endothelial NRP1 and NRP2 said composition provided in a pharmaceutically acceptable carrier,the composition includes any one of peptide, protein, monoclonal antibody, small molecule, oligonucleotide or antisense oligonucleotide.

12. A composition according to claim 11 wherein the composition includes any one or any combination of NRPa-308, EG00229 or Sunitinib.

13. A composition according to claim 11 wherein composition includes CEND-1 cyclic peptide.

14. A composition according to claim 11 wherein the composition includesanti-NRP antibody ASP1948.

15. A composition according to claim 11 wherein the composition includeshumanized monoclonalantibody ATYR2810.