Endothelial VEGFR Coreceptors Neuropilin-1 and Neuropilin-2 Are Essential for Tumor Angiogenesis

Neuropilin (NRP) expression is highly correlated with poor outcome in multiple cancer subtypes. As known coreceptors for VEGFRs, core drivers of angiogenesis, past investigations have alluded to their functional roles in facilitating tumorigenesis by promoting invasive vessel growth. Despite this, it remains unclear as to whether NRP1 and NRP2 act in a synergistic manner to enhance pathologic angiogenesis. Here we demonstrate, using NRP1ECKO, NRP2ECKO, and NRP1/NRP2ECKO mouse models, that maximum inhibition of primary tumor development and angiogenesis is achieved when both endothelial NRP1 and NRP2 are targeted simultaneously. Metastasis and secondary site angiogenesis were also significantly inhibited in NRP1/NRP2ECKO animals. Mechanistic studies revealed that codepleting NRP1 and NRP2 in mouse-microvascular endothelial cells stimulates rapid shuttling of VEGFR-2 to Rab7+ endosomes for proteosomal degradation. Our results highlight the importance of targeting both NRP1 and NRP2 to modulate tumor angiogenesis. Significance: The findings presented in this study demonstrate that tumor angiogenesis and growth can be arrested completely by cotargeting endothelial NRP1 and NRP2. We provide new insight into the mechanisms of action regulating NRP-dependent tumor angiogenesis and signpost a novel approach to halt tumor progression.


Introduction
Angiogenesis is a critical driver of tumor growth and metastatic dissemination. Without the expansion of a vascular network to supply oxygen and nutrients to the tumor, growth cannot proceed past a few millimeters (1)(2)(3). VEGF-dependent stimulation of VEGFR-2 represents a major signaling pathway promoting angiogenesis, yet the clinical benefits of targeting the VEGF/VEGFR-2 axis remain modest. Only minimal increases in progressionfree survival rates for various tumor types, including lung, breast, kidney, and colon cancers, have been reported following treatment (3). Only when combined with chemotherapy have such therapies become recognized as an effective strategy against cancer growth, antiangiogenics acting to selectively have since been demonstrated to influence extracellular matrix (ECM) attachment by regulating biodirectional integrin signaling, suggesting the existence of an endothelial-initiated, autocrine regulation of angiogenic responses (22,23).
Owing to their ability to associate with a diverse range of receptors, in turn forming holoreceptors to propagate a plethora of downstream proangiogenic signaling cascades, NRPs are promising targets for antitumor therapies (3). For example, the NRP1-specific small-molecule inhibitors EG and ATWLPPR have been demonstrated to inhibit NRP1-VEGFR-2 signaling, and impair both tumor angiogenesis and tumor growth in vivo (28)(29)(30). Tandem-virtual screening and cell-based screening have since been utilized by Borriello and colleagues, to identify a series of non-peptide VEGF-NRP antagonists, notably NRPα-47 and NRPα-308, which display antiangiogenic and antiproliferative capabilities in vitro, in addition to antitumorigenic effects on breast cancer in vivo (31,32). More recently, the antitumor potential of NRPα-308 was employed against clear-cell renal cell carcinoma (ccRCC), a highly vascularized cancer arising from the overexpression of VEGF-A 165 . Compared with the tyrosine kinase inhibitor sunitinib, the current reference treatment for ccRCC, NRPα-308 was found to suppress ccRCC cell proliferation, migration, and invasiveness to a greater extent. Genetic depletion studies supported these findings, and alluded to the fact that both NRP1 and NRP2 should be completely inhibited to obtain maximal therapeutic effect (33).
Currently, there have been limited studies comparing the antiangiogenic 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. Utilizing these models, we demonstrate that in multiple models of cancer, cotargeting the endothelial expression of both NRP1 and NRP2 severely inhibits primary and secondary tumor growth and angiogenesis, to a much greater extent than when either NRP receptor is targeted alone. The depletion of both NRP1 and NRP2 severely impairs fibronectin containing extra domain-A (EDA-FN) secretion in vivo and in vitro, which likely impedes pathologic vessel stability and growth. We also demonstrate that NRP depletion stimulates the rapid degradation of VEGFR-2, metering surface receptor availability for VEGF-A 165 -induced proangiogenic responses. 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. PCR confirmation of Cre-recombinase status was performed using the following oligonucleotide primers (Thermo Fisher Scientific): Forward primer:  -GCCGCCGGGATCACTCTC- , Reverse primer:  -CCAGCCGCCGTCGCAACT- . NRP1 flfl and NRP2 flfl mice were bred with PDGFb.iCreER T2 mice to generate NRP1 flfl .Pdgfb-iCreER T2 and NRP2 flfl .PDGFb.iCreER animals. NRP1 flfl .Pdgfb-iCreER T2 and NRP2 flfl .PDGFb.iCreER mice were subsequently bred to generate NRP1 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 of littermate controls.

CMT19T Tumor Growth Assays
Mice received intraperitoneal injections of tamoxifen (T5648, Sigma; 75 mg/kg bodyweight, 2 mg/mL stock in corn oil) thrice weekly for the duration of the experiment from day minus 4 (D-4) to day 17 (D17) to induce target deletion. CMT19T lung carcinoma cells (CR-UK Cell Production; 1 × 10 6 ) were implanted subcutaneously into the flank of mice at D0 and allowed to develop until D18. On D18, mice were killed, and tumor volumes and weights measured. Tumor volume was calculated according to the formula: length × width 2 × 0.52. For all tumor studies, <passage 15 cells were thawed from frozen stocks and expanded no more than three passages prior to administration.
Mycoplasma testing to confirm negative status was performed on cancer cells on a 6-monthly basis. Cell-line authentication was not performed.

Intervention Tumor Growth Assays
CMT19T lung carcinoma cells (1 × 10 6 ) or B16-F10 melanoma cells (ATCC; RRID:CVCL_U240; 4 × 10 5 ) were implanted subcutaneously into the flank of mice at D0 and allowed to develop until D18/D24. PyMT-BO1 cells (provided by Katherine Weilbaecher (Washington University, St. Louis, MO; 1 × 10 5 in matrigel) were implanted orthotopically into the inguinal mammary fat pad at D0 and allowed to develop until D15. For both tumor models, mice received intraperitoneal 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/D24, mice were killed, and tumor volumes and weights measured. For all tumor studies, <passage 15 cells were thawed from frozen stocks and expanded no more than three passages prior to administration. Mycoplasma testing to confirm negative status was performed on cancer cells on a 6-monthly basis. Cell-line authentication was not performed.
EC stimulation was achieved using 30 ng/mL VEGF-A 164 (VEGF-A; mouse equivalent of VEGF-A 165 ) after 3 hours incubation in serum-free medium (Op-tiMEM; Thermo Fisher Scientific). VEGF-A was made in-house as previously described by Krilleke and colleagues (39).

Metastasis Experiments
Luciferase + -tagged B16-F10 melanoma cells (1 × 10 6 ) were intravenously injected into the tail vein of mice at D0 and allowed to disseminate until D14. Mice received intraperitoneal injections of tamoxifen (75 mg/kg bodyweight, 2 mg/mL stock) thrice weekly for the duration of the experiment from D3 to induce target deletion. On D14, mice were killed, and lungs removed for bioluminescence imaging and subsequent immunofluorescence analysis of sections.

Biotin-surface Protein Labeling
ECs were washed twice on ice with Soerensen buffer (SBS) pH 7.8 (14.7 mmol/L KH 2 PO 4 , 2 mmol/L Na 2 HPO 4 , and 120 mmol/L Sorbitol pH 7. , and placed on ice as described previously (40). Lysates were cleared by centrifugation at 12,000 × g for 20 minutes at 4°C, then quantified using the DC Bio-Rad protein assay. Equivalent protein concentrations were immunoprecipitated with Protein G Dynabeads (10004D, Thermo Fisher Scientific) coupled to a mouse anti-biotin primary antibody. Immunoprecipitated biotin-labeled proteins were separated by SDS-PAGE and subjected to Western blot analysis.

Immunocytochemistry
ECs were seeded onto acid-washed, oven sterilized glass coverslips for 3 hours.
Following VEGF-A stimulation, ECs were fixed in 4% PFA, washed in PBS, blocked and permeabilized with 10% goat serum in PBS 0.3% triton X-100.

Statistical Analysis
The graphic illustrations and analyses to determine statistical significance were generated using GraphPad Prism 9 software (GraphPad Software, ver 9.1.0) and Student 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 SEM (±SEM). Asterisks indicate the statistical significance of P values: NS (not significant), P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.

Data Availability Statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

Dual Targeting of Endothelial Expressed NRPs Inhibits Primary Tumor Growth and Angiogenesis
As coreceptors for VEGF family receptors, endothelial NRPs are becoming increasingly recognized as candidate targets for suppressing 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 (35) with mice expressing a tamoxifen-inducible Pdgfb-iCreER T2 promoter (41), we previously showed that endothelial NRP2 (NRP2 EC ) promotes pathologic angiogenesis to support the progression of primary tumors in a lung-carcinoma model. Acute, endothelial-specific depletion of NRP2 impaired tumor development and vascularization approximately 2fold, revealing a novel, effective therapeutic strategy against cancerous growth (42). Indeed, Kaplan-Meier plots indicate a significantly reduced overall patient survival following diagnosis with lung carcinoma when either NRP1 or NRP2 mRNA expression is elevated (43; Supplementary Fig. S1A).  Fig. S1D and S1E). While iCreER T2 -induced gene recombination is still active in mature adult endothelium (41), this confirms that NRP receptor targeting only affects actively growing vasculature rather than quiescent vessels.
To determine whether cotargeting endothelial NRP1 and NRP2 impedes tumor growth in already established tumors, we next performed intervention CMT19T allograft studies in our NRP1 flfl NRP2 flfl .EC KO animals, delaying tamoxifen administrations until 7 days after cell implantation (Fig. 1H). By doing so, we aimed to provide a more clinically relevant study design, where treatment is initiated once a cancer has become vascularized. Following this regimen, we observed a similarly severe impediment to tumor growth and angiogenesis following the combined loss of endothelial NRP1 and NRP2 ( Fig. 1I-L). Again, no changes in mean animal weight were observed between groups (Supplementary Fig. S1F). NRP1 flfl NRP2 flfl .EC KO tumors displayed significant reductions in the density of endomucin + (Fig. 1M and N), CD31 + , and ERG + vasculature (Supplementary Fig. S1G and S1H), in addition to significantly fewer Ki67 + proliferating cells compared with control tumors (Fig. 1M and N).
Finally, to exclude tumor size as a statistical confounder, and therefore assess whether the loss of endothelial NRP1 and NRP2 directly influences pathologic angiogenesis, tamoxifen administration was suspended further until 12 days after CMT19T cell implantation ( Supplementary Fig. S1I). Tumor growth was tracked from D7, and a subset of size-matched tumors (∼350 mm 2 ) was harvested from both control and NRP1 flfl NRP2 flfl .EC KO animals on D16. Immunolabeling of endomucin + vessels revealed an approximately 50% reduction in tumor vascularity despite no significant differences in tumor volume, tumor cell proliferation, or apoptosis ( Supplementary Fig. S1J-S1Q). Alongside a reduction in tumor vessel density, we observed a large increase in the incidence of vessel regression in these NRP1 flfl NRP2 flfl .EC KO Fig. S1I-S1K). These data suggests that by targeting endothelial NRP expression, growth of large, fully vascularized tumors is inhibited completely over time.

NRP.EC KO Tumor Vasculature Displays Reduced Pericyte Coverage and EDA-FN Fibrillogenesis
The ECM component EDA-FN is a known marker of tumor vasculature, and is essential for the development of a metastatic microenvironment (20,(45)(46)(47).
As both NRP1 and NRP2 have been reported to regulate FN fibrillogenesis in ECs in the past (40,42), we considered whether the deposition of EDA-FN around tumor vessels would be perturbed in our knockout models. Compared with respective Cre-negative control tumors, only those depleted for both NRP1 and NRP2 saw a significant reduction in EDA-FN coverage around tumor vasculature ( Fig. 2A and B), suggesting that both endothelial NRPs facilitate tumor angiogenesis by promoting vessel stability. Deoxycholate fractionation of both soluble and insoluble EDA-FN in siRNA-transfected WT mouse-lung ECs subsequently validated these findings; combined depletion of both NRP1 and NRP2 resulting in a significant reduction in EDA-FN expression from both unstimulated and VEGF-A 165 -stimulated insoluble fractions ( Fig. 2C and D).
Another important regulator of angiogenesis and vascular stability is the ability for ECs to recruit pericytes. These perivascular mesenchymal cells secrete proangiogenic growth factors to stimulate vessel growth in tumors, and regulate vessel permeability (48,49). As NRP1 flfl NRP2 flfl .EC KO tumor vessels exhibited significantly reduced EDA-FN outgrowth, we subsequently assessed pericyte coverage by co-immunolabeling NG2 + mural cells with endomucin + blood vessels (48). Compared with control tumors, we observed a significant reduction in the number of vessel-associated pericytes in NRP1 flfl NRP2 flfl .EC KO tumor vasculature (Fig. 2E and F), concomitant with a significant increase in the frequency of regressed vessels, again, determined by the presence of endomucin − , collagen IV + basement membrane sleeves (44; Fig. 2G and H). It is likely therefore that the reduction in vascular density observed in NRP1 flfl NRP2 flfl .EC KO tumors arises as a result of increased tumor vessel regression following pericyte dropout. A significant reduction in tumor vessel diameter was also observed (Fig. 2I).

Primary Tumor Development and Angiogenesis is Susceptible to the Effects of Cotargeting Endothelial NRP1 and NRP2 in Multiple Cancer Models
As the endothelial codepletion of NRP1 and NRP2 was found to effectively impair primary lung carcinoma growth and angiogenesis, we proceeded to assess the efficacy of their codepletion in other paradigms of cancer. To investigate whether the loss of NRP expression influences melanoma development, B16-F10 melanoma cells were subcutaneously implanted and allowed to grow for a period of 18 days, following our initial intervention-based tamoxifen regime as previously described in Fig. 1H (Fig. 3A). From D11, NRP1 flfl NRP2 flfl .EC KO tumors grew significantly smaller than their control counterparts, and when excised were found to have developed to only approximately 10% the size of control tumors. Indeed, a small number of tumors were found to have regressed entirely (Fig. 3B-E). No changes in mean animal weight were observed between control and NRP1 flfl NRP2 flfl .EC KO mice (Fig. 3F).
In a similar manner, we assessed the impact of codepleting endothelial NRP1 and NRP2 on a luminal B model of breast cancer. PyMT-BO1 cancer cells (50) were orthotopically implanted into the fourth inguinal mammary gland of NRP1 flfl NRP2 flfl and NRP1 flfl NRP2 flfl .EC KO mice, and allowed to grow over a period of 15 days. Again, tamoxifen administration was delayed until D7 to allow for palpable tumors to develop prior to the onset of target depletion (Fig. 3G). Compared with NRP1 flfl NRP2 flfl control tumors, those grown in NRP1 flfl NRP2 flfl .EC KO animals developed approximately 65% smaller by D15 (Fig. 3H-K), alongside no significant alterations in mean animal weight (Fig. 3L). Unlike the B16-F10 tumors, which failed to grow more than approximately 2 mm in size in our NRP1 flfl NRP2 flfl .EC KO mice, we were able to process our PyMT-BO1 tumors for immunofluorescence imaging analysis. NRP1 flfl NRP2 flfl .EC KO PyMT-BO1 tumors were found to be approximately 70% less vascularized than respective Cre-negative control tumors ( Fig. 3M  and N; Supplementary Fig. S2A and S2B), corroborating our CMT19T studies, and confirming that the expression of NRP1 and NRP2 is essential for tumor angiogenesis in multiple cancer models.

Endothelial NRP1 and NRP2 Codepletion Reduces the Metastatic Potential of Circulating Melanoma Cells
Not only are murine B16-F10 cells a well-established, aggressive tumor model for preclinical investigations into melanoma progression, they are also known to preferentially metastasize to the lungs of C57/BL6 mice (51).  suppressing hematogenous metastasis, we measured pulmonary seeding 14 days after intravenous injection of luciferase + -tagged B16-F10 cells (Fig. 4A).
NRP1 flfl NRP2 flfl .EC KO mice were found to develop significantly fewer metastatic lung nodules than control mice, subsequently confirmed by bioluminescence imaging ( Fig. 4B and C). Immunofluorescence staining of lung metastases revealed a robust expression of both NRP1 and NRP2 colocalizing to endomucin + vasculature in control nodules, but not in lung nodules of NRP1 flfl NRP2 flfl .EC KO mice (Fig. 4D). Furthermore, lung metastases of NRP1 flfl NRP2 flfl .EC KO mice were observed to be significantly smaller and less vascularized than their control counterparts ( Fig. 4E and 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 angiogenesis and growth.

Endothelial NRPs Regulate VEGFR-2 Turnover to Sustain Proangiogenic Signaling Responses
Sustained hyperactivation of VEGFR-2 is largely considered one of the most critical aspects of pathologic angiogenesis during tumor growth. Both NRP1 and NRP2 are also known coreceptors of VEGFRs and their respective VEGF signaling moieties (8,9). We therefore examined whether VEGFR-2 signaling would be perturbed in tumor vasculature depleted for NRP1 and NRP2 by measuring VEGFR-2 and phosphorylated-VEGFR-2 Y1175 localization to endomucin + vessels. While NRP1 flfl .EC KO and NRP2 flfl .EC KO CMT19T tumors saw reductions in VEGFR-2 localization of approximately 30% and 10%, respectively, we observed a compounded reduction of over 50% in NRP1 flfl NRP2 flfl .EC KO tumors ( Fig. 5A and B). Likewise, simultaneous depletion of both endothelial NRP1 and NRP2 resulted in an equivalent loss of colocalized phosphorylated-VEGFR-2 Y1175 expression from tumor vessels ( Fig. 5C; Supplementary Fig. S3A).
To further elucidate how endothelial NRPs co-operate to influence VEGFR-2 activity, we examined VEGFR-2 dynamics in vitro. First, we established VEGFR-2 surface expression levels in Ctrl siRNA-treated ECs remained intact up to 5 minutes after stimulation with VEGF-A 165 (Fig. 5D) by biotin labeling. Lysates from Ctrl, NRP1, NRP2, and NRP1/2 siRNA-treated ECs were subsequently analyzed by Western blotting to assess changes in VEGFR-2 expression following an acute 5-minute period of VEGF-A 165 stimulation. This revealed that total VEGFR-2 expression was significantly diminished in stimulated ECs depleted for both NRP1 and NRP2 compared with unstimulated knockdown ECs (Fig. 5E-H).
Following receptor stimulation and internalization, VEGFR-2 is shuttled from Rab5 + early endosomes to either Rab4/Rab11 + recycling endosomes, or is rapidly degraded via Rab7 + late endosomes and the proteosome. We therefore proceeded to determine any changes in the fraction of VEGFR-2 localizing to Rab7 + punctae following 5 minutes of VEGF-A 165 stimulation. siNRP1/2 depleted ECs displayed a significantly greater proportion of VEGFR-2 present in Rab7 + vesicles compared with siCtrl ECs (Fig. 5I and J), suggesting that NRP1 and NRP2 promote VEGFR-2-induced proangiogenic responses by moderating receptor turnover.
Previous studies have demonstrated that NRP2 also acts as a coreceptor for the lymphangiogenic factor VEGF-C to promote VEGFR-3-mediated lymphatic vessel sprouting (12,57). NRP1 has also been shown to interact with VEGF-C, possibly to influence proangiogenic signaling cascades (58). As VEGFR-3 was found to be expressed in vessels of control CMT19T tumors ( Supplementary  Fig. S3D), we thought it pertinent to likewise consider whether NRP depletion would influence VEGFR-3 shuttling to Rab7 + endosomes in a similar manner to above. In nonstimulated ECs, NRP depletion was found to consistently favor VEGFR-3 localization to Rab7 + endosomes, suggesting that both NRP1 and NRP2 regulate VEGFR-3 expression and activity. Following 5-minute incubation with VEGF-C, however, only in siNRP1 ECs did we observe a significant increase in the number of Rab7 endosomes positive for VEGFR-3; neither siNRP2 nor siNRP1/2 ECs were found to respond to VEGF-C stimulation (Supplementary Fig. S3E and S3F).

Discussion
Pathologic angiogenesis is a core driver of aggressive tumorigenesis, yet the clinical benefits of targeting principle regulators of proangiogenic cascades have, thus far, shown limited efficacy (3). We demonstrate that the endothelial-specific cotargeting of both NRP receptors, NRP1 and NRP2, provides effective inhibition against tumor growth and secondary site metastasis in multiple cancer models, likely by potentiating the rapid delivery of VEGFR-2 to late-endosomes for degradation. Importantly, we highlight the importance of targeting the expression of both NRPs simultaneously for maximum therapeutic effect.

FIGURE 5
Endothelial NRPs regulate VEGFR-2 turnover to sustain proangiogenic signaling responses. A, Representative tumor sections from Cre-negative and Cre-positive CMT19T tumors showing colocalization between VEGFR-2 and endomucin + blood vessels. Scale bar = 100 μm. Note: PDGFb.iCreER − endomucin image panels in Fig. 2A (24). Equally, genetic silencing of Sema3F, which binds exclusively to NRP2, was shown to reduce VEGFR-2 phosphorylation and signaling (21,71). In direct conflict to this, reports have also shown Sema3A signaling to reduce cell adhesion to the ECM (70), and to impair the invasiveness of both breast and prostate cancer cells in vitro (72,73). Similarly, Sema3F has primary functions as a tumor suppressor (70), inhibiting the attachment and metastatic spread of lung, breast, and melanoma cells (15,(74)(75)(76). Further studies delineating the effects of silencing the expression of either or both NRPs on semaphorin signaling are certainly required to explicate the molecular mechanisms by which Sema3s regulate EC behavior, particularly in the arena of tumor progression.
As NRP1 and NRP2 are canonical coreceptors for VEGFR-2 in endothelial cells, we hypothesized that their codepletion would provide effective inhibition of VEGFR-2-induced responses. NRP1/NRP2 knockout tumor vasculature was found to express significantly less VEGFR-2 than either NRP1 or NRP2 knockout tumors, in addition to reduced phosphorylated VEGFR-2 expression. Mechanistic studies utilizing siRNA transfected WT 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-A 165 stimulation, likely resulting in a severely moderated VEGFR-2 response. This work supports that of Ballmer-Hofer and colleagues, who delineated that in the absence of NRP1, or in ECs stimulated with a non-NRP1-binding VEGF-A isoform, VEGFR-2 is rerouted to the degradative pathway specified by Rab7 vesicles. Importantly, this was found to occur only following 30 minutes VEGF-A stimulation (77), suggesting that the rate of VEGFR-2 degradation is accelerated when NRP1 and NRP2 are lost in tandem, as we observed changes after only 5 minutes.
In conclusion, our findings show that the activity of endothelial NRPs together is required for sustained tumor angiogenesis, and support a hypothesis that maximum antiangiogenic efficacy can be achieved by cotargeting both NRP1 and NRP2 rather than targeting either receptor individually. Dual loss of both NRPs was found to severely abrogate tumor development and tumor angiogenesis in multiple models of cancer, in addition to secondary tumor development. This work provides strong evidence for the need to develop novel targeted therapeutics specific for both endothelial NRP1 and NRP2 receptors, against pathologies characterized by uncontrolled vascular expansion.

Study Limitations
This study utilizes transgenic mice that express a tamoxifen-inducible form of Cre-recombinase (iCreER T2 ), under the control of an endothelial-specific Pdgfb promoter gene (Pdgfb-iCreER T2 ). While this model has been shown on multiple occasions as a powerful tool to manipulate the expression of endothelial targets (41,(78)(79)(80), we recognize that alternative Cre-models, such as Cdh5-iCre, exhibit a lower degree of expression variability (Pdgfb is neither exclusively nor ubiquitously expressed in murine vasculature), and have been used as such to positive effect. As with other inducible Cre alleles however, while Cdh5-iCre exhibits a greater degree of endothelial specificity, experimental variability in recombination efficiency has been reported, in addition to its downregulated expression in mature quiescent vascular beds (80,81). Other AACRJournals.org Cancer Res Commun; 2(12) December 2022 models include Esm1-Cre, which while commonly associated with the study of sprouting angiogenesis in the postnatal retina, reports have demonstrated its ability to study tumor angiogenesis in the Lewis lung carcinoma model. Its differential expression in mature vasculature however, has yet to be determined (80,82). While beyond the range of this study, we believe there would be instructive benefit from confirming the findings presented using another relevant Cre system.
Furthermore, this study does not detail the precise in vitro mechanisms by which NRP depletion results in the severe impairments to EDA-FN deposition. While pertinent to explore in future works, it is likely that this arises as a consequence of both NRP1 and NRP2's ability to promote intracellular traffic of α5 integrin (40,83), a crucial event that expedites EDA-FN fibrillogenesis. As others have demonstrated previously, any disruption to the cellular machineries that support recycling of active α5 integrin results in reduced EDA-FN secretion and polymerization in vitro and in vivo (84).