Nucleolin promotes angiogenesis and endothelial metabolism along the oncofetal axis in the human brain vasculature

Glioblastomas are among the deadliest human cancers and are highly vascularized. Angiogenesis is dynamic during brain development, almost quiescent in the adult brain but reactivated in vascular-dependent CNS pathologies, including brain tumors. The oncofetal axis describes the reactivation of fetal programs in tumors, but its relevance in endothelial and perivascular cells of the human brain vasculature in glial brain tumors is unexplored. Nucleolin is a regulator of cell proliferation and angiogenesis, but its roles in the brain vasculature remain unknown. Here, we studied the expression of Nucleolin in the neurovascular unit in human fetal brains, adult brains, and human gliomas in vivo as well as its effects on sprouting angiogenesis and endothelial metabolism in vitro. Nucleolin is highly expressed in endothelial and perivascular cells during brain development, downregulated in the adult brain, and upregulated in glioma. Moreover, Nucleolin expression correlated with glioma malignancy in vivo. In culture, siRNA-mediated Nucleolin knockdown reduced human brain endothelial cell (HCMEC) and HUVEC sprouting angiogenesis, proliferation, filopodia extension, and glucose metabolism. Furthermore, inhibition of Nucleolin with the aptamer AS1411 decreased brain endothelial cell proliferation in vitro. Mechanistically, Nucleolin knockdown in HCMECs and HUVECs uncovered regulation of angiogenesis involving VEGFR2 and of endothelial glycolysis. These findings identify Nucleolin as a neurodevelopmental factor reactivated in glioma that promotes sprouting angiogenesis and endothelial metabolism, characterizing Nucleolin as an oncofetal protein. Our findings have potential implications in the therapeutic targeting of glioma.


Introduction
inside and outside the CNS. In peripheral tissues, NCL was shown to be upregulated at the cell surface of ECs in angiogenic vessels in breast tumors (51), with regulatory effects on carcinogenesis and angiogenesis, and it was shown to regulate EC motility and tube formation in vitro (52). Moreover, targeting endothelial NCL induced endothelial apoptosis and vessel normalization in a pancreatic tumor mouse model (53,54). However, the role of NCL on angiogenesis and EC function in the developing human brain and in human gliomas remains poorly understood. Here, using a variety of in vivo and in vitro assays, we show that NCL is an oncofetal protein in human gliomas regulating sprouting angiogenesis and endothelial metabolism.

Results
NCL is a neurodevelopmental protein of the oncofetal axis that is silenced in the healthy adult brain and reactivated in the NVU/PVN of glial brain tumors. To investigate whether NCL constitutes a neurodevelopmental protein that is reactivated in brain tumors, we performed immunofluorescence microscopy of the main NVU/PVN cellular components of human fetal brain, of human normal adult brain, and of human glial brain tumors. NCL was highly expressed during fetal forebrain neocortex development, at gestational week 18 (GW18) and GW22, significantly downregulated in the adult brain, and upregulated in brain tumors, as revealed by immunofluorescence staining against NCL and the nuclear marker TO-PRO-3 (55) (Figure 1, A-C and P). Moreover, NCL expression was present throughout the nucleoplasm during fetal development but was restricted to the nucleolus in the adult human brain (Figure 1, A and B). In GBM, NCL expression was detected across the entire nucleoplasm and appeared similar to the pattern observed during fetal development ( Figure 1, A and C). Within the NVU, NCL was expressed in both endothelial and perivascular cells (Figure 1, D-O). NCL was highly expressed in cells labeled with the endothelial marker cluster of differentiation 31 (CD31) during brain development, where 84% of CD31 + ECs showed NCL expression across the entire nucleoplasm ( Figure 1, D and Q). NCL was significantly downregulated in the adult brain, with 16% of the CD31 + cells being NCL + ECs (predominant nucleolar expression) (Figure 1, E and Q), but it was significantly upregulated in GBM ECs, where 67% of the CD31 + were NCL + (nucleoplasm and nucleolar expression), similar to its expression in fetal brain (Figure 1, D, F, and Q). NCL was also highly expressed in CD105 + angiogenic ECs during fetal brain development and in GBM, with 75% and 74% CD105 + /NCL + double-positive ECs, respectively ( Figure 1, G, I, and R), whereas only 13% CD105 + /NCL + ECs could be observed in adult brain slices (Figure 1, H and R), consistent with the reported quiescence of ECs in the adult normal brain (11,56,57).
Taken together, these data reveal that NCL is highly expressed in endothelial and certain NVU cells (astrocytes > pericytes) during fetal brain development, is subsequently downregulated in the adult brain, and is reactivated in GBM. This characterizes NCL as an oncofetal protein that is reactivated in human glial brain tumors after downregulation in the quiescent adult NVU (18)(19)(20)(21)(22)(23)(24)(25).
NCL expression within the NVU correlates with glial brain tumor malignancy and progression. In order to address the expression of NCL in glial brain tumor progression (from WHO low-to high-grade tumors; refs. 62-64), we referred to tissue microarrays (TMAs) of human glioma stained by IHC for NCL and the nuclear marker Mayer's hemalum (Figure 2, A-D). NCL expression was markedly upregulated in human glial brain tumors as compared with the adult normal brain (Figure 2, A-E). Moreover, NCL expression was significantly increased during glial tumor progression, ranging from 32% of NCL + cells in WHO grade I glioma to 57% in glioma grade IV (GBM) ( Figure 2E). NCL showed a significant upregulation from lowgrade (WHO grade I and II) to high-grade glioma (WHO grade III) as well as a significant increase from WHO grade III to WHO grade IV glioma (Figure 2 showed a slight but significant decrease as compared with primary GBM ( Figure 2E). NCL expression correlated well with the established proliferation marker Ki-67 (65) in all glioma grades ( Figure 2G).
Based on NCL expression within perivascular cells of the developmental and tumoral NVU, we next addressed its effects on tumoral cell proliferation. To determine whether NCL promotes GBM cell proliferation, NCL was knocked down in the human GBM cell lines LN-229 (66) and  as well as in freshly isolated primary human GBM cells (GBM-1) using siRNA ( Figure 2, I-K). Cell proliferation of LN-229, LN-18, and GBM-1 was inhibited by siRNA-mediated knockdown of NCL ( Figure 2H) compared with scrambled controls, in agreement with the previously reported strong proproliferative effect of NCL in human GBM cells (42,68).
Next, we examined the expression of NCL in tumor blood vessels using spatial transcriptomics and IHC. In agreement with our immunofluorescent data in GBMs (Figure 1, F and Q), exploratory spatial transcriptomics in human GBMs showed cooccurrence of NCL and various endothelial markers including CD31 (PECAM1), CD105 (ENG), CLDN5, and VWF (Supplemental Figure 1, A-L). Moreover, NCL was indeed present in the wall of tumor blood vessels, showing an increased expression during glial tumor progression (WHO I-IV; Figure 2, L-O), further suggesting a regulatory effect on human glial brain tumor angiogenesis.
Taken together, these data indicate that NCL expression is reactivated in tumor cells and tumor ECs within the NVU during human astrocytic tumor progression.
NCL is expressed within the NVU and in sprouting ETCs, and it promotes the number of tip cell filopodia during brain development in vivo. NCL has been shown to affect tumor and blood vessel growth in peripheral tissues (42,51,52,68,69), but whether it regulates sprouting angiogenesis during brain development remains unknown. To assess whether NCL affects sprouting angiogenesis and ETCs during brain development, we addressed NCL expression in the vicinity of sprouting blood vessels in the human fetal brain. CD105-labeled ETCs with their typical, finger-like protruding filopodia could be recognized in GW18 and GW22 human fetal brain forebrains ( Figure 3, A-D). NCL was expressed in CD105 + endothelial tip, stalk, and phalanx cells (Figure 3, A-D) as well as in perivascular cells surrounding sprouting capillary ETCs (filopodia) (Figure 3, A-D). We observed NCL in nuclei of CD105 + endothelial tip, stalk, and phalanx cells but not on the (endothelial and perivascular) cell surface or in filopodia protrusions ( Figure 3, A-D).
We examined whether NCL expression affected the number of ETC filopodia and observed a lower number of filopodia in ETCs with low NCL expression ( Figure 3, E and F) and a higher number of filopodia in ETCs with high NCL expression ( Figure 3, G and H). Accordingly, we quantified the number of filopodia per NCL + /CD105 + ETCs and assessed NCL expression for each ETC. Indeed, the number of filopodia positively correlated with NCL expression in the ETCs, as revealed by median fluorescence intensity ( Figure 3I). These results strongly suggest that NCL positively regulates the number of ETC filopodia in the human fetal brain.
NCL promotes HCMEC and HUVEC sprouting angiogenesis in vitro. Based on these expression studies suggesting a role for NCL in sprouting angiogenesis and ETC filopodia in vivo, we next investigated the functional role of NCL in human angiogenic EC sprouting in vitro. We used siRNA to knock down NCL in human cerebral microvascular EC/D3 (HCMEC/D3, hereafter referred to as HCMEC) and HUVECs (Figure 4  . Nucleolin is expressed in endothelial and perivascular cells during human brain development, is downregulated in the adult brain, and is reactivated in glial brain tumors in vivo. Sections (20 μm) of human fetal (GW [18][19][20][21][22] and adult brains as well as sections from human GBMs were stained for Nucleolin, the vascular endothelial cell markers CD31 and CD105, the astrocytic marker GFAP, the pericyte marker NG2, and TO-PRO-3 nuclear counterstaining. (A-C and P) Nucleolin (green) is highly expressed in the nuclei of the developing human fetal brain (A) and of human brain tumors (C), but it shows a significant downregulation in the adult normal/healthy brain (B and P). (D-I, Q, and R) Nucleolin (green) is highly expressed in CD31 + blood vessel endothelial cells (red) in the human fetal (D and Q) and pathological brain (F and Q), but it is significantly downregulated in endothelial cell of the quiescent adult brain (E and Q). (G-I and R) Nucleolin shows a high expression in CD105 + activated endothelial cells (red) in the human fetal brain (G and R) and in glioblastoma (I and R) but is significantly downregulated in the quiescent adult normal brain (H and R), with a very low number of CD105 + endothelial cells in the quiescent adult brain (H and R). (J-L and S) Nucleolin (green) is highly expressed in GFAP + neural precursors cells (red) in the fetal brain (J) and in tumoral astrocytes in glioblastoma (L), but it is significantly downregulated in adult normal brain (K and S). (M-O and T) In human fetal and adult brains, NG2 + pericytes (red) partially express low levels of Nucleolin (M and N); Nucleolin expression is highly upregulated in human brain tumor NG2 + pericytes (O and T). Data represent mean ± SEM of 2-3 patients (2-3 sections per patient, based on tissue availability). For statistical analysis, 1-way ANOVA with Tukey's post hoc test were performed. *P < 0.05, **P < 0.01, ****P < 0.0001. Scale bars: 25  To test the effects of NCL on sprouting angiogenesis in vitro, we referred to an in vitro spheroid angiogenesis assay (70). HCMECs and HUVECs in the sprouting spheroid assay grew vessel-like sprouts composed of multiple branches in the control group ( Given the important role of NCL in cell proliferation (37,39,40), we next assessed whether NCL affects EC proliferation in a 3 HT proliferation assay. Indeed, HCMEC and HUVEC proliferation was significantly reduced upon siRNA-mediated NCL knockdown ( Figure 4N and Supplemental Figure 2N), indicating a positive regulatory role for NCL on HCMEC and HUVEC proliferation, reminiscent of stalk cell behavior in vivo (11,27,57).
Because we observed NCL expression in brain ECs ( Figure 1, D-F, and Figure 4, A-D), based on its positive effects on (brain) EC proliferation ( Figure 4N and Supplemental Figure 2N), and given that NCL has been targeted in cancer cells and retinal ECs using the aptamer AS1411(45-50, 71, 72), we next tested the effects of the NCL-specific aptamer AS1411 on brain EC proliferation. Treatment of HCMECs using 1.25, 5, and 10 μM of AS1411 dose-dependently inhibited their proliferation after 96 hours ( Figure 4O). Taken together, these results suggest that endothelial NCL is a positive regulator of sprouting angiogenesis, endothelial proliferation, and filopodia formation in the brain that can potentially be targeted in brain ECs using aptamers.
NCL regulates HCMEC and HUVEC lamellipodia and filopodia formation and actin cytoskeleton orientation. Lamellipodia and filopodia are composed of actin and myosin fibers, and they are essential components of in vivo sprouting angiogenesis (28). Therefore, to further assess the effects of NCL on HCMEC and HUVEC angiogenesis in vitro, we addressed cell shape and morphology as well as actin orientation after NCL knockdown ( Figure 5 Figure  3, C and F). Accordingly, the distribution of actin orientation showed a classical peak in controls, whereas in the HCMEC NCL KD and HUVEC NCL KD , the actin orientation was more randomly distributed ( Figure 5K and Supplemental Figure 3K), indicating the HCMEC NCL KD and HUVEC NCL KD had poorly orientated stress fibers, as opposed to well-aligned stress fibers of control cells.
To assess the effects of NCL on HCMECs and HUVECs and their filopodia, we cultured these cells on a substrate consisting of nanopillar arrays (73, 74) ( Figure 6 Next, we examined the movement of NCL siRNA-treated HCMECs and HUVECs on the nanopillar surface. In HCMEC NCL KD and HUVEC NCL KD , the mean displacements of the nanopillars were significantly These data indicate that NCL is important for actin orientation and polarization, which is required for EC lamellipodia and filopodia formation, structures that are crucial for migration, proliferation, and sprouting of vascular ECs in vivo.  Figure 5D). In HCMECs NCL KD , genes involved in inflammatory responses including IL17D and CCL2 (75) were among the top-regulated genes ( Figure 7E and Supplemental Results).
Next, to address the molecular pathways regulated by NCL in ECs, we performed gene set enrichment analyses (GSEA) (76) between siNCL-treated HCMECs/HUVECs and the control HCMECs/ HUVECs. In HCMEC NCL KD , GSEA followed by pathway visualization using cytoscape (77) revealed that upregulated genes were mainly involved in regulation of angiogenesis and immune-related processes, while the downregulated genes were linked to regulation of metabolic processes and translation ( Figure 7F). Indeed, angiogenesis and vascular development were significantly enriched upon NCL depletion in HCMECs, whereas oxidative phosphorylation and glycolysis showed significant enrichment in control HCMECs (Figure 7, F-J).
Based on the observed regulatory effect of NCL on sprouting angiogenesis, we next analyzed the expression of main regulators of the key angiogenic pathways VEGF-A-VEGFR2/VEGFR3-Dll4-Jagged-Notch and Hippo-YAP-TAZ. We observed regulation of the central VEGF-A-VEGFR2 pathway, upregulation of the antiangiogenic Dll4-Notch pathway as well as downregulation of the proangiogenic Hippo-YAP-TAZ pathway upon NCL knockdown (Figure 8, C-E; Supplemental Figure 6, C-E; and Supplemental Results), supporting the proangiogenic role of NCL in brain and peripheral ECs.
Finally, given its central role in angiogenesis and vascular growth and based on its regulation in the bulk RNA-Seq data, we validated the alteration of transcriptional expression of VEGFR2 at the protein level using immunofluorescence in both cell types. We observed decreased expression of the phosphorylated form of VEGFR2 (p-VEGFR2) in HCMEC NCL KD and HUVEC NCL KD (Figure 8, F-O, and Supplemental Figure 6, F-O), thereby indicating a positive regulatory effect of NCL on VEGFR2 and suggesting a potential crosstalk between the VEGF-A-VEGFR2 and NCL pathways in brain and peripheral ECs. Taken together, these results suggest that NCL's positive regulatory effects on CNS sprouting angiogenesis and  Metabolomics confirm regulation of endothelial glucose metabolism upon NCL knockdown. Endothelial metabolism is a crucial regulator of sprouting angiogenesis, ETC formation, and endothelial lamellipodia and filopodia dynamics (87)(88)(89)(90). Moreover, EC glycolysis regulates the rearrangement of ECs by promoting filopodia formation and by reducing intercellular adhesion (90). Based on the regulation of metabolic pathways upon NCL knockdown in our bulk RNA-Seq data as well as on the observed regulatory effects of NCL on sprouting angiogenesis, ETC (filopodia), and the actin cytoskeleton, we next investigated whether NCL affected endothelial glucose and fatty acid metabolism (89). Therefore, we performed unbiased metabolic profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (91) in HCMEC NCL KD /HUVEC NCL KD compared with HCMEC Control KD /HUVEC Control KD (Figure 9, A-F, and Supplemental Figure 9, A-F). Hierarchical clustering revealed that metabolite levels of HCMEC NCL KD and HUVEC NCL KD clearly separated from HCMEC Control KD and HUVEC Control KD , revealing significantly regulated metabolites between the groups (Figure 9, A and B, and Supplemental Figure 9, A and B). This analysis showed 747 and 383 metabolites altered by the knockdown of NCL in HCMECs and HUVECs, respectively ( Figure 9B and Supplemental Figure 9B). Principal component analysis (PCA) further identified specific groups of metabolites, including those involved in endothelial glucose metabolism to be different in HCMEC NCL KD and HUVEC NCL KD as compared with the control groups ( Figure 9, C and D, and Supplemental Figure 9, C and D).
Next, we examined the metabolic pathways regulated by NCL in ECs. GSEA (76) revealed glycolysis and fatty acid metabolism pathways to be downregulated in HUVEC NCL KD and fatty acid metabolism pathways to be downregulated in HCMEC NCL KD ( Figure 9E and Supplemental Figure  9E). Moreover, glycolysis was downregulated (even though not significantly) in HCMEC NCL KD (Figure 9E). We next analyzed the abundance of metabolites involved in glycolysis and other metabolic pathways important for EC homeostasis and activation (89) (Figure 9F, Supplemental Figure 9F, and Supplemental Figure 10). Interestingly, we found that, among the 8 glucose metabolites detected in HCMECs and HUVECs, glyceraldehyde 3-phosphate (G3P), phosphoenolpyruvate (PEP), and lactate (Lac) were significantly decreased in HCMEC NCL KD , whereas Lac was significantly decreased in HUVEC NCL KD ( Figure 9F and Supplemental Figure 9F). Notably, Lac levels were decreased by 35% in HCMEC NCL KD versus HCMEC Control KD and by 55% in HUVEC NCL KD versus HUVEC Control KD ( Figure 9F and Supplemental Figure 9F). Moreover, the NADH/NAD + ratio indicating for metabolic activity (92,93) showed a decrease (although not significant) upon NCL KD in both cell types ( Figure 9F and Supplemental Figure 7F). Together, these results indicate that NCL exerts its positive regulatory effects on CNS sprouting angiogenesis via positive regulation of endothelial (glucose) metabolism.
NCL regulates endothelial glucose metabolism but not fatty acid metabolism. To further examine the observed effects of NCL on endothelial metabolism, we next performed functional metabolic assays addressing endothelial glucose and fatty acid metabolism in HCMECs and HUVECs. Using a glycolytic flux assay (94), siRNA-mediated knockdown of NCL resulted in a significant reduction of glycolysis as compared with the control HCMECs and HUVECs ( Figure 10A and Supplemental Figure 11A). Similarly, HCMEC NCL KD and HUVEC NCL KD showed reduced glucose uptake and decreased Lac production as compared with the HCMEC NCL KD and HUVEC Control KD (Figure 10, B and C, and Supplemental Figure 11, B and C), indicating a positive regulatory effect of NCL on HCMEC and HUVEC glucose metabolism.
6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and HK2 have been shown to be key regulators of endothelial glucose metabolism (88,89). To address whether NCL affected the expression patterns of these genes in HCMECs and HUVECs, we performed qPCR and Western blots of HCMEC NCL KD , HUVEC NCL KD , HCMEC Control KD , and HUVEC Control KD . HK2 was significantly downregulated upon NCL knockdown on the mRNA level ( Figure 10D and Supplemental Figure 11D), whereas PFKFB3 showed no significant change on either the mRNA or the protein levels ( Figure 10, E-J, and Supplemental Figure 11, E-J).
Next, we assessed whether NCL also regulates endothelial fatty acid oxidation (FAO), which is known to exert crucial effects on endothelial stalk cell proliferation (89,95) and to be upregulated in quiescent ECs as a protection against oxidative stress (96). NCL knockdown in HCMECs and HUVECs did not affect endothelial FAO ( Figure 10K and Supplemental Figure 11K). Carnitine palmitoyltransferase 1A (CPT1A) has been shown to be a key regulator of endothelial fatty acid metabolism (88,89). As expected, qPCR and Western blot analysis of HCMEC NCL KD and HUVEC NCL KD showed no significant differences of CPT1A as compared with the HCMEC Control KD and HUVEC Control KD groups at either the mRNA levels and protein levels ( Figure 10, L-N, and Supplemental Figure 11, L-N).
Taken together, these data reveal that KD of NCL decreases endothelial glucose metabolism without affecting endothelial fatty acid metabolism, indicating a positive regulatory role of NCL on sprouting angiogenesis via promoting endothelial glucose metabolism.

Discussion
Here, using in vitro and in vivo approaches, we show that NCL is a positive regulator of angiogenesis in the human fetal brain. Our results suggest that NCL promotes brain endothelial sprouting, proliferation, and filopodia formation, potentially via interaction with the VEGF-A-VEGFR2 pathway and positively regulates brain endothelial glucose metabolism via the regulation of glycolytic enzymes, including HK2. We propose that, by acting on the cytoskeleton of CNS endothelial (tip and stalk) cells and their filopodia, and by regulating vascular endothelial metabolism, NCL controls the sprouting and filopodia extension of growing CNS blood vessels during human fetal brain development and presumably in human brain tumors. Importantly, the characterization of NCL as an oncofetal protein in the brain tumor vasculature and its inhibition using aptamers identifies NCL as a potential pharmaceutical target for gliomas.
NCL may be a putative NVU/PVN-derived oncofetal signal to regulate developmental brain and brain tumor (vascular) growth. Most of the evidence regarding the molecular regulation of sprouting angiogenesis during brain development is based on murine studies (97,98), whereas less knowledge exists regarding how the vascularization and ETCs are regulated in the human brain. Interestingly, angiogenesis is highly dynamic during brain development and almost quiescent in the adult healthy brain (11,14,57), but it is reactivated in a variety of angiogenesis-dependent CNS pathologies such as brain tumors, brain vascular malformations, or stroke (4,5,14), thereby activating endothelial-and perivascular cells of the NVU (7,11,57). In our study, we not only observed a reactivation of NCL in angiogenic endothelial and perivascular cells within glial brain tumors, but we also observed a positive correlation of NCL expression with astrocytic tumor progression, in agreement with ref. 99, suggesting a crucial role of NCL as a PVN-derived signal in both angiogenic and tumor growth. The PVN has been shown to activate tumor growth in mouse and zebrafish models of breast cancer (100). Strikingly, endothelial-derived thrombospondin-1 in the stable microvasculature induced sustained breast cancer cell quiescence, but this suppressive cue was lost in sprouting neovasculature, where ETC-derived active TGF-β1 and periostin promoted breast tumor growth (100). These characterized the stable microvasculature as a "dormant perivascular (tumor) niche" in contrast to the sprouting neovasculature, which constitutes an "activated perivascular (tumor) niche" in which ETCs exert crucial roles. In light of these studies, the exploration of NCL's angiogenic function within the perivascular tip cell niche in vivo promises to be an exciting avenue for future investigations.
Here, we found that NCL is an important positive regulator of angiogenesis and ETC filopodia in human fetal brain. The expression of NCL in endothelial and perivascular cells such as astrocytes and pericytes within the NVU of the human fetal brain, its downregulation in the adult healthy brain and reactivation in brain tumors characterizes NCL as an oncofetal protein and suggests an integral role once reactivated during brain cancer. Its high expression in CD105 + angiogenic blood vessel ECs in both human fetal brain and human GBM (but not in the adult healthy brain) supports this presumed role in active (developmental, tumor) versus stable (adult healthy) brain angiogenesis. Accordingly, our in vitro results suggest that, by exerting stimulatory effects on sprouting angiogenesis, filopodia  A and B) Heatmap showing the expression of the top 15 genes driving the enrichment of "angiogenesis" pathways in HCMEC NCL KD and the top 50 genes driving the enrichment of "oxidative phosphorylation and glycolysis" pathways in HCMEC Control KD . (C-E) VEGFA expression was not differentially regulated, and VEGFR2 expression was upregulated (C) in HCMECs NCL KD as compared with HCMECs Control KD . NCL knockdown induced a significant upregulation of the Dll4-Jagged-Notch signaling pathway, including in HES1, NOTCH1, NOTCH4, and Jagged1 (JAG1), while HES2 and DLL4 were not differentially regulated upon NCL knockdown (D). siNCL treatment caused a significant downregulation of the YAP-TAZ gene YAP1 as well as the YAP-TAZ downstream effector gene CTGF but not of the YAP-TAZ downstream effector gene CYR61 (E). (F-O) HCMECs were stained for NCL (gray), F-actin (green, stained with phalloidin), VEGFR2 (red, in F-I), or p-VEGFR2 (red, in K-N) and the general nuclear marker DAPI (blue). VEGFR2 expression was not regulated (J, n = 3), but pVEGFR2 expression was significantly downregulated (O, n = 3) upon siRNA-mediated NCL knockdown in HCMECs as compared with the control condition. Data represent mean ± SEM. Wald test corrected for multiple testing using the Benjamini-Hochberg method (C-E) and 2-tailed unpaired Student's t test (J and O) were performed. **P < 0.01, ***P < 0.001. The boxed areas  extension, and glucose metabolism of vascular ECs, NCL might promote the sprouting of angiogenic blood vessel ECs into the brain parenchyma. The latter is also strongly suggested by the positive correlation between NCL expression and the number of ETC filopodia in the fetal brain parenchyma in vivo. Other neurodevelopmental regulators such as VEGF-A and GPR124 are downregulated in the adult healthy CNS and are reactivated in vascular-dependent CNS pathologies such as brain tumors or stroke (101)(102)(103). However, in contrast to those angiogenic factors, NCL is one of the first to have been directly compared in both human fetal brain and human gliomas. Furthermore, NCL endothelial and perivascular expression within the fetal-and tumor PVN and its presumed different roles on the involved cell types (angiogenesis versus tumor growth) characterize NCL as an important developmental signal reactivated during brain tumor growth and, consequently, as an oncofetal protein.
NCL is a regulator of glucose but not fatty acid metabolism and may, therefore, have different effects on endothelial tip and stalk cells during sprouting angiogenesis. Endothelial metabolism has recently emerged as a crucial regulator of sprouting angiogenesis during development and in tumors (87-89, 95, 104, 105). Moreover, it was suggested that ETCs mainly rely on glycolysis, whereas endothelial stalk cells also use fatty acid metabolism to support proliferation (87-89, 95, 104, 105). Here, we found that NCL positively regulates endothelial glycolysis but not fatty acid metabolism in vitro, indicating that NCL's main effect might be on tip cells, but NCL's precise roles on both tip and stalk cells inside and outside the CNS -for instance, in the embryonic or postnatal brain (26,57) or in the postnatal retina (106) -need to be investigated in vivo (Supplemental Discussion).
As also demonstrated by our bulk RNA-Seq, pathways regulated downstream of NCL were linked to angiogenesis and endothelial glucose metabolism. While the observed regulatory effects of NCL on endothelial glucose metabolism are in line with an important role of glycolysis in angiogenesis and vascular biology inside and outside the CNS (88,89,104,107,108), we cannot exclude that NCL regulates other metabolic pathways that participate in (brain) angiogenesis and (brain) EC biology. Thus, given the crucial role of endothelial metabolism for vessel sprouting in development and disease (87,88,104) as well as of tumor metabolism in gliomas (35), investigating the precise role of NCL on EC metabolism and angiogenesis in the human brain vasculature along the oncofetal axis promises to be exciting.
A putative role for NCL in angiogenesis-dependent CNS pathologies via molecular crosstalk with the VEGF-VEG-FR signaling axis. Angiogenesis and the PVN exert crucial roles in the pathophysiology of various vascular-dependent CNS diseases such as brain tumors, vascular malformations, and stroke (6,7,17,49,50,56). With regard to brain tumors, glycosylated surface NCL has been shown to increase with the malignancy grade of human gliomas (99). A high expression of NCL may, therefore, promote vascularization of astrocytoma and thereby promote brain tumor growth (Supplemental Discussion). Furthermore, the anticancer aptamer AS1411 -that binds specifically to NCL -has shown promising clinical activity and is being widely used as a tumor-targeting agent (72) as well as an inhibitor of pathological angiogenesis in the retina (49,50). In line with these reports, we find AS1411-mediated inhibition of brain EC proliferation in vitro, indicating that AS1411 could be tested to target the brain tumor vasculature in vivo. Interestingly, antibody-and peptide-mediated targeting of NCL induced normalization of tumor vasculature in pancreas and breast cancer models (53,69), further suggesting that strategies targeting NCL might affect the GBM vasculature. During (glial) brain tumor progression, vascular dysfunction is partially mediated via angiogenic factors including VEGF-VEGFR (17,(109)(110)(111), and blocking VEGF-VEGFR signaling results in transient normalization of the immature and leaky brain tumor vasculature and leads to survival benefits in patients with newly diagnosed as well as recurrent GBM (17,112,113). Here, we observed a positive regulatory effect of NCL on the VEGF-A-VEGFR2 pathway in vitro, indicative of a molecular Figure 10. NCL positively regulates endothelial glucose metabolism via glycolytic enzymes, including HK2, but does not affect fatty acid oxidation in human brain endothelial cells. (A-C) Metabolic assays of HCMECs, upon NCL downregulation with siRNA targeting NCL. NCL knockdown decreased the glycolytic flux (A, n = 3), glucose uptake (B, n = 4), and Lac production (C, n = 4) in HCMECs as compared with the tested controls. (D) qPCR revealing a significant downregulation of about 30% of hexokinase-2 (HK2) mRNA expression by siRNA-targeted NCL knockdown. PFKFB3 expression showed slight but no significant increase (n = 3). (E and F) Western blot using antibodies against PFKFB3 revealed no significant regulation of PFKFB3 expression by NCL knockdown (n = 3). (G-J) HCMECs were stained for NCL (green), PFKFB3 (red), and the general nuclear marker DAPI (blue). No difference in PFKFB3 expression could be seen between NCL knockdown HCMECs (G and H) and the HCMECs treated with a control siRNA (I and J). (K) NCL knockdown in HCMECs did not affect fatty acid oxidation (n = 3). (L and M) Western blot using antibodies against carnitine palmitoyltransferase 1A (CPT1A) showed no difference in CPT1A protein expression between NCL knockdown HCMECs and the control condition (n = 3). (N) qPCR showed no significant regulation of CPT1A mRNA expression upon siRNA-targeted NCL knockdown (n = 3). Data represent mean ± SEM. Two-tailed unpaired Student's t test were performed. *P < 0.05, **P < 0.01. Scale bars: 20 μm in G-J. crosstalk between these 2 signaling axes. Given that both pathways regulate angiogenesis and vascular normalization, combinatorial targeting of NCL and VEGF-A-VEGFR2 to normalize the brain tumor vasculature may be a promising antiangiogenic strategy for GBM patients. Taken together, these literature indications in concert with our data suggest that -in addition to its effect on tumor cell proliferationoncofetal NCL may regulate brain tumor vascularization and could be a candidate for targeted therapy on both tumor and ECs in human gliomas.

Methods
Supplemental Methods are available online with this article.
Study approval. Tissue samples from human fetal brains were obtained from postmortem fetuses derived from spontaneous abortions and received by the Department of Pathological Anatomy, University of Bari School of Medicine. Tissue preparation and storage were performed as previously described. The study was approved by the Ethics Committee of the University of Bari Medical School and complied with the principles stated in the Declaration of Helsinki.
Tissue samples from human patients with GBM and controls from temporal lobes after selective temporal lobectomy in patients with chronic pharmacoresistant mesial temporal lobe epilepsy were obtained during surgery at the Department of Neurosurgery, University Hospital Zurich. Written informed consent was obtained from patients before study entry. All procedures were conducted in accordance with the Declaration of Helsinki, and the study was approved by the Ethics Committee of the Canton Zurich.