Abnormal tumor vessels promote metastasis and impair chemotherapy. Hence, tumor vessel normalization (TVN) is emerging as an anti-cancer treatment. Here, we show that tumor endothelial cells (ECs) have a hyper-glycolytic metabolism, shunting intermediates to nucleotide synthesis. EC haplo-deficiency or blockade of the glycolytic activator PFKFB3 did not affect tumor growth, but reduced cancer cell invasion, intravasation, and metastasis by normalizing tumor vessels, which improved vessel maturation and perfusion. Mechanistically, PFKFB3 inhibition tightened the vascular barrier by reducing VE-cadherin endocytosis in ECs, and rendering pericytes more quiescent and adhesive (via upregulation of N-cadherin) through glycolysis reduction; it also lowered the expression of cancer cell adhesion molecules in ECs by decreasing NF-κB signaling. PFKFB3-blockade treatment also improved chemotherapy of primary and metastatic tumors.

Anti-angiogenic drugs blocking VEGF signaling are used for anti-cancer treatment, but their success is limited. Tumor vessel normalization (TVN) is emerging as a therapeutic anti-cancer approach, capable of reducing metastasis while improving delivery and response to chemotherapy, but strategies promoting TVN by targeting tumor endothelial cell (TEC) metabolism have not been tested. Here, we report that reduction of EC glycolysis by haplodeficiency of the glycolytic activator PFKFB3 in ECs decreased metastasis at least in part by promoting TVN. Treatment with a low dose of the pharmacological PFKFB3-blocker 3PO, which did not affect cancer cell proliferation, induced similar therapeutic benefit and increased the delivery and response to chemotherapy. These findings merit further consideration of blocking TEC glycolysis for anti-cancer therapy.

In contrast to traditional anti-angiogenic therapy that aims to inhibit tumor vessel growth, an emerging paradigm is to normalize tumor vessels in order to restore perfusion, and thereby to reduce metastasis while improving chemotherapy (). This involves normalization of the endothelial layer, basement membrane, and mural cells. However, all vessel normalization strategies focus on targeting angiogenic growth factors and downstream signaling. Here, we assessed if targeting PFKFB3 in ECs affects tumor vessels.

Tumor vessels are structurally and functionally abnormal (). They are irregular in shape and size, tortuous, and morphologically heterogeneous. This impairs perfusion, which deprives cancer cells from oxygen and nutrients, thus creating a hostile milieu from where cancer cells escape via invasion and metastasis (). Tumor vessels have a leaky EC barrier, facilitating intravasation and dissemination of cancer cells. They also have fewer, more detached pericytes, which further destabilizes vessels (). The perfusion defects also impair the delivery and efficacy of chemotherapy, as the latter often relies on conversion of oxygen to radicals, and oxygen supply in tumors is limited ().

Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases.

Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases.

Endothelial cell (EC) metabolism has gained attention as a therapeutic target for inhibiting angiogenesis. We reported that blocking the glycolytic activator PFKFB3 or the fatty acid oxidation regulator CPT1a reduced angiogenesis in ocular and inflammatory disorders (), but did not study the effect of the PFKFB3-blocker 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) on tumor vessels. Tumor endothelial cells (TECs) are activated cells (), but it is unknown if they have an altered glycolytic metabolism, and if targeting glucose metabolism in TECs offers therapeutic benefit.

The improved chemotherapeutic effect was accompanied by more efficient delivery of CPt. Indeed, when treating B16-F10 tumor-bearing mice with 3PO before administering a single dose of CPt (10 mg/kg), we observed more CPt-DNA adducts in cancer cells treated with 3PO compared with controls ( Figure 8 L), indicating that CPt delivery was increased. Similar results were observed in Pfkfb3mice ( Figures S7 D and S7E). The increased anti-tumor effect of the combination of 3PO plus CPt in vivo was not due to an increase in chemosensitivity of cancer cells to CPt in the presence of 3PO ( Figure 8 M).

We then explored if treatment of B16-F10 tumor-bearing mice with 3PO enhanced the anti-tumor growth effect of a dose of cisplatin (CPt) (2.5 mg/kg body weight; three times per week), which affected tumor growth only minimally ( Figure 8 I). When mice were pretreated with 3PO to induce TVN before administration of CPt and then continued 3PO treatment, CPt inhibited tumor growth more substantially ( Figure 8 I). Of note, CPt monotherapy did not significantly reduce metastatic burden in control mice, but nearly completely prevented metastasis in combination with 3PO ( Figure 8 J). 3PO treatment also enhanced the cytotoxic effect of a clinically relevant standard (maximal) dose of CPt in liver and lung metastasis models upon, respectively, injection of B16-F10 cancer cells into the portal or tail vein ( Figures 8 K and S7 C). 3PO monotherapy only tended to reduce metastasis, presumably because 3PO treatment was initiated only 3 days after cancer cell injection, thus phenocopying the lack of an effect of 3PO on primary tumor growth (see above). However, the finding that 3PO treatment reduced metastasis only when 3PO was administered 3 days before—not after—systemic cancer cell injection further supports an effect of 3PO on cancer cell extravasation.

Given that 3PO treatment improved pericyte coverage more than EC deficiency of PFKFB3 ( Figure 7 O versus Figure 7 L), we explored if pericytes were also a target of 3PO. We first characterized their glucose metabolism, and observed that pericytes are highly glycolytic ( Figure 8 A ). When calculating the theoretical amount of ATP generated by glycolysis, glucose oxidation, glutamine oxidation, and fatty acid oxidation () in pericytes, glycolysis contributed for up to 85%, with minimal contribution by other pathways ( Figure S7 A). Treatment of pericytes with 3PO reduced glycolysis ( Figure 8 B), and impaired proliferation and migration, while increasing quiescence ( Figures 8 C and S7 B). In line, there were fewer proliferating NG-2pericytes around tumor vessels in 3PO-treated tumors ( Figures 8 D and 8E), indicating that they were less activated. Pericytes, pretreated with 3PO, also adhered better to control ECs ( Figures 8 F and 8G), which can be explained by findings that 3PO upregulated N-cadherin levels in pericytes ( Figure 8 H). These effects can explain why the more sedentary, quiescent, and adhesive pericytes stay put and cover ECs in tumors better upon 3PO treatment.

Scale bars, 10 μm (D), 75 μm (F), 100 μm (L). All data are means ± SEM.p < 0.05. For (B), (C), and (H), p values were calculated by mixed model statistics (Kenward-Roger test). In (I), the p value refers to CPt + 3PO versus control. See also Figure S7

(M) Proliferation of cultured B16-F10 cancer cells upon treatment with 25 μM CPt in the presence of increasing concentrations of 3PO (n = 4).

(L) Images of B16-F10 tumor sections, stained for CPt-DNA adducts from tumors treated with vehicle (ctrl) or 3PO upon administration of a single dose of CPt (10 mg/kg). Quantification of CPt-DNA adducts is indicated (n = 19).

(K) Quantification of metastatic liver area upon portal vein injection of B16-F10 cancer cells in mice treated with vehicle (ctrl), 3PO (25 mg/kg daily for 5 consecutive days, initiated on the third day after cancer cell injection, followed by 2 day drug holiday and 5 days of daily treatment), and a standard (maximal) dose of CPt (10 mg/kg administered on days 5 and 11 after cancer cell injection), alone and together with 3PO (n = 10–11).

(J) Metastatic index of s.c. implanted B16-F10 cancer cells disseminating to the lungs from mice in (I).

(I) Growth curve of s.c. B16-F10 tumor upon treatment with vehicle (ctrl), 3PO (25 mg/kg; initiated three times per week when tumors reached a volume of 100 mm 3 ) and a submaximal dose of CPt (2.5 mg/kg administered every other day during the last week before the termination of the experiment), alone or together with 3PO (n = 20–24).

(H) Immunoblot of protein levels of N-cadherin (N-cadh) in control and 3PO-treated pericytes. Densitometric quantification is indicated (normalized to α-tubulin loading control; n = 3).

(D) Micrographs of sections of s.c. B16-F10 tumors from control and 3PO-treated mice stained for EC marker isolectin B4 (IB4), pericyte marker NG-2, and proliferation marker Ki67. Nuclei are counterstained with DAPI. Arrows, Ki67 + pericyte nuclei. Dotted lines in zoom-in panels, vessel wall.

(C) Effect of 3PO on pericyte proliferation (left bar; n = 6) and quiescence (flow cytometric analysis upon staining for EdU, right bar; n = 3).

Effect of PFKFB3 Inhibition on Pericytes and Delivery and Efficacy of Chemotherapy

Figure 8 Effect of PFKFB3 Inhibition on Pericytes and Delivery and Efficacy of Chemotherapy

In healthy microvessels, quiescent pericytes stay put and cover ECs, but in tumor vessels, pericytes are activated and proliferate more actively, yet become detached from tumor ECs (resulting in reduced pericyte coverage), partly because of reduced adhesive properties (due to lower levels of neural (N)-cadherin, mediating adhesion between ECs and pericytes) and increased motility (). In addition, unstable tumor vessels are continuously remodeled so that their basement membrane (deposited partly by quiescent pericytes) is incomplete or even absent. Double staining for CD31 and pericyte marker NG-2 showed that more pericytes covered ECs in B16-F10 tumors of Pfkfb3mice ( Figures 7 K and 7L). A substantial fraction of tumor vessels in WT mice consisted of naked ECs without or with little laminin-positive basement membrane, while tumors in Pfkfb3mice contained more mature CD31lamininvessels ( Figure 7 M). Similar results of improved vessel maturation were obtained when analyzing LLC tumors in Pfkfb3mice ( Figures S6 H and S6I) and B16-F10 or Panc02 tumors upon treatment with 3PO ( Figures 7 N, 7O, S6 J, and S6K). PFKFB3 blockade by 3PO also increased maturation of tumor vessels in liver metastases upon portal vein injection of B16-F10 cancer cells ( Figures S6 L and S6M). Mechanistically, pretreatment of ECs with 3PO increased adhesion of pericytes to ECs ( Figure S6 N) and elevated N-cadherin mRNA levels in these cells ( Figure S6 O), contributing to pericyte recruitment upon cell-autonomous endothelial PFKFB3 inhibition.

We also explored if PFKFB3 inhibition changed other EC characteristics that could reduce cancer cell intravasation. Of note, the finding that metastasis was reduced only when 3PO was initiated before—not after (see below)—systemic cancer cell injection suggests that PFKFB3 inhibition also impaired cancer cell extravasation. We considered if PFKFB3 inhibition rendered ECs less adhesive for cancer cells. Indeed, PFKFB3 blockade reduced the number of cancer cells adhering to and migrating across ECs, pre-activated with interleukin-1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α) ( Figures 7 A–7C , S6 A, and S6B). Notably, the reduced adhesion was due to an altered EC pro-inflammatory signature. Indeed, upon activation with IL-1β, PFKFB3-inhibited ECs expressed lower levels of VCAM-1, ICAM-1, and E-selectin ( Figures 7 D and S6 C–S6E), adhesive molecules involved in cancer cell intra/extravasation (). In accordance, ICAM-1 immunoreactive levels in TECs were lower in tumors of Pfkfb3mice ( Figure 7 E). We then assessed if PFKFB3 inhibition decreased nuclear factor κB (NF-κB) signaling in ECs, because of several reasons: (1) IL-1β induces signaling partly via NF-κB; (2) the glycolytic product lactate stimulates NF-κB signaling in ECs (); (3) PFKFB3 inhibition lowers lactate levels in ECs (see above and); and (4) IL-1β and TNF-α treatment of ECs increased glycolysis and, in accordance, PFKFB3 levels ( Figures S6 F and S6G), while PFKFB3 blockade counteracted this increase and lowered the elevated levels of glycolysis ( Figure S6 F). Indeed, PFKFB3 inhibition reduced NF-κB signaling ( Figure 7 F). In response to cytokine activation, IκBα, a chaperone that keeps the NF-κB subunits p65 and p50 inactive in the cytosol, becomes phosphorylated before degradation, resulting in release and activation (phosphorylation) of p65 and p50. In agreement, IL-1β stimulation increased levels of phospho-p65 and -IκBα ( Figures 7 G–7I). Notably however, upon PFKFB3 blockade, levels of phospho-p65 and -IκBα were only minimally elevated ( Figures 7 G–7I). In agreement, phospho-p65 immunoreactive levels appeared lower in ECs of tumors in 3PO-treated mice ( Figure 7 J).

(N) Micrographs of sections of s.c. B16-F10 tumors from control or 3PO-treated mice stained for CD31 and NG-2. Nuclei are counterstained with DAPI.

(M) Micrographs of B16-F10 tumor sections from WT and PFKFB3 +/ΔEC mice, stained for CD31 and laminin to visualize the basement membrane. Quantification of % of laminin + vessels is indicated (n = 10–19).

(K) Micrographs of sections of s.c. B16-F10 tumors from WT and Pfkfb3 +/ΔEC mice, stained for CD31 and NG-2. Nuclei are counterstained with DAPI.

(J) Representative micrographs of p-p65 staining of tumor vessels in s.c. B16-F10 tumors of control versus 3PO-treated mice and quantification of p-p65 + signal (average pixel intensity expressed in a.u.) (n = 5). Arrows, ECs. The top and the bottom small images at the right show the p-p65 + DAPI channels only, with a higher magnification of the boxed areas. Dotted lines, vessel wall.

(I) Densitometric quantifications of p-IκBα (relative to total IκBα) in (G) (n = 3). ND, not detectable.

(G) Immunoblot of protein levels of phosphorylated p65 (p-p65) and total p65 (upper blot), and of phosphorylated IκBα (p-IκBα) and total IκBα (bottom blot) in control and 3PO-treated ECs upon treatment with vehicle, 3PO (20 μM), IL-1β (1 ng/mL) alone and together with 3PO (20 μM). β-Tubulin was used as loading control.

(F) Quantification of NF-κB luciferase reporter activity in ECs upon treatment with vehicle (ctrl), 3PO (20 μM), IL-1β (1 ng/mL) alone and together with 3PO (n = 4).

(E) Representative micrographs of B16-F10 tumor sections from control and 3PO-treated mice co-stained for CD31 and ICAM1 with quantification of ICAM + area (% of total vessel area) (n = 5–6). Right panels, ICAM1 signal channel only.

(D) RT-PCR of mRNA expression levels of VCAM-1, ICAM-1, and E-selectin in ECs upon treatment with IL-1β alone (1 ng/mL) or together with 3PO (20 μM). Values are expressed relative to IL-1β-stimulated cells (n = 4–5).

(C) Quantification of transendothelial cancer cell migration through the EC monolayer, stimulated with IL-1β (1 ng/mL) or TNF-α (10 ng/mL) without or with 3PO (10 μM) (n = 5).

(B) Quantification of number of adherent cancer cells per well in (A) (n = 4).

(A) Micrographs of B16-F10 cancer cells (green) adhering to an EC monolayer upon single or combined treatment with vehicle (ctrl), 3PO (10 μM), IL-1β (1 ng/mL), and 3PO plus IL-1β.

We then explored how PFKFB3 blockade increased plasma membrane VE-cadherin levels. Given that VE-cadherin is internalized via clathrin-mediated endocytosis (), and this process relies on ATP-consuming actin-myosin remodeling (), we hypothesized that PFKFB3-driven glycolysis was necessary for VE-cadherin endocytosis. Indeed, using phRodo-dextran, a pH-sensitive fluorescent reporter, the intensity of which intensifies with increasing acidity (as in acidified endocytic vesicles), we observed that PFKFB3 blockade reduced internalization of phRodo-dextran in ECs ( Figures 6 G and 6H). Similarly, incubation of ECs with a fluorescently labeled anti-VE-cadherin antibody, followed by mild acid wash to remove cell surface bound antibody, revealed cytosolic accumulation of fluorescent label in response to VEGF stimulation of NECs ( Figures 6 I and 6J). Notably, 3PO, which alone did not have an effect, largely abrogated the increase in VE-cadherin endocytosis induced by VEGF in NECs ( Figures 6 I and 6J).

We assessed how PFKFB3 inhibition regulated VE-cadherinadherens junctions (AJs) in cultured NECs and human umbilical venous ECs (HUVECs, abbreviated as ECs). Two types of AJs can be distinguished: (1) continuous stable AJs in a quiescent EC network, associated with parallel cortical actin bundles and in which VE-cadherin is localized linearly along cell-cell borders; and (2) discontinuous fragmented AJs in ECs with reduced network integrity, attached to radial stress fibers, and in which VE-cadherin is distributed in short linear structures perpendicular to cell-cell borders (). PFKFB3 blockade increased the length of continuous VE-cadherinAJs, while reducing the fraction of discontinuous VE-cadherinAJs, thus indicating that 3PO promoted vascular integrity and EC interconnectivity ( Figures 6 C and 6D). PFKFB3 blockade also reduced the number of intercellular gaps, further evidence for a tighter, more connected monolayer with increased integrity ( Figure 6 E). Also, 3PO increased the trans-EC electrical resistance, a measure of barrier tightness, and largely abrogated the permeability increase induced by vascular endothelial growth factor (VEGF) ( Figure 6 F).

Endothelial alpha-parvin controls integrity of developing vasculature and is required for maintenance of cell-cell junctions.

We explored if a tightened EC barrier could contribute to the reduced cancer cell intravasation in Pfkfb3mice. Disorganized tumor vessels have lower levels of the junctional protein VE-cadherin, while normalized tumor vessels tighten their EC barrier by upregulating VE-cadherin (). RT-PCR showed that VE-cadherin (Cdh5) mRNA levels were lower in eTECs ( Figure S1 A). Whole-mount staining of B16-F10 tumor sections for CD31 and VE-cadherin showed that VE-cadherinadherens junctions were more abundant and more strongly stained in Pfkfb3mice ( Figure 6 A ). Also, scanning electron microscopy showed a regular, smooth, orderly formed, flat surface of a monolayer of ECs in tumor vessels of Pfkfb3mice, in contrast to the more irregular, chaotically organized, and often discontinuous EC lining in WT mice ( Figure 6 B). Similar results were obtained in LLC tumors in Pfkfb3mice ( Figures S5 A and S5B) and upon 3PO treatment of s.c. B16-F10 tumors ( Figures S5 C and S5D) and Panc02 tumors ( Figure S5 E), as well as in B16-F10 liver metastases ( Figure S5 F).

Scale bars, 10 μm (A, B, G), 20 μm (C, I). Data in (D), (E), (F), (H), and (J) are means ± SEM.p < 0.05. See also Figure S5

(G) Micrographs of ECs showing internalized phRodo-dextran (red) upon treatment with vehicle (ctrl), 3PO, or endocytosis blocker dynasore. Cells were visualized in blue by CFP.

(F) Quantification of transendothelial electrical resistance (TEER) of ECs upon treatment with vehicle (ctrl), 3PO (5 μM), VEGF (100 ng/mL), or VEGF plus 3PO. Data are presented as cumulative change over 4 hr in monolayer resistance normalized to TEER value of control (n = 4).

(A) Micrographs of thick sections of s.c. B16-F10 tumors from WT or Pfkfb3 +/ΔEC mice, double stained for VE-cadherin (VE-cadh) and CD31.

Comparable results were obtained when using LLC tumors in Pfkfb3mice and treating B16-F10 or Panc02 tumor-bearing mice with 3PO, although we adapted the vascular analysis according to how vessels formed. Indeed, in LLC tumors, we observed a contiguous network of tiny vessels, such that individual vessel profiles could not be detected, precluding us from counting vessel densities; we therefore quantified total CD31vessel area. In LLC and Panc02 tumors, the vessel lumen was too small to reliably quantify lumen size; hence, as an alternative of the perfusable area, we quantified the lectinvessel area. Regardless, qualitatively similar results of comparable vessel density, vascular enlargement, reduced EC proliferation matched by reduced EC death, fewer ECs per vessel length, increased perfusion and reduced hypoxia were obtained for LLC tumors in Pfkfb3mice ( Figures S4 A–S4F), and for B16-F10 or Panc02 tumors upon 3PO treatment ( Figures 5 A –5L and S4 G–S4N). Notably, 3PO treatment also induced TVN in metastatic liver tumors upon portal vein injection of B16-F10 cancer cells ( Figures S4 O–S4Q).

(K) Micrographs of PIMO staining (brown zones within dotted lines) of hypoxic zones in s.c. B16-F10 tumors from control and 3PO-treated mice.

(I) Micrographs of lectin-FITC-perfused and CD31-stained vessels in s.c. B16-F10 tumors from control and 3PO-treated mice.

(G) Micrographs of confocal images of CD31-stained sections of s.c. B16-F10 tumor from control and 3PO-treated mice. Nuclei are counterstained with DAPI.

(F) Quantification of % of apoptotic TUNEL + CD31 + ECs in s.c. B16-F10 tumors from control and 3PO-treated mice (n = 7–8).

(E) Quantification of % of proliferating PHH3 + CD31 + ECs in s.c. B16-F10 tumors from control and 3PO-treated mice (n = 5–7).

(D) Quantification of total perfusable area (sum of lumen area of all vessels, % of tumor area) in s.c. B16-F10 tumors from control and 3PO-treated mice (n = 5).

(C) Quantification of vessel lumen size in s.c. B16-F10 tumors from control and 3PO-treated mice (n = 5).

As hypoxia promotes cancer cell invasion and metastasis (), we investigated tumor perfusion and oxygenation. Injection of fluorescein isothiocyanate-conjugated lectin (labeling perfused vessels) and staining for CD31 to identify all tumor vessels showed that the perfused tumor vessel area was increased in Pfkfb3mice ( Figures 4 K and 4L). Staining for the hypoxia marker pimonidazole revealed reduced hypoxic B16-F10 tumor area in Pfkfb3mice ( Figures 4 M and 4N).

We explored if PFKFB3 haplodeficiency altered structural and functional properties of tumor vessels by staining for CD31. Compared with WT mice, B16-F10 tumors in Pfkfb3mice had a comparable vessel density ( Figures 4 A and 4B ); however, the vessel lumen size and total perfusable area (sum of lumen area of all vessels) were increased ( Figures 4 C and 4D). This was surprising given that EC proliferation was reduced in tumor vessels of Pfkfb3mice ( Figure 4 E). However, EC apoptosis was also decreased ( Figure 4 F), thus balancing off impaired EC growth. Moreover, ECs were arranged/positioned differently in Pfkfb3tumor vessels, which could further explain the vessel enlargement. Indeed, CD31 staining of thick tumor sections revealed that the vascular architecture in tumors of Pfkfb3mice was less irregular, tortuous, and disorganized ( Figures 4 G and 4H). When analyzing cross-sectional vessel profiles, the EC layer in WT mice appeared thick, irregular, packed, and protruded extensions into the lumen, while the EC layer in Pfkfb3mice was thinner, more elongated, and stretched, appearing more regular and flattened with a smoother surface ( Figure 4 I). As a result of this morphogenic rearrangement, tumor vessels in Pfkfb3mice contained fewer EC nuclei per micrometer of cross-sectional vessel length ( Figure 4 J).

(M) Micrographs of H&E and pimonidazole (PIMO) staining (brown zones within dotted lines) of hypoxic zones in s.c. B16-F10 tumors from WT and Pfkfb3 +/ΔEC mice.

(K) Micrographs of lectin-fluorescein isothiocyanate (FITC)-perfused and CD31-stained vessels in s.c. B16-F10 tumors from WT and Pfkfb3 +/ΔEC mice.

(I) Micrographs of confocal images of CD31 + s.c. B16-F10 tumor sections from WT and Pfkfb3 +/ΔEC mice. Nuclei are counterstained with DAPI.

(G) Micrographs of confocal images of CD31-stained thick sections of s.c. B16-F10 tumors from WT and Pfkfb3 +/ΔEC mice.

(F) Quantification of the % of apoptotic TUNEL + CD31 + ECs in s.c. B16-F10 tumors from WT and Pfkfb3 +/ΔEC mice (n = 11).

(E) Quantification of the % of proliferating PHH3 + CD31 + ECs in s.c. B16-F10 tumors from WT and Pfkfb3 +/ΔEC mice (n = 5).

(C) Quantification of vessel lumen size in s.c. B16-F10 tumors from WT and Pfkfb3 +/ΔEC mice (n = 6).

To mimic PFKFB3 haplodeficiency, we used a low dose of 3PO (25 mg/kg; three times per week), which reduced glycolysis in vivo ( Figure 3 A ). This dose did not affect cancer cells, as 3PO treatment did not alter tumor growth and cancer cell proliferation ( Figures 3 B and 3C). In line, cultured NECs and eTECs were more sensitive to PFKFB3 blockade than cancer cells ( Figures 3 D–3G and S3 A–S3D). 3PO treatment phenocopied the genetic effects in both B16-F10 and Panc02 tumor models ( Figures 3 H–3N and S3 E–S3J). 3PO treatment, initiated 3 days prior to cancer cell injection and continued thereafter, also reduced pulmonary metastases upon intravenous (i.v.) injection of B16-F10 cancer cells ( Figure 3 O).

Scale bars, 100 μm (C), 50 μm (J, K). All data are means ± SEM.p < 0.05. See also Figure S3

(O) Quantification of metastatic area in lungs (area of metastatic lesions, % of total lung area) upon tail vein injection of B16-F10 cancer cells in mice pretreated 3 days before cancer cell injection with vehicle (ctrl) or 3PO (n = 5).

(N) Quantification of B16-F10 cancer cell colonies, obtained upon isolation and culturing of circulating B16-F10 cancer cells from s.c. tumor-bearing mice treated with vehicle (ctrl) or 3PO (n = 3 biological repeats of 3–9 pooled individual animals each).

(K) Micrographs of s.c. B16-F10 tumor sections from control and 3PO-treated mice, stained for CD31 and endoglin to assess intraluminal CD31 – endoglin + cancer cells (arrows).

(J) Micrographs of H&E-stained B16-F10 tumor sections in control and 3PO-treated mice. Dotted line, border between tumor and surrounding muscle; arrows, residual muscle tissue.

(G) Dose-response analysis of the effect of 3PO on proliferation of Panc02 cells (n = 3).

(F) Dose-response analysis of the effect of 3PO on proliferation of B16-F10 (n = 3).

(E) Dose-response analysis of the effect of 3PO on proliferation of eTECs (n = 3 biological repeats of pooled ECs isolated from 10 to 15 individual animals each).

(D) Dose-response analysis of the effect of 3PO on proliferation of NECs (n = 3 biological repeats of pooled ECs isolated from 10 to 15 individual animals each).

(C) Micrographs of sections of B16-F10 tumors from control or 3PO-treated mice, stained for proliferation marker Ki67. Nuclei are counterstained with DAPI. Quantification of Ki67 + cells (Ki67 + nuclei, % of total) is indicated (n = 3).

(A) Gas chromatography-mass spectrometry (GC-MS) analysis of blood [ 13 C]-lactate levels upon i.v. injection of [U- 13 C]-glucose in control (ctrl) and 3PO-treated mice (n = 7–8).

Double staining for CD31 and endoglin, labeling both ECs and B16-F10 cells, showed that the fraction of tumor vessels containing cancer cells in their lumen, and the number of cancer cells inside vessels were decreased in Pfkfb3mice, suggesting impaired cancer cell intravasation ( Figures 2 L–2N), as confirmed by colony formation of circulating cancer cells ( Figure 2 O). We also explored if PFKFB3 inhibition affected later stages of metastasis. Upon tail vein injection of B16-F10 cells, metastasis in the lungs was reduced in Pfkfb3mice ( Figure 2 P).

B16-F10 tumor volume, end-stage tumor weight, cancer cell proliferation, and apoptosis were comparable with WT and Pfkfb3mice ( Figures 2 G and 2H, and S2 D–S2F). In contrast, tumor necrosis, metastasis, and invasion were reduced, and tumors had more sharply demarcated borders in Pfkfb3mice ( Figures 2 I–2K, S2 G, and S2H). Similar results were obtained in the LLC tumor model ( Figures S2 I–S2M).

To elucidate the role of PFKFB3 in tumor angiogenesis, we used a genetic and pharmacological approach to inhibit PFKFB3. For the genetic approach, we crossed vascular endothelial (VE)-cadherin (PAC)-Cremice () with Pfkfb3mice (), and treated 8-week-old progenies with tamoxifen to obtain Pfkfb3mice, haplodeficient for PFKFB3 in ECs. We then subcutaneously (s.c.) implanted syngeneic B16-F10 melanoma (“s.c. B16” model) or Lewis lung carcinoma (LLC) cells (“s.c. LLC” model) in Pfkfb3mice. PCR analysis showed that the Pfkfb3 allele was correctly recombined ( Figure S2 A), while RT-PCR confirmed that Pfkfb3 mRNA levels were reduced in NECs from PFKFB3mice ( Figure S2 B). Pfkfb3mice were healthy, fertile, gained normal body weight, and had normal organ vessel densities ( Figure S2 C). Glycolysis was lower in NECs and eTECs from Pfkfb3mice, but hypoxia increased glycolysis in both genotypes ( Figures 2 E and 2F; blue bars). To assess the effects of pharmacological PFKFB3-blockade by 3PO treatment, we used two tumor models, i.e., orthotopic implantation of pancreatic Panc02 and the s.c. B16 model ().

Metabolic pathway analysis further revealed that glycolytic flux was nearly 3-fold higher in eTECs ( Figure 2 A ), while glucose oxidation and oxygen consumption linked to ATP production were not altered ( Figures 2 B and 2C). Also, eTECs incorporated moreC label fromC-glucose into RNA and DNA ( Figure 2 D), implying that eTECs utilized more glucose carbons for biomass production. Hypoxia (omnipresent in tumors []) increased glycolysis in NECs and eTECs ( Figures 2 E and 2F; gray bars). These data render glycolysis, and the glycolytic activator PFKFB3, in eTECs an attractive target.

(P) Quantification of metastatic area in lungs (area of metastatic lesions, % of total lung area) upon tail vein injection of B16-F10 cancer cells in WT and Pfkfb3 +/ΔEC mice (n = 6–9).

(O) Quantification of B16-F10 cancer cell colonies upon isolation and culturing of circulating B16-F10 cancer cells from s.c. B16-F10 tumor-bearing WT and Pfkfb3 +/ΔEC mice (n = 3 biological repeats of 4–6 pooled individual animals each).

(L) Micrographs of s.c. B16-F10 tumors from WT and Pfkfb3 +/ΔEC mice, stained for CD31 and endoglin to assess intraluminal CD31 – endoglin + cancer cells (arrows).

(K) Micrographs of H&E-stained B16-F10 tumor sections of cancer cell invasion in WT and Pfkfb3 +/ΔEC mice. Dotted line, border between tumor and surrounding muscle; arrows, residual muscle tissue.

(I) Micrographs of H&E staining of necrotic areas (asterisks within dotted lines) in B16-F10 tumors in WT and Pfkfb3 +/ΔEC mice; quantification of necrotic area is indicated (% of total tumor area; n = 7–11).

(F) Glycolytic flux in eTECs from WT and Pfkfb3 +/ΔEC mice exposed to normoxia (21% oxygen) or hypoxia (0.5% oxygen) (n = 5–10); values normalized to flux in normoxic WT cells.

(E) Glycolytic flux in NECs from WT and Pfkfb3 +/ΔEC mice, exposed to normoxia (21% oxygen) or hypoxia (0.5% oxygen) (n = 5); values normalized to flux in normoxic WT cells.

(D) Incorporation of 14 C-glucose label in DNA and RNA in NECs and eTECs (n = 4–5); dpm, disintegrations per minute.

Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases.

Using liquid chromatography-mass spectrometry (LC-MS), we measured steady-state levels of a defined set of metabolites of glycolysis, PPP, and nucleotide synthesis in eTECs to further establish the importance of these pathways. Correlation heatmap analysis, hierarchical clustering, and principal component analysis showed that NECs and eTECs cluster in two separate groups (p < 0.05), suggestive of a distinct metabolic profile. Comparing levels of measured metabolites by metabolite set analysis using ROAST confirmed that eTECs differed from NECs (FDR-adjusted p value of 0.0104) ( Figures 1 G and S1 B). Levels of some metabolites of these pathways tended to be higher or were elevated in eTECs ( Figure 1 H; Table S4 ). Further characterization showed that glucose consumption and lactate excretion in the medium were increased ( Figures 1 I and 1J).

Subsequent pathway mapping and heatmap analysis revealed that transcripts of most glycolytic genes were upregulated in eTECs ( Figures 1 D and 1E; Table S2 ), including PFKFB3, the glucose transporter GLUT1 (Slc2a1), and rate-limiting enzymes of glycolytic side pathways, such as the pentose phosphate pathway (PPP) (glucose-6-phosphate dehydrogenase [G6pdx]; hexose-6-phosphate dehydrogenase [H6pd]) and the serine biosynthesis pathway (SBP) (phosphoglycerate dehydrogenase [Phgdh]), involved in biomass (nucleotide) synthesis ( Figures 1 D and 1E). A similar analysis revealed upregulation in eTECs of genes involved in nucleotide synthesis ( Figure 1 F; Table S3 ). RT-PCR confirmed mRNA upregulation in eTECs of enzymes involved in glycolysis (including PFKFB3) and nucleotide synthesis, as well as in cell proliferation ( Figure S1 A). As the data suggest that eTECs increase glycolysis to support proliferation and biomass production, we focused on glucose metabolism.

When performing RNA sequencing of eTECs and NECs, we focused on the 1,255 metabolic genes detectable in ECs. Correlation heatmap analysis and hierarchical clustering revealed that NECs and eTECs group into distinct metabolic clusters, indicating that their metabolic gene signatures differed ( Figure 1 C). Testing ten focused self-contained gene sets in central carbon metabolism using the rotation gene set testing (ROAST) tool to assess the significance of changes in metabolic pathways as a unit () showed that glycolysis was upregulated and had the highest fraction of upregulated genes of all pathways in central metabolism (false discovery rate [FDR]-adjusted p value of 0.023) ( Table S1 ).

We isolated TECs and characterized their metabolic profile. We injected B16-F10 melanoma cells in the portal vein (“p.v. B16 [liver]” model) of wild-type (WT) mice to induce tumor growth in the liver. After 14 days, we isolated ECs from the tumor-infested livers and, as controls, normal endothelial cells (NECs) from livers of healthy mice. As tumors represented 70%–80% of the tissue volume in tumor-infested livers, the isolated EC population was highly enriched in TECs (eTECs), but still contained a minor fraction of NECs. Compared with NECs, eTECs proliferated and migrated more actively ( Figures 1 A and 1B ). To assess which metabolic pathway was more active in eTECs versus NECs, we performed exploratory RNA sequencing, which suggested that eTECs were hyperglycolytic, and then confirmed these findings with targeted metabolomics analysis.

All data are means ± SEM.p < 0.05. For (H), p values were calculated by one sample t test. See also Figure S1

(H) Steady-state metabolite levels of metabolites of glycolysis, PPP, and nucleotide synthesis in eTECs, relative to NECs (n = 10). Dotted line, expression level in NECs.

(G) Correlation heatmap and cluster analysis of metabolites (shown in [H]) of glycolysis, PPP, and nucleotide synthesis in eTECs versus NECs (numbers refer to individual samples, n = 5). For color scale and clustering, see (C).

(F) Pathway map showing changes in transcript levels in eTECs (relative to NECs) of genes of nucleotide synthesis (n = 4; green: upregulated by at least 15%; gray: unchanged, fold change <15%). Ribonucleotides (red) and deoxyribonucleotides (blue).

(E) Heatmap and cluster analysis of transcript levels of genes in glycolysis and side pathways in eTECs versus NECs (numbers in panel refer to individual samples, n = 4). For color scale and clustering, see (C).

(D) Pathway map showing changes in transcript levels in eTECs (relative to NECs) of genes involved in glycolysis and side pathways (n = 4; green: upregulated by at least 15%; gray: unchanged, fold change <15%).

(C) Correlation heatmap and hierarchical cluster analysis of transcript levels of 1,255 metabolic genes in NECs and eTECs (numbers in panel refer to individual samples, n = 4). Color scale: red, high correlation; blue, low correlation. Hierarchical clustering: color differences in dendrogram indicate significant clustering (p < 0.05; multiscale bootstrap analysis).

(A) Proliferation of eTECs, expressed relative to NECs (n = 3 biological repeats of pooled ECs isolated from 10 to 15 mice).

Discussion

The key findings of our study are: (1) TECs are hyperglycolytic and targeting TEC glycolysis induces TVN, thereby reducing cancer cell invasion, intravasation, and dissemination; (2) pharmacological blockade of PFKFB3 at a dose that does not affect cancer cell proliferation, promotes TVN and improves delivery of and response to a standard dose of chemotherapy; and (3) mechanistically, PFKFB3 inhibition induces these effects partly by impairing VE-cadherin endocytosis and inflammation in ECs, and by rendering pericytes more quiescent and adhesive via glycolysis reduction.

De Bock et al., 2013a De Bock K.

Georgiadou M.

Carmeliet P. Role of endothelial cell metabolism in vessel sprouting. Fan et al., 2012 Fan T.W.

Tan J.

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Lane A.N. Stable isotope resolved metabolomics analysis of ribonucleotide and RNA metabolism in human lung cancer cells. 14C-glucose incorporation into RNA and DNA. Hence, eTECs divert more glucose carbons into these pathways for enhanced nucleotide synthesis to sustain rapid proliferation, although glucose carbons can also contribute to nucleotide synthesis via other pathways. In agreement, eTECs expressed higher mRNA levels of enzymes involved in nucleotide synthesis. Hypoxia, angiogenic factors such as VEGF, basic fibroblast growth factor, known to upregulate PFKFB3 levels ( De Bock et al., 2013a De Bock K.

Georgiadou M.

Carmeliet P. Role of endothelial cell metabolism in vessel sprouting. De Bock et al., 2013b De Bock K.

Georgiadou M.

Schoors S.

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et al. Role of PFKFB3-driven glycolysis in vessel sprouting. Transcriptomic and metabolic analysis showed that eTECs have a hyperglycolytic metabolism, which they need in part for high ATP-demanding processes (increased motility, active proliferation) (). Proliferating cells use glucose carbons, partly via the PPP and SBP, for nucleotide synthesis (). In agreement, eTECs, which proliferated more actively, expressed higher transcript levels of enzymes involved in glycolysis, PPP and SBP, and increasedC-glucose incorporation into RNA and DNA. Hence, eTECs divert more glucose carbons into these pathways for enhanced nucleotide synthesis to sustain rapid proliferation, although glucose carbons can also contribute to nucleotide synthesis via other pathways. In agreement, eTECs expressed higher mRNA levels of enzymes involved in nucleotide synthesis. Hypoxia, angiogenic factors such as VEGF, basic fibroblast growth factor, known to upregulate PFKFB3 levels (), and cytokines contribute to hyperglycolysis of eTECs. Since ECs heavily rely on glycolysis to meet bioenergetics needs, even more so than certain cancer cells (), they are very sensitive to changes in PFKFB3 gene dosage or activity.

Jain, 2014 Jain R.K. Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia. Mazzone et al., 2009 Mazzone M.

Dettori D.

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et al. Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization. De Bock et al., 2013b De Bock K.

Georgiadou M.

Schoors S.

Kuchnio A.

Wong B.W.

Cantelmo A.R.

Quaegebeur A.

Ghesquiere B.

Cauwenberghs S.

Eelen G.

et al. Role of PFKFB3-driven glycolysis in vessel sprouting. Carmeliet and Jain, 2011 Carmeliet P.

Jain R.K. Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases. Jain, 2014 Jain R.K. Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia. Mazzone et al., 2009 Mazzone M.

Dettori D.

Leite de Oliveira R.

Loges S.

Schmidt T.

Jonckx B.

Tian Y.M.

Lanahan A.A.

Pollard P.

Ruiz de Almodovar C.

et al. Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization. All previous TVN strategies focused on targeting angiogenic signals, but targeting EC metabolism as an approach for promoting TVN remained uncharted territory. Our data show that lowering of TEC glycolysis can induce TVN. As TECs are hyperactivated (), rendering them more quiescent by lowering glycolysis counteracts their hyper-proliferative and -migratory behavior () and contributes to TVN. PFKFB3 inhibition did not affect tumor vessel density, but enlarged tumor vessel lumen, smoothened EC surfaces, and stabilized tumor vessels by depositing a more prominent basement membrane and increasing coverage with pericytes. While TVN can be accompanied by pruning of immature vessels (for instance, when using anti-VEGF agents), other studies report that tumor vessel densities remain unchanged during TVN ().

Notably, even though PFKFB3 inhibition reduced eTEC proliferation, tumor vessels were enlarged. This is likely due to several reasons: (1) the reduced TEC proliferation was balanced off by reduced TEC death; and (2) TEC alignment and arrangement were different, i.e., TECs appeared more stretched and aligned more uniformly, forming a thinner EC layer with fewer cells per vessel length. It is tempting to speculate that the latter phenomenon relates to the higher expression of VE-cadherin in TECs. Indeed, according to Laplace's law, vessel wall tension is proportional to the radius of the vessel. Hence, stronger expression of VE-cadherin, functionally reflected by increased electrical resistance, in PFKFB3-inhibited ECs may allow these cells to cope with a greater wall tension, thus allowing them to enlarge their lumen, a hypothesis requiring confirmation.

+ adherens junctions and elevated VE-cadherin protein levels at the plasma membrane, and consistent herewith, reduced the number of intercellular gaps, while increasing the trans-EC electrical resistance, all signs of barrier tightening and monolayer integrity. Tightening of EC junctions contributes to vessel integrity, vascular barrier tightness, induction of an EC “phalanx” phenotype (smooth, streamlined cobblestone monolayer of interconnected adherent ECs aligned in the direction of flow), possibly also vessel enlargement, and reduced vessel tortuosity—all processes that improve tumor vessel perfusion and oxygenation. Mechanistically, PFKFB3 inhibition impaired VE-cadherin endocytosis induced by VEGF. Given that VE-cadherin is internalized via clathrin-mediated endocytosis ( Xiao et al., 2005 Xiao K.

Garner J.

Buckley K.M.

Vincent P.A.

Chiasson C.M.

Dejana E.

Faundez V.

Kowalczyk A.P. p120-Catenin regulates clathrin-dependent endocytosis of VE-cadherin. Boulant et al., 2011 Boulant S.

Kural C.

Zeeh J.C.

Ubelmann F.

Kirchhausen T. Actin dynamics counteract membrane tension during clathrin-mediated endocytosis. PFKFB3 inhibition in ECs promoted the formation of stable, continuous VE-cadherinadherens junctions and elevated VE-cadherin protein levels at the plasma membrane, and consistent herewith, reduced the number of intercellular gaps, while increasing the trans-EC electrical resistance, all signs of barrier tightening and monolayer integrity. Tightening of EC junctions contributes to vessel integrity, vascular barrier tightness, induction of an EC “phalanx” phenotype (smooth, streamlined cobblestone monolayer of interconnected adherent ECs aligned in the direction of flow), possibly also vessel enlargement, and reduced vessel tortuosity—all processes that improve tumor vessel perfusion and oxygenation. Mechanistically, PFKFB3 inhibition impaired VE-cadherin endocytosis induced by VEGF. Given that VE-cadherin is internalized via clathrin-mediated endocytosis (), and this actin-myosin contraction-dependent process requires ATP (), our findings suggest that ECs use glycolytic ATP for VE-cadherin endocytosis. This could explain why a reduction of glycolysis elevated VE-cadherin levels at the cell surface, and hence tightened EC junctions.

Armulik et al., 2011 Armulik A.

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Betsholtz C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Carmeliet and Jain, 2011 Carmeliet P.

Jain R.K. Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases. Jain, 2014 Jain R.K. Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia. Mazzone et al., 2009 Mazzone M.

Dettori D.

Leite de Oliveira R.

Loges S.

Schmidt T.

Jonckx B.

Tian Y.M.

Lanahan A.A.

Pollard P.

Ruiz de Almodovar C.

et al. Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization. Francescone et al., 2014 Francescone R.

Ngernyuang N.

Yan W.

Bentley B.

Shao R. Tumor-derived mural-like cells coordinate with endothelial cells: role of YKL-40 in mural cell-mediated angiogenesis. Compared with healthy vessels, tumor vessels are more devoid of pericytes, which makes them more leaky, and facilitates cancer cell intravasation and dissemination (). Immature tumor vessels without pericytes are also poorly perfused (). Hence, promoting pericyte coverage to stabilize tumor vessels not only improves perfusion, but also increases barrier tightness, which impairs cancer cell intravasation and metastasis (). Contrary to quiescent pericytes in healthy vessels, pericytes in tumors are activated and proliferate more actively, yet are detached from tumor vessels, partly because of reduced N-cadherin-dependent adhesive properties and increased motility (). Thus, although pericyte proliferation is increased, pericyte numbers around tumor vessels are reduced, indicating that their reduced adhesiveness and increased motility rather than their proliferation rate determine pericyte coverage.

Tillet et al., 2005 Tillet E.

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Huber P. N-cadherin deficiency impairs pericyte recruitment, and not endothelial differentiation or sprouting, in embryonic stem cell-derived angiogenesis. Blindt et al., 2004 Blindt R.

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Vogt F. Downregulation of N-cadherin in the neointima stimulates migration of smooth muscle cells by RhoA deactivation. Our data indicate that pericytes are highly glycolytic. Hence, inhibition of PFKFB3 in pericytes (as occurs during 3PO treatment) rendered these cells less motile and more quiescent and sedentary, and increased their adhesion to ECs, all changes that improve pericyte coverage of ECs. The elevated expression of N-cadherin, mediating adhesion between ECs and pericytes (), might be due to the fact that pericytes proliferate less and are more quiescent, conditions known to elevate N-cadherin expression (). Hence, by rendering pericytes more quiescent and adhesive, glycolysis reduction in these cells (upon 3PO treatment) improves pericyte coverage, and hence TVN.

Schoors et al., 2014 Schoors S.

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et al. Partial and transient reduction of glycolysis by PFKFB3 blockade reduces pathological angiogenesis. Marx et al., 1994 Marx M.

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et al. p75(NTR)-dependent activation of NF-kappaB regulates microRNA-503 transcription and pericyte-endothelial crosstalk in diabetes after limb ischaemia. Hutter-Schmid and Humpel, 2016 Hutter-Schmid B.

Humpel C. Platelet-derived growth factor receptor-beta is differentially regulated in primary mouse pericytes and brain slices. Cell-autonomous endothelial deletion of PFKFB3 also promoted pericyte coverage. This is likely the result of a crosstalk from the ECs (normalized by PFKFB3 inhibition) toward the pericytes. Indeed, PFKFB3 inactivation in ECs promotes EC quiescence (), which is known to favor pericyte recruitment via enhanced expression of N-cadherin (as observed in this study) (). Further, PFKFB3 inhibition reduces NF-κB signaling, a pathway known to inhibit pericyte coverage (). Also, the improved oxygenation and enhanced blood flow, resulting from EC layer normalization, can stimulate pericyte coverage (). The improved pericyte coverage then further stabilizes the normalized EC layer to promote normalization of the whole tumor vessel wall. Overall, while PFKFB3 inhibition in pericytes directly promotes TVN, cell-autonomous endothelial inhibition of PFKFB3 can also indirectly promote TVN through an EC → pericyte crosstalk.