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Genetic Deletion of Vascular Endothelial Growth Factor Receptor 2 in Endothelial Cells Leads to Immediate Disruption of Tumor Vessels and Aggravation of Hypoxia
Department of Anatomy, Keio University School of Medicine, Shinjuku-ku, Tokyo, JapanDepartment of Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan
Department of Anatomy, Keio University School of Medicine, Shinjuku-ku, Tokyo, JapanDepartment of Orthopedic Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan
Address correspondence to Yoshiaki Kubota, M.D., Ph.D., or Ikue Tai-Nagara, Ph.D., Department of Anatomy, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Address correspondence to Yoshiaki Kubota, M.D., Ph.D., or Ikue Tai-Nagara, Ph.D., Department of Anatomy, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Vascular endothelial growth factor (VEGF) blockers are used widely in clinics to target various types of human cancer. Although VEGF blockers exert marked tumor suppressive effects, the therapeutic effects can be limited. Moreover, accumulating evidence shows that VEGF acts not just on endothelial cells but also on various nonendothelial cells, including tumor and immune cells, suggesting a need to revisit the bona fide action of VEGF on endothelial cells using specific genetic mouse models. Herein, tamoxifen-inducible endothelial-specific knockout mice lacking VEGF receptor 2 (Vegfr2), the major signal transducer for VEGF, were used. The initial event resulting from cessation of endothelial Vegfr2 signaling was vascular truncation and fragmentation, rather than maturation of abnormalized vessels. Although deletion of endothelial Vegfr2 suppressed intratumor hemorrhage, it enhanced hypoxia in tumor cells and reduced the number of infiltrating cytotoxic T cells, suggesting a profound reduction in intratumor blood flow. In various tissues, deletion of endothelial Vegfr2 induced regression of healthy capillaries in intestinal villi, substantiating intestinal perforation, which is one of the most common adverse effects of VEGF blockade in humans. Overall, the data suggest that some of the known effects of VEGF blockers on tumor vessels are caused by partial cessation of VEGF signaling, or by actions on nonendothelial cells. The results increase the understanding of the mechanisms underlying anti-angiogenic therapy.
Vascular endothelial growth factor (VEGF) is the strongest mitogen that stimulates growth of blood vessels; thus, it is crucial for physiological and pathologic angiogenesis.
Since the US Food and Drug Administration approved VEGF neutralizing antibodies as anti-angiogenic drugs for cancer therapy in 2004, significant tumor-suppressive effects against various types of human cancers (when combined with other anticancer drugs) have been documented. However, in many cases, the effects of VEGF blockers in humans are modest when compared with those observed in experimental animal models.
Moreover, indiscriminate VEGF blockade damages healthy vessels outside tumors, causing severe adverse effects, such as cerebral hemorrhage and intestinal perforation.
An important aspect of VEGF blockers is that they transiently normalize the abnormal structure and function of tumor vessels to improve delivery of oxygen and drugs.
This is why VEGF blockers are more effective when administered in combination with conventional cytotoxic agents than when administered as single agents.
Initially, pharmacologic blockade of VEGF receptor 2 (VEGFR2), the main receptor for VEGF, ameliorates the structural and functional abnormality of the tumor vessels before destroying them in the long-term.
Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors.
The short period during which VEGFR2 blockade normalizes rather than damages tumor vessels is called the normalization window, and is known to last for up to 6 days after administration in animal models.
Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, angiopoietin-1, and matrix metalloproteinases.
Classically, it is proposed that VEGF acts primarily on vascular endothelial cells (ECs) through VEGFR2, as expression of this receptor is largely restricted to ECs. In some tumors, including glioblastoma multiforme, colorectal cancer, and squamous skin tumor, tumor cells also express VEGF receptors, and their growth and stemness are supported by VEGF signaling.
However, VEGFR2 expressed by CD8+ T cells increases expression of inhibitory immune checkpoints, such as programmed death receptor-1, resulting in suppression of tumor immunity.
On the other hand, VEGFR2 decreases recruitment of immunosuppressive monocytes to tumors, suggesting a mechanism underlying resistance to anti-VEGF therapies.
Thus, there is a need to examine the precise action of VEGF on tumor ECs using specific genetic mouse models. For this purpose, mice with tamoxifen-inducible endothelial-specific knockout of Vegfr2 and syngeneic mouse tumor models were used. Histologic examination during and after the normalization window indicated that the initial event resulting from cessation of endothelial Vegfr2 signaling affected vascular truncation and fragmentation, leading to perivascular hypoxia. Moreover, examination of various tissues outside tumors showed that deletion of endothelial Vegfr2 elicited immediate regression of healthy capillaries in intestinal villi, a finding in agreement with the intestinal perforation observed in patients treated with VEGF blockers. Overall, the current data clarify the direct consequences resulting from genetic deletion of Vegfr2 in ECs, thereby increasing the understanding of anti-angiogenic therapy.
Materials and Methods
Mice
Animal care was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Keio University, and experiments were performed in accordance with the Guidelines of Keio University for Animal and Recombinant DNA experiments. The CAG-LSL-GFP,
Cdh5-BAC-CreERT2Vegfr2flox/+ mice were used as littermate controls. The mutant mice were crossed >10 times with C57BL/6J mice. For adult mouse experiments involving tamoxifen-inducible expression of Cre, 1 mg of tamoxifen (Sigma-Aldrich, St. Louis, MO; H5648) was injected subcutaneously at the indicated time points.
Tumor Models
B16 mouse melanoma cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Next, 2 × 106 cells were implanted subcutaneously into the backs of 6-week–old male mice. Tumors were measured daily using a caliper, and volume was calculated using the following formula: volume = tumor length × width × height/2.
Preparation of Tissue Sections and Whole-Mount Samples
Surgically dissected tissues were fixed overnight in 4% paraformaldehyde in phosphate-buffered saline (PBS). Frozen sections cut from those samples were used for immunohistochemical analysis. All tumor samples were sectioned with a 14-μm thickness at the plane of the maximum cut surface. To prepare whole-mount samples, tissues were dissected and fixed overnight in 4% paraformaldehyde in PBS. All section and whole-mount tissues were stained as described below.
Immunostaining
Immunohistochemical analyses of whole-mount samples or tissue sections were performed as described previously.
The primary monoclonal antibodies used were as follows: anti-CD31 (Chemicon, Temecula, CA; MAB1398Z; 1:1000), anti-CD4 (BD Pharmingen, Franklin Lakes, NJ; 550280; 1:50), anti-CD8a (BD Pharmingen; 1:50), α-smooth muscle actin–Cy3 conjugated (Sigma-Aldrich; C6198; 1:500), anti-Ter119 (R&D Systems, Minneapolis, MN; MAB1125; 1:500), and F4/80 (Serotec, Oxford, UK; MCA497R; 1:500). The primary polyclonal antibody used was anti–green fluorescent protein (GFP)–Alexa Fluor 488 conjugated (Molecular Probes, Eugene, OR; A21311; 1:500). Secondary antibodies were Alexa Fluor 488–conjugated IgGs (Molecular Probes; A11034, A11006, and A11055; 1:500) and Cy3/Cy5 DyLight549/DyeLight649-conjugated IgGs (Jackson ImmunoResearch, West Grove, PA; 711-165-152, 112-165-167, 127-165-160, 711-605-152, 112-605-167, and 127-605-160; 1:500). The Hypoxyprobe-1 Plus Kit (Chemicon; HP2-100) was used to detect hypoxic cells. In brief, 60 mg kg−1 of pimonidazole was injected intraperitoneally into mice 30 minutes before euthanasia. Then, tumors were harvested, sectioned, and stained with Hypoxyprobe Mab1–fluorescein isothiocyanate. An 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay using a Click-iT EdU Imaging Kit (Invitrogen, Carlsbad, CA) was used to analyze cell proliferation in vivo. Briefly, 50 μL of EdU dissolved in dimethyl sulfoxide/PBS (final concentration, 0.5 mg/mL) was injected intraperitoneally into mice 2 hours before sacrifice. For nuclear staining, specimens were treated with DAPI (Molecular Probes; D-1306).
Confocal Microscopy
Fluorescent images were visualized under a confocal laser-scanning microscope (FV1000; Olympus, Tokyo, Japan). Quantification of cells or parameters of interest was conducted using three images, each with a 1270 × 1270 μm field of view, per sample (to count EdU+ cells, CD4+ cells, and CD8+ cells), or entire sections of tumor (to count tumor vessels, and to measure hemorrhagic, hypoxic, and vascularized areas). ImageJ software version 1.52a (NIH, Bethesda, MD; https://imagej.nih.gov/ij, last accessed October 12, 2019) was used for quantification of the indicated areas.
X-Gal Staining of Tissue Sections
Dissected tumors were fixed for 10 minutes in 1% glutaraldehyde in PBS, and hemispheres were cut. After post-fixation overnight, samples were snap frozen in OCT compound (Sakura Finetechnical Co Ltd, Tokyo, Japan), sectioned (14 μm thick) at the plane of the maximum cut surface of the tumor, and incubated for 1 hour at 37°C with 1 mg/mL of X-Gal in a standard X-Gal reaction buffer (35 mmol/L potassium ferrocyanide, 35 mmol/L potassium ferricyanide, 2 mmol/L MgCl2, 0.02% Nonidet P-40, and 0.01% sodium deoxycholate in PBS).
Quantitative RT-PCR Analysis
Total RNA was prepared from tumor tissues, and reverse transcription was performed using Superscript II (Invitrogen). Quantitative PCR assays were performed with an ABI 7500 Fast Real-Time PCR System using TaqMan Fast Universal PCR master mix (Applied Biosystems, Foster City, CA) and TaqMan Gene Expression assay mix of Tnf (Mm00443258_m1), Il6 (Mm00446190_m1), Pdcd1 (Mm01285676_m1), and Cd274 (Mm00452054_m1); A mouse β-actin (Mm00607939_s1) assay mix served as an endogenous control. Data were analyzed using 7500 Fast System SDS software version 1.3.1.
Statistical Analysis
Results are expressed as the means ± SD. Comparison of the averages from two groups was made using a two-tailed t-test. P < 0.05 was considered statistically significant.
Results
Tumor and Normal ECs Show Opposite Expression Patterns of Vegfr1 and Vegfr2
In general, VEGFR2 is expressed preferentially by vascular ECs of growing vessels; VEGFR1 expressed by the same or neighboring ECs sequesters VEGF to fine-tune signaling via VEGFR2.
To examine the expression patterns of VEGFR1 and VEGFR2 precisely during tumor progression, Vegfr1-BAC-DsRed+Vegfr2-BAC-GFP+ mice were implanted subcutaneously with B16 melanoma cells, followed by examination of the immunoreactivity of red fluorescent protein from Discosoma sp. and GFP in the resultant tumors 14 days after transplantation. GFP (Vegfr2) was expressed abundantly in ECs lining blood vessels growing into tumors, whereas expression of DsRed (Vegfr1) in these vessels was low (Figure 1, A–C and E ). This expression pattern was opposite to that in normal vessels in the surrounding skin; expression of DsRed was high, whereas that of GFP was low (Figure 1, A–D). Both GFP and DsRed were detected only in ECs, and expression was below detectable levels in non-ECs, such as hematopoietic cells (Figure 1, D and E). Higher expression of Vegfr2 in tumor vessels than in dermal vessels was confirmed by X-Gal staining of B16 tumors transplanted into Vegfr2+/lacZ knock-in mice (Figure 1, F–H). To delete genes from tumor ECs, Cdh5-BAC-CreERT2 mice
were to be used; therefore, the efficiency of these mice was tested by crossing with an indicator line, CAG-LSL-GFP. Although a previous report indicates that another Cdh5-CreERT2 line recombines genes in hematopoietic cells, such as myeloid cells,
Cre expression was detected only in ECs of tumors grown in Cdh5-BAC-CreERT2 mice (Figure 1, I–N).
Figure 1Opposite Vegfr1 and Vegfr2 expression patterns in tumor and normal endothelial cells. A–E: Immunohistochemistry in B16 tumor sections. Dotted lines indicate the border between tumors and normal skin. D: Enlarged view of the skin, boxed (solid) area in C. E: Enlarged view of the tumor, boxed (dotted) area in C. Both green fluorescent protein (GFP) and DsRed are below detectable levels in non–endothelial cells (arrows). F–H: X-Gal staining in B16 tumor sections. G: Enlarged view of the skin, boxed (solid) area in F. H: Enlarged view of the tumor, boxed (dotted) area in F. I: Protocol for tamoxifen injection and tumor inoculation. J–N: Immunohistochemical analysis of B16 tumor sections. K–N: Enlarged view of the tumor, boxed (dotted) area in J. Scale bars: 200 μm (A–C, F, and J); 50 μm (D, E, G, H, and K–N).
Intermittent Endothelial Vegfr2 Deletion Destroys Tumor Vessels and Increases Hypoxia
Next, tamoxifen-inducible endothelial-specific Vegfr2 knockout mice (Cdh5-BAC-CreERT2Vegfr2flox/flox; hereafter referred to as Vegfr2iΔEC mice) were generated to examine the role of endothelial Vegfr2 in the B16 mouse melanoma model. Clinically, VEGF blockers are typically administered after tumors have grown to a detectable level; therefore, an intermittent deletion experiment was performed by delaying initiation of Cre induction by tamoxifen (Figure 2A). Five days after Cre induction (day 16), tumor growth in Vegfr2iΔEC mice began to be suppressed, followed by gradual shrinkage (Figure 2, B–E). On day 11 after tamoxifen induction in Vegfr2iΔEC mice, immunohistochemical analysis of tumor sections showed reduced vessel density and hemorrhage, whereas the total number of tumor vessels was not changed significantly (Figure 2, F–N). Considering intratumor variability with respect to the amount of blood vessels,
all vessels in the entire field in the plane of the maximum cut surface of the tumor were counted rather than vessels in high-magnification views only. Interestingly, vessel truncation and fragmentation were apparent in the tumors of Vegfr2iΔEC mice (Figure 2, H and K). In these truncated vessels in Vegfr2iΔEC mice, α-smooth muscle actin–positive pericytes were apparently detached as in tumor vessels of control mice, in contrast with healthy skin vessels tightly covered with pericytes (Figure 2, O–Q). Interestingly, some vessels were fully covered with, while others were devoid of, pericytes in Vegfr2iΔEC mice (Figure 2Q), suggesting that abnormalized vessels were truncated together with associated pericytes. These tumors showed increased hypoxia and reduced tumor proliferation, particularly in perivascular spaces (Figure 2, R–W). Taken together, the data suggest that deletion of endothelial Vegfr2, even if intermittent, destroys tumor vessels and enhances hypoxia.
Figure 2Intermittent deletion of endothelial Vegfr2 destroys tumor vessels and increases hypoxia. A: Protocol for tamoxifen injection and tumor inoculation. B–D: Mice at 22 days post-transplantation of B16 cells into the back skin (arrowheads) and resected tumors. E: Quantification of tumor diameter. F–N: Immunohistochemical analysis of tumor sections and quantification. Intratumoral hemorrhage (open arrowheads) in tumors from control (Cont) mice is greater than that in Vegfr2iΔEC mice. Vascular fragmentation (arrows) is apparent in tumors from Vegfr2iΔEC mice. G and J: Enlarged view of the tumor, boxed area in F and I. H and K: Enlarged view of the tumor, boxed area in G and J. O–Q: Immunohistochemical analysis of the tumor and skin. Some vessels (closed arrowhead) are fully covered with but others (open arrowheads) are devoid of pericytes in Vegfr2iΔEC mice. R–W: Immunohistochemical analysis of tumor sections and quantification. Tumors in Vegfr2iΔEC mice show enhanced hypoxia (arrowheads) and reduced proliferation (arrows). Data are expressed as the means ± SD (E, L–N, T, and W). n ≧ 13 (E); n ≧ 7 (F–N); n ≧ 4 (R–W). ∗P < 0.05, ∗∗P < 0.01. Scale bars: 1 cm (B–D); 1 mm (F and I); 200 μm (G, J, R, S, U, and V); 50 μm (H, K, and O–Q). ASMA, alpha-smooth muscle actin; BV, blood vessel; EdU, 5-ethynyl-2′-deoxyuridine.
vessel truncation and fragmentation in the tumors of Vegfr2iΔEC mice may cause different outcomes. Immunohistochemical analysis showed that both CD4+ and CD8+ T cells were more abundant in the margins of growing tumors than in the submargins (Figure 3, A–E, K, and L ). The difference of vessel density between the tumor core and periphery may reflect the number of T cells. Interestingly, the number of CD8+ T cells, but not that of CD4+ T cells, in Vegfr2iΔEC mice was reduced significantly in the marginal and submarginal areas (Figure 3, A–L), suggesting a differential response to acute vessel disruption. Overall, tumor growth in Vegfr2iΔEC mice was inhibited without an apparent increase in the number of infiltrating T cells.
Figure 3Deletion of endothelial Vegfr2 reduces infiltration of CD8+ T cells into tumors. A–L: Immunohistochemical analysis of tumor sections and quantification. Mice were treated with tamoxifen on days 11, 13, and 15, and sacrificed on day 22, after transplantation of B16 cells. The number of CD8+ T cells (closed arrowheads), but not CD4+ T cells (open arrowheads), reduced significantly in marginal and submarginal areas of Vegfr2iΔEC mice. B and G: Enlarged view of the marginal zone, boxed (solid) area in A and F. C and H: Enlarged view of the submarginal zone, boxed (dotted) area in A and F. Data are expressed as the means ± SD (K and L). n = 3 (A–L). ∗P < 0.05, ∗∗P < 0.01. Scale bars: 200 μm (A and F); 50 μm (B–E and G–J). Cont, control.
Therefore, the appearance of vascular structures in various tissues of Vegfr2iΔEC mice, including lung, heart, liver, kidney, intestine, retina, and tail skin, was examined (Figure 4). The results showed regression of capillaries in the intestinal villi of Vegfr2iΔEC mice (Figure 4, E and J). No apparent defects in vascular structures were observed in other tissues (Figure 4, A–D, F–I, and K–R).
Figure 4Deletion of endothelial Vegfr2 induced regression of normal capillaries in intestinal villi. Immunohistochemical analysis of sections (A–J) or whole-mount (K–R) samples from mice treated with tamoxifen on days 11, 13, and 15, and sacrificed on day 22, after transplantation of B16 cells. Vegfr2iΔEC mice show regression of capillaries running in the intestinal villi (arrowheads). Scale bar = 50 μm (A–R). ASMA, alpha-smooth muscle actin.
The Initial Event Resulting from Cessation of Endothelial Vegfr2 Signaling Is Vascular Truncation Rather than Maturation
Next, the immediate response of tumor vessels to endothelial Vegfr2 deletion during the so-called normalization window was examined. Considering the time lag between tamoxifen administration and reduction of Vegfr2 protein expression, the time of analysis was set at 4 days after tamoxifen injection (Figure 5A). At this time, tumor size was not significantly different between control and Vegfr2iΔEC mice (Figure 5B), suggesting that initial vascular changes could be examined before tumor growth was affected. Immunohistochemical analysis revealed apparent vascular truncation and fragmentation, rather than maturation, of abnormalized vessels in Vegfr2iΔEC mice (Figure 5, C–H). There were no significant changes in vessel density or number, but the hemorrhage area and tumor proliferation were reduced, and perivascular hypoxia was aggravated (Figure 5, I–M). Relative expression of Tnf, Il6, Pdcd1, and Cd274, evaluated by real-time quantitative PCR assay, showed no significant difference between control and Vegfr2iΔEC mice, suggesting vessel fragmentation was not caused by altered inflammatory conditions (Figure 5, N–Q). These data suggest that vascular truncation and fragmentation in Vegfr2iΔEC mice, even if it occurred only in a population of tumor vessels, disrupted the hierarchical system of the vascular networks, resulting in an immediate reduction in blood flow. In addition, such fragmentation and truncation were not accompanied by physical rupture of blood vessels because hemorrhage was somewhat suppressed. Next, mice were transplanted with Lewis lung carcinoma cells rather than B16 melanoma cells. Lewis lung carcinoma cells grew faster than B16 cells in both control and Vegfr2iΔEC mice (Figure 6, A and B ). However, at 4 days after tamoxifen induction (the time point of analysis), tumor size was not significantly different between control and Vegfr2iΔEC mice (Figure 6B). Immunohistochemical analysis of Lewis lung carcinoma tumors from Vegfr2iΔEC mice showed immediate vascular truncation and fragmentation, with no effect on vessel number or density (Figure 6, C–F, I, and J), as seen in the experiment with B16 cells. Both the area of hemorrhage and tumor proliferation in Lewis lung carcinoma tumors from Vegfr2iΔEC mice were reduced, and perivascular hypoxia was aggravated (Figure 6, G, H, and K–M), in accordance with the results in B16 cells.
Figure 5The initial event resulting from cessation of endothelial Vegfr2 signaling is vascular truncation rather than maturation. A: Protocol for tamoxifen injection and B16 tumor inoculation. B: Quantification of tumor diameter. C–M: Immunohistochemical analysis of tumor sections and quantification. Intratumoral hemorrhage (open arrowheads), which is abundant in tumors from control (Cont) mice, is reduced in tumors from Vegfr2iΔEC mice. Vascular fragmentation (arrows) is apparent in tumors from Vegfr2iΔEC mice. D and G: Enlarged view of the tumor, boxed area in C and F. E and H: Enlarged view of the tumor, boxed area in D and G. N–Q: Relative expression of Tnf, Il6, Pdcd1, and Cd274, quantified by real-time quantitative PCR analysis, on tumor samples at day 15 after transplantation. Data are presented as the means ± SD (B and I–Q). n ≧ 4 (B); n ≧ 3 (C–M). ∗P < 0.05. Scale bars: 1 mm (C and F); 200 μm (D and G); 50 μm (E and H). BV, blood vessel; EdU, 5-ethynyl-2′-deoxyuridine.
Figure 6The vascular response of Lewis lung carcinoma (LLC) tumors in response to deletion of endothelial Vegfr2 is similar to that of B16 tumors. A: Protocol for tamoxifen injection and LLC tumor inoculation. B: Quantification of tumor diameter. C–M: Immunohistochemical analysis of tumor sections and quantification. Intratumoral hemorrhage (open arrowheads), which is abundant in tumors from control (Cont) mice, is reduced in tumors from Vegfr2iΔEC mice. Vascular fragmentation (arrows) is apparent in tumors from Vegfr2iΔEC mice. Tumors from Vegfrr2iΔEC mice show increased hypoxia (closed arrowheads). D and F: Enlarged view of the tumor, boxed area in C and E. Data are presented as the means ± SD (B and I–M). n ≧ 7 (B); n ≧ 4 (C–M). ∗P < 0.05. Scale bars: 200 μm (C, E, G, and H); 50 μm (D and F). BV, blood vessel; EdU, 5-ethynyl-2′-deoxyuridine.
The data from two syngeneic mouse models presented herein show that the primary and immediate effect caused by cessation of endothelial Vegfr2 signaling in tumors is vascular truncation and fragmentation rather than vessel maturation. Accordingly, deletion of endothelial Vegfr2 reduced infiltration of cytotoxic T cells and increased hypoxia in tumor cells, suggesting a profound reduction in blood flow in tumors caused by disrupted blood vessel hierarchies.
Evidence from both clinical and preclinical settings supports the existence of vessel normalization during treatment with VEGF blockers.
Unexpectedly, apparent vessel maturation was not observed in this study, not even in the early phase after Vegfr2 deletion. The difference between previous studies and the current one might be ascribed to differences in pharmacologic or genetic targeting. From a pharmacologic perspective, the inhibitory effect could be partial but still transduce a weak VEGFR2 signal.
Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors.
Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, angiopoietin-1, and matrix metalloproteinases.
In contrast to full cessation after genetic deletion, such a partial reduction may lead to transient vessel maturation. In support of this assumption, in vivo imaging revealed that a high dose of the anti-human VEGFR2 antibody ramucirumab reduces tumor vessel numbers without inducing maturation.
Moreover, the earliest action of anti-VEGF drugs against vascular changes caused by adenovirus expressing VEGF-A is collapse of large vessels and formation of glomeruloid microvascular proliferations.
However, the time of analysis in this study is not relevant to this issue because there were no observed vascular changes, including vessel maturation and fragmentation (data not shown), before day 4 after tamoxifen administration, likely because Cre-mediated reduction of Vegfr2 did not occur.
Since the US Food and Drug Administration approved the VEGF-specific monoclonal antibody bevacizumab in 2004 as a first-line treatment for metastatic colorectal cancer, several receptor tyrosine kinase inhibitors (which target VEGFR2 non-specifically), such as sunitinib and sorafenib, have been approved for use against various cancers.
Indirect comparisons suggest that sunitinib is superior to bevacizumab plus interferon in terms of progression-free survival in patients with metastatic renal cell carcinoma.
Sunitinib and bevacizumab for first-line treatment of metastatic renal cell carcinoma: a systematic review and indirect comparison of clinical effectiveness.
Although direct comparison of the tumor-suppressing effects of these drugs in clinical settings is unrealistic, current preclinical data shown in this study could be a theoretical basis for selection of drugs that are most suitable in each case.
In conclusion, the present data on the role of endothelial Vegfr2 in mouse tumor models will help to distinguish the effects of VEGF blockers on ECs and non-ECs. Accumulating evidence shows that the consequences of VEGF inhibition are complicated, variable, and context dependent. Better understanding of the molecular and cellular mechanisms underlying VEGF/VEGF receptor–mediated tumor angiogenesis may improve the current limitations of anti-angiogenic cancer therapy.
Author Contributions
I.T.-N. and Y.Ku. designed the experiments; Y.Ki., T.A., T.I., and I.T.-N. performed experiments and analyzed the data; M.E. provided experimental materials; Y.Ki. edited the article; and I.T.-N. and Y.Ku. wrote the article.
Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors.
Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, angiopoietin-1, and matrix metalloproteinases.
Sunitinib and bevacizumab for first-line treatment of metastatic renal cell carcinoma: a systematic review and indirect comparison of clinical effectiveness.
Supported by Grants-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan 18H05042, 18K19553, and 19H03397 (Y.Ku.); by Japan Agency for Medical Research and Development-PRIME JP20gm6210017h0002 and 21gm6210017h0003 (Y.Ku.); by Japan Science and Technology Agency (MoonshotR&D) JPMJMS 2024 (Y.Ku.); and by research grants from Inamori Foundation, Kao Foundation for Arts and Culture, Takeda Science Foundation, Mochida Memorial Foundation, Mitsubishi Foundation, Cell Science Research Foundation, SENSHIN Medical Research Foundation, Sumitomo Foundation, Daiichi Sankyo Foundation of Life Science, Naito Foundation, Uehara Memorial Foundation, and Toray Science Foundation (Y.Ku.).
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