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Abstract

  1. Top of page
  2. Abstract
  3. Abnormal vascular structure in cancers
  4. Vascular regression and hypoxia may induce the invasive phenotype of cancer cells
  5. Persistent normalization of tumor vessels improves tumor microenvironment and inhibits metastasis
  6. Additional benefits of normalization of tumor vessels
  7. Concluding remarks
  8. Acknowledgments
  9. Disclosure statement
  10. References

Angiogenesis is recognized as one of the principal hallmarks of cancers. Cancers contain newly formed immature vessels devoid of firm coverage by pericytes. Several drugs targeting vascular endothelial growth factor signals are now in clinical use for anti-angiogenic cancer treatment. Those drugs transiently normalize tumor vessels and ultimately provoke vascular regression. This regression causes tumor hypoxia, which could trigger certain cancer cells to become more invasive and metastatic. Normalized vessels do not induce tumor hypoxia, and may protect from cancer cell intravasation and enhance anticancer treatment with chemotherapeutic agents, radiation, or immune therapy. Thus, persistent vascular normalization could be an alternative goal of anti-angiogenic cancer treatment. (Cancer Sci 2011; 102: 1253–1256)

Angiogenesis or neovascularization, the formation of neovessels, is a fundamental process observed under both physiological and pathological conditions, and is now recognized as one of the principal hallmarks of cancers.(1) Folkman(2) first proposed the possibility of anti-angiogenic therapy for the treatment of cancer. This initial proposal was considered unrealistic. However, the continuous effort by Folkman(3) and his colleagues proved its credibility. Perhaps the most important work in the field of angiogenesis research has been the discovery of vascular endothelial growth factor (VEGF).(4) Ferrara(5) and his colleagues isolated VEGF, and showed its essential role in vascular development in the embryo and angiogenesis in the adult, including cancers. More importantly, they raised the blocking anti-VEGF mAb and exploited it as the therapeutic agent. As expected, VEGF receptors (VEGFRs) also became recognized as valuable therapeutic targets, and various tyrosine kinase inhibitors targeting VEGFRs, such as sorafenib, sunitinib, and pazopanib have since been developed for cancer treatment (Table 1).(6)

Table 1.   Anti-angiogenic drugs that target vascular endothelial growth factor (VEGF) signals
DrugType of agentClinical application
  1. GIST, gastrointestinal stromal tumor; NSCLC, non-small cell lung carcinoma; TKI, tyrosine kinase inhibitor.

BevacizumabAnti-VEGF mAbMetastatic colon cancer
Metastatic NSCLC
Metastatic breast cancer
Metastatic renal cancer
Glioblastoma
SunitinibTKIGIST
Metastatic renal cancer
SorafenibTKIMetastatic renal cancer
Metastatic hepatoma
PazopanibTKIMetastatic renal cancer

The effect of anti-VEGF antibody in preclinical studies is remarkable. Anti-VEGF antibody as a single agent shows a significant antitumor effect in mice by inhibiting tumor angiogenesis.(7) However, when humanized anti-VEGF mAb bevacizumab was applied in clinical trials of cancer patients, it was found to be effective when combined with chemotherapeutic agents. Bevacizumab was approved as the first anti-angiogenic drug for cancer treatment based on the results of a randomized clinical trial in metastatic colon cancers, in which the addition of bevacizumab to irinotecan plus fluorouracil/leucovorin improved progression-free survival and overall survival.(8) Bevacizumab is now approved for treatment of metastatic colon cancer, non-small-cell lung cancer, breast cancer, renal cancer, and glioblastoma.(9)

The requirement of chemotherapeutic agents for the anti-angiogenic cancer therapy with bevacizumab needs to be rationalized. One of the most plausible mechanisms offered concerning its requirement of combined chemotherapy is vascular normalization. Jain(10) proposed that bevacizumab transiently normalizes tumor vessels and thereby improves the tumor environment and blood flow, and that facilitates the delivery of chemotherapeutic agents to the tumor tissue.

Abnormal vascular structure in cancers

  1. Top of page
  2. Abstract
  3. Abnormal vascular structure in cancers
  4. Vascular regression and hypoxia may induce the invasive phenotype of cancer cells
  5. Persistent normalization of tumor vessels improves tumor microenvironment and inhibits metastasis
  6. Additional benefits of normalization of tumor vessels
  7. Concluding remarks
  8. Acknowledgments
  9. Disclosure statement
  10. References

The vasculature is primarily composed of luminal endothelial cells (ECs) and surrounding mural cells (smooth muscle cells in large vessels and pericytes in capillaries). The tight association of mural cells to ECs makes vessels mature and resistant to angiogenic stimuli. This composition of ECs and mural cells defines vessels as being normal or mature. Angiogenesis includes the following sequential steps: detachment of surrounding mural cells for initiation of angiogenesis; ECM degradation by endothelial proteases; migration of ECs at the tip; proliferation of ECs at the stalk; tube formation by ECs; and redistribution and tight association of mural cells to ECs for vascular normalization. The excess synthesis of angiogenic factors, including VEGF, initiates the process of angiogenesis. Because of the continuous and excessive synthesis of VEGF in cancer tissue, tumor vessels remain immature, lacking tight association of mural cells to endothelial tubes. These immature tumor vessels display high vascular permeability, and thus the tumor tissue is edematous, containing extravasated plasma components (Fig. 1). In addition to edema, the expansion of cancer tissue results in increased interstitial pressure, causing impaired tumor blood flow.(11) Tumor-associated ECs differ from normal ECs. They occasionally have excess centrosomes and are aneuploid, which may contribute to the morphologic and functional abnormalities of tumor vessels.(12) A recent report suggests that excessive VEGF signaling causes this centrosome abnormality.(13)

image

Figure 1.  Illustration of abnormal tumor vessels. Pericytes firmly attach to endothelial tube in normal vessels. Cancers contain newly formed immature vessels devoid of firm coverage by pericytes. Those tumor vessels have numerous sproutings and show high vascular permeability. EC, endothelial cells.

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Anti-angiogenic drugs targeting VEGF signals normalize tumor vessels. However, as VEGF acts as a survival factor of ECs, this normalization of tumor vessels is transient, and tumor vessels would finally regress. Jain(10) refers to this limited period as the vascular normalization window.

Vascular regression and hypoxia may induce the invasive phenotype of cancer cells

  1. Top of page
  2. Abstract
  3. Abnormal vascular structure in cancers
  4. Vascular regression and hypoxia may induce the invasive phenotype of cancer cells
  5. Persistent normalization of tumor vessels improves tumor microenvironment and inhibits metastasis
  6. Additional benefits of normalization of tumor vessels
  7. Concluding remarks
  8. Acknowledgments
  9. Disclosure statement
  10. References

The benefit of anti-angiogenic drugs targeting VEGF signals does not last long, as many patients encounter progression of cancers. This drug resistance can be explained by the recurrence of tumor angiogenesis through the compensatory production of angiogenic factors other than VEGF or recruitment of bone marrow-derived angiogenic cells.(14) However, the recurrence of tumor angiogenesis might not be the only reason for tumor progression.

Hypoxia due to the regression of tumor vessels could alter the property of cancer cells through the induction of hypoxia inducible factor-1 (HIF-1), as HIF-1 is reported to be involved in the induction of genes that elicit invasive and metastatic phenotypes of cancer cells.(15) With regard to this occurrence, important studies have reported the activation of the invasive cancer phenotype after the blockade of VEGF signaling. For example, Paez-Ribes et al.(16) applied blocking VEGFR2 antibodies to mouse models of pancreatic neuroendocrine carcinoma and glioblastoma, and found that cancers adapted to the treatment with blocking VEGFR2 antibodies by showing heightened invasiveness or metastasis. Ebos et al.(17) applied sunitinib, a tyrosine kinase inhibitor targeting VEGFRs, to a mouse xenograft model of melanoma cells, and found that transient treatment with sunitinib accelerated metastasis. Indeed, the acquisition of the invasive phenotype in humans has been reported in cases of glioblastoma during the course of treatment with anti-angiogenic drugs (Fig. 2).(18)

image

Figure 2.  Sequential changes of tumor vessels by the blockade of vascular endothelial growth factor (VEGF) signals. The blockade of VEGF signals transiently normalizes tumor vessels and ultimately provokes vascular regression. However, the benefit of this treatment does not last long, as many patients encounter progression of cancers. This tumor progression can be explained by the recurrence of tumor angiogenesis or sustained tumor hypoxia that causes cancer cells to become more invasive and metastatic.

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Persistent normalization of tumor vessels improves tumor microenvironment and inhibits metastasis

  1. Top of page
  2. Abstract
  3. Abnormal vascular structure in cancers
  4. Vascular regression and hypoxia may induce the invasive phenotype of cancer cells
  5. Persistent normalization of tumor vessels improves tumor microenvironment and inhibits metastasis
  6. Additional benefits of normalization of tumor vessels
  7. Concluding remarks
  8. Acknowledgments
  9. Disclosure statement
  10. References

The targeting of the VEGF signaling pathway is not the only way to inhibit tumor angiogenesis. A number of endogenous angiogenesis inhibitors are found in the body, and they can also be applied to anti-angiogenic therapy. Although the majority of these angiogenesis inhibitors are extrinsic to the vasculature, the ECs themselves have been found to produce intrinsic angiogenesis inhibitors. For instance, semaphorin 3A (SEMA3A) is expressed in ECs, whose expression is downregulated in cancers. Maione et al.(19) introduced the SEMA3A gene into the adeno-associated virus (AAV) vector, and applied it to a mouse model of pancreatic neuroendocrine carcinoma, the same model used by Paez-Ribes et al. Continuing supplementation with SEMA3A significantly decreased both tumor vascular area and diameter of tumor vessels, and maintained the remaining tumor vessels in the normalized state. During the course of this AAV-SEMA3A treatment, tumor hypoxia was evident in the acute phase, but it disappeared in the chronic phase because of the persistence of normalized tumor vessels. Importantly, as the treatment with SEMA3A did not cause regression of tumor vessels, the acquisition of the invasive cancer phenotype was not evident.(19) We have isolated vasohibin-1 (VASH1) as an endothelium-produced negative feedback regulator of angiogenesis.(20) Endogenous VASH1 is mainly produced by ECs in the termination zone of angiogenesis and stops the process, whereas exogenous VASH1 efficiently inhibits sprouting.(21) When applied to cancers, VASH1 inhibits tumor angiogenesis and makes tumor vessels mature.(22,23) Importantly, VASH1 maintains the vessels and does not induce vascular regression.(24)

In addition to the application of angiogenesis inhibitors, a novel target has been identified for vascular normalization. Mazzone et al.(25) recently reported that tumors in mice with a haplodeficiency of prolyl hydroxylase domain protein-2 (PHD2) were less invasive and metastatic. PHD2 is one of a group of proteins that hydrolyze critical residues in HIF-1 for its degradation. Haplodeficiency of PHD2 did not affect tumor vessel density or luminal size. Alternatively, in these haplodeficient mice, sprouting ECs were redirected to a more quiescent cell type, causing them to become arrayed in a “phalanx” of tightly apposed, regularly ordered cobblestone ECs. Importantly, this normalization of tumor vessels in PHD2+/− mice improved perfusion and oxygenation, thus rendering tumor cells less invasive and metastatic.

These observations show that persistent normalization of tumor vessels not only offers better delivery of chemotherapeutic agents to cancer tissue but also renders cancer cells less invasive and metastatic.

Additional benefits of normalization of tumor vessels

  1. Top of page
  2. Abstract
  3. Abnormal vascular structure in cancers
  4. Vascular regression and hypoxia may induce the invasive phenotype of cancer cells
  5. Persistent normalization of tumor vessels improves tumor microenvironment and inhibits metastasis
  6. Additional benefits of normalization of tumor vessels
  7. Concluding remarks
  8. Acknowledgments
  9. Disclosure statement
  10. References

There are additional benefits of the normalization of tumor vessels that improve the efficacy of anticancer treatment.

Vascular normalization enhances radiosensitivity.  The radiosensitivity of cancer cells is influenced by various factors, and one of them is oxygenation. As radiation therapy requires proper oxygenation to express its cytotoxic effect, severely hypoxic cancer tissues are resistant, thus requiring a higher dose of radiation to achieve the same level of cellular killing. Pre-clinical evidence indicates that inhibition of VEGF may increase local control of tumor growth after radiation. Several mechanisms have been postulated to explain this phenomenon including increased oxygenation secondary to vascular normalization.(26)

Vascular normalization enhances tumor immunity.  Tumor immunity is dependent on the recruitment of tumor-specific effector cells to the tumor parenchyma, and this process is controlled by microenvironmental factors that regulate leukocyte–endothelium interaction necessary for leukocyte extravasation. However, one of the obstacles is impaired interaction of leukocytes with abnormal tumor vessels for extravasation.(27) This escape of tumor immunity can be overcome by the normalization of tumor vessels. Several anti-angiogenic agents were reported to improve leukocyte–endothelium interaction and influx of leukocytes into the tumor parenchyma.(28)

Regulator of G protein signaling (RGS) proteins represent a group of molecules that play a pivotal role in influencing G protein-coupled receptor signals. RGS5, one member of the RGS family, has been implicated in tumor angiogenesis. Hamzah et al.(29) showed that loss of the RGS5 gene resulted in normalization of tumor vessels and a marked reduction in tumor hypoxia and hyperpermeability. Importantly, when combined with adoptive transfer of ex vivo activated T-lymphocytes, the loss of the RGS5 gene resulted in a significant improvement in the influx of immune effector cells in, and survival of, tumor-bearing mice.(29)

Concluding remarks

  1. Top of page
  2. Abstract
  3. Abnormal vascular structure in cancers
  4. Vascular regression and hypoxia may induce the invasive phenotype of cancer cells
  5. Persistent normalization of tumor vessels improves tumor microenvironment and inhibits metastasis
  6. Additional benefits of normalization of tumor vessels
  7. Concluding remarks
  8. Acknowledgments
  9. Disclosure statement
  10. References

This mini-review summarized the consequences of the normalization of tumor vessels. One may claim that vascular normalization induces resistance to anti-angiogenic treatment, as normalized tumor vessels are resistant to vascular regression.(30,31) However, recent observations suggest that vascular regression might not be an optimal goal. Vascular regression induces tumor hypoxia, a condition that may make cancer cells more invasive and metastatic. Tumors with normalized vessels are not hypoxic. Moreover, such normalization can protect vessels from cancer cell intravasation and enhance anticancer treatment with chemotherapeutic agents, radiation or immune therapy (Fig. 3).

image

Figure 3.  Merits of persistent vascular normalization for anticancer therapy. Persistent vascular normalization can be achieved by semaphorin 3A, vasohibin-1, inhibition of prolyl hydroxylase domain protein-2 (PHD2), or inhibition of regulator of G protein signaling (RGS5). This vascular normalization might protect from cancer cell intravasation and enhance anticancer treatment with chemotherapeutic agents, radiation, or immune therapy.

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Targeting of VEGF signaling induces transient vascular normalization, but it ultimately causes vascular regression. Therefore, we need to consider an alternative. Several approaches applying endogenous angiogenesis inhibitors or targeting PHD2 or RGS5 can be used for the normalization of tumor vessels. In addition, angiopoietin1-Tie2 signaling is involved in vascular quiescence and normalization,(32,33) and bone marrow-derived mononuclear cells can normalize tumor vessels.(34) Nevertheless, we need to consider further feasible methodologies to achieve persistent normalization of tumor vessels in humans, and to validate its possible combined effect with chemotherapeutic agents, radiation, or immune therapy.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Abnormal vascular structure in cancers
  4. Vascular regression and hypoxia may induce the invasive phenotype of cancer cells
  5. Persistent normalization of tumor vessels improves tumor microenvironment and inhibits metastasis
  6. Additional benefits of normalization of tumor vessels
  7. Concluding remarks
  8. Acknowledgments
  9. Disclosure statement
  10. References

The author is supported by a Grant-in-Aid (22112006) for Scientific Research on Innovative Areas “Integrative Research on Cancer Microenvironment Network” from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References

  1. Top of page
  2. Abstract
  3. Abnormal vascular structure in cancers
  4. Vascular regression and hypoxia may induce the invasive phenotype of cancer cells
  5. Persistent normalization of tumor vessels improves tumor microenvironment and inhibits metastasis
  6. Additional benefits of normalization of tumor vessels
  7. Concluding remarks
  8. Acknowledgments
  9. Disclosure statement
  10. References