f o c u s o n Ta r g e t i n g a n g i o g e n e s i s
REVIEWS VEGF-targeted therapy: mechanisms of anti-tumour activity Lee M. Ellis* and Daniel J. Hicklin‡
Abstract | Several vascular endothelial growth factor (VEGF)-targeted agents, administered either as single agents or in combination with chemotherapy, have been shown to benefit patients with advanced-stage malignancies. VEGF-targeted therapies were initially developed with the notion that they would inhibit new blood vessel growth and thus starve tumours of necessary oxygen and nutrients. It has become increasingly apparent, however, that the therapeutic benefit associated with VEGF-targeted therapy is complex, and probably involves multiple mechanisms. A better understanding of these mechanisms will lead to future advances in the use of these agents in the clinic.
*Departments of Surgical Oncology and Cancer Biology, Unit 444, University of Texas M.D. Anderson Cancer Center, PO BOX 301402, Houston, Texas 77230–1402, USA. ‡ Oncology Discovery, Schering-Plough Research Institute, Schering-Plough Corporation, 2,015 Kenilworth, Galloping Hill Road, New Jersey 07033, USA. e-mails:
[email protected];
[email protected] doi:10.1038/nrc2403 Published online 3 July 2008
Studies over the past 30 years have provided significant insights into the angiogenic process and its role in cancer biology, with over 17,000 papers published on the topic. The cloning of vascular endothelial growth factor (VEGF) in 1989 (Refs 1,2) was a major milestone in our understanding of tumour angiogenesis. A mere 14 years later, this paradigm of bench-to-bedside research culminated with the first VEGF-targeted agent, the antiVEGF monoclonal antibody bevacizumab (Avastin, Genentech), showing clinical benefit in patients with metastatic colorectal cancer (CRC) when combined with chemotherapy3. Since then, additional phase III studies have shown the benefits of bevacizumab as well as other VEGF-targeted therapies, either as single agents or when combined with chemotherapy4–8. Despite extensive studies with VEGF-targeted therapy in both the laboratory and the clinic, the mechanisms responsible for the anti-tumour activity of these agents are not fully understood. Emerging data suggest that multiple mechanisms account for the efficacy of VEGF-targeted therapies in patients with cancer, which was originally thought to be a result of their ability to block new blood vessel growth. With this in mind, it is important to emphasize that not all patients benefit from VEGFtargeted therapy. Thus, insights into the mechanisms associated with the anti-tumour activity of VEGFtargeted therapy might help improve current therapy with these agents, uncover mechanisms of intrinsic or acquired resistance, and identify predictive markers for therapy. For each proposed mechanism of action, we will discuss the data supporting each hypothesis, and where appropriate, data refuting each hypothesis. It is not possible to incorporate the multiplicity of data reported in
abstract form, so when referring to clinical trial data we will only comment on those reports published in reputable, peer-reviewed journals. The related topic of inherent and/or acquired resistance to VEGF-targeted therapy is discussed by Douglas Hanahan and Gabriel Bergers in this issue of Nature Reviews Cancer9.
VEGF biology The mammalian VEGF family consists of five glycoproteins referred to as VEGFA, VEGFB, VEGFC, VEGFD (also known as FIGF) and placenta growth factor (PlGF, also known as PGF)10,11. The best characterized of the VEGF family members is VEGFA (commonly referred to as VEGF), which is expressed as various isoforms owing to alternative splicing that leads to mature 121-, 165-, 189- and 206-amino-acid proteins, although proteolytic cleavage of these isoforms can lead to other, smaller isoforms. VEGF165 is the predominant isoform and is commonly overexpressed in a variety of human solid tumours. The VEGF ligands bind to and activate three structurally similar type III receptor tyrosine kinases, designated VEGFR1 (also known as FLT1), VEGFR2 (also known as KDR) and VEGFR3 (also known as FLT4) (FIG. 1). The assortment of VEGF ligands have distinctive binding specificities for each of these tyrosine kinase receptors, which contributes to their diversity of function. In response to ligand binding, the VEGFR tyrosine kinases activate a network of distinct downstream signalling pathways12. VEGFR2 expression is restricted primarily to the vasculature and is the key mediator of VEGF-induced angiogenesis. VEGFR1 is expressed on
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RE V IE W S At a glance • Vascular endothelial growth factor (VEGF) mediates numerous changes within the tumour vasculature, including endothelial cell proliferation, migration, invasion, survival, chemotaxis of bone marrow-derived progenitor cells, vascular permeability and vasodilation. • There are several approaches to inhibiting VEGF signalling, including neutralization of the ligand or receptor by antibodies, and blocking VEGF receptor (VEGFR) activation and signalling with tyrosine kinase inhibitors. • VEGF-targeted therapy has been shown to be efficacious as a single agent in renal cell carcinoma and hepatocellular carcinoma, whereas it is only of benefit when combined with chemotherapy for patients with metastatic colorectal, non-small-cell lung and metastatic breast cancer. • VEGF-targeted therapy affects numerous cell types within the tumour microenvironment, including endothelial cells, haematopoietic progenitor cells, dendritic cells and tumour cells. • VEGF-targeted therapy has multiple mechanisms of action that might be dependent on tumour type. • VEGF-targeted therapy affects vascular function (flow and permeability) in addition to blocking further new blood vessel growth.
VEGF trap A fully human soluble decoy receptor protein that consists of a fusion of the second immunoglobulin (Ig) domain of human VEGFR1 and the third Ig domain of human VEGFR2 with the constant region (Fc) of human IgG1. VEGF trap has a high affinity for all isoforms of VEGFA, as well as PlGF.
the vasculature as well but is also expressed on several other types of cells. The exact role of VEGFR1 on tumour endothelium remains to be elucidated. VEGFR1 has a tenfold higher binding affinity to VEGF, but exerts less activation of intracellular signalling intermediates than VEGFR2 (Ref. 13). VEGFR1 can function as a negative regulator of angiogenesis, by binding VEGF and preventing its binding to VEGFR2 (Ref. 14). VEGFR3 preferentially binds VEGFC and VEGFD and its expression in the adult is primarily on lymphatic endothelial cells. More recent data has demonstrated the expression and function of VEGFR3 on vascular endothelial cells15. VEGFR3 is important in cardiovascular development and remodelling of primary vascular networks during embryogenesis, and has a crucial role in post-natal lymphangiogenesis16,17. The neuropilins (NP1 and NP2, also known as NRP1 and NRP2) act as co-receptors for the VEGFRs, increasing the binding affinity of VEGF to VEGFR tyrosine kinase receptors18–22. NP1 and NP2 have been postulated to signal independently of their association with VEGFR tyrosine kinase receptors, but the role of VEGF activation of NP-mediated signalling is not fully understood. In fact, recent studies suggest that dual targeting of the vasculature with antibodies to VEGF and NP1 is more effective than single-agent therapy23. Differences in VEGFR biology are important, as various VEGF-targeting approaches differ in their ability to block receptor function. VEGF promotes tumour angiogenesis, and hence blood flow, through several mechanisms, including enhanced endothelial cell proliferation and survival; increased migration and invasion of endothelial cells; increased permeability of existing vessels, forming a lattice network for endothelial cell migration; and enhanced chemotaxis and homing of bone marrowderived vascular precursor cells (both endothelial cells and pericytes) 24,25. In addition to having proangiogenic effects, VEGF has several important functions that are independent of vascular processes,
including autocrine effects on tumour cell function (survival, migration, invasion), immune suppression, and homing of bone marrow progenitors to ‘prepare’ an organ for subsequent metastasis26. Interestingly, some of the diverse effects of VEGF may not always lead to an increase in tumour blood flow, which is presumed to be associated with angiogenesis. For example, an increase in vessel dilation can lead to turbulent and inefficient blood flow. Alternatively, increased permeability of vessels due to VEGF may lead to increased interstitial pressure and secondary constriction of vessels27,28. As stated elsewhere in this Review, the number of vessels in a defined area of a tumour does not always correlate with an increase in blood flow (that is, function).
VEGF-targeted approaches Recognition of the VEGF pathway as a key regulator of angiogenesis has led to the development of several VEGF-targeted agents that include neutralizing antibodies to VEGF or VEGFRs, soluble VEGF receptors or receptor hybrids and tyrosine kinase inhibitors (TKIs) with selectivity for VEGFRs29. Thus far, the anti-VEGF monoclonal antibody bevacizumab and the VEGFR TKIs sorafenib (Nexavar, Bayer/Onyx) and sunitinib (Sutent, Pfizer) are currently approved by the US Food and Drug Administration (FDA) for clinical use23,30,31. Bevacizumab is a humanized monoclonal antibody with a circulating half-life of ~20 days that is currently approved by the FDA for patients with metastatic CRC, non-small cell lung cancer and metastatic breast cancer in combination with chemotherapy3,6,8 (TABLE 1). The neutralization of VEGFA by an antibody or soluble receptor construct (VEGF trap) can prevent its binding to, and activation of, VEGFR1, VEGFR2, NP1 and NP2. The TKI sorafenib has shown single-agent efficacy in patients with advanced renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC)4,5. Sunitinib has also been shown to be efficacious as a single agent in patients with RCC 7. It should be emphasized that, owing to their mode of action at the ATP binding pocket, TKIs are selective rather than specific for a particular kinase(s). Thus, TKIs designed to target VEGF receptors are actually considered ‘multi-kinase’ inhibitors. For example, sorafenib and sunitinib also have significant activity against Raf, platelet-derived growth factor receptor (PDGFRb), fibroblast growth factor receptor (FGFR), FLT3, KIT and FMS (also known as CSF1R) receptors32. In this Review, we make the assumption that the activity of the VEGFR TKIs observed in the clinic is related primarily to VEGFR targeting. However, we do recognize that these agents are selective rather than specific VEGFR inhibitors. Therefore, it is likely that some of the clinical activity can be attributed to activity on other tyrosine kinase receptors32. Notably, some VEGFR-targeted TKIs significantly inhibit the activity of PDGFRs, whereby a dual attack on the vasculature (VEGFR on endothelial cells and PDGFR on pericytes) in preclinical studies leads to greater efficacy than inhibiting a single receptor family.
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f o c u s o n Ta r g e t i n g a n g i oRE g eVnIE eW siS s PlGF
VEGFC
VEGFB
VEGFA
VEGFD
S–S
VEGFR1
NP1 or NP2
Vasculogenesis Angiogenesis
VEGFR2
S–S
VEGFR3
NP2
Lymphangiogenesis
Figure 1 | Vascular endothelial growth factor (VEGF) family members and | Cancer receptors. The mammalian family of VEGF ligands consists of fiveNature familyReviews members, VEGFA, VEGFB, VEGFC, VEGFD and placental growth factor (PlGF). The three tyrosine kinase (TK) VEGF receptors have specific binding capabilities. Neuropilin 1 (NP1) and NP2 were initially hypothesized to function only as co-receptors for VEGF (increasing the binding affinity of the ligands to their TK receptors), although recently it has been hypothesized that these receptors can signal independently of the TK receptors. VEGFA binds to both VEGF receptor 1 (VEGFR1) and VEGFR2. VEGFB and PlGF bind exclusively to VEGFR1. Heterodimers of VEGFA and PlGF have been identified that can bind to and activate VEGFR2. VEGFR3 is a specific receptor for VEGFC and VEGFD. VEGFC and VEGFD can be proteolytically processed to allow binding to VEGFR2 as well. NP1 and NP2 can also act as co-receptors for certain VEGF–VEGFR complexes and, along with other molecules such as integrins and VE-cadherin (CDH5), can modulate VEGF–VEGFR activation and signalling11,18,19,107–109. Adapted, with permission, from Ref. 11 American Society of Clinical Oncology (2005).
Mechanisms of action of VEGF-targeted therapy The inhibition of VEGF signalling may affect tumour growth through several mechanisms, some of which are discussed below. These mechanisms should not be considered as mutually exclusive; indeed, in some tumours VEGF-targeted therapy may act through parallel mechanisms. It is also plausible that different mechanisms have a more or less important role depending on tumour type. This is a crucially important point, as in certain cancers (for example, RCC)4,7 single-agent VEGF-targeted therapy has significant activity, whereas in other tumour types VEGF-targeted therapy has considerably less clinical benefit (if any). Angiogenesis in RCC is presumed to be highly VEGF-dependent, in part owing to high frequency inactivation of the von Hippel–Lindau tumour suppressor gene, VHL, in these tumours. Interestingly, several studies using anti-VEGF therapy in RCC have failed to convincingly confirm that mutational status of VHL was predictive of efficacy. By contrast, mechanisms of anti-tumour activity are likely to be more complex when anti-VEGF therapy is combined with chemotherapy. These observations support the view that the efficacy (or lack thereof) might be dependent upon specific effects of VEGF-targeted therapy on distinct tumour types33,34.
The study of VEGF-targeted therapies and their mechanisms of action in preclinical models has led to the successful translation of these therapies to the clinic. However, preclinical models have limitations and thus we may not be able to appreciate the full extent of mechanisms associated with inhibiting VEGF in these types of studies. Typically, preclinical models of tumour growth are quite rapid and are supported by vascular structures that likewise grow rapidly. Thus, tumour vessels in murine models tend to be more plastic and responsive to anti-angiogenic therapy. Although functional changes in the vasculature of humans receiving anti-VEGF therapy have been clearly observed with sophisticated imaging techniques, the tumour response (if any) is much less dramatic than observed in mice23,35–37. Therefore, preclinical findings should only be extrapolated to clinical situations with caution. It is important to emphasize that clinical validation of hypotheses regarding VEGF-targeted therapy requires carefully controlled randomized clinical trials. Although there are interesting reports from trials with small numbers of patients, these small trials do not provide definitive answers. However, these trials can be considered ‘hypothesis-generating’ trials that warrant further study in prospective phase III randomized trials that are adequately powered to confirm or disprove findings from smaller studies.
Anti-angiogenic effects. The process of tumour angiogenesis was initially defined as the outgrowth of post-capillary venules from pre-existing vessels: classical ‘sprouting angiogenesis’24. However, this definition requires refinement. The current view of tumour angiogenesis, and more specifically angiogenesis secondary to VEGF, involves a complex series of events including classic sprouting angiogenesis, the loss of pericyte–endothelial cell adhesion, increased permeability, vasodilation and the incorporation of bone marrow-derived endothelial progenitor cells10,29,38–40. Although the concepts of tumour angiogenesis are well-accepted, in certain organs one has to question whether or not this angiogenesis occurs at all. For example, the liver, lung and brain are highly vascular organs, and vessel cooption may have an important role in the vascularization of these tumours. It is well-recognized that metastatic tumours to the liver have distinct growth patterns. Metastatic CRC often replaces the liver parenchyma, rather than displacing it. This might occur because CRC cells express CD95 ligand (also known as FAS ligand (FASLG)),which in turn mediates hepatocyte cell death as these cells express the ligand receptor, CD95 (FAS), on their cell surface41. In fact, careful studies of the pathology of liver and lung metastasis have determined that there are different growth patterns, including an ‘angiogenic’ and ‘non-angiogenic’ growth pattern42,43. By contrast, tumours such as carcinoids and HCC displace the hepatic parenchyma and therefore angiogenesis is necessary for these tumours to continue to grow. Interestingly, radiographic computed tomography (CT) findings support this hypothesis: CRC
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RE V IE W S Table 1 | Completed anti-VEGF therapy phase III trials Trial design
Disease type
Line of reaction
Primary Change in response endpoint rate with addition of anti-VEGF therapy (%) met?*
Change in progression-free survival (months)
Combination therapy trials Capecitabine +/– BEV*
mBreast cancer
Refractory
No
10
0.7
Paclitaxel +/– BEV
mBreast cancer
First line
Yes
22
5.9
Docetaxel +/– BEV
mBreast cancer
First line
Yes
11
0.7–0.8
Carboplatin + paclitaxel +/– BEV
NSCLC
First line
Yes
15
1.9
Gemcitabine + cisplatin +/– BEV
NSCLC
First line
Yes
10–14
0.4–0.6
5-Fluorouracil + leucovorin +/– SU5416
mCRC
First line
No
Not reported
Not reported
IFL +/– BEV
mCRC
First line
Yes
10
4.4
FOLFOX +/– PTK787/ ZK222584
mCRC
First line
No
–4
0.2
XELOX + FOLFOX +/– BEV
mCRC
First line
Yes
0
1.4
FOLFOX +/– BEV
mCRC
Refractory
Yes
14
2.6
FOLFOX +/– PTK787/ ZK222584
mCRC
Refractory
No
1
1.5
Gemcitabine +/– BEV*
Pancreatic cancer
First line
No
1
0
Interferon +/– BEV*
RCC
First line
Yes
18
4.8
Single-agent therapy trials Sorafenib versus placebo
RCC
Refractory
Yes
8
2.7
Sunitinib versus interferon
RCC
First line
Yes
31
5.9
Sorafenib versus placebo
HCC
First line
Yes
2
3.0
Sorafenib versus placebo
HCC
First line
Yes
2
1.4
Data subject to change based on subsequent analyses. *Primary endpoints vary from trial to trial, but typically involve overall survival or progression-free survival. BEV, bevacizumab; CRC, colorectal cancer; FOLFOX, folinic acid (leucovorin), fluorouracil, oxaliplatin; Gem, gemcitabine; HCC, hepatocellular carcinoma; IFL, irinotecan, fluorouracil and leucovorin; m, metastatic; NSCLC, non-small-cell lung cancer; RCC, renal cell carcinoma; VEGF, vascular endothelial growth factor; XELOX, capecitabine (Xeloda) and oxaliplatin.
metastases in the liver often exhibit poor blood flow, whereas both metastatic carcinoid tumours and HCC exhibit marked increases in blood flow (hypervascular) relative to surrounding liver. These observations raise important issues regarding the use of anti-angiogenic therapies in specific diseases and sites of tumour growth. However, at present there are no clinical data to suggest that the site of tumour growth influences the response to VEGF-targeted therapies. Thus, understanding the process of angiogenesis has become much more of a challenge than it was during the infancy of this field, owing to the specific nature of individual tumour types growing in specific microenvironments. Owing to the complexity of the development and morphogenesis of the tumour blood supply, defining ‘anti-angiogenesis’ has likewise become a challenge. If we revisit the original definition of angiogenesis — the
induction of new blood vessel growth from existing vessels — then it follows that anti-angiogenic therapy should be the opposite: the inhibition of new blood vessel growth, with subsequent induction of tumour dormancy or cytostasis. However, agents initially hypothesized to be truly anti-angiogenic (intended to specifically target endothelial cells), such as TNP-470 or endostatin, have not yet led to any documented benefit to patients in randomized phase III trials, or even modest activity in phase II trials44,45. Admittedly, these agents have not been well-studied in combination with chemotherapy. However, these agents, despite being available for several years, have not shown clinical activity of note, whereas anti-VEGF therapy has demonstrated evidence of singleagent activity in specific tumour types4,5. So, is there evidence for inhibition of angiogenesis by VEGF-targeted therapies?
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f o c u s o n Ta r g e t i n g a n g i oRE g eVnIE eW siS s
Progression-free survival (PFS). The length of time that patients are free from any significant increase in tumour size or development of new tumours. PFS is measured as the time from start of treatment to the first measurement of cancer growth (by pre-defined criteria).
Inhibition of new vessel growth. Although vessel count or microvessel density has been shown to be a reliable prognostic marker, vessel counts have not been shown to correlate with efficacy of VEGF-targeted or anti-angiogenic activity. In fact, the value of vessel counts as a measure of anti-angiogenic activity has been questioned46. Folkman and colleagues stated that as vessels are destroyed or vessel growth is inhibited the intercapillary distance remains the same. Thus, vessel counts and/or density remain unchanged even in the face of effective therapy. Although markers of angiogenic activity on endothelial cells have been investigated, none have shown any correlation to clinical response to therapy. Changes in the function of the vascular bed are likely to be much more important a parameter of anti-angiogenic activity than simple vessel presence and are better studied with non-invasive imaging techniques. In fact, recent studies with anti-DLL4 therapy (targeting the Notch pathway on endothelial cells) have shown that the vessel count is increased, whereas tumour blood flow and growth are decreased47–51. This supports the notion that the function of the vasculature is more important than the morphology (that is, vessel count). There are no adequately powered clinical trials that have investigated the effects of single-agent bevacizumab on vessel counts before and after therapy. This is probably due, in part, to the difficulties in obtaining patient tumour specimens for study. The logistics of obtaining serial tumour biopsy samples in clinical trials are challenging owing to various factors including cost, ethics (discomfort, potential complications), sampling error, inadequately powered trials and the time points chosen for biopsy. Although in phase I studies in various tumour types investigators have noted decreases in vessel counts in tumours biopsied before and after VEGF-targeted therapy, this finding is not universal52,53. There are no known reliable markers in humans of anti-angiogenic activity, thus the value of sequential biopsies to evaluate anti-angiogenic activity is unproven at this time. If biopsies are to be done, it is crucial that investigators provide rationale before initiation of any study. Have molecular or immunohistochemical studies been validated in preclinical models and in exploratory analyses from human tissues? Are the studies to be done based on tested biological principles, and not performed with a ‘shotgun’ approach? Can results from these studies be correlated to non-invasive imaging studies, so that in the future biopsies are not necessary? Will information obtained from biopsies lead to continued therapy or a change in therapy? And lastly, will the data collected be robust enough for statistical analyses? Biopsies are associated with risk and discomfort, and investigators must adhere to the clinical rule of “primum non nocere” (First, do no harm). There is indirect evidence that VEGF-targeted therapy is cytostatic for blood vessel growth, as was first hypothesized54. One example is from patients with advanced RCC receiving sorafenib who experienced a progression-free survival of 5.5 months, which was significantly longer than that of patients treated with placebo4. Although a higher percentage of patients had apparent
decreases in tumour size, only 10% met standard criteria for objective response (response evaluation criteria in solid tumours (RECIST)). The improvement in progression-free survival in this study was greater than would be expected with the relatively low response rate of 10%. These clinical results suggest a cytostatic effect of VEGF-targeted therapy, that is, tumour growth was delayed, but there was little evidence of tumour shrinkage, which could be attributable to inhibition of vessel growth. However, VEGF-targeted therapy may be cytostatic by other mechanisms. Induction of endothelial cell apoptosis. VEGF mediates numerous pro-survival pathways in endothelial cells including induction or activation of BCL2, Akt, survivin and inhibitor of apoptosis proteins (IAPs) as well as others55,56. As VEGF mediates endothelial cell survival functions, loss of VEGF signalling has been proposed to lead to endothelial cell apoptosis. Although haploinsufficiency of a single VEGF allele leads to embryonic death, this could be due to lack of progressive vessel development rather than the death of endothelial cells. Using an inducible VEGF expression system in glioma cells, Benjamin and Keshet demonstrated that VEGF was required for endothelial cell survival in tumour xenografts57. More recently, autocrine endothelial signalling of VEGF has been found to be important in endothelial cell survival and homeostasis. Many studies using VEGF-targeted therapies in murine models demonstrated that inhibition of VEGF signalling could lead to tumour endothelial cell apoptosis, although rarely did such therapies lead to regression of established tumours58–60. The induction of endothelial cell apoptosis owing to VEGF-targeted therapy is indirectly supported by clinical trial results. This mechanism of action is probable in tumour types in which single-agent VEGF-targeted therapy leads to significant response rates, such as those observed in patients with RCC. In a phase III trial of patients with metastatic RCC randomized to sunitinib or interferon α, response rates were 31% in the sunitinib arm and 6% in the interferon arm7. In a phase II trial in patients treated with axitinib, a relatively selective VEGF receptor TKI, responses were observed in 44% of patients7. Interestingly, there were cases where patients did not exhibit a defined response by standard clinical criteria, but imaging studies demonstrated tumour necrosis. These observations suggest that vessel destruction by VEGF-targeted therapy might be occurring in this particular tumour type, although patients eventually become refractory to treatment in the vast majority of cases. Differences in response rates in clinical trials investigating anti-VEGF therapy in RCC might be due to numerous factors, including the distinct targeting profile of the drug, pharmacokinetics and patient selection (first-line therapy versus cytokine-refractory patients). In addition, RECIST criteria might not tell the whole story, as there are examples of anti-VEGF-induced cavitation and loss of viable tumour mass without significant alteration of tumour measurements4. Thus, as hypothesized during the development era of targeted therapies, current
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RE V IE W S Pre-metastatic niche
Early metastatic disease
Advanced disease
VEGF
VEGFR1+ myeloid cells
VEGFR1+ bone marrow-derived cells are recruited to future organ sites of metastases
VEGFR2+ EPCs HSCs Bone marrow
VEGFR2+ EPCs recruited to sites of tumour growth contribute to neoangiogenesis and incorporate into tumour vasculature
Figure 2 | Blockade of incorporation of haematopoietic and endothelial progenitor cells. Tumours secrete vascular endothelial growth factor (VEGF) family ligands that Nature Reviews | Cancer are chemotactic for bone marrow-derived cells including haematopoietic and + endothelial progenitor cells. VEGF receptor 1 (VEGFR1) cells can migrate to the premetastatic niche and prepare an organ for metastasis. In addition, VEGFR2+ progenitor cells can be incorporated into the growing vascular bed and contribute to tumour angiogenesis. Blockade of VEGF signalling can prevent these progenitor cells from contributing to tumour angiogenesis and metastasis. EPC, endothelial progenitor cell; HSC, haematopoietic stem cell.
response criteria may not be a good indicator of clinical benefit and one must rely on progression-free survival and/or overall survival for evaluation of efficacy61.
Vasculogenesis This term is used for the de novo formation of new blood vessels from haematopoietic progenitor cells and normally takes place during embryogenesis. During tumour angiogenesis, EPCs migrate to sites of tumour growth and differentiate into tumour endothelial cells participating in new blood vessel growth.
VEGF-targeted therapy used in combination with lowdose metronomic chemotherapy. Tumour endothelial cells, when dividing, become sensitive to chemotherapy (similarly to other dividing cells). The use of continuous, low-dose (‘metronomic’) chemotherapy to target dividing endothelial cells was first studied by Browder and colleagues in the Folkman laboratory62. Kerbel and colleagues have expanded on this work and explored a combination metronomic therapy approach where continuous, low-dose chemotherapy is administered concurrent to VEGF-targeted therapy as a dual attack on the tumour vascular bed63. The principle behind this approach is that the chemotherapy attacks the proliferating endothelial cells while VEGF-targeted therapy diminishes survival signalling in endothelial cells, making them more susceptible to chemotherapy-induced apoptosis64. In addition, metronomic chemotherapy and/or VEGF-targeted therapy may also decrease the number of endothelial precursor cells that can be incorporated into the growing vascular bed (see below)65. This approach is currently being explored in clinical trials66. Blockade of incorporation of haematopoietic and endothelial progenitor cells. VEGF can stimulate vasculo genesis in tumours by recruiting bone marrow-derived haematopoietic progenitor cells (HPCs) and endothelial progenitor cells (EPCs)25,67,68 (FIG. 2). More recently, EPCs have been referred to as ‘vascular modifying cells’ based on their characteristics described below. Markers
used to identify these cell types vary from laboratory to laboratory, so the reader is encouraged to review each manuscript carefully for techniques. VEGFR1+ HPCs home to sites of distant metastases before seeding of tumour cells establishing a so-called ‘pre-metastatic niche’26. Following dissemination of tumour cells within the pre-metastatic niche, VEGFR2 + EPCs are then recruited to promote vasculogenesis. EPCs can proliferate and migrate from the bone marrow to the circulation, where they are referred to as circulating EPCs (CEPs). The recruitment and incorporation of VEGFR2+ EPCs into vessels is supported by perivascular VEGFR1+ HPCs68. VEGFR1-expressing cells have been shown to be essential for extravasation and retention of VEGFR2+ EPCs69. Selective inhibition of VEGFR1+ HPCs eliminates the pre-metastatic niche, reducing the formation of micrometastases. Selective targeting of VEGFR2+ EPCs results in the formation of micrometastases without vascularization26. The chemotaxis of EPCs is largely under the influence of VEGF, although other cytokines such as granulocyte colony stimulating factor (G-CSF) and stromal cell-derived factor 1 (SDF1, also known as CXCL12) have also been shown to be chemotactic to EPCs70. Preclinical studies, as well as early-phase clinical studies, have shown that VEGF-targeted therapy can inhibit the mobilization of EPCs and their presumed incorporation into the tumour vasculature53. However, the dynamics of this process remain to be determined, as EPCs may decrease soon after anti-VEGF therapy, but may increase with continued therapy. Further studies are needed to determine whether EPC levels are biomarkers of response or resistance. The importance of EPCs is dependent on the per cent of the vessels within the vasculature that incorporate EPCs. Preclinical models of tumour growth are characterized by rapidly growing tumours that far exceed the rate of tumour growth that occurs in humans. In these preclinical models the rate of incorporation of EPCs is relatively high, with >90% of the endothelial cells comprising the tumour vasculature in certain tumours25, although the range is typically 5–50% for most studies. Even in preclinical models, such as an orthotopic model of glioma, the per cent of endothelial cells derived from the bone marrow is low (4%), although over time 54% of tumour vessels were composed of at least one bone marrow-derived progenitor cell40. In humans, the per cent of tumour vessels derived from bone marrow precursors is much lower. In one study in which patients developed tumours subsequent to bone-marrow transplant where marrow was derived from donors of the opposite sex (and thus the vessels or endothelial cells could be evaluated with sex chromosome-specific probes), <12% of tumour vessels contained a bone-marrow-derived cell71. A recent preclinical study by Gao and colleagues demonstrated that this low percentage of incorporated EPCs (~12%) is sufficient and necessary for the conversion of avascular micrometastases to progressive metastatic tumours72. Apparently, although the incorporation rate of EPCs into tumour vasculature may be low, these cells provide a crucial function in sustaining angiogenesis
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f o c u s o n Ta r g e t i n g a n g i oRE g eVnIE eW siS s a Baseline Baseline
to those studies described above, as well as others. Therefore, this mechanism of VEGF-targeted therapy will undoubtedly be vigorously studied and debated in the future.
Day post bevacizumab bevacizumab Day22— – after
Baseline Baseline
b 20
Blood flow
Blood volume
Permeability
10
3%
0
–4%
(%)
–10
–2%
–3%
–2%
–4%
–20 –30 –40 –50 –60
–24%
–28%
–49%
BEV PEG
Day 2 Wk 18 Wk 9 Wk 18
–34%
–33%
Day 2 Wk 18 Wk 9 Wk 18
Day 2 Wk 18 Wk 9 Wk 18
–40%
Nature Reviews Figure 3 | Vascular endothelial growth factor (VEGF)-targeted therapy and| Cancer vasoconstriction. VEGFA, through VEGF receptor 1 (VEGFR1) and VEGFR2, induces nitric oxide and prostacyclin, two potent vasodilators. a | Using computerized tomography (CT) scanning of carcinoid liver metastases, one can determine blood flow (upper left panel heat map: red represents high blood flow, blue represents low blood flow). This CT scan shows a patient with metastatic carcinoid tumour in a randomized phase II trial where patients received either bevacizumab (BEV) or pegylated interferon (PEG). After a single dose of bevacizumab, tumour blood flow is markedly decreased (right upper panel). This rapid decrease in blood flow is unlikely to be due to vessel destruction as this time frame is too rapid for this to occur. This rapid decrease in blood flow is probably due to vasoconstriction. b | Graphical representation of data80. Reproduced, with permission, from Ref. 80 American Society of Clinical Oncology (2008). Wk, week.
in growing tumours. Additionally, the contribution of EPCs in promoting tumour angiogenesis may be much more important in certain cancers that grow rapidly, such as lymphoma. Thus, blockade of VEGFR1+ HPCs and VEGFR2+ EPC-mediated vasculogenesis in tumours may be an important mechanism of VEGF-targeted therapy in selected tumour types. It is important to point out that the role of BMderived cells in tumour angiogenesis remains controversial. Purhonen et al. recently reported that BM-derived, VEGFR2-positive cells or other endothelial cell precursors do not contribute to the angiogenic tumour vasculature73. Conclusions from this study are in contrast
Effects on vessel function Vascular constriction. VEGF can increase expression of nitric oxide (NO), prostacyclins and other soluble mediators that lead to vasodilation74. The role of VEGF regulation of NO and regulation of vessel tone was addressed by Isner and colleagues in non-tumour-bearing rats. In this model, VEGF administration induced hypotension, which was blocked by pre-treatment with an NO inhibitor75. Although some investigators hypothesize that vasodilation leads to inconsistent blood flow due to spatial and temporal heterogeneity38, others suggest that vasodilation of tumour vessels leads to increased blood flow23,76. In clinical trials, shortly after the administration of VEGF-targeted therapy (within 48 h), marked changes are observed on radiographic studies37,77–80. It is important to emphasize that standard dynamic contrast-enhanced magnetic resonance imaging (MRI) studies typically measure the transfer constant (ktrans) that reflects both permeability and flow. Although certain centres have the expertise and ability to refine such studies, reproducibility among various centres remains a challenge78. Typically ktrans rapidly decreases in tumours following administration of VEGF-targeted therapy (within 48 h), but definitive statements regarding actual blood flow cannot be made78. By contrast, functional CT scanning can provide distinct quantitation of tissue perfusion, permeability and relative blood volume, although resolution and sensitivity can be poor at times. Several studies with VEGF-targeted therapy have noted a decrease in tumour perfusion soon after administration of the agent37,77,79 (FIG. 3). Although some investigators interpret this as normalization of the tumour vasculature (see below), this can also be viewed as simple vasoconstriction, possibly leading to ischaemia and in some cases tumour necrosis. This may be particularly relevant in RCC, one of the most vascular tumours known, which responds well to single-agent VEGFtargeted therapy. The fact that this observed decrease in perfusion occurs so rapidly suggests that this is not due to vessel destruction, but more probably effects on the integrity (permeability) and function (vessel perfusion) of the vascular bed. As stated above, it is important to note that the effect of anti-VEGF therapy on the vasculature may be time-dependent (that is, having early effects on vascular function and late effects of endothelial cell survival and proliferation). The acute effects on blood flow are probably due to decreases in production of the vasodilators NO and prostacyclin, which we hypothesize are chronically increased in tumour vessels owing to high levels of VEGF. Inhibition of VEGF activity can lead to a decrease in production of these vasoactive intermediates, leading to a relative vasoconstriction. This vasoconstriction is not limited to the tumour vasculature, as one of the most common adverse effects of VEGF-targeted therapy is hypertension. In fact, some investigators have hypothesized that hypertension might be a surrogate
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Figure 4 | Vascular endothelial growth factor (VEGF)-targeted Nature therapy induced Reviews | Cancer normalization of the tumour vasculature. a | Depiction of vascular normalization with VEGF-targeted therapies. Normal vascular networks have a hierarchal structure with evenly spaced vessels and efficient blood flow. By contrast, the tumour vasculature is abnormal with increased permeability, high interstitial pressure, and dilated and tortuous vessels, and bidirectional flow can occasionally be seen. This abnormal vascular network is inefficient and is characterized by an inefficient blood supply. VEGF-targeted therapy can limit the permeability induced by VEGF, constrict dilated vessels and, in turn, theoretically, improve blood flow. Improved blood flow can increase delivery of chemotherapy and oxygen (increasing the efficacy of irradiation). Most studies demonstrating normalization observed a window of time for which this occurs. b | A magnetic resonance imaging study of a patient with a glioblastoma receiving a tyrosine kinase inhibitor targeting VEGF receptors. The dynamic contrast-enhanced magnetic resonance imaging demonstrates a rapid decrease in vascular permeability23. Reproduced, with permission, from Ref. 23 Cell Press (2007).
marker of activity7. This principle is being investigated in a phase III trial in patients with metastatic breast cancer who received bevacizumab and paclitaxel. Preliminary evidence from this study suggests that those patients who developed hypertension appeared to benefit more than patients who did not. Normalization. In most tumours, the vasculature is characterized by a relatively inefficient blood supply that differs in many respects from normal vascular networks. The tumour vascular abnormalities include increased permeability, vessel dilatation, tortuosity, abnormal spacing, decreased/abnormal pericyte coverage and irregular basement membrane structures38,81,82. Owing to the fact that many of the abnormalities of the tumour vascular network are secondary to VEGF, Jain has hypothesized that VEGF-targeted therapy can ‘normalize’ the vasculature of tumours38. This normalization of the tumour vascular network can theoretically lead to making the blood-flow more uniform with subsequent increased delivery of chemotherapy and oxygen (FIG. 4). Several studies from Jain’s group have characterized this process that occurs with VEGF-targeted therapy including the identification of a window of normalization in murine tumour models. Indeed, this window of normalization in mice is relatively short (days) and occurs soon after initiation of VEGF-targeted therapy. In a phase II study in patients with glioblastoma, the use
of the VEGFR TKI AZD2171 as a single agent led to a decrease in cerebral oedema and normalized the functional and structural aspects of the tumour vasculature, as measured by MRI23. This normalization window varied from patient to patient, and in some patients lasted months. Eventually, however, the window of normalization was lost, and the exact time frame was difficult to determine owing to the prescheduled intervals of MRI studies. The definition of ‘normalization’ requires some attention. If one defines ‘normalization’ as simply reversing the abnormalities of the vascular network induced by VEGF (that is, increased permeability, vasodilation, tortuosity, and so on) then many investigators would agree that this occurs following VEGFtargeted therapy. However, the term normalization has also been interpreted as consistently redistributing blood flow and increasing overall delivery of chemotherapy and oxygen during the normalization window. In this case, investigators in the field of angiogenesis have diverse views. Although it is likely that some degree of vascular normalization occurs with VEGFtargeted therapy in nearly all tumours, the concept that VEGF-targeted therapy leads to redistribution in blood flow and increased delivery of anti-neoplastic agents is probably not universal. For example, although some investigators have noted an increase in delivery of chemo therapy to tumours with VEGF-targeted therapy83,84, others have not noted any change in oxygenation (representing improved blood flow) over time85. In a recent study, investigators noted that VEGF-targeted therapy decreased permeability of the blood–brain barrier (BBB) in a murine glioma model and this impaired the effect of concurrent chemotherapy, “presumably by restoration of the BBB and obstruction of chemo-distribution to tumour cells”86. In human tumours one must take into account the characteristics of individual tumour types. For example, human pancreatic cancer is characterized by a high stromal content, and the stromal compartment can either directly contribute to angiogenesis or actually impede blood flow. This decrease in blood flow in human pancreatic adenocarcinoma is frequently observed with CT imaging. Thus, the ‘plasticity’ of the vasculature in pancreatic cancer is likely to be less than that of other tumours that have less of a stromal component. In addition, some tumours such as HCC and neuroendocrine tumours are inherently hypervascular with a vascular ‘flush’ seen on CT imaging with intravenous contrast. Thus, if one extrapolates ‘normalization’ to include an increase in drug delivery, this must be considered in the context of the tumour type and stage of tumour growth, as multiple tumours within the same patient may have different normalization windows. In contrast to the normalization hypothesis and expected increase in delivery of chemotherapy to tumours, Kasman, Baghri and associates87 have recently reported that an anti-VEGF antibody does not increase drug delivery to human tumour xenografts in mouse models. Instead, these investigators showed that VEGF antibody led to increased vascular damage when
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study showed that nitroglycerine decreased levels of hypoxia-inducible factor and VEGF expression in the tumour, suggestive of vasodilation76.
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Figure 5 | A direct effect of vascular endothelial growth | Cancer factor (VEGF)-targeted therapy onNature VEGFReviews receptor 1 (VEGFR1) expression has been observed on human tumour cells implanted in mice. a | Immunostaining of human breast invasive ductal carcinoma tissue with antiVEGFR1 monoclonal antibody (MAb) shows expression of VEGFR1 on subsets of tumour cells (brown staining)110. b | The direct effect of VEGF on human tumour cells can be investigated with species-specific MAbs. In this study, mice were implanted with human breast cancer xenografts that express VEGFR1. Mice were treated with MAbs that specifically recognize human VEGFR1 (IMC18F1). The use of this species-specific MAb led to decreased tumour growth, confirming a functional role for VEGFRs on tumour cells. Part a reproduced, with permission, from REF.110 Wiley-Liss, Inc. (2006). Part b reproduced, with permission, from Ref. 91 American Association for Cancer Research (2006).
combined with chemotherapy. Interestingly, these findings are in agreement with proposed anti-angiogenic mechanisms of metronomic chemotherapy, suggesting that VEGF-targeted therapy may sensitize the tumour vascular compartment to cytotoxic agents63. In contrast to the theory that VEGF-targeted therapy leads to vasoconstriction and improved blood flow, there are studies to suggest that vasodilators may actually increase the delivery and efficacy of chemotherapy. In a randomized phase II study in patients with non-smallcell lung cancer, patients were randomized to chemotherapy or chemotherapy plus nitroglycerine. Patients receiving nitroglycerine were observed to have an increased response rate (72% versus 42%)76. A follow-up
Direct effects on tumour cells Expression of all VEGFRs, as well as NP1 and NP2, has been detected on tumour cells88. Therefore, in addition to its effects on tumour vasculature, VEGF-targeted therapy may have direct effects on tumour cells that impair tumour growth and/or metastasis. However, direct clinical evidence to support this hypothesis has been difficult to obtain. VEGF mediates numerous tumour cell functions, including cell survival, migration and invasion. VEGF provides a survival signal for breast carcinoma cells in vitro and blockade of VEGF results in apoptosis of these cells89. In vitro studies using VEGFR1-expressing human colon cancer cells showed that a monoclonal antibody to VEGFR1 blocked tumour cell migration, invasion and colony formation as a surrogate marker of survival90. In human pancreatic cancer cells the activation of VEGFR1 leads to an alteration in cell phenotype, from an epithelial phenotype to a mesenchymal phenotype (the epithelial–mesenchymal transition)19. More recently, Wu et al. demonstrated that anti-VEGFR1 monoclonal antibody could block intracellular signalling and growth of human breast cancer xenografts91 (FIG. 5). These and other studies suggest that VEGF-targeted therapy may have a role in directly inhibiting tumour growth. A phase II clinical trial in patients with inflammatory breast cancer found that VEGFR2 was present and activated on tumour cells and that treatment with bevacizumab blocked the activation of tumour cell VEGFR2 (Ref. 52) . Recently, NP2 on CRC cells was found to mediate tumour cell migration, invasion and survival92. In vivo studies with liposomal small interfering RNA led to a decrease in tumour growth and metastasis, although it is unclear whether this is mediated through a VEGFdependent pathway. Thus, VEGF receptors are present and functional on tumour cells and VEGF-targeted therapy might have a direct inhibitory effect on tumour cells in addition to its effect on the vasculature. Immune modulation Accumulating evidence suggests that VEGF has an important role in establishing immune privilege of tumours by blocking dendritic cell (DC) differentiation93,94. DCs are bone marrow-derived antigen-presenting cells that have a crucial role in initiating immune responses against foreign pathogens95–98 and the presentation of tumour antigens to various immune effector populations. Factors released from the tumour microenvironment, including VEGF, stimulate recruitment of immature DCs from the bone marrow and peripheral tissues to sites of tumour growth99,100 (FIG. 6). Once in the tumour microenvironment, tumour-associated immature DCs capture tumour antigens derived from apoptotic cells and migrate to draining lymph nodes where they present these antigens to naive T cells. Inefficient presentation of tumour antigens can result in immune tolerance and immune privilege of tumour cells in the host.
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Figure 6 | Tumour-derived vascular endothelial growth factor (VEGF) inhibits maturation of dendritic cells (DCs). VEGF and other tumour-derived factors recruit immature DCs (iDCs) from the bone marrow and peripheral tissues Nature to sitesReviews of tumour | Cancer growth. Apoptotic tumour cells in the tumour microenvironment release tumour antigens that are engulfed by iDCs, resulting in their activation and differentiation into mature dendritic cells (mDCs). These mDCs migrate to peripheral lymph nodes where they present tumour antigens in the context of major histocompatibility complex (MHC) class I and II antigens to CD4+ and CD8+ T cells. VEGF released by tumour cells inhibits the maturation of of iDCs to mDCs, resulting in inefficient presentation of tumour antigens and immune privilege of tumour cells. VEGF-targeted therapy has the potential to reverse the negative effects of VEGF on iDC maturation, resulting in more efficient presentation of tumour antigens to the host immune system. GM–CSF, granulocyte macrophage colony-stimulating factor; HPC, haematopoietic progenitor cell; IL3, interleukin 3; TCR, T-cell receptor; VEGFR, VEGF receptor.
A role for VEGF in abnormal DC differentiation was first demonstrated in vitro93 and addition of neutralizing VEGF antibodies resulted in normal maturation of DCs93. These initial in vitro findings were confirmed in vivo. Administration of recombinant VEGF to tumourfree mice resulted in defective DC development and an accumulation of Gr-1+ (also know as LY6G+) immature myeloid-derived suppressor cells101. Furthermore, pretreatment of mice with VEGF inhibited FLT3 ligand stimulation of DC differentiation from bone marrow progenitor cells102. Treatment of tumour-bearing mice with neutralizing VEGF antibodies resulted in an increased number of spleen and lymph node DCs and improved DC differentiation101,102. Treatment with VEGF-targeted antibodies was also shown to improve anti-tumour peptide cytotoxic T-lymphocyte responses and efficacy of tumour immunotherapy in mouse models102. Thus, VEGF is a strong negative modulator of DC function in the tumour microenvironment, which contributes to immune privilege of tumours in the host. The potential pathways through which VEGF can inhibit DC differentiation have been investigated (BOX 1).
These preclinical findings suggest that blockade of VEGF signalling could improve anti-tumour responses in patients through improvement in DC function and immune recognition of tumour cells. Although intriguing, this hypothesis has been studied only to a limited extent clinically. In a phase I study of VEGF trap, DC and immune function was tested in 15 patients103,101. VEGF trap did not affect the total population of DCs, their myeloid or plasmacytoid subsets, myeloid-derived suppressor cells or regulatory T cells. VEGF trap did appear to significantly increase the fraction of mature DCs, suggesting that DC differentiation was improved in these patients. However, VEGF trap treatment was not associated with an overall increase in non-specific or antigen-specific T-cell responses. As these results were generated in a small patient cohort, it is difficult to draw conclusions from these data. Clearly, additional studies in larger patient populations are warranted to fully understand the importance of VEGF-targeted therapy on immune function in cancer patients. It will also be important to study VEGF-targeted therapy in combination with other tumour immunotherapy strategies, such as anti-CTLA4 and tumour vaccine therapies, that directly aim to enhance the immune response against tumours.
Counteracting VEGF and/or EPC upregulation One mechanism by which VEGF-targeted therapy may be of benefit to patients is by counteracting the upregulation of VEGF expression following genotoxic stress induced by chemotherapy or radiation therapy. VEGF expression is upregulated by variations in the microenvironment that are associated with stress, such as hypoxia, low pH and nutrient deprivation. Genotoxic stress induced by chemotherapy and radiation therapy has also been found to induce VEGF expression. Human melanoma cells treated with dacarbazine led to an increase in secreted VEGFA and interleukin 8 (IL8)102. In these studies, the authors found an induction of VEGF levels and increased promoter activity. In a follow-up study, this group showed that dacarbazineresistant melanoma cell lines demonstrated increased growth in vivo with increased microvessel density103. Others have shown that ultraviolet irradiation or photodynamic therapy can increase tumour cell VEGF secretion from keratinocytes or prostate cancer cells, respectively104. Lastly, irradiated tumour cells were shown to have increased expression levels of VEGF. Importantly, sublethal irradiation actually led to an induction of in vivo tumour growth hypothesized to be secondary to increased VEGF secretion 102. Our laboratory has recently shown that oxaliplatin induces VEGFA and other members of the VEGF family of ligands including PlGF and VEGFC. We also studied the effect of acute exposure (6–24 h) of oxaliplatin in induction of VEGF receptors on tumour cells, and found that VEGFR1 is induced by oxaliplatin exposure. Thus, one proposed mechanism of action of VEGFtargeted therapy is to offset induction of VEGF signalling, presumed to be survival signals for tumour cells.
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f o c u s o n Ta r g e t i n g a n g i oRE g eVnIE eW siS s Box 1 | VEGF effects on dendritic cell differentiation Vascular endothelial growth factor (VEGF)-stimulated activation of VEGF receptor 1 (VEGFR1) signalling is responsible for inhibition of immature dendritic cell (DC) maturation through a nuclear factor κB (NFκB)-dependent pathway93,101,104. The role of VEGFR1 activation in DCs was further demonstrated using DCs derived from Id1–/– mice that are defective in VEGFR1 signalling105. Inhibition of VEGFR1 signalling using a neutralizing VEGFR1-specific monoclonal antibody restored DC function105. Treatment of human monocyte-derived DCs with the VEGFR1-specific ligand placenta growth factor (PlGF) downregulates expression of CD80, CD86, CD83, CD40 and HLA-DR23. PlGF also inhibits tumour necrosis factor α (TNFα) production by DCs in response to lipopolysaccharide stimulation and suppresses CD4+ T-cell proliferation in an allogeneic mixed lymphocyte response assay. Thus, VEGF signalling through VEGFR1 inhibits NFκB activation in immature DCs, as well as downregulating costimulatory molecules, leading to defective functional maturation that may contribute to inefficient induction of immunity in cancer patients. It should be noted that a recent report has shown that VEGF may inhibit the antigen-presenting function of mature DCs by a VEGFR2 signalling mechanism106.
Another mechanism by which VEGF-targeted therapy might be effective is its role in blocking chemotaxis of bone marrow-derived progenitor cells by cytotoxic agents. There is evidence that tumour cell repopulation following administration of cytotoxic therapy is supported by mobilization of bone marrow-derived EPCs that home to sites of angiogenesis and promote tumour growth105. Mobilization and recruitment of bone marrow-derived circulating EPCs to tumour vasculature is rapidly induced following administration of vascular disrupting agents, and is probably due to VEGF induced chemotaxis106. This so-called ‘vasculogenic rebound’ can be blocked by VEGF-targeted treatments.
Allogeneic Allogeneic refers to cells or tissues from two different individual sources that are the same strain, but differ genetically in their major histocompatibility complex.
1. 2.
3.
4. 5.
Challenges and conclusions A large number of elegant preclinical studies have generated several hypothetical mechanisms for the clinical activity observed with VEGF-targeted agents. However, confirmation of these mechanisms in clinical research has proved challenging. We have outlined some of the obstacles with biomarker and tissue studies throughout this Review. In addition to those challenges stated earlier, one of the most important shortcomings in the field of VEGF-targeted therapy is the failure to identify and validate predictive markers (a priori, markers of drug activity that predict different degrees of benefit from a specific therapy). In order to identify predictive
Keck, P. J. et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 246, 1309–1312 (1989). Leung, D. W., Cachianes, G., Kuang, W. J., Goeddel, D. V. & Ferrara, N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246, 1306–1309 (1989). Hurwitz, H. et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N. Engl. J. Med. 350, 2335–2342 (2004). This seminal paper reports the results of the first randomized phase III clinical trial demonstrating the benefits of bevacizumab in combination with chemotherapy in patients with metastatic colorectal cancer. Escudier, B. et al. Sorafenib in advanced clear-cell renalcell carcinoma. N. Engl. J. Med. 356, 125–134 (2007). Llovet, J. et al. Sorafenib improves survival in advanced hepatocellular carcinoma (HCC): results of a phase III
markers it is necessary to understand the mechanisms of VEGF-targeted agents as it is likely that the predictive markers may be specific for a disease type and mechanism of action of VEGF-targeted therapy for that particular disease or angiogenic process. For example, CEPs are unlikely to serve as a predictive marker in tumours in which there is little uptake of CEPs into the growing vascular bed. In short, despite extensive investigation, there are no known predictive markers of VEGF-targeted therapy. In patients for whom VEGF-targeted therapy is found to be efficacious, it is also unfortunate that the duration of activity is relatively short, being on the order of 3–8 months with single-agent therapy. Preclinical investigations have provided insights into mechanisms of VEGFtargeted drug resistance (see the Review by Hanahan and Bergers in this issue)9. We also need to develop better agents and develop combination regimens that synergize with VEGF. Preclinical studies suggest that targeting other members of the VEGF family such as PlGF may be efficacious when resistance develops to therapies targeting the VEGF ligand 107. In addition, there are ongoing clinical trials investigating the effects of ‘vertical pathway inhibition’; for example, bevacizumab combined with a VEGFR TKI. Furthermore, preclinical studies have shown that targeting the VEGF ligand and neuropilin can enhance efficacy in preclinical models23. Targeting complementary angiogenic pathways, such as the anti-VEGF and anti-DLL4 combination, has also been shown to improve efficacy in preclinical models. There are currently over 20 different VEGF-targeted agents in clinical trials. Monoclonal antibodies selectively target components of the VEGF pathway and this approach has perhaps less toxicity owing to the lack of off-target effects. However, TKIs that have off-target effects can be an advantage because they target multiple kinases involved in tumour growth and angiogenesis, albeit at the cost of potentially higher and more frequent toxicities. As stated throughout this Review, it is essential to test these hypotheses in well-conducted clinical trials. Results from clinical trials must then be interpreted without bias in order to fully understand how targeting VEGF affects the tumour microenvironment as well as normal host tissues.
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11. Hicklin, D. J. & Ellis, L. M. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J. Clin. Oncol. 23, 1011–1027 (2005). A comprehensive review of VEGF biology. 12. Kowanetz, M. & Ferrara, N. Vascular endothelial growth factor signaling pathways: therapeutic perspective. Clin. Cancer Res. 12, 5018–5022 (2006). 13. Waltenberger, J., Claesson-Welsh, L., Siegbahn, A., Shibuya, M. & Heldin, C. H. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J. Biol. Chem. 269, 26988–26995 (1994). 14. Hiratsuka, S., Minowa, O., Kuno, J., Noda, T. & Shibuya, M. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc. Natl Acad. Sci. 95, 9349–9354 (1998).
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Competing interests statement
The authors declare competing financial intererests: see web version for details.
DATABASES Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene BCL2 | CD4 | CD40 | CD80 | CD83 | CD86 | CDH5 | CSF1R | CTLA4 | CXCL12 | DLL4 | FAS | FASLG | FIGF | FLT1 | FLT3 | FLT4 | Id1 | IL8 | KDR | KIT | LY6G | NFκB | NRP1 | NRP2 | PDGFRB | PGF | TNF | VEGFA | VEGFB | VEGFC | VHL National Cancer Institute: http://www.cancer.gov/ breast cancer | colorectal cancer | pancreatic cancer National Cancer Institute Drug Dictionary: http://www.cancer.gov/drugdictionary/ axitinib | bevacizumab | endostatin | interferon-α | oxaliplatin | paclitaxel | sorafenib | sunitinib | TNP-470
FURTHER INFORMATION Angiogenesis inhibitors in clinical trials: http://www.cancer.gov/clinicaltrials/developments/antiangio-table All links are active in the online pdf
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volume 8 | august 2008 | 591 © 2008 Macmillan Publishers Limited. All rights reserved.
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