以肿瘤血管为靶标的癌症治疗研究

以肿瘤血管为靶标的癌症治疗研究
　　李鲁远（美国乔治城大学医学院，华盛顿2000 7）
　　［摘要］异常活跃的血管发育是恶性肿瘤生长的一个重要条件。肿瘤血管有 3个要素使它成为开发抗癌药物的一个较好的靶标。第一，同基本处于静止状态的正常血管相比，肿瘤血管内皮细胞处于高度生长状态，因此成为突出目标。第二，肿瘤血管细胞是从正常组织进人肿瘤的正常细胞，基因组稳定，不象基因组极不稳定的癌细胞那样容易产生多种抗药性，因此可能较易控制。第三，尽管各种肿瘤细胞差异极大，其血管细胞因为是正常细胞，差异较小，因此同一药物如果针对肿瘤血管则可能对不同肿瘤均有疗效。本文对目前国际上对肿瘤血管发育机理的了解，常用的研究手段，及抗血管生长药物开发等方面的
概况作一简介。
　　【关键调】肿瘤血管；肿瘤；治疗
　　［中囹分类号］ R730．5［文献标识吗］ A
Targeting Tumor Vasculature for Anti-Cancer Therapy
Lu-Yuan H (tombardi Cancer Center, ffeorgetown University Medical Center, 3970 Reservoir Road, washington
DC, 20007, USA).
Introduction
Tumor vascularisation is a vital process for the 
progression of all solid tumors from a small, localized focus to
an enlarging tumor with the capability to metastasize. This
process, illustrated in Figure 1, involves the tumor 
inducing capillary sprouts from surrounding vasculature by 
altering the balance of angiogenic promoters and inhibitors.
This leads to the formation of a large anastamosing vascular
network, whieh is structurally and physiologieally different
from native vessels. This allows for tumor intrasavation,
embolisation and metastatic spread. Tumors that either
fail, or are prevented from undergoing that process remain
at the stage of a small 2 ~ 3 rum3 focus. There is great 
interest in developing new therapeutic approaches that target
tumor neo-vasculature, given that the overall standardized
mortality from the majority of solid tumors has altered only
marginally over the last two decades with traditional 
treatment regimens.
Anti-angiogenesis as a therapeutic concept was largely
developed in the early 1970s after a body of experimental
work had confirmed the biological importance of tumor 
angiogenesis. Therapeutic approaches aimed at preventing
the process of neo-vascularisation were termed anti-
angiogenic, and excluded therapies that would target existing
tumor vasculature by infarction or vasoconstriction. 
Vascular attack is a term used to describe therapeutic strategies
thst target differences between normal vasculature and
tabor vasculature. Such differences include the greatly
increased rate of endothelial cell proliferation in tumor 
vasculature as compared to quiescent normal vasculature and
approaches directed at differential expression of specific
endothelial markers on tumor and normal vessels.
Importance of vascularity in tllmor development
It has long been recognized that tumors possess an 
abundant vasculature and that this vasculature plays an 
important role in the biology of a growing cancer. In 1904,
Ribbert postulated that Tumors have a superior vascular
supply and in 1908 Bowen theorized that eomplete 
obliteration of the blood supply to a tumor means sphacelation,
separation and cure. AlgUire drew attention to this 
phenomenon as being an active process instigated by a tumor,
noting that an outstanding characteristic of the rapidly
growing tumor is its capacity to elicit the production of a
new eapillary endothelium from the host. The process of
tumor vascularisation was visualized in experimental 
animals by transplanting tumors into transparent chambers in
the cheek or ear, or directly into the aqueous humor of the
eye. Warren visualized the growth of tumor vessels into
transplanted metastatic melanoma and noted that these 
vessels were morphologically distinct from host vessels, being
tortuous, thin walled, with sluggish flow. Using such
models, the phenomenon of tumor dorfnancy was 
described. Tannock demonstrated that the mitotic index of
tumor cells decreased with distance from the supplying
blood vessel, by measuring cell division by tritiated 
thymidine uptake in a murine tumor with a partieularly orderly
vascular tree. By altering ambient oxygen tensions he 
demonstrated that a mathematical model of oxygen diffusion
could predict the change in mitotic index. The overall rate
of tumor growth approximated to the rate of endothelial cell
proliferation, and not to the much higher rate of tumor cell
proliferation. He concluded that the nutrient supply to the
tumor, as governed by the proliferation of the 
endotheliuml limited tumor size by balancing tumor cell 
proliferation with concomitant tumor necrosis.
Folkman noted that tumors transplanted into isolated
perfused organ systems such as the rabbit thyroid or dog
intestinal segment, were uniformly size limited. Though
not immediately aPParent, the size limitation occurred 
because the perfusate in these systems prevented neo-
vascularisation due to the lack of platelets. By comparing the
groWth of transplanted autologous tumors in the avascular
aqueous humor with the vascular iris he could show 
distinct avascular and vascular phases of tumor growth, Which
was confirmed by intravenous fluroscein injections. 
Commencement of vascularisation in the iris coincided with
tumor growth beyond 2 ~ 3 nun3 and an increase in the rate
of tumor doubling size by approximately-20 fold. Tumors
implanted in the avascular aqueous humor failed to grow
beyond 2 ~ 3 mms. He concluded that vascularisation of a
tumor was an essential step in the progression of a tumor
beyond a small primary focus and inferred that Preventing
this process, which he termed anti-angiogenesis was a 
viable therapeutic approach.
Angiogenesis assays
The are a number of bioassays that are used for 
measuring experimental and clinical angiogenesis. As noted 
above transparent chambers techniques for visualizing tumor
vascularization were developed in the early twentieth 
century. The long-term cultivation of human and bovine 
capillary endothelial cells allowed for assays of groWth, 
proliferation, chemotaxis and tubule formation assays. 
Development of chorio~allantoic membrane (CAM) assay and the
intra-comeal assay allowed for measurement of the total
angiogenic response to purified angiogenic proteins or 
inhibitors. In the CAM assay, a chick embryo can be 
maintained in a petri dish and angiogenie agents or tissue 
samples can be placed directly onto the avascular allantoic
membrane where new blood vessel formation can be 
quantitated. In the intra-comeal assay a small pocket is created
surgically in the eornea of an experimental animal and the
sample to be assayed is inserted in this pocket. New vessel
growth can readily be observed. Other angiogenesis models
have been reviewed comprehensively elseWhere.
Angiogenic switch in tumor development
Adult endothelium is essentially quiescent. In 
response to both physiological stimuli (as seen in the 
proliferative endometrium and ovary) and pathological 
circumstances (injury, tumor growth and diabetic retinopathy)
this quiescent endothelium can alter to a rapidly 
proliferating and organizing population of cells. Physiological 
angiogenesis can also be rapidly curtailed indicating that the
process is held in check physiologically and can be 
activated in response to appropriate stimuli I in a fashion 
analogous to the clotting cascade. A considerable number of
positive and negative regulatory molecules have been 
implicated in the control of this process.'
By contrast the vasculature of a tumor is not quiescent
but is rapidly proliferating. Endothelial cell turnover in
tumor vasculature has been shown to be up to fifty fold
greater than that seen in quiescent adult vasculature. A
tumor induces this proliferative vascular response from host
vessels by altering the balance of positive and negative 
innuences in its local vicinity and may utilize a number of
different strategies to effect this change. The acquisition of
a pro-angiogenic phenotype has been termed the 
angiogenic switch and may he a rate-limiting step in tumor 
progression.
Evidence exists that tumors undergo a switch from a
non-angiogenic Phenotype to an angiogenic phenotype as
they progress from pre-malignant stages to frankly invasive
cancers. In human tumors the development of vascularity
within a tumor can be associated with progression of a
tumor from a non-invasive pre-malignant stage through 
defined stages to an invasive carcinoma. In cervical 
carcinoma staining of micro-vessels with an antibody to Von 
Willebrand factor shows a clear increase in the number of
micro-vessels apposed to the basement membrane between
cervical intra-epithelial neoplasia (CIN) l&II as compared
with CIN Ill lesions. Furthermore, micro-vessel density,
as a surrogate for angiogenic phenotype, is a powerful 
independent prognostic indicator both of distant metastasis
and survival. After the initial reports of micro-vessel 
density as a prognostic indicator in melanoma and breast 
carcinomal a substantial body of evidence now exists that 
micro-vessel density in a primary tumor can predict 
metastasis and survival in a variety of cancers j including 
nonsmall cell lung carcinoma, prostate carcinoma, bladder
carcinoma, head and neck carcinoma, rectal carcinoma
and CNS tumors.
Angiogeulc factors
At least thirteen angiogenic polypeptides that have
positive effects on standard assays have now been 
identified, not all of which have been imPlicated in the 
of tumor angiogenesis. The two positive regulators that are
implicated most commonly in the neo-vascularisation of
tumors are vascular endothelial gtowth factor (VEGF) and
basic fibroblast growth factor (FGF-2 ). In addition to
their active soluble forms, both VEGF and FGF-2 appear
'to exist as inactive molecules sequestered in extra-cellular
matrix, presumably facilitating their physiological 
regulation. Tumors can locally up-regulate these factors both by
increased production and by release of the sequestered 
inactive molecules.
i) Vascular endothelial growth factor (VEGF)
VEGF is an angiogenic factor that has been strongly
implicated in tumor neo-vascularisation. This protein was
first identified as a vascular permeability factor that played
a role in ascites formation. Its more general angiogenic
properties were identified later when it was fully 
characterized. VEGF is a secreted protein that is mitogenic for 
endothelial cells and yet has no appreciable mitogenic 
activity on other cell types. It has a wide range of angiogenic
effects on endothelial cells,- including ehemotaxis and 
capillary tubule formationl'n VI'ho. It is positively angiogenic
in the CAM and comeal pocket assay. The biology of
VEGF is interesting in that it exists as four different 
isoforms of various lengths in their primary sequences
(VEGFlzl, VEGF16s, VEGFlso, VEGFzo6 ). These 
isoforms are all products of the same gene but are produced
by differential messenger RNA splicing. The significance
of these isoforms is that the larger peptides bind with 
higher sanity to heparan like proteoglycans and thus are 
effectively sequestered by the extra-cellular matrix. They can
however be cleaved into soluble shorter molecules by 
proteases such as plasmin. This may be an important 
physiological means of regUlation, and it may be particularly 
important in the setting of a tumor where proteolytic activity
is up-regUlated. Two high affinity VEGF receptors with
tyrosine kinase activity have now been identified (fib-l,
KDR) and the expression of these is restricted to vascular
endothelium.
n) Fibroblast growth factors
The fihroblast growth factors (FDFs) are a family of
structurally homologous cytokines now totaling 12 in 
number. They are functionally diverse, with roles in embryonic
development, neo-vascularisation and wound healing. The
prototype molecule, basic fibroblast growth factor 
(FGF2) was initially identified from bovine brain and pituitary
extracts and later purified from tumor cell lines as a 
capillary endothelial growth factor. FGF-2 1 and the other 
prototype molecule acidic fibroblast growth factor (FGF-1 ),
are potently mitogenic for endothelial cells and for a wide
variety of other cell types of mesodermal and neuro-
ectodermal lineage. They are chemotactic for endothelial cells
and fibroblasts and induce differentiation a variety of
cells.
The importance of this family of growth factors in
tumor biology is demonstrated partly by the fact that later
additions to the FGF family (FGF-3, FGF4 & FGF-5 )
are oncogene products that can transform cells 1'n VI'ho.
Also transfecting cDNA coding for FGF- 1 and FGF4 into
poorly tumorigenic non metastatic cell lines will convert
them into angiogenic metastasizing clones. Elevated levels
of basic FGF have been demonstrated in serum and urine
in a significant proportion of patients with a variety of
cancersl ranging from 29% in breast cancer patients to
60% in sarcoma patients.
Both basic and acidic FGF lack a secretion peptide
' but nevertheless are transported outside of the cells. These
factors are ubiquitously present in the extra-cellular 
matrix, bound to heparin-like proteoglycans. The 
transportation mechanism of fibroblast groWth factors is not yet clear.
There are a number of mechanisms by which tumors 
release FGF s from tumor cells or the inactive extra-cellular
reservoir to induce anglogenesis. Firstly FGF-3, FGF4&
FGF-5 are all pl.oducts of oncogenes and possess secretion
peptides and are secreted by tumor cells as mature 
glycosylated soluble angiogenic factors. Sdly in mouse 
fibrosarcomas t the tumor develops the ability to release soluble
and active FGF-2, that normally is bound to 
proteoglycans, in a signal peptide independent fashion. This
change in phenotype coincides both with progression to an
angiogenic, invasive tumor from non-invasive fibromatosis.
Thirdly heparanases can degrade heparin like 
proteoglycans, thus releasing active FGF from the extracellular 
matrix.
Anti-angiogenic factors
As noted earlier, normal vasculature is essentially
quiescent and physiological angiogenesis is often swiftly
terminated, whereas pathological angiogenesis is often 
prolonged and difficult to terminate. This would imply the 
existence of an endogenous negative regulatory system that
may be defective or overridden in tumor angiogenesis. An
increasing number of endogenous compounds have been 
identified that have inhibitory activity on endothelial cells
assessed byin Vjtro andin VI'ro assays.
i) Throlnbospondin
Thrombospondin is a 160 Kd adhesive glycoprotein,
found in platelet alpha granules involved in platelet 
aggregation. A 140 Kd fragment of thrombospondin is a specific
anti-angiogenic factor and also is under transcriptional
control of the tumor suppressor gene p53. Thrombospondin
was identified in the conditioned media of a hamster cell
line that is one step away from being tumorigenic and can
be transformed into a tumorigenic cell line by addition of
carcinogens. The parental cell line is non-angiogenic in a
comeal assay but the transformed cell line is. The 
conversion to the angiogenic phenotype is caused by the loss of a
single allele of p53, which caused loss of production of an
inhibitory glycoprotein, which was shown to be a fragment
of thrornbospondin. Subsequently other fragments of 
thrombospondin have been shown to have inhibitory activity.
n) Angiostatin and endostatin
Angiostatin was identified from a lung carcinoma cell
line that is particularly effective in suppressing metastatic
growth until the primary tumor is removed. Sera and urine
from animals with this primary tumor have anti-angiogenic
activity in in VJ'tro assays. Animals bearing this primary
tumor inhibit the growth of blood vessels to an angiogenic
stimulus (FGF-2 ) implanted into the cornea, but when
the primary tumor wus removed, vessel growth in response
to FGF-2 was preserved. A circulating angiogenic 
inhibitor I subsequently named angiostatin, was purified from the
urine of these animals, and found to be a 38 Kd fragment
of plasminogen. Purified angiostatin when given 
systemically can reproduce the inhibitory effect of the primary
tumor after it has been surgically removed. The anti-
metastatic effects of systemic angiostatin have been coallrmed
in a number of cell lines with little evidence of toxicity. A
prostate cancer cell line, which also demonstrates this 
inhibitory phenomenon I can enzymatically cleave 
plasminogen to form angiostatin, which is active inin aam and in
vial angiogenesis assays.
Another circulatory inhihitor I endostatin I was 
identified using the same strategy. Endostatin is a fragment of
collagen XVlll, a form of collagen that exists exclusively
in blood vessels. Once again this eompound can inhibit
angiogenesis in standard assays and cause complete 
regression of a variety of formed tumors in experimental animals I
measuring up to l% of body mass. Repeated intermittent
treatment with endostatin, after tumors have re-grown from
their regressed dormant state, will induce regression with
no sign of drug resistance.
iii) Vascular endothelial growth inhibitor
Vascular endothelial cell groWth inhibitor (VEGI) is
a recently described cytokine that has been implicated in
maintenance of normal vascular quiescence and is also as a
novel anti-angiogenic agent. The physiology of this protein
is interesting in that it is produced almost exclusively by
endothelial cells and the target cells for activity I through a
presumed cell surface receptor, are also endothelial cells.
The cytokine was identified by a subtractive technique
whereby messenger RNAs (mRNA) that were expressed
only in endothelial cells were identified and cloned. VEGI
mRNA codes for a 174-amino acid protein that exhibits
considerable structural homology to tumor necrosis family
(TNF ) family members. Subsequent northern blotting 
analysis confirmed expression only in endothelial cells.
However VEGI mRNA is widely expressed in many adult
human tissues suggesting a physiological role in adult 
vasculature. The primary sequence of the VEGI protein 
reselnLbles a type 11 membrane protein with a brief 
intracellular segment and most of the protein being an extracelhllar
domain, similar to most TNF family m6mbers. Since many
TNF family members are cleaved from the membrane to
function as soluble paracrine factors on target cells with
appropriate receptors, a similar mechanism of activation is
assumed for VEGI. A recombinant soluble form of this
protein was found to have highly potent anti- angiogenic
activity in endothelial proliferation assays, while having no
effect on the proliferation of non-endothelial cells. 
Furthermore tubule formation on collagen gels and vessel 
formation on the CAM in response to either FGF-2 or VEGF
were all inhibited by VEGI.
To mimic the presumed physiological mechanism of
activity where the extracellular domain of the protein acts
as a soluble factor, a secreted form of VEGI was over-
expressed in murine colon cancer cells. The transfected
cancer cells had greatly decreased tumorigenicity when 
implanted in C57/BL syngenic mice and inhibition of tumor
angiogenesis was evident from much decreased intra-
tumoral microvessel density. Similar anti-tumor effects were
found when this secreted form of VEGI was expressed in
non tumorigenic Chinese hamster ovary cells that were then
co-inoculated into athymic mice with aggressive breast
cancer cell lines. The resulting inhibition of growth of the
breast cancer cells indicates a paracrine anti-tumor effect
by VEGI presumably by inhibition of tumor 
vascularisation. Taken together, these findings suggest that the 
protein functions physiologically as an endogenous mediator of
vascular quiescence via a juxtacrine or paracrine 
mechanism, and also implicate VEGI as an attractive candidate
molecule for anti-angiogenic therapy.
Tumor endothelial cells as a therapeutic target
Another anti-vascular approach is to direct toxic 
agents at the tumor endothelial cell. This approach has a
number of theoretical advances over conventional cytotoxic
therapy whose principal mechanism of action is directed
principally at the tumor cell. First, endothelial cells are
not transformed and as such are unlikely to acquire 
mutations resulting in drug resistance. Second, as native host
cells, therapy directed at endothelial cells would be 
applicable to solid tumors generally, irrespective of tumor cell
origin. Third endothelial cells are uniquely exposed to
blood borne agents, circumventing a problem of drug 
delivery to the center of a tumor, which is a major hurdle in
conventional treatment.
A variety of exogenous anti angiogenic agents that are
under clinical evaluation appear to have direct anti-
proliferative effects on endothelial cells. Although some 
synthe\ic compounds exert anti-endothelial effects through 
endogenous growth factors (e. g. pentosan polysulfate binds to
heparin binding groWth factors) such as FGF, other have
direct ant-endothelial activity. The fumagillin derivative
TNP470 is a potent synthetic anti-angiogenic compound
that prevent'endothelial cell entry into the cell cycle.
Carboxyamido-triazole inhibits signal transduction of 
endothelial cell receptor tyrosine kinases. Other anti-
angiogenic compounds, such as thalidoide, have nuclear 
mechanisms of action.
There are potential endogenous target molecules for
such an approach. As tumor vessels are actively 
proliferating, a number Of molecules are over-expressed on actively
dividing endothelial cells whilst absent or weakly 
expressed L. quiescent vessels. The integrin av P3 is 
specifically expressed on proliferating vessels in healing
wounds and granulation tissue and also in tumor 
vasculature while not on quiescent vasculature. A monoclonal 
antibody to integrin av P3 prevent angiogenesis in the CAM
assay, and can cause melanoma tumor regression in 
animals as a result of endothelial cell apoptosis. Other 
candidate molecules that may be specific to tumor vasculature
include the VEGF receptors, the EN7/44 antigen and 
endosialin.
Condusions
The process of neo-vascularisation is a pre-requisite
for tumor development beyond a small primary focus. The
biology of the humoral mechanisms underlying this process
is increasingly understood and is the basis for a number of
novel approaches to treatment of solid tumors. As a result
of this research, many compounds that have a partial or
complete anti angiogenic mechanisms of action are now 
under assessment in clinical trials. Future clinical research
will focus on the potential for synergistic action with 
conventional ehemotherapy and also new regimens for the 
administration of anti angiogenic drugs. Given the role of 
angiogenic inhibitors in the maintenance of tumor dormancy,
it is possible that these agents may be effective in the 
maintenance of long term remissions, an approach not 
currently used for solid tumors.