-
[CANCER RESEARCH 36, 2807-2812, August 1976]
of other tissues (5). The vitreous normally lacks blood yessels.
In diabetes, however, blindness can result from theproliferation of
retinal capillaries into the substance of thevitreous (14).
Because of the possibility that a diffusible
vasoformativesubstance, similar to TAF, mediates this process, we
implanted tumors into the vitreous at various distances fromthe
retina. Tumor angiogenesis, unexpectedly, proceededdifferently than
in previous experimental sites. In the vitreous, tumors remained in
a prolonged avascular state despite proximity to retinal
capillaries; they became vascularized only when contiguous with the
retinal surface.
MATERIALS AND METHODS
Tumor Stock. Two long-term lines of experimental tumors, the
rabbit V2 carcinoma (30) and the mouse ependymoblastoma (40), were
transferred s.c. in homologous animals every 2 to 3 weeks. The
vascular edge of a palpable 1-to 2-cm nodule was passed through a
cytosieve and thecells were suspended in 2 ml of cooled lactated
Ringer'ssolution. Viability exceeded 90% by trypan blue
exclusion.
Host Animals. Tumor cells were transplanted to the vitreous of
either the rabbit or the dog. The New Zealand Whiterabbit was used
because (a) the V2 carcinoma is homologous to it, (b) the rabbit's
retinal vessels lie in direct contactwith the vitreous gel, free
from surrounding glial tissue, and(C) the vessels are restricted to
a limited zone of the retinal
surface, so that comparisons could be made between vascularized
and nonvascularized areas (13). The retina of thedog more closely
resembles that of the human because it iscompletely vascularized
and has an inner limiting membrane between the vitreous and the
retinal vessels. Theretinal vessels of young puppies, however, can
proliferateinto the vitreous in response to an experimental
stimulus(hyperoxia) (32).
Intravitreal Implantation. General anesthesia was induced with
i.v. pentobarbital (Pitman-Moore, Inc., Washington Crossing, N. J.)
for adult albino rabbits, and i.m.fentanyl-droperidol (Veterinary
Laboratories, Inc ., Lenexa,Kans.) for adult and neonatal dogs. The
pupils were dilatedwith tropicamide (Alcon Laboratories, Inc., Fort
Worth,Texas). After topical anesthesia, a 27-gauge needle waspassed
through the superior-temporal sclera, 4 mm postenor to the limbus.
The needle tip could be advanced to anypoint between the lens and
the retina with the guidance ofan indirect ophthalmoscope. Facility
with this instrument
2807AUGUST 1976
Prolonged Tumor Dormancy by Prevention of Neovascularizationin
the Vitreous1
Steven Brem,2 Henry Brem, Judah Folkman, Daniel Finkelstein, and
Arnall Patz3
Retinal Vascular Service, Wilmer Ophthalmological Institute,
Johns Hopkins Hospital, Johns Hopkins University School of
Medicine, Baltimore, Maryland21205 (S. B. , D. F., A. P.]; National
Cancer Institute, Bethesda, Maryland 20014 (S. B.J; and Department
of Surgery, Children's Hospital Medical Center andHarvard Medical
School, Boston, Massachusetts 02115 (H. B. , J. F.)
SUMMARY
Tumors release a diffusible substance that
stimulatesneovascularization. To study the neovascularization
thatoccurs in diabetic retinopathy, we implanted V2 carcinomasand
mouse ependymoblastomas into the vitreous of experimental animals.
In the vitreous, unlike previous sites, thetumors failed to
stimulate neovascularization. They grew forweeks as small,
unvascularized, three-dimensional aggregates of cells. Explosive
growth into a large, vascularizedmass occurred when the avascular
tumors reached the retinal surface. The vitreous proved to be a
valuable model forobserving the in vivo growth of small, solid
tumors. Xenografts survived for months without evidence of
immunerejection. The consequence of the prolonged avascularstate is
the restriction of tumor size. The normal vitreousmay act to
inhibit capillary proliferation. An understandingof the mechanism
for maintaining the avascular state maylead to therapeutic blockade
of neovascularization. Thiswould be important in the management of
diabetic retinopathy and neoplasia.
INTRODUCTION
Capillary proliferation is important in the pathogenesis ofboth
neoplasia (1, 10, 16, 21) and diabetic retinopathy (2,33, 38). For
tumors, growth occurs in 2 phases: the avascular phase, in which
growth is limited by di'ffusion of nutrients, followed by the
vascular phase, in which rapid growthis associated with
neovascularization (19). A potent signalfor capillary proliferation
diffuses from the tumor (26), stimulating capillaries at distances
as far as 2- to 5 mm from theneoplasm (12, 24, 25). A wide variety
of animal and humantumors contain this biochemical message, called
TAF4 (23,37).
We report here that the vitreous chamber of the eyeprovides a
unique model to study the in vivo behavior oftumors in the
avascular phase. The vitreous, the spacebetween the lens and the
retina, consists of collagen, glycosaminoglycans, and proteins
similar to the ground substance of connective tissue or the
interstitial compartment
1 Supported by Grants EY-205 and EY-O1368 from the National Eye
Insti
tute and CA-14019 from the National Cancer Institute.2 To whom
requests for reprints should be addressed , at Baltimore Cancer
Research Center, 22 S. Greene Street, Baltimore, Md. 21201.3
Recipient of a Career Award from THE SEEING EYE, Inc.
4 The abbreviation used is: TAF, tumor angiogenesis factor.
Received November 26, 1975; accepted April 26, 1976.
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-
S.Brem etal.
was learned rapidly; a standard, readily available, 20-diopter
convex lens and a light source was used (35). Fifty @I,containing
iO@to 106cells, were injected gently. Each inoculation took less
than 1 mm and was atraumatic; the vitreous remained transparent.
Contact with the lens or retinawas avoided to prevent a cataract or
retinal tear.
Ophthalmoscopy. Serial observations were made withthe indirect
ophthalmoscope, supplemented by slit-lampphotography and
fluorescein angiography. Tumor size wasestimated by comparison with
injected microspheres ofknown diameters (45 ±5, 97 ±7, and 325
±9 .tm, aluminummicrospheres from the Particle Information
Service, Grant'sPass, Oreg.).
Histology. At the completion of the experiments, someanimals
received an intravitreal injection of [3Hjthymidine(specific
activity, 14 Ci/mmole; Schwarz/Mann, Orangeburg, N. V.) for
autoradiography (15); others received anintracarotid infusion of
colloidal carbon (Guenther-Wagner,Hannover, Germany) to outline the
microvasculature (25).All eyes were enucleated , fixed in 10%
buffered formalin,embedded in paraffin, and stained with
hematoxylin andeosin for routine histological examination.
RESULTS
Chart 1. Tumors within the vitreous remain unvascularized. A, a
spheroidal colony 1 week after implantation; B, slow tumor growth
for severalweeks as a cylindrical stalk directed towards the
retinal vessels. Although thetumor advances close to the retina,
angiogenesis occurs only after the tumoris contiguous with the
retina in C. Two weeks later, D shows a large,vascularized mass
protruding from the retina.
l5Oi
@ 100
a:0
@ 50I—
z4Lu
0
Rabbit Carcinoma. Tumor growth was observed in 66 of75 rabbit
eyes. For as long as the tumors were within thevitreous, up to 100
days, they remained unvascularized.Proliferation of retinal vessels
never occurred until the tumors were contiguous with the
retina.
After the 1st week, the eyes contained generally 1 but asmany as
12 tumors displaying 2 patterns of growth: (a)spheroidal nodules;
these grew slowly, remained avascular,and achieved maximal
diameters of 0.25 to 0.50 mm, (b)cylindrical stalks; these had
similar cross-sectional diameters but grew as far as 3 to 7 mm
along a path directedtowards the retinal vessels on the optic disc.
This pathfollowed a posteriorly directed movement of fluid
normallypresent in the rabbit vitreous (28). Eventually, many of
thespheroidal tumors also produced a linear stalkdirected towards
the optic disc (Chart 1, A and B).
Once the tumor stalks reached the retinal surface, thetumors
became vascularized by proliferating retinal vessels(Chart 1C). The
vascularized tumors entered a new, explosive phase of growth (Chart
2). Within 2 weeks, a largeexophytic mass, representing
approximately a 19,000-foldincrease in volume, grew along the
vascularized portion ofthe retina and protruded into the vitreous
(Chart iD). Afterlocal invasion into the retina and optic nerve,
the tumorsinfiltrated the choroid and the sclera.
Exponential growth was also observed in 11 of 75 eyesthat
developed vascularized carcinomas at the injection siteon the
scleral surface. The intravitreal portion of thesetumors, along the
injection tracks, remained avascular withalmost no change in
size.
Ocular changes were absent in 20 eyes that receivedinjections of
controls: 0.9% NaCI solution, India ink, aluminum microspheres,
fresh liver homogenates, or boiled V2carcinomas. Liver homogenates
and boiled tumors disappeared after a few weeks. The surrounding
vitreous media
VASCULAR TUMOR
/
.---. -. -.---s--—-.------.
2 4 6 8 0 II
WEEKSChart 2. Differences in tumor growth in the avascular and
vascular state.
Three rabbit carcinomas were implanted into the vitreous at a
distance of 2mm from the retinal vessels. For over 9 weeks, the
tumors remained dormantin the avascular phase, initial volume of
3.3 ±2.4 (S.D.) x 1O@cu mm slowlyincreased to 7.0 ±6.5 x 1O@@cu
mm. Arrow, onset of retinal neovascularization. Two weeks later, in
the vascular phase tumor volume increased about19,000-foldto126
±19cu mm.
remained transparent throughout the study, as it had in theeyes
with the viable tumor. Retinal vascular changes wereabsent in all
of the control eyes.
Histologically, the vascularized tumors contained anaplastic
cells with mitotic figures as well as proliferatingcapillaries. By
contrast, the unvascularized tumors showedan outer layer of 10 to
20 viable cells and an inner necroticcenter (Fig. 1). Incorporation
of [3Hjthymidine was restricted to the tumor cells at the periphery
of the tumor; theendothelial cells of the retina failed to
incorporate[3H]thymidine in the eyes with the unvascularized
tumor.These tumors and the surrounding vitreous were free
offibroblasts, leukocytes, or other cells from the host.
In another set of experiments, xenografts of rabbit V2carcinoma
were transplanted to the vitreous of the dog.During 6 months of
observation, these tumors grew veryslowly in 5 of 13 puppy and 3 of
4 adult eyes. The tumorsremained close to the lens and did not
approach the retinal
2808 CANCERRESEARCHVOL. 36
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DISTANCE OFI TUMORFROM
R@RL@@5@*mZAhb0N@ 7 DAYS@ 42 DAYS
Prolonged Tumor Dormancy
surface; the retinal vessels appeared normal. Histologically,the
tumors resembled the unvascularized nodules seen inthe rabbit
vitreous. Viable tumor cells were clumped together in small
colonies; host cells or vascular elementswere absent (Fig. 2).
Mouse Brain Tumor. The mouse ependymoblastomaformed spheroidal
colonies in 8 of 12 rabbit eyes. Thesetumors were observed for more
than 4 months. The tumorsremained unvascularized and at a distance
of greater than 2mm from the retinal surface (Fig. 3A). As in the
previousexperiment with xenografts, there was no gross or
microscopic evidence of an immune rejection. Histologically,
thecells appeared viable, especially at the periphery of thecolony
(Fig. 3B).
DISCUSSION
These studies show that: (a) the vitreous provides a valuable
model to investigate the growth of transplantable tumors; (b) this
model displays the longest demonstration ofin vivo avascular tumor
growth; and (c) tumor angiogenesisproceeds differently in the
vitreous than in previously studied models. This difference
suggests that vitreous may actas an inhibitor of
neovascularization.
The advantages of the vitreous over conventional in vivoand in
vitro systems are: (a) the vitreous chamber is virtuallyacellular
so that growth can be observed without contamination by host cells;
(b) the clarity of the media permitsimmediate, direct observation
of tumor colonies as small as0.02 mm in diameter. Small tumors of
this size might beuseful in studies of early neoplastic events or
micrometastases (34); (C) the viscosity of the vitreous gel and its
collagenous matrix enable the tumor implants to remain in
arelatively fixed position for repeated observations; (d)
thetechniques are simple. The vitreous contains its own nutrient
media, obviating the complexities and artifacts inherentin tissue
culture and in perfusion of organ cultures (20).
The vitreous appears to be an immunologically privilegedsite
since incompatible grafts survive over prolonged penods.
Characteristics of vitreous that may account for itsimmune
privileged status include avascularity and an intercellular matrix
that prevents the host immune cells fromattacking the transplant
(5). Mouse brain tumors survivedfor over 4 months in the vitreous,
compared to less than 3weeks in the cornea (9), which is a
previously describedimmune privileged site (6, 24).
There is not only a prolonged survival of transplantabletumors
but also a prolonged survival of tumors in the avascular state. The
consequence of prolonged growth in theavascular state is the
restriction of tumor size. This principleis illustrated in Chart 2
by the difference in the growth rate ofthe unvascularized
carcinomas in the vitreous and the vascularized carcinomas on the
retinal surface. Growth of populations of cells living in
3-dimensional aggregates is limited by the diffusion of nutrients;
cells at the surface have anabundant supply and replicate; cells in
the center die ofmalnutrition. The balance between replication and
necrosisaccounts for tumor dormancy (22). When capillaries
penetrate the dormant nodule, the perfusion of nutrients resultsin
rapid growth. The same principle applies to the clinical
presentation of human eye tumors, e.g. , retinoblastoma.Large
vascularized masses appear on the retinal surface,but intravitreal
metastases are dormant nodules, remainavascular, and rarely exceed
a diameter of 1 mm (18).
In the vitreous, tumor angiogenesis proceeds differentlyfrom
previous sites where tumors consistently
stimulateneovascularization at distances up to 2 to 4 mm (12, 24,
25).In the cornea, implants of V2 carcinoma 1 mm from thelimbus
attract new vessels within 4 days, and the tumors arevascularized
after 7 days. In the anterior chamber, similar tothe vitreous in
its avascularity and paucity of cells, tumorsstimulate vessels at a
distance of several mm. By contrast,in the vitreous, tumors remain
unvascularized for an average of 42 days (Chart 3). Even at a
distance as close as 0.1mm, blood vessels fail to proliferate
toward the tumor.Vascularization occurs only when the growing edge
of thetumor contactsthe retinalsurface.
The vitreous, therefore, may interfere with the transfer ofa
diffusible, vasoproliferative stimulus from the tumor to thehost
endothelial cells. It could exert this effect by 1 of 2plausible
mechanisms: (a) a direct inhibition of endothelialcell
proliferation or (b) a limitation of the diffusion of TAFfrom the
tumor to the retinal vessels. Vitreous containshuge aggregates of
negatively charged protein-polysaccharides that can act as
molecular sieves, trap large macromolecules, and block their
diffusion (31, 36). Other explanations, such as direct toxicity to
the tumor cells (4) or inabilityof proliferating capillaries to
penetrate the vitreous areshown to be unlikely by the results of
autoradiographicstudies. Tumor cells in the vitreous within 0.1 mm
of theretina incorporated [3Hjthymidine but endothelial cells ofthe
retina did not. This implies that vitreous is not toxic totumor
cells and that the retinal vessels are not proliferating.
These studies support the concept that blockade of angiogenesis,
i.e. , “antiangiogenesis'â€c̃ould arrest tumorgrowth at a tiny
size (16). Antiangiogenesis has been suggested as a potential
therapeutic adjunct (17). Recently, aninhibitor of tumor
angiogenesis has been demonstrated inneonatal rabbit cartilage (7,
8). Cartilage is a relatively avascular tissue that, like vitreous,
consists almost entirely of anextracellular matrix composed of
water, collagen, and protein-polysaccharide complexes (11, 39).
Why are these tissues avascular? Developmental studiesof
cartilage and vitreous in humans have shown that bothtissues are
vascularized in the embryo but that vessels
Chart 3. Prevention of vascularization in the vitreous. V2
carcinomas,implanted in the rabbit cornea at 1 mm from the nearest
vessels, stimulatethe ingrowth of capillaries and are vascularized
after 7 days. In the vitreous,these tumors fail to stimulate
retinal neovascularization; vascularization occurs only when the
tumors contact the retinal surface, e.g. , in these experiments
after 42 ±22 days.
AUGUST1976 2809
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2810 CANCERRESEARCHVOL. 36
S.Brem etal.
regress after birth (27, 29). If an inhibitor of capillary
proliferation exists in cartilage or vitreous, its normal
biological 15.role could be the regulation of this vascular
regression. Thiswould be important in understanding the mechanism
and 16.possible control of retinal neovascularization in diabetes.
17.Further experiments on the vitreous-vascular and vitreoustumor
interactions might provide additional clues leading to 18.the
blockade of neovascularization. Such blockade of neo-
19.vascularization would have therapeutic implications in diabetic
retinopathy and pathological corneal neovasculariza- 20.tion as
well as in neoplasia.
ACKNOWLEDGMENTS
We thank Dr. Dianna Ausprunk for preparation and interpretation
of theautoradiographs, Sylvia Weinberg for histological sections,
Dr. Chung-HoChen for cooperation, Stephen P. Miller for technical
assistance, JanisCirulis and Gary Lees for the illustrations, and
Carl Cobb for help in preparingthemanuscript.
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Prolonged Tumor Dormancy
V 1A
Fig. 1. A, diagonal section of an unvascularized V2 carcinoma,
100 days after transplantation to the rabbit vitreous, growing as a
thin, linear stalk. H & E, x64. B, histology of the same stalk.
H & E, x 300.
Fig. 2. Histology of the V2 carcinoma, 5 months after
transplantation, growing as an avascular spheroid in the dog
vitreous. H & E, x 1000.
AUGUST1976 2811
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. a
C
@—-----
2812 CANCER RESEARCH VOL. 36
,
.@
S
3A
a
@3B@@
Fig. 3. A, an avascular colony of mouse ependymoblastoma cells
(upper left), 47 days after transplantation to the rabbit vitreous,
approximately 2.5 mmfrom the retinal surface (lower right). H &
E, x 40. B, histology of the periphery of the mouse tumor showing
many small cells with nuclear pleomorphism andprominent chromatin.
H & E, x 1.000.
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1976;36:2807-2812. Cancer Res Steven Brem, Henry Brem, Judah
Folkman, et al. Neovascularization in the VitreousProlonged Tumor
Dormancy by Prevention of
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