U.S. patent application number 10/173358 was filed with the patent office on 2002-10-17 for three-demensional ex vivo angiogenesis system.
Invention is credited to Gulec, Seza A., Woltering, Eugene A..
Application Number | 20020150879 10/173358 |
Document ID | / |
Family ID | 26985078 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020150879 |
Kind Code |
A1 |
Woltering, Eugene A. ; et
al. |
October 17, 2002 |
Three-demensional ex vivo angiogenesis system
Abstract
An in vitro tissue angiogenesis and vasculogenesis system is
disclosed that allows the outgrowth of microvessels from a
three-dimensional tissue fragment implanted in a matrix. The matrix
may, for example, be a fibrin- or collagen-based matrix fed by a
growth medium, for example, a mixture of tissue culture medium,
serum, or a layer of growth medium containing a defined mixture of
growth factors. This system, which may be used with human or other
mammalian or animal tissues, may be used in assaying tumor
angiogenic potential, or in promoting angiogenesis in other
tissues, e.g., promoting angiogenesis prior to transplantation of a
tissue. The angiogenic potential of a tissue can be determined by
measuring the growth of microvessels into the matrix. The
three-dimensional structure of the tumor or other tissue is
maintained in the matrix, including blood vessels. In another
aspect, the method allows for the proliferation of a tissue
specimen, thus increasing the mass of cells available for
subsequent transplant; and the method also provides for the
proliferation of blood vessels from the tissue mass, thus enhancing
the chance of successful engraftment.
Inventors: |
Woltering, Eugene A.;
(Kenner, LA) ; Gulec, Seza A.; (New Orleans,
LA) |
Correspondence
Address: |
PATENT DEPARTMENT
TAYLOR, PORTER, BROOKS & PHILLIPS, L.L.P
P.O. BOX 2471
BATON ROUGE
LA
70821-2471
US
|
Family ID: |
26985078 |
Appl. No.: |
10/173358 |
Filed: |
June 17, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10173358 |
Jun 17, 2002 |
|
|
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09866296 |
May 25, 2001 |
|
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60325758 |
May 30, 2000 |
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Current U.S.
Class: |
435/4 ;
435/40.5 |
Current CPC
Class: |
G01N 33/5088 20130101;
G01N 33/6863 20130101 |
Class at
Publication: |
435/4 ;
435/40.5 |
International
Class: |
C12Q 001/00; G01N
033/48 |
Goverment Interests
[0002] The development of this invention was subject to a contract
between the Board of Supervisors of Louisiana State University and
Agricultural and Mechanical College, and the United States
Department of Veterans Affairs. The Government has certain rights
in this invention.
Claims
We claim:
1. A method for assaying angiogenesis ex vivo, said method
comprising the steps of: (a) embedding a three-dimensional
mammalian tissue sample in a matrix, wherein the tissue sample has
at least one cut surface exposing blood vessels; (b) supplying to
the embedded tissue sample a medium that supports the growth of the
tissue sample; (c) incubating the embedded tissue sample in the
medium for a time sufficient to allow angiogenic vessels, if any,
to grow into the matrix surrounding the tissue sample; and (d)
observing or measuring the angiogenic vessels, if any, that grow
into the matrix surrounding the tissue sample.
2. A method as recited in claim 1, wherein the medium comprises a
serum-free medium that supports the growth of the tissue sample;
wherein the medium contains substantially no exogenous
angiogenesis-enhancing factors and substantially no exogenous
angiogenesis-suppressing factors.
3. A method as recited in claim 1, wherein the medium comprises
serum.
4. A method as recited in claim 1, wherein the medium comprises an
angiogenesis-enhancing factor.
5. A method as recited in claim 4, wherein the
angiogenesis-enhancing factor is selected from the group consisting
of platelet-derived growth factor, vascular endothelial growth
factor, epidermal growth factor, fibroblast growth factor, and
transforming growth factor .beta..
6. A method as recited in claim 1, wherein the matrix comprises
fibrin.
7. A method as recited in claim 1, wherein the matrix comprises
collagen.
8. A method as recited in claim 1, wherein the matrix comprises
gelatin.
9. A method as recited in claim 1, wherein the matrix comprises
agarose, agar, alginate, or silica gel.
10. A method as recited in claim 1, wherein the matrix comprises
Matrigel.
11. A method as recited in claim 1, wherein the tissue sample is a
tumor fragment.
12. A method as recited in claim 1, wherein the tissue sample is
not a tumor fragment, and wherein the tissue sample is not an
isolated segment of an artery or vein.
13. A method as recited in claim 1, additionally comprising the
step of supplying an additional factor to the embedded tissue
sample, and measuring the difference in angiogenesis for the tissue
sample as compared to the angiogenesis of an otherwise identical
and otherwise identically-treated control tissue sample that is not
supplied with the factor; whereby the difference in observed
angiogenesis is a measure of the angiogenic enhancement or
angiogenic suppression characteristics of the supplied factor.
14. A method for growing a tissue ex vivo, said method comprising
the steps of: (a) embedding a three-dimensional mammalian tissue
sample in a matrix, wherein the tissue sample has at least one cut
surface exposing blood vessels; (b) supplying to the embedded
tissue sample a medium that supports the growth of the tissue
sample; and (c) incubating the embedded tissue sample in the medium
for a time sufficient to allow angiogenic vessels to grow into the
matrix surrounding the tissue sample; and to allow the number of
cells in the tissue to proliferate, so that the tissue's
suitability for transplant is improved.
15. A method as recited in claim 14, wherein the medium comprises
serum.
16. A method as recited in claim 14, wherein the medium comprises
an angiogenesis-enhancing factor.
17. A method as recited in claim 16, wherein the
angiogenesis-enhancing factor is selected from the group consisting
of platelet-derived growth factor, vascular endothelial growth
factor, epidermal growth factor, fibroblast growth factor, and
transforming growth factor .beta..
18. A method as recited in claim 14, wherein the matrix comprises
fibrin.
19. A method as recited in claim 14, wherein the matrix comprises
collagen.
20. A method as recited in claim 14, wherein the matrix comprises
gelatin.
21. A method as recited in claim 14, wherein the matrix comprises
agarose, agar, alginate, or silica gel.
22. A method as recited in claim 14, wherein the matrix comprises
Matrigel.
23. A method as recited in claim 14, wherein the tissue sample is
selected from the group consisting of skin tissue, parathyroid
tissue, thyroid tissue, pituitary tissue, adrenal tissue, pancreas
tissue, cardiac muscle tissue, skeletal muscle tissue, retina
tissue, kidney tissue, liver tissue, and prostate tissue.
24. A method as recited in claim 14, additionally comprising the
subsequent step of transplanting the incubated embedded tissue
sample with angiogenic vessels into a host in need of such a
transplant.
25. A method as recited in claim 14, wherein said incubating step
is conducted for a time sufficient for the mass of the tissue to
increase by at least about 25%.
26. A method as recited in claim 25, additionally comprising the
subsequent step of transplanting the incubated embedded tissue
sample with angiogenic vessels into a host in need of such a
transplant.
27. A tissue with angiogenic vessels produced by the method of
claim 14.
28. A tissue with angiogenic vessels produced by the method of
claim 15.
29. A tissue with angiogenic vessels produced by the method of
claim 16.
30. A tissue with angiogenic vessels produced by the method of
claim 17.
31. A tissue with angiogenic vessels produced by the method of
claim 18.
32. A tissue with angiogenic vessels produced by the method of
claim 19.
33. A tissue with angiogenic vessels produced by the method of
claim 20.
34. A tissue with angiogenic vessels produced by the method of
claim 21.
35. A tissue with angiogenic vessels produced by the method of
claim 22.
36. A tissue with angiogenic vessels produced by the method of
claim 23.
37. A tissue with angiogenic vessels produced by the method of
claim 25.
Description
[0001] The benefit of the May 30, 2000 filing date of provisional
application .alpha.______ (which is a conversion of nonprovisional
application 09/580,894) is claimed under 35 U.S.C. .sctn.
119(e).
[0003] This invention pertains to methods to promote ex vivo
angiogenesis in tissues, for example, in tissues to be
transplanted. This invention also pertains to methods to assay
angiogenesis in tissues, for example tumor tissues, and to assess
the effects of inducers and inhibitors of angiogenesis. Such
information can be of use, for example, in making a prognosis for a
tumor, or in evaluating the likely effect in vivo of
anti-angiogenic factors on a tumor.
[0004] "Neovascularization," "vasculogenesis," and "angiogenesis"
are terms that describe the formation of new capillaries.
Angiogenesis is a normal physiological process, the generation of
new capillary blood vessels from pre-existing vessels. Angiogenesis
rarely occurs in physiologically normal adult tissues. Exceptions
include the ovary, the endometrium, the placenta, wound healing,
and inflammation. Angiogenesis is an important step in ovulation
and also in implantation of the blastula after fertilization.
Angiogenesis is sometimes distinguished from vasculogenesis, the
emergence of blood vessels de novo from a subpopulation of
mesenchymal cells known as angioblasts, which differentiate into
endothelial cells.
[0005] The identification of several angiogenic factors and the
isolation and culture of capillary endothelial cells (ECs) have led
to a greater understanding of the cellular and biochemical bases of
new vessel growth. Until recently ECs have been the focus of most
studies of microvascular growth. However, capillaries are not
simply tubes of ECs; they also contain a second cellular component,
the mural cell, or pericyte. Angiogenesis involves the differential
growth and sprouting of endothelial tubes, and the recruitment and
differentiation of mesenchymal cells into vesicular smooth muscle
cells and pericytes. Communication between the endothelium and the
mesenchyme is important for angiogenesis. Three such communication
pathways have been identified:
[0006] (1) Mesenchymal cells signal endothelial cells via the
angiopoietin/Tie-2 signaling pathway. See Suri et al., Cell 87:
1171 (1996); T. Sato et al. Nature 376, 7074 (1995); Maisonpierre
et al., Science 277: 55 (1997).
[0007] (2) Endothelial cells induce differentiation of pericytes
through the platelet-derived growth factor (PDGF) signaling
pathway. See Lindahl et al. Science 277: 242 (1997); Soriano, Genes
Dev. 8: 1888 (1994).
[0008] (3) An endoglin-mediated pathway of endothelial-mesenchymal
communication was reported by Li et al. Science. 284: 1534-1537
(1999).
[0009] In normal adult mammals, angiogenesis occurs infrequently,
yet it can be rapidly induced in response to various stimuli. The
normal rate of capillary endothelial cell turnover in adult mammals
is typically measured in months or years. However, when the
normally quiescent endothelial cells lining venules are stimulated,
they will degrade their basement membrane and proximal
extracellular matrix, migrate directionally, divide, and organize
into new functioning capillaries with new basal lamina within a
matter of days. This dramatic amplification of the microvasculature
of a tissue is temporary, for as rapidly as they are formed the new
capillaries virtually disappear, returning the tissue's vasculature
to its previous state.
[0010] Among the most extensively studied of normal angiogenic
processes is wound repair. Important characteristics of
wound-associated angiogenesis are that it is local, rapid,
transient, tightly controlled, and that it promptly regresses back
to a steady-state level. The abrupt termination of angiogenesis
following wound repair apparently results from two control
mechanisms, mechanisms that are not mutually exclusive. First, due
to factors that are not well understood, there appears to be a
marked reduction in the synthesis or elaboration of angiogenic
mediators. Second, there appears to be a simultaneous increase in
levels of substances that inhibit new vessel growth. The control of
angiogenesis thus depends on a balance of several positive and
negative regulators.
[0011] Recent research has begun to uncover the genetic mechanisms
controlling angiogenesis. See Maswell et al. Nature 399, 271-275
(1999); Stebbins et al., Science 284, 455-461 (1999); Kaumra et al.
Science 284, 662-665.
[0012] Angiogenesis is regulated by both angiogenic and angiostatic
factors. The role of inhibitors in angiogenesis was first suggested
by observations that hyaline cartilage appeared to be particularly
resistant to vascular invasion. It was later observed that many
other cell and tissue extracts also contain inhibitors of
angiogenesis. Several natural and artificial angiogenic inhibitors
have been identified, including: inhibitors of basement membrane
biosynthesis, placental RNase inhibitor, lymphotoxin, interferons,
prostaglandin synthetase inhibitors, heparinbinding fragments of
fibronectin, protamine, angiostatic steroids, several
anti-neoplastic and anti-inflammatory agents, platelet factor-4,
thrombospondin-1, angiostatin, integrin antagonists, and certain
forms of thrombin.
[0013] Gasparini, Drugs July;58(1):17-38(1999) discusses the
possible use of angiogenesis inhibitors to intervene into
neoplastic processes. The basic idea is to use inhibitory agents to
block angiogenesis, thereby causing tumor regression in various
types of neoplasia. Therapeutic candidates include naturally
occurring angiogenesis inhibitors (e.g., angiostatin, endostatin,
platelet factor-4), specific inhibitors of endothelial cell growth
(e.g., TNP-470, thalidomide, interleukin-12), agents that
neutralize angiogenic peptides (e.g., antibodies to fibroblast
growth factor or vascular endothelial growth factor, suramin and
its analogs, tecogalan, agents that neutralize receptors for
angiogenic factors, agents that interfere with vascular basement
membrane and extracellular matrix (e.g., metalloprotease
inhibitors, angiostatic steroids), and anti-adhesion molecules
(e.g., antibodies such as anti-integrin alpha v beta 3). Rosen L,
Oncologist; 5 Suppl 1:20-7 (2000) discusses strategies for the
application of antiangiogenic therapies to cancer.
[0014] Other compounds that have been described as inhibitors of
angiogenesis include the cartilage-derived inhibitor TIMP,
thrombospondin, laminin peptides, heparin/cortisone, minocycline,
fumagillin, difluoromethyl omithine, and sulfated chitin
derivatives.
[0015] Of particular interest is the new class of antiangiogenic
substances called METH proteins. Their enzymatic activity makes
this class of agents candidates for possible control by small
molecules, a goal that has eluded pharmacotherapy. See Vazquez F.
et al. J Biol Chem Aug 13;274(33):23349-57 (1999). The angiotensin
II type 2 receptor is another example of a receptor that mediates
an antiangiogenic response, and that may be amenable to regulation
by small molecules.
[0016] Hypoxic conditions can induce angiogenesis. Conversely, when
newly-formed vessels bring oxygen to the tissue, the proteins
involved in induction of angiogenesis are marked for destruction
and angiogenesis ceases.
[0017] Numerous factors have also been identified that induce
vessel formation in vitro or in vivo in animal models. These
include: .alpha.FGF, .beta.FGF, TGF-.alpha., TNF-.alpha., VPF,
VEGF, PDGF, monobutyrin, angiotropin, angiogenin, hyaluronic acid
degradation products, and AGE-products.
[0018] Monitoring angiogenic processes can provide valuable
information on tumor progression, metastasis and prognosis (Szabo
and Sandor, Eur J Surg Suppl;(582):99-103 (1998)). There is an
unfilled need for improved methods of monitoring angiogenesis to
support the development and application of antiangiogenic
interventions. The ability to monitor angiogenesis will also assist
the discovery of new antiangiogenic agents.
[0019] Diseases Associated with Angiogenesis.
[0020] Abnormal angiogenesis occurs when improper control of
angiogenesis causes either excessive or insufficient blood vessel
growth. For example, conditions such as ulcers, strokes, and heart
attacks may result in some cases from levels of angiogenesis
insufficient for normal healing. Conversely, excessive blood vessel
proliferation may favor tumor growth and spread, blindness, and
arthritis. Diseases that have been associated with
neovascularization include, for example, diabetic retinopathy,
macular degeneration, sickle cell anemia, sarcoid, syphilis,
pseudoxanthoma elasticum, Pagets disease, vein occlusion, artery
occlusion, carotid obstructive disease, chronic uveitis/vitritis,
mycobacterial infections, Lyme disease, systemic lupus
erythematosis, retinopathy of prematurity, Eales disease, Bechets
disease, infections causing retinitis or choroiditis, presumed
ocular histoplasmosis, Bests disease, myopia, optic pits, Stargarts
disease, pars planitis, chronic retinal detachment, hyperviscosity
syndrome, toxoplasmosis, trauma, and post-laser complications.
Other angiogenic-related diseases may include, for example,
diseases associated with rubeosis (neovascularization of the
angle), and diseases caused by abnormal proliferation of
fibrovascular or fibrous tissue, including all forms of
proliferative vitreoretinopathy. An improved ability to monitor
angiogenesis can assist in developing improved methods of
intervention, diagnosis, and prognosis of such diseases.
[0021] Angiogenesis in Solid Tumor Formation and Metastasis.
[0022] Angiogenesis is prominent in solid tumor formation and
metastasis. Several experimental studies have concluded that
primary tumor growth, tumor invasiveness, and metastasis all
require neovascularization. The process of tumor growth and
metastasis is complex, involving interactions among transformed
neoplastic cells, resident tissue cells (e.g., fibroblasts,
macrophages, and endothelial cells), and recruited circulating
cells (e.g., platelets, neutrophils, monocytes, and lymphocytes). A
possible mechanism for the maintenance of tumor growth is an
imbalance, or disregulation, of stimulatory and inhibitory growth
factors in systems within the tumor. Disregulation of multiple
systems allows the perpetuation of tumor growth and eventual
metastasis. Angiogenesis is one of many systems that is
disregulated in tumor growth. In the past it has been difficult to
distinguish between disregulation of angiogenesis and disregulation
of other systems affecting a developing tumor. As another
complicating factor, Maniotis A J et al. Am J Pathol September
;155(3):739-52 (1999) have noted that aggressive human melanomas
mimic vasculogenesis by producing channels of patterned networks of
interconnected loops of extracellular matrix, in which red blood
cells, but not endothelial cells, are detected. These channels may
facilitate perfusion of tumors, independent of perfusion from
angiogenesis.
[0023] A tumor cannot expand without a blood supply to provide
nutrients and remove cellular wastes. Tumors in which angiogenesis
is important include solid tumors, and benign tumors including
acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas.
Inhibiting angiogenesis could halt the growth of these tumors.
Angiogenic factors have been reported as being associated with
several solid tumors, including rhabdomyosarcoma, retinoblastoma,
Ewing sarcoma, neuroblastoma, and osteosarcoma.
[0024] Angiogenesis has also been associated with some non-solid
tumors, including blood-born tumors such as leukemias, various
acute or chronic neoplastic diseases of the bone marrow marked by
unrestrained proliferation of white blood cells, usually
accompanied by anemia, impaired blood clotting, and enlargement of
the lymph nodes, liver, and spleen. It is believed that
angiogenesis may play a role in the abnormalities in the bone
marrow that give rise to leukemias.
[0025] Tumor Growth Beyond 1 to 2 mm Diameter is Dependent on
Angiogenesis.
[0026] Angiogenesis in normal wound repair appears to be under
strict control, and is self-limited. By contrast,
neovascularization is exaggerated and is not well-controlled during
neoplastic transformation. It appears that tumors continually renew
and alter their vascular supply. Normal vascular mass is
approximately 20% of total tissue mass, while tumor vascular mass
may comprise as much as 50% of the total tumor. Neovascularization
is both a marker of pre-neoplastic lesions, as well as a condition
that perpetuates tumor growth.
[0027] Several studies have found a correlation between the
magnitude of tumor-derived angiogenesis and metastasis in melanoma,
prostate cancer, breast cancer, and non-small cell lung cancer.
These studies support the conclusion that tumor-associated
angiogenesis is disregulated, with an imbalance that favors either
the expression of local angiogenic factors or the suppression of
angiostatic factors. Also, the degree of angiogenic response in a
tumor is related to the prognosis; i.e., the higher the degree of
angiogenesis, the worse the prognosis.
[0028] Experimental Models of Angiogenesis.
[0029] A source of angiogenic stimulation can be either endogenous
or exogenous to the vessel-sprouting tissue. Exogenous stimulation
requires two types of tissue, the stimulating tissue and the
responding or sprouting tissue. Endogenous stimulation requires
only one tissue, since both the stimulus and the response occur
within the same tissue.
[0030] Several in vivo angiogenesis models have been developed. The
corneal pocket assay involves the surgical implantation of polymer
pellets containing angiogenic factors in the cornea of larger
animals such as rabbits. Quantitation is difficult, and few such
tests have apparently been conducted. The chick chorioallantoic
membrane assay involves the removal and transfer of a chick embryo
from the shell to a cup. The angiogenic material is suspended in a
vehicle, typically a solution of methyl cellulose, and is then
dried on a glass cover slip and placed on the chorioallantoic
membrane. The appearance of new vessels is observed. The rabbit ear
chamber assay requires the surgical insertion of a glass or plastic
viewing device, and the measurement of capillary migration by
microscopy. However, it is difficult to apply angiogenic materials
in this assay. The rat dorsal air sac assay involves implants of
stainless steel chambers containing angiogenic factors and is
difficult to quantitate. The alginate assay involves the
subcutaneous injection into mice of tumor cells encased in
alginate.
[0031] The endothelial cell proliferation assay relies on
measurements of cell proliferation. It is typically performed in
96-well tissue culture plates.
[0032] The endothelial cell migration assay assesses migration of
endothelial cells toward a stimulus. Inhibition of angiogenesis is
shown by blockage of migration in the presence of the inhibitor.
See Dameron et al., Science, 265, 1582-84 (1994).
[0033] In the endothelial cell tube formation assay, human
umbilical vascular endothelial cells (HUVECs) are plated on gels of
a matrix such as Matrigel.TM.. See Schnaper et al., J. Cell.
Physiol, 156, 235-246 (1993). Matrigel.TM. is described in U.S.
Pat. No. 5,382,514. Baatout S, and Cheta N, Rom J Intern Med 1996
July-December ; 34(3-4):263-9 describe Matrigel.TM. as a mixture of
basement membrane proteins including laminin, type IV collagen,
entactin/nitrogen and proteoheparan sulfate, and various growth
factors. Matrigel.TM. induces endothelial cells to differentiate,
as evidenced both by morphologic changes and by a reduction in
proliferation. It therefore offers a convenient system to study
biochemical and molecular events associated with angiogenesis.
Further, Matrigel.TM. permits one to study the roles of the
extracellular matrix in angiogenesis. Sprouts from vessels in
adjacent tissue penetrate into the gel within days of connecting it
to the external vasculature.
[0034] Maldonado et al., Pathol Oncol Res; 4:225-9 (1998),
developed an angiogenesis model that demonstrated that human
metastatic prostate cancer cells appeared to induce HUVECs to
translocate across a Matrigel-coated membrane.
[0035] The corneal micropocket assay is widely accepted as being
generally predictive of clinical usefulness. In this assay, an
angiogenic agent is a factor that is seen to consistently act to
promote the ingrowth of one or more blood vessels within the
cornea, preferably without evidence of the influx of
leukocytes.
[0036] The rodent mesenteric-window assay is a model that exploits
the virtually avascular membranous rodent mesentery. After
experimental treatment, angiogenesis is quantified in the mesentery
histologically as the number of vessels per unit length of
mesentery. See Norrby et al. "Mast-cell-mediated angiogenesis: a
novel experimental model"; Virchows Arch B Cell Pathol Incl Mol
Pathol; 52:195-206 (1986).
[0037] In chemotactic chamber assays, millipore chambers containing
tumors are implanted in an animal such as a hamster. Once such
device is known as a "Boyden chamber." The Boyden chamber contains
an upper well and a blind lower well, separated by a semipermeable
membrane. Chemoattractants are placed in the lower well. See, e.g.,
U.S. Pat. No. 4,912,057.
[0038] In the alginate-entrapped tumor cell assay, tumor cells
entrapped in alginate are implanted in an animal. See Plunkett and
Hailey, Laboratory Investigation, 62:510517 (1990).
[0039] In the microbead assay magnetic microbeads are incubated
with capillary endothelial cells, such that 10-15 microbeads are
internalized per cell. Cells containing the ingested beads are
subjected to various stimuli and allowed to proliferate,
distributing the ingested beads into daughter cells. Quantification
and distribution of the average number of beads in individual cells
allows one to monitor endothelial stimulation and inhibition. See
Cao Y, et al., Lab Invest August ;78(8):1029-30 (1998).
[0040] In a three-dimensional co-culture system, capillary-like
structures are induced in a structure containing sandwiched layers
of collagen gels and fibrin gels. Each layer can be seeded with
cells, such as fibroblasts or cancer cells. It has been reported
that in the absence of fibroblasts, endothelial cells do not
survive in this system. See Janvier et al. Anticancer Research
17:1551-1558 (1997).
[0041] There have also been exogenous models of angiogenesis using
serum supplements. Explants of muscular and adipose tissue, minced
into small fragments and embedded in a three-dimensional matrix of
fibrin or collagen, in the presence of serum, gave rise to an
extensive outgrowth of branching and anastomosing capillary-like
tubes. See Montesano et al. Cell Biology International Reports, 9:
869-875 (1985). This system was not autoregulatory, however, since
regulatory substances were provided in the serum.
[0042] In each of these assays, tumors are modeled either by the
activity of single cells, or of a group of cells that induces the
formation of blood vessels originating from tissue exogenous to the
implanted tumor, and then penetrating the tumor from without.
[0043] Endogenous Angiogenesis Models
[0044] By contrast to the exogenous angiogenesis assays described
above, endogenous angiogenesis assays have been used to observe
whether particular conditions promote the endogenous sprouting of
new vessels from tissue into a surrounding cell-free matrix in a
serum-free medium.
[0045] One endogenous assay is the aortic ring assay. Preexisting
blood vessels can generate new vessels in the absence of exogenous
angiogenic stimuli, because the vessel wall is autoregulatory
through autocrine, paracrine, and juxtacrine mechanisms.
("Juxtacrine" signaling occurs when the ligand and its receptors
are both anchored in the cell membrane.) The vessel wall produces
growth factors, proteolytic enzymes, matrix components, cell
adhesion molecules, and vasoactive factors. Thus, rat aortic or
venous explants cultured in collagen gels under serum-free
conditions will sprout new vessels induced by the combined effect
of injury and exposure to collagen. See Nicosia R F, et al. Int Rev
Cytol, 185:1-43 (1999).
[0046] Another endogenous angiogenesis assay is the placental
explant assay. The endometrium expresses interacting peptide and
non-peptide growth factors during endometrial renewal, factors that
include epidermal growth factor, transforming growth factors (e.g.
TGF-.beta.), platelet-derived growth factor/thymidine
phosphorylase, tumor necrosis factors, and vascular endothelial
growth factor (VEGF). See Smith S K, Hum Reprod Update 4:509-19
(1998).
[0047] In the angiogenesis assay described by Brown, et al. Lab
Invest 75:539-55 (1996), a fragment of human placental blood vessel
embedded in a fibrin gel in microculture plates gave rise to a
complex network of microvessels during a period of 7 to 21 days in
culture. This method is also described in Australian patent AU-B
17500/95. This group has recently published a study of tumor
inhibitors using this assay. See Parish et al., Cancer Res; 59:
3433-41 (1999).
[0048] Prior Tumor Cell Angiogenesis Models Have Been
Exogenous.
[0049] Unlike normal ovary, endometrium, and placenta, most tumor
tissue is not specialized to function as an angiogenic organ.
Neither does tumor tissue possess autoregulatory angiogenic
capacity, as does the aorta. Thus, in all known prior models of
tumor angiogenesis, the tumor is an angiogenic stimulus to which
the surrounding tissue responds by sprouting new vessels toward and
into the tumor. While tumor cell invasion and angiogenesis share
several similarities, there are also important differences. The
initiation of both processes requires attachment to a basement
membrane, followed by disruption of the membrane and migration
through the defect. After the invading cell crosses the basement
membrane barrier, cell proliferation produces either a new vessel
lumen or metastatic foci. It is likely that the two processes are
mutually stimulating, since vascularization allows tumor growth,
and tumor growth requires vascularization. The two processes
operate in opposing directions, however. Tumor cell invasion occurs
when cells move from a tumor into surrounding tissue, whereas
tumor-induced angiogenesis is the sprouting of new vessels from the
surrounding tissue toward the tumor.
[0050] Quantitating Angiogenesis.
[0051] Several methods have been used to quantitate angiogenesis or
perfusion. See, e.g., Hoffman et al., Cancer Res September 1;
57(17): 3847-51 (1997); and Cancer Res September
1;57(17):3847-51(1997). Okada et al., Jpn J Cancer Res September ;
87(9): 952-7 (1996) described the measurement of hemoglobin as a
surrogate for direct angiogenesis measurement.
[0052] Conrad et al., Lab Invest March ;70(3):426-34 (1994);
Iwahana et al., Int. J Exp Pathol 77:109-14 (1996); Rohr et al.,
Nouv Rev Fr Hematol 34:287-94 (1992); and Nikiforidis et al., Eur J
Radiol 29: 168-79 (1999) disclose the use of computer image
analysis to quantitate angiogenesis.
[0053] Matrices and Extracellular Matrices.
[0054] As used in the specification and claims, the term "matrix"
refers to a porous, composite, solid or semi-solid substance, for
example a gel, having pores or spaces sufficiently large for cells
to populate. Depending on context, the term "matrix" can also refer
to matrix-forming materials, i.e., materials that will form a
matrix under suitable conditions. Matrix-forming materials may, for
example, require the addition of a polymerizing agent to form a
matrix, e.g., adding thrombin to a solution containing fibrinogen
to form a fibrin matrix. Other matrix materials include collagen
(all types), combinations of collagen and fibrin, agarose (e.g.,
Sepharose.TM.), and gelatin.
[0055] Extracellular matrices include, for example, collagen,
fibrin, fibronectin, and hyaluronic acid. Artificial, biocompatible
extracellular matrices include, for example, dextran polymers,
polyvinyl chlorides, polyglycolic acids, polylactic acids,
polylactic coglycolic acids, and silicone. Synthetic extracellular
matrices are described in Putnam and Mooney, Nat Med 1996
Julu;2(7):824-6.
[0056] Matrices useful in the compositions and methods of this
invention may be pre-formed. or they may be formed in situ, for
example, by polymerizing compounds and compositions such as
fibrinogen to form a fibrin matrix. Matrices that may be preformed
include those made from the following components, or various
mixtures of the following components: collagen, collagen analogs or
collagen mimics (e.g., collagen sponges and collagen fleece),
chemically modified collagen, gelatin beads or sponges, gel-forming
or composite substances comprising a biocompatible matrix material
that will allow cells to populate the matrix, and collagen
complexed with other compounds to enhance collagen's ability to
polymerize, maintain its structure, or resist degradation. See,
e.g., U.S. Pat. Nos. 5,830,492; 5,824,331; 5,834,005; and
5,922,339.
[0057] In addition to fibrin gels, Matrigel, alginate, agarose, and
biological-molecule-impregnated polyester have been used as
matrices to enhance angiogenesis. See Fournier and Doillon,
Biomaterials 17:1659-65 (1996). Zimrin A B et al., Biochem Biophys
Res Commun 1995August 15;213(2):630-8, noted that there were some
differences between endothelial cells cultured in the presence of
fibrin versus those cultured in Matrigel.
[0058] U.S. Pat. No. 5,830,504 discloses an artificial bioactive
matrix comprising cooperative combinations of ligands within a
matrix.
[0059] Kim B S et al., Biotechnol Bioeng January 1998 5;
57(1):46-54, describe the use of polyglycolic acid as an
extracellular matrix.
[0060] Changes in extracellular matrix structure and composition
can have important regulatory effects on cell behavior. For
example, Kanzawa et al., Ann Plast Surg March 1993; 30(3): 244-51,
examined angiogenesis in a three-dimensional model in vitro, using
HUVECs cultured in a collagen gel. An abundant, capillary-like
network with a lumen structure was seen histologically, forming at
a collagen density less than 0.15% for either type I or type III
collagen. At the same density, type III collagen induced a
capillary-like network with HUVECs at an earlier stage of culture
than did type I collagen. Thus, both collagen density and type can
influence angiogenesis.
[0061] Endothelial growth medium is a serum-free medium that
supports the growth and maintenance of vascular endothelial cells.
See, e.g., Gorfien et al. (1993) Exp. Cell Res. 206, 291; and
Gorfien et al. (1992) Focus 14: 14. The high levels of serum
supplementation that are often used in endothelial cell culture may
create problems in experimental design or in interpretation of
results.
[0062] Gorman L et al., Nutrition April 1996; 12(4):266-70, further
refined the growth requirements of endothelial cells. These authors
reported that M199 medium that is deficient in amino acids but
supplemented with glutamine was superior to M199 complete medium
(medium 199 (Gibco BRL, Grand Island, N.Y.)).
[0063] U.S. Pat. Nos. 6,139,574 and 6,176,874 disclose solid
free-form (e.g., polymeric) fabrication methods for manufacturing
devices for tissue regeneration, in a matrix having a network of
lumens said to be functionally equivalent to the naturally
occurring vasculature of tissue, which can be lined with
endothelial cells and coupled to blood vessels at the time of
implantation.
[0064] Published international application WO 95/23968 discloses a
method for obtaining angiogenesis by culturing a blood vessel
fragment with a physiological gel and nutrients. The physiological
gel was said to preferably be fibrin, collagen, Matrigel, or
similar.
[0065] "Cell treatment could help doctors make old hearts young
again," internet article available at
http://www.cnn.com/2000/HEALTH/11/12/heart.- repair/index.html
(November 2000) is an account in the popular press of treating
damaged hearts by injecting isolating skeletal myoblasts around the
area of a scar on the heart tissue. Similar approaches using marrow
stromal cells and circulating immature endothelial cells were also
mentioned.
[0066] No prior reports are known of angiogenesis assays for tumors
or other tissue in which the intact three-dimensional structure of
the tissue is maintained during the assay--as opposed to, for
example, reports of an assay conducted on an isolated artery or
vein.
[0067] No prior reports are known in which angiogenesis has been
promoted in three-dimensional tissues ex vivo prior to
transplantation.
[0068] We have discovered an in vitro tissue angiogenesis and
vasculogenesis system that allows the outgrowth of microvessels
from a three-dimensional tissue fragment implanted in a matrix. The
matrix may, for example, be a fibrin- or collagen-based matrix fed
by a growth medium, for example, a mixture of tissue culture
medium, serum, or a layer of serum-free medium with defined growth
factors. This system, which may be used with human or other
mammalian or animal tissues, may be used in assaying tumor
angiogenic potential, or in promoting angiogenesis in other
tissues, e.g., promoting angiogenesis prior to transplantation of a
tissue. The angiogenic potential of a tissue can be determined by
measuring the growth of microvessels into the matrix. The system is
based on endogenous angiogenesis, vasculogenesis,
neovascularization, or tissue perfusion, independent of tumor
angiogenesis or other tissue angiogenesis. By contrast, tumor
angiogenesis per se results from the formation of patterned
networks of interconnected loops of extracellular matrix through
which tumor perfusion may occur. The three-dimensional structure of
the tumor or other tissue is maintained in the matrix, including
its blood vessels, supportive stromal elements such as fibroblasts,
and neural and endothelial cells. In another aspect, the method
allows for the proliferation of a tissue specimen, thus increasing
the mass of cells available for subsequent transplant; and the
method also provides for the proliferation of blood vessels from
the tissue mass, thus enhancing the chance of successful
engraftment. The mass of the tissue to be transplanted is
preferably increased by at least about 25%, more preferably by at
least about 50%, most preferably by at least about 100%.
[0069] Unless otherwise clearly indicated by context, the
appearance of new vessels in the novel system, whether by
angiogenesis or vasculogenesis, is considered as a measure of the
angiogenic potential of a tumor or other tissue. Classification as
"angiogenesis," "vasculogenesis," or "neovascularization" may help
promote understanding, but should not be interpreted to limit the
scope of the present invention. Moreover, for the purposes of the
present specification and claims, unless otherwise clearly
indicated by context, the term "angiogenesis" should be interpreted
also to include the processes of vasculogenesis and
neovascularization.
[0070] The novel system displays several unique and surprising
characteristics that are not found in any known prior tissue
angiogenesis model. Intact tissue architecture is maintained,
including supportive stromal elements (e.g., fibroblasts), neural
tissues, and endothelial tissues. The inclusion of such elements is
important, as the presence of these tissues and of the supporting
fibrin matrix better provide the framework required for
angiogenesis and growth of tumors or other tissues. Vessel growth
rate typically exceeds the rate of tissue growth, meaning that the
growth rate of angiogenic vessels may be measured without
interference from tissue growth. The ability to independently and
accurately measure the growth of angiogenic vessels is particularly
surprising, because no known prior model has provided this
important capability. The differential growth pattern of tissue
cells and angiogenic vessels in a fibrin gel matrix separates the
angiogenic vessels and the tissue stroma into independently
observable regions of interest (vessel and tissue compartments).
The compartmental structure of the novel system allows the
measurement of differential effects of various anti-tumor or tissue
stimulatory therapies on tissue and angiogenic vessel
components.
[0071] The present invention may be used to observe angiogenesis in
any type of solid tumor, or to promote angiogenesis in any type of
normal, vascularized tissue. If desired, results maybe expressed in
a semi-quantitative or quantitative manner; quantification may be
conducted, for example, by direct examination, computer-assisted
image analysis, or measurements of surrogate indicators of the
creation of perfusion channels. Examples of such surrogate
indicators include tritiated-thymidine uptake, gene up regulation,
and .sup.125I-bromodeoxyuridine uptake.
[0072] Methods of cell culture, gel formation, vessel quantitation,
and matrix preparation are well known in the art. Thus, most
methods of cell culture or gel formation that will support growth
of cells embedded within a matrix may be used to practice the
present invention, including by way of example those described in
the present application. Moreover, most matrices capable of
supporting angiogenesis may be used to practice the present
invention, including by way of example those described in the
present application. Also, any method of vessel quantitation,
including but not limited to those described in the present
specification, may be used to practice the invention.
[0073] Test compounds, angiogenesis factors, or sera are preferably
layered over or incorporated into the feeding layer in an
appropriate concentration. The compounds or sera then diffuse into
the fibrin matrix to produce effects on the tissue fragment and its
sprouting angiogenic vessels.
[0074] Evaluation of Neovessel Initiation.
[0075] The initiation fraction may be computed by counting the
number of wells that develop an angiogenic response, and dividing
by the total number of wells plated.
[0076] Angiogenesis Initiation Rate.
[0077] The initiation rate equals the slope of the curve of a plot
of the fraction of angiogenesis initiation in culture against
time.
[0078] Evaluation of Neovessel Proliferation/Promotion.
[0079] For subjective scoring, the discs are divided into four
quadrants and rated on a 0-4 scale for the amount of angiogenic
growth. Using a 0-4 rating scale in each of four quadrants, a total
score of 0-16 may be determined for each well. If desired, a more
objective measurement may be obtained, for example, by using
optical microscopy and digital image analysis to measure the total
surface area of angiogenic sprouting. By measuring total surface
area as a function of time, the rate of change may be
determined.
[0080] Viability Measurements.
[0081] Cellular viability may be evaluated using any of various
methods known in the art. A convenient method is a colorimetric
assay such as the MTT assay (Promega, Madison, Wis.). This assay is
based on the cellular conversion of a tetrazolium salt into a blue
formazan product. The MTT assay can be performed at the end of a
specified time period on both the tissue fragment and on angiogenic
sprouts. This assay can be used, for example, to compare
drug/sera-treated and untreated wells.
[0082] Proliferation Measurements.
[0083] Any of various methods known to the art may be used to
measure proliferation of cells. For example, uptake of nonspecific
tracers such as .sup.3H-thymidine or .sup.125I-UDR, which
incorporate only into actively dividing cells, may be used to
compare uptakes in treated wells versus untreated wells. Use of
specific receptor-mediated tags can also be used to assess
tissue-versus-vessel uptake in treated and untreated wells.
Statistically significant differences in uptake are attributed to
effects of the drug, serum, or other treatment.
[0084] Tumor and Other Tissue Sources.
[0085] Monolayer cell lines, solid tumor fragments, or other
tissues may be harvested from or grown in a suitable host animal. A
suitable host for many experimental purposes is the nude mouse.
Tumors, for example, are harvested upon reaching a size of 1-2 cm,
which is sufficient to provide an adequate number of tumor discs.
For clinical purposes, fresh surgical specimens may be used to
assess the angiogenic potential of a particular tumor or other
tissue. Exposing a cut surface within the tumor or other tissue,
i.e., exposing cut blood vessels, is believed to enhance the
tissue's angiogenic response by inducing hypoxia in the transected
vessel edges.
[0086] Assays.
[0087] The novel system may be used in various assays to test the
effects of different agents on angiogenesis. Examples of such
agents include growth factors, growth factor inhibitors, serum
(including autologous serum), chemotherapeutic agents, external
beam radiation, in-situ radiation therapy (such as that delivered
via radiopharmaceutical targeting compounds, for example
radiolabeled somatostatin, monoclonal antibodies, and peptides),
growth factors, growth factor inhibitors, steroid and peptide
hormones or their analogs, and chemotherapeutic agents.
[0088] Monolayers of various tumor cells lines can be placed into
or onto a solid/semi-solid feeder layer to test the effects on
angiogenesis of mediators released from the cells.
[0089] In vitro Metabolic Manipulations.
[0090] The tissue-specific metabolism of different soluble
substances may be evaluated by implanting cells, for example
hepatocyte clusters or liver fragments, into the solid/semi-solid
feeder layer. The effects of soluble factors in circulating blood
may be evaluated by replacing the liquid feeder layer with serum,
including autologous serum from the same patient.
[0091] Non-Oncological Applications.
[0092] In addition to evaluating responses in tumors, this
invention allows the evaluation or the promotion of angiogenic
responses in other tissues or organs undergoing physiologic or
pathophysiologic changes. Such other applications include, for
example, the evaluation of embryologic tissues, the promotion of
angiogenesis in wounds, in cardiac muscle; or conversely the
evaluation of the inhibition of angiogenesis in inflamed tissues of
rheumatic disorders, or in skin conditions such as psoriasis. Other
applications include the induction of angiogenesis in a tissue
transplant, including an autologous transplant; diseases such as
parathyroid reimplantation in the forearm following total
parathyroidectomy, or reimplantation of pituitary, adrenal,
pancreatic, other endocrine tissues, or other peptide- or
amine-producing tissues. The inhibitors and stimulators of
angiogenesis in any tissue may be studied using an assay in
accordance with the present invention. Tissue may be allowed to
grow in assay conditions until the host tissue proliferation
increases significantly above the mass of tissue originally
implanted in the system.
[0093] In Vivo Systemic Assays Using the Present Invention.
[0094] The present invention maybe used in conjunction with an in
vivo systemic assay. Tumor growth is initiated in a suitable host
such as the nude mouse or rat; the tumors are allowed to grow to
1-2 cm; and the tumors are then challenged systemically with the
test compound or radiation treatment of interest. Following
treatment, the animal is sacrificed and a tumor is harvested. A
tumor harvested prior to the systemic test serves as a control. The
tumors are both processed as per the 3DTAM protocol (S. Gulec et
al., "A new in vitro angiogenesis assay with spatially intact human
tumor architecture. The 3D tumor angiogenesis model (3DTAM),
preprint 2001). Both sets of tumor fragments are evaluated for
their angiogenic response. This approach allows one to assess the
effects of in vivo therapy in the presence of biologic variables
that affect drug pharmacokinetics, such as liver metabolism and
renal excretion, as well as humoral interactions at the plasma or
tissue level.
[0095] Multi-Compartment Techniques.
[0096] Multiple compounds or radiation treatments can be evaluated
simultaneously with multiple wells, separated from one another by
dialysis membranes. Multi-compartment procedures can also be
conducted with compartments or wells comprising a non-toxic,
water-soluble or water-insoluble gel. Such gels include, for
example, collagen, other collagen-based materials as previous
discussed, agarose, agar, alginate, silica, or protein-based gels
such as gelatin. The wells are loaded with fibrin, or with a soft
gel containing tissue samples. In this embodiment, the compartments
or wells may optionally be sealed, for example with a layer of
agarose, before the wells are filled. Adjacent wells may be used
for sera, tumor, or tissue to provide comparative data. A
multi-compartment system separated by semipermeable membranes or
gels may be used to evaluate the ability of a tumor, serum, or
other factor to induce a directional angiogenic response.
Optionally, one may harvest all or a portion of the gel separating
different wells. The harvested portions may then be assayed for
specific diffusible substances responsible for inducing a
directional angiogenic response.
[0097] Advantages of the Novel System
[0098] The invention allows a tumor or other tissue to induce an
angiogenic response while maintaining an intact three-dimensional
architecture.
[0099] The present invention offers several advantages. It allows
the evaluation of a tumor or other tissue's angiogenic response
while maintaining an intact three-dimensional architecture. Tumor
(or other tissue) compartments maybe evaluated simultaneously or
separately. The novel system allows the evaluation of drugs that
require activation in vivo and drugs that are active ex vivo. One
advantage of this invention is that it may be used to provide a
functional (as opposed to histological) angiogenic index. A
functional angiogenic index may help to reveal tumors with a poor
prognosis due to a high functional angiogenic index, even though
they may have a low histological angiogenic index. A disparity
between functional and histological angiogenic indices may occur if
circulating anti-angiogenic substances (such as
angiostatin/endostatin) mask the angiogenic potential of a tumor.
Culturing tumors in a serum-free environment may better "unmask"
angiogenic suppressors or stimulators, and thus better reveal their
true angiogenic potential. In lieu of a serum-free environment, a
low serum environment (e.g., less than about 20% or less than about
10% serum) may be used. This may demonstrate that removal or
debulking of tumors that secrete a suppressor is not warranted and
may be harmful.
[0100] Conversely, using this system in the presence of high serum
levels (greater than about 50% serum) may unmask angiogenesis
suppressors that are present in some serum types, such as those
from nude mice implanted with Lewis lung carcinoma.
[0101] The invention may also be used to develop prognostic tests
for a patient's resistance or susceptibility to the future
development of malignancy or angiogenesis-related diseases.
[0102] An important aspect of the invention is its use in ex vivo
angiogenesis to develop a blood supply in a tissue to be engrafted,
thus decreasing the time needed for adequate microcirculation to
develop after implantation. This method also promotes the
proliferation of tissue, which may increase the cell population
available to engraft subsequently. Such cell population increase
may be desirable for implantation of various tissues, for example
endocrine tissue (e.g., thyroid, adrenal glands, pancreas,
pituitary, parathyroid), muscle tissues (e.g., cardiac or skeletal
muscle), kidney, liver, skin, prostate, retina, and other
tissues.
[0103] The invention may also be used to evaluate the up or down
regulation of a specific gene by a tumor or tissue, thus allowing
treatments to be based on gene expression.
EXAMPLES
[0104] As initial examples, we studied receptor mediated cytotoxic
effects of various radiolabeled somatostatin analogs.
[0105] This initial study observed significant in vitro cytotoxic
effects on human tumors and their angiogenic vessels by
somatostatin analogs labeled with .sup.111In, .sup.125I, or
both.
[0106] We used the novel compartmental angiogenesis system to study
the differential effects of somatostatin receptor subtype 2
("sst-2") mediated, in situ radiation therapy on tumors and their
angiogenic vessels in a way that could not have been accomplished
with prior angiogenesis models. The most dramatic results were
obtained with IMR-32 (human neuroblastoma) tumors, in which both
the tumor and the vascular compartments expressed sst-2. Tumor
dissolution and angiogenic vessel disruption were seen in all
fragments that were treated with a radiolabeled somatostatin
analog. Conversely, we observed no effect of radiolabeled
somatostatin analogs on the MDA (human breast carcinoma) tumor
fragments. Watson, J. C. et al., Surgery August ; 122(2): 508-13
(1997) demonstrated similar differences in the cytotoxicity of
somatostatin analogs labeled with Auger emitters in tumor cell
monolayer cultures.
[0107] Somatostatin analogs containing an Auger electron-emitting
label provided an excellent test of the invention. Auger electron
treatment represents true in situ radiation therapy, in which
radiation is delivered to a target following the specific high
affinity binding of a radiolabeled ligand (e.g., a somatostatin
analog) to its receptor (e.g., a somatostatin receptor). Auger
electrons emitted by radioisotopes such as .sup.111In or .sup.125I
have a very short range (on the order of 50 .ANG.), and are
therefore only effective if the radioisotope can be delivered
intracellularly, preferably to the nucleus itself. The use of Auger
electron-emitting, targeted radiopharmaceuticals limits collateral
radiation damage to normal cells by limiting cytotoxicity to those
cells that bind and internalize the radioligand. Moreover, since
the somatostatin receptor sst-2 is uniquely overexpressed in
angiogenic blood vessels, labeled somatostatin analogs will bind
only to angiogenic blood vessels, but not to their normal
counterparts.
[0108] We chose the somatostatin analogs .sup.111In-pentetreotide
(Mallinckrodt Medical, St. Louis, Mo.), JIC2DL, and DTPA-JIC2DL.
(For the latter two compounds, see D. Coy, W. Murphy, E. Woltering,
J. Fuselier, and G. Drouant, "Hydrophilic Somatostatin Analogs,"
U.S. patent application Ser. No. 09/196,259, filed Nov. 19, 1998.)
The analogs were labeled with either .sup.111In or .sup.125I, or in
some cases were dually labeled with both isotopes. JIC2DL has a
sub-nanomolar binding affinity to the somatostatin receptor sst-2
(personal communication, David Coy, Tulane University, New Orleans,
La.). JIC2DL can be iodinated on its two-tyrosine residues, while
DTPA-JIC2DL can be labeled with .sup.111In, .sup.125I, or both.
[0109] We hypothesized that tumor xenograft explants expressing the
sst-2 receptor would show cytotoxic changes when treated with
radiolabeled somatostatin analogs, while those without sst-2 would
not. We also hypothesized that treatment with radiolabeled
somatostatin analogs would inhibit angiogenic blood vessel growth,
independent of the tumor's sst-2 status.
[0110] We cultured two human carcinoma cell lines obtained from the
American Tissue Culture Collection (ATCC). One cell line (IMR-32)
expressed the sst-2 receptor and the other (MDA-MB-231 did not). We
implanted these cell lines into nude mice to create human tumor
xenografts. Subsequently we harvested the xenografts and embedded
tumor fragments in fibrin gel matrixes. These tumor-containing gels
were treated with radiolabeled somatostatin analogs to determine
whether these compounds would destroy tumor cells or angiogenic
blood vessels.
[0111] We demonstrated that the IMR-32 human neuroblastoma cell
line expressed sst-2 as expected from its neuroendocrine
differentiation, while the MDA-MB-231 human breast adenocarcinoma
cells did not express sst-2. Angiogenic vessels also express sst-2,
while other blood vessels do not. We tested the following two
compartment pairs with these cell lines: (1) sst-2 (+) tumor, sst-2
(+) neovessels; and (2) sst-2 (-) tumor, sst-2 (+) neovessels.
[0112] The human breast carcinoma cell line, MDA-MB-231, was
maintained in Lebowitz's L-15 medium (Life Technologies Inc, Grand
Island, N.Y.), supplemented with 10% fetal bovine serum (FBS) (Life
Technologies Inc, Grand Island, N.Y.). The human neuroblastoma cell
line, IMR-32, was maintained in Minimum Essential Medium (Life
Technologies, Inc, Grand Island, N.Y.), supplemented with 15% FBS,
non-essential amino acids (Life Technologies Inc, Grand Island,
N.Y.), L-glutamine (Cellgro, Va.), and antibiotics. Cells were
harvested at subconfluence and resuspended in Hank's balanced salt
solution (Life Technologies Inc, Grand Island, N.Y.).
[0113] While the initial examples described here report results
obtained with fresh human surgical tumors or with tumors derived
from tumor cell lines, the same general technique will also work to
promote angiogenesis ex vivo in tissue explants intended for
transplantation. Such tissues may, for example, be autologous, or
they may be obtained during harvest from operative specimens, or
brain dead donors--all in accordance with applicable statutes,
regulations, and Institutional Review Board procedures. The tissues
will often proliferate in culture in parallel with angiogenesis,
further enhancing the usefulness of the tissue in transplantation.
The ability to transplant intact tissues with pre-formed angiogenic
vessels in this manner should provide substantial clinical benefits
as compared to the infusion of individual cells, or the transplant
of tissue that has not been allowed to develop an angiogenic
response.
[0114] Nude Mice and Creation of Human Tumor Xenografts.
[0115] All animal experiments reported in this specification were
approved by the Louisiana State University Health Sciences Center
animal care committee. BALB/c Harlan Sprague Dawley nude mice
(Indianapolis, Ind.) were injected with 1.5.times.10.sup.7 cells
subcutaneously in both flank regions. The mice invariably grew
solid tumors at the site of injection over a period of 4-6 weeks.
The tumors were allowed to reach a size of 1.5-2 cm. (Continued
growth to larger tumor sizes would often be accompanied by central
tumor necrosis.) Tumors were harvested using a sterile technique
under inhalation anesthesia with methoxyflurane. The mice were
euthanized immediately after tumor harvest.
[0116] Preparation of Tumor Fragments.
[0117] Fresh tumors were processed immediately after harvesting.
Tumor fragments 2 mm diameter and 1 mm thick were prepared, and
then embedded in a fibrin gel. The fibrin gels were prepared in 96
well-plates using a specific tumor supporting medium as described
below.
[0118] Preparation of the Tissue Supporting Medium.
[0119] A serum-free, basic growth medium comprising a balanced salt
solution, an antibiotic-antifungal solution, and an endothelial
growth medium was buffered to a pH of 7.4. Specifically, 9.5 g of
medium 199 (Gibco BRL, Grand Island, N.Y.) was dissolved in 980 mL
deionized H.sub.2O. 10 mL of antibiotic-antimycotic solution (Gibco
BRL, Grand Island, N.Y.) containing 10,000 U of penicillin base,
10,000 U of streptomycin base and 25 .mu.g of Amphotericin B was
added. The pH was then adjusted by adding 2.2 g of Na HCO.sub.3 (EM
Science, Gibbston, N.J.), and was further titrated with 1N NaOH if
needed to reach a pH of 7.4. This solution was mixed with
endothelial growth medium (EGM) (Gibco BRL, Grand Island, N.Y.) in
a 3:1 ratio, and was sterilized by passing it through a 0.22-micron
filter.
[0120] Preparation of Fibrin Matrix Components for Tumor Fragment
Embedding.
[0121] A pro-coagulation solution was prepared by dissolving
fibrinogen (0.12 g )(Sigma, St. Louis, Mo.) and 0.2 g of
.epsilon.-amino caproic acid in 40 mL endothelial growth medium.
Human thrombin (2 .mu.l) (Sigma, St. Louis, Mo.) was placed in the
bottom of each well of a 96 well plate. Endothelial growth medium
is a serum-free medium designed for the growth and maintenance of
vascular endothelial cells. See Gorfien et al. (1993) Exp. Cell
Res.206: 291; and Gorfien et al. (1992) Focus 14: 14.
[0122] Final Assembly of the Fibrin Matrix Tumor System, and
Maintenance of the Well-Plates.
[0123] One tumor disc was placed in the center of each
thrombin-treated well. 0.2 mL procoagulation solution was carefully
layered over the tumor fragments in each of the wells. Fibrin clot
formation took place within 20-30 minutes at 37.degree. C. The
plates were kept at 37.degree. C. in a 5% CO2/95% air humidified
atmosphere.
[0124] Radiolabeled Somatostatin Analogs, Treatment and Evaluation
Protocol.
[0125] The radiolabeled somatostatin analogs used in the
experiments with the IMR-32 tumor line were (1) .sup.111In
pentetreotide (Mallinckrodt Medical St. Louis, Mo.); (2)
.sup.111In-DTPA JIC2DL; (3) .sup.125I-JIC2DL and (4) .sup.111In-
and .sup.125I-DTPA JIC2DL. Support medium containing the
radiopharmaceutical was added over the fibrin clots in the well
plates bearing the tumor fragments, at 100 .mu.Ci/well.
Concentrations for the .sup.111In-labeled analogs and the
.sup.125I-labeled analogs were 7.2.times.10.sup.-9 M and
3.9.times.10.sup.-8 M, respectively. Treatments were administered
on the first day of tumor implantation. Each treatment group
contained 30 tumor fragments. IMR-32 tumor fragments were treated
with all 4 radiopharmaceuticals tested (i.e., n=(30/treatment
group).times.4 groups=120 total). .sup.111In-DTPA JIC2DL was the
only radiolabeled somatostatin analog used in the experiments with
the MDA-MB-231 tumor line (n=30). Control groups were given the
support medium only (n=30 for each of the two tumor types).
Capillary sprouting was monitored visually for 14 days.
[0126] The percentage of wells in which new angiogenic vessel
growth initiated was observed. For subjective angiogenic scoring,
the discs were divided into four quadrants and rated on a 0-4 scale
for the amount of angiogenic growth. Using this 0-4 rating scale in
each of four quadrants, a total score of 0-16 was given for each
tumor fragment. The mean .+-. standard deviation of the angiogenic
score for each treatment group was calculated. Means for control
and treatment groups were compared for statistical significance
(P<0.05) using the two-tailed Student t-test.
Results
[0127] Angiogenic Initiation.
[0128] The angiogenic initiation fraction for all cultures was
similar, regardless of tissue type or treatment. Untreated IMR
fragments (24/30; 80%) and untreated MDA tumor fragments (25/30;
83%) demonstrated angiogenic growth. The initiation fraction in
.sup.111In-DTPA-JIC2DL treated IMR and MDA tumor fragments were
25/30 (83%) and 24/30 (80%) respectively. The differences in
initiation fractions of treated and untreated tumor fragments in
both groups were not statistically significant. The initiation
fractions for the IMR tumor fragments treated with the other
radiopharmaceuticals were not significantly different from control,
ranging from 21/30 (70%) to 25/30 (83%).
[0129] Angiogenic Response.
[0130] The endpoints used to evaluate the compartmental tumor
angiogenesis system included the response of the tumor
(regression/disintegration) and the angiogenic response. The
angiogenic response endpoints comprised the total angiogenic score,
the full angiogenic response fraction, the angiogenic inhibition
pattern, primary-secondary failure, and architectural disruption.
Angiogenic scores were calculated for each group of tumor fragments
on day 14 using a visual rating system. Fragments that did not show
angiogenic initiation were excluded from this portion of the
analysis. Mean angiogenic scores for the control groups of IMR and
MDA tumor fragments were similar (11.9.+-.3.3 and 12.4.+-.3.9,
respectively). In the treated IMR lines, groups were observed with
severe architectural disruption associated with tumor necrosis. No
comparable scoring, therefore, was possible for this treatment. In
the treated MDA group, the mean angiogenic score was 6.4.+-.2.9.
This score was significantly different from the mean angiogenic
score of the MDA control group (12.4.+-.3.9) (p<0.0001). 20/24
(83%) of the tumor fragments in the MDA treatment group showed
architectural disruption and evidence of vessel destruction.
[0131] Tumor Response.
[0132] All tumor fragments in the control groups for both tumor
cell lines remained intact on day 14. In the treated IMR groups,
all tumor fragments showed degenerative changes ranging from
vacuolization to nearly complete tumor lysis. No significant
differences were seen among the anti-tumor effects of the 4
different radiopharmaceuticals. In the treated MDA group, all tumor
fragments remained intact, with no evidence of cytotoxic
changes.
[0133] The most dramatic results were seen with the IMR-32 tumors,
in which both the tumor and the vascular compartments expressed
sst-2. Tumor dissociation and angiogenic vessel degradation were
seen in all fragments that received the experimental treatment.
Conversely, no effect was seen on the MDA tumor fragments. However,
angiotoxicity was seen in 92% of the experimental MDA fragments.
Sparing of the MDA (sst-2 negative) tumor compartment from the
effects of the in situ radiation was strong evidence of the highly
selective nature of the Auger emitter treatment.
[0134] Definition
[0135] Any biological system will, in a literal sense, be
three-dimensional. However, as used in the specification and
claims, a tissue or tissue fragment is considered to be
"three-dimensional" if it has multiple layers of cells comprising
blood vessels and other cells of the tissue, and if the
architecture of the tissue or tissue fragment (including, for
example, the blood vessels, supportive stromal elements such as
fibroblasts, neural and endothelial cells) is substantially intact
and has not been disrupted as compared to the comparable tissue in
vivo. As examples, a tumor, tumor sample, other tissue, or other
tissue sample is considered "three-dimensional" within the scope of
this definition if its structure has not been disrupted. It may be
sliced or reduced in thickness, so long as multiple layers of cells
are retained, and so long as the relative structure and relation of
blood vessels and other cells to one another is maintained.
[0136] As examples, the following would not be considered
"three-dimensional" within the scope of the above definition: an
isolated vein; an isolated artery; isolated cells from a disrupted
tumor or other tissue; or an agglomerations of cells grown in
culture--even an agglomeration that has substantial thickness and
is "three-dimensional" in the ordinary sense--if the agglomeration
lacks the architecture of the comparable tissue in vivo--such as an
agglomeration of tumor cells grown in culture without any
vascularization.
[0137] The complete disclosures of all references cited in this
specification are hereby incorporated by reference. In the event of
an otherwise irreconcilable conflict, however, the present
specification shall control. Also incorporated by reference are the
complete disclosures of the following unpublished papers, none of
which is prior art to the present invention: S. Gulec et al.,
"Antitumor and antiangiogenic effects of somatostatin
receptor-targeted in situ radiation with .sup.111In-DTPA-JIC2DL,"
J. Surg. Res., vol.97, pp.131-137 (2001); and S. Gulec et al.,
"Antiangiogenic treatment with somatostatin receptor-mediated in
situ radiation," American Surgeon (in press, 2001); S. Gulec et
al., "A new in vitro angiogenesis assay with spatially intact human
tumor architecture. The 3D tumor angiogenesis model (3DTAM)
(preprint 2001). Also incorporated by reference is the complete
disclosure of the priority provisional application No. ______
(which is a conversion of nonprovisional application Ser. No.
09/580,894), filed May 30, 2000.
* * * * *
References