Title: Three-dimensional 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.
Patent Number: 6,893,812 Issued on 05/17/2005 to Woltering, et al.
| Inventors:
|
Woltering; Eugene A. (Kenner, LA);
Gulec; Seza A. (New Orleans, LA)
|
| Assignee:
|
Board of Supervisors of Louisiana State University and Agricultural and Mechanical College (Baton Rouge, LA)
|
| Appl. No.:
|
866296 |
| Filed:
|
May 25, 2001 |
| Current U.S. Class: |
435/4; 435/1.1; 435/29; 435/325; 435/375; 435/395 |
| Intern'l Class: |
C12Q 001/00; C12N001/00; C12N001/02; C12N005/06; C12N005/08 |
| Field of Search: |
435/4,11,29,325,375,395,25,28,525
|
References Cited [Referenced By]
U.S. Patent Documents
Other References
Barendsz-Janson et al. Journal of Vascular Research. 1998, 35:109-114.*
Guyton A. Textbook of Medical Physiology. 1991. 8 edition. W.B. Sauders Company.
p. 190.*
Brown et al. Laboratory Investigation. 1996. vol. 75, No. 4, pp. 539-555.*
Montesano et al. Cell Biology International Reports. 1985. vol. 9, No. 10, pp. 869-875.*
Lugasy et al. C R Acad Sci Ser III Sci Vie. (1991), 313 (1), 37-44.*
Gulec, S. et al., "Antitumor and antiangiogenic effects of somatostatin receptor-targeted
in situ radiation with 111In-DTPA-JIC 2DL," J. Surg. Res. vol. 97, pp.
131-137 (2001).
Knott, R.M. et al., "A Model System for Study of Human Retinal Angiogenesis:
Activation of Monocytes and Endothelial Cells and the Association with the Expression
of the Monocarboxylate Transporter Type 1 (MCT-1), " Diabetologia, vol. 42, pp.
870-877 (1999).
|
Primary Examiner: Afremova; Vera
Attorney, Agent or Firm: Runnels; John H.
Goverment Interests
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.
Parent Case Text
The benefit of the May 30, 2000 filing date of provisional application 60/325,758
is claimed under 35 U.S.C. §119(e).
Claims
1. A method for assaying the angiogenic potential of a particular tumor in a
mammal; said method comprising the steps of:
(a) embedding a three-dimensional tissue sample in a matrix, wherein the tissue
sample is taken from a particular tumor in a mammal; wherein the tissue sample
has at least one cut surface exposing blood vessels; wherein the three-dimensional
tissue sample comprises multiple layers of cells comprising blood vessels, supportive
stromal elements, neural cells, and endothelial cells; wherein the architecture
of the tissue sample is substantially intact and has not been disrupted as compared
to that of comparable tissue in vivo; and wherein the three-dimensional tissue
sample does not consist of an isolated artery or an isolated vein;
(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 any angiogenic vessels to grow into the matrix surrounding the tissue
sample; and
(d) observing or measuring any angiogenic vessels that grow into the matrix surrounding
the tissue sample;
whereby:
the growth or any angiogenic vessels into the matrix is a measure of the angiogenic
potential of the particular tumor from which the tissue sample was taken.
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 β.
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™ matrix.
11. A method as recited in claim 1, additionally comprising the step of supplying
a 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.
12. A method as recited in claim 1, wherein said method additionally comprises
the step of supplying an angiogenic suppression factor to the 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 measured difference
in angiogenesis between the samples is a measure of the angiogenic suppression
characteristics of the supplied factor against the tumor from which the sample
was taken.
13. A method as recited in claim 1, wherein said method additionally comprises
the step of supplying an angiogenic stimulation 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 measured
difference in angiogenesis between the samples is a measure of the angiogenic stimulation
characteristics of the supplied factor for the tissue from which the sample was taken.
Description
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.
"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.
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:
(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).
(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).
(3) An endoglin-mediated pathway of endothelial-mesenchymal communication was
reported by Li et al. Science. 284: 1534-1537 (1999).
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.
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.
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.
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,
heparin-binding 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.
Gasparini, Drugs Jul; 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.
Other compounds that have been described as inhibitors of angiogenesis include
the cartilage-derived inhibitor TIMP, thrombospondin, laminin peptides, heparin/cortisone,
minocycline, fumagillin, difluoromethyl ornithine, and sulfated chitin derivatives.
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 August 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.
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.
Numerous factors have also been identified that induce vessel formation
in vitro or in vivo in animal models. These include: αFGF, βFGF, TGF-α,
TNF-α, VPF, VEGF, PDGF, monobutyrin, angiotropin, angiogenin, hyaluronic
acid degradation products, and AGE-products.
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.
Diseases Associated with Angiogenesis.
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.
Angiogenesis in Solid Tumor Formation and Metastasis.
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.
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.
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.
Tumor Growth Beyond 1 to 2 mm Diameter is Dependent on Angiogenesis.
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.
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.
Experimental Models of Angiogenesis.
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.
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.
The endothelial cell proliferation assay relies on measurements of cell proliferation.
It is typically performed in 96-well tissue culture plates.
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).
In the endothelial cell tube formation assay, human umbilical vascular endothelial
cells (HUVECs) are plated on gels of a matrix such as Matrigel™. See Schnaper
et al., J. Cell. Physiol, 156, 235-246 (1993). Matrigel™ 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™ as a mixture of basement membrane proteins
including laminin, type IV collagen, entactin/nitrogen and proteoheparan sulfate,
and various growth factors. Matrigel™ 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™ 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.
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.
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.
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).
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.
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).
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).
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).
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.
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.
Endogenous Angiogenesis Models
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.
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).
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-β), 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).
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).
Prior Tumor Cell Angiogenesis Models have been Exogenous.
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.
Quantitating Angiogenesis.
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.
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.
Matrices and Extracellular Matrices.
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™),
and gelatin.
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 July; 2(7):824-6.
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 pre-formed 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.
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 AB et al., Biochem Biophys Res
Commun 1995 August 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.
U.S. Pat. No. 5,830,504 discloses an artificial bioactive matrix comprising
cooperative combinations of ligands within a matrix.
Kim B S et al., Biotechnol Bioeng Jan. 5, 1998; 57(1):46-54, describe the use
of polyglycolic acid as an extracellular matrix.
Changes in extracellular matrix structure and composition can have important
regulatory effects on cell behavior. For example, Kanzawa et al., Ann Plast Surg
1993 March; 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.
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.
Gorman L et al., Nutrition 1996 April; 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.)).
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.
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.
"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.
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.
No prior reports are known in which angiogenesis has been promoted in three-dimensional
tissues ex vivo prior to transplantation.
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, maybe 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%.
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.
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 maybe 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.
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 may be 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
125I-bromodeoxyuridine uptake.
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.
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.
Evaluation of Neovessel Initiation.
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.
Angiogenesis Initiation Rate.
The initiation rate equals the slope of the curve of a plot of the fraction of
angiogenesis initiation in culture against time.
Evaluation of Neovessel Proliferation/Promotion.
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.
Viability Measurements. 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.
Proliferation Measurements.
Any of various methods known to the art may be used to measure proliferation
of cells. For example, uptake of nonspecific tracers such as
3H-thymidine
or
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.
Tumor and Other Tissue Sources.
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.
Assays.
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.
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.
In Vitro Metabolic Manipulations.
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.
Non-Oncological Applications.
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.
In Vivo Systemic Assays Using the Present Invention.
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.
Multi-compartment Techniques.
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.
Advantages of the Novel System
The invention allows a tumor or other tissue to induce an angiogenic response
while maintaining an intact three-dimensional architecture.
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 de-bulking of tumors that secrete a suppressor
is not warranted and may be harmful.
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.
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.
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.
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
As initial examples, we studied receptor mediated cytotoxic effects of various
radiolabeled somatostatin analogs.
This initial study observed significant in vitro cytotoxic effects on human
tumors and their angiogenic vessels by somatostatin analogs labeled with
111In,
125I, or both.
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.
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
111In or
125I have a very short range (on the order
of 50 Å), 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 over-expressed in angiogenic
blood vessels, labeled somatostatin analogs will bind only to angiogenic blood
vessels, but not to their normal counterparts.
We chose the somatostatin analogs
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
111In or
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
111In,
125I,
or both.
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.
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.
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.
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.).
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.
Nude Mice and Creation of Human Tumor Xenografts.
All animal experiments re
