# Patent

## Title
Generation and use of vasculogenic tumors and products derived from same

## Abstract

Tumors can be directed into a vasculogenic program and used to regenerate tissues in a verterbrate host, identify factors originating from the tumor that are useful for tissue regeneration, and to generate such factors for use in a subject apart from the tumor itself.

## Background
RELATED APPLICATIONS
This application claims benefit under 35 U.S.C. 119(e) of the filing date of U.S. Ser. No. 60/728,033 filed on Oct. 18, 2005, the entire disclosure of which is incorporated herein by reference.


GOVERNMENT SUPPORT
The present invention was supported in part by grants from the United States National Cancer Institute CA-78496 and CA-86302. The U.S. Government may retain certain rights in the invention.


FIELD OF THE INVENTION
The present invention relates generally to tissue regeneration.
BACKGROUND OF THE INVENTION
The therapeutic need to generate nerves, arteries and veins is enormous. Fifty-eight million people in the United States have cardiovascular disease (Ingham, P. W. et al, Genes Devel. 15, 3059-87 (2001)). Another quarter of a million people have spinal cord injuries. In addition, damage to peripheral nerves often results in loss of muscle function, impaired sensation, and painful neuropathies (Evans, G. R. Semin. Surg. Oncol. 19(3), 312-318 (2000)). Progress in addressing these pathologies has been mainly along two fronts—the graft and the bioengineered construct. At present, the “gold standard” in nerve and vessel replacement is still the autologous graft (Evans, G. R. Semin. Surg. Oncol. 19(3), 312-318 (2000)). Understanding how to harness and amplify the regenerative capacities of adult tissues stands to provide answers to many regeneration problems and augment bioengineering solutions (Fuchs, E. et al. Cell 116, 769-778 (2004). To this end, there is a great unmet need to provide mechanistic insight into development and to recover latent capacities for arteriogenic and neurogenic activity.
SUMMARY OF THE INVENTION
It is widely believed that, in order to obtain the oxygen and nutrient supply they need to survive, tumors invade surrounding tissues and generate a number of factors to stimulate an angiogenic program. It has now been discovered according to the present invention how to recapitulate the embryogenesis program in adult tissue, using tumors as a source of signals involved in directing tissue generation, including tissue regeneration. The invention is based in part on the surprising discovery by the inventors that a developing tumor can be utilized to elicit large-gauge neurovascular bundles, including nerves, arteries, and veins in a supporting fascial sheath, which reached up to 30 mm in length and homed to the tumor. The embryonic developmental morphogen Sonic hedgehog (Shh) (Lum, L. et al. Science 304, 1755-9 (2004); Chiang, C. et al., Nature 383, 407-13 (1996); Marti, E. et al., Nature 375, 322-325 (1995); Ingham, P. W. et al., Genes Devel. 15, 3059-87 (2001)) was detected throughout the neurovascular bundle and surrounding sheath and found to be essential for bundle induction.
The invention is based in part upon the surprising discovery by the inventors of the regenerative potential of the cellular components of arteries, veins, and nerves in a mature animal, and the ability of tumors to unlock this potential. The invention is also based in part upon the surprising demonstration by the inventors, using this discovery, of induction of neurovascular bundles of unprecedented scale. The invention is also based in part on the surprising discovery by the inventors of a critical role for Shh signaling in promoting these structures through a recapitulation of embryogenic fate determination and developmental programs.
It has been discovered according to the invention that following tumor implantation there can be a remodeling of the vascular hierarchy of the host, with the tumor acquiring a large-gauge vascular accommodation reminiscent of that to an organ. The confinement of influx to and efflux from the tumor to a coupled artery and vein is likened to that seen in encapsulated organs, e.g., the kidney. Strikingly, nerve induction accompanies the artery and vein, together forming a neurovascular bundle. The ability of a tumor to elicit directed arteriogenesis and neurogenesis in mature animals has ramifications for the exploitation of regenerative potential in the adult setting.
It has also been discovered according to the invention that the large vascular structures induced by the implanted tumor can be used to access information about tumor influx and tumor efflux. More particularly, the invention provides methods for identifying tumor products and for assessing tumor uptake and metabolism of various agents including drugs.
The invention in one aspect is a method for generating a tumor characterized by a vasculogenic program. The method according to this aspect of the invention includes the steps of implanting an intact tumor, or a nondispersed portion thereof, into a living vertebrate; and growing the implanted tumor or portion thereof in the vertebrate for at least a selected time, to generate a tumor characterized by a vasculogenic program. In one embodiment the steps of implanting and growing are repeated at least one time.
In one embodiment according to this and other aspects of the invention, the vasculogenic program comprises an artery and a vein.
In one embodiment according to this and other aspects of the invention, the intact tumor is a tumor characterized by a vasculogenic program.
In one embodiment according to this and other aspects of the invention, the intact tumor is derived from a tumor cell line.
In one embodiment according to this and other aspects of the invention, the implanting is implanting into a subcutaneous site.
In one embodiment according to this and other aspects of the invention, the implanting is implanting into a peritoneal site.
In one aspect the invention is a method for isolating a selected type of living tissue from a tumor. The method according to this aspect of the invention includes the steps of implanting an intact tumor, or a nondispersed portion thereof, into a living vertebrate; growing the implanted tumor or portion thereof in the vertebrate for at least a selected time, to generate a tumor characterized by a vasculogenic program; and isolating a selected type of living tissue from the tumor characterized by the vasculogenic program.
In one embodiment according to this and other aspects of the invention, the selected type of living tissue is selected from artery and vein.
In one embodiment according to this and other aspects of the invention, the selected type of living tissue is nerve.
In one embodiment according to this and other aspects of the invention, the selected type of living tissue is a stem cell.
In one aspect the invention is a composition comprising a selected type of living tissue prepared according to the method described above, namely, implanting an intact tumor, or a nondispersed portion thereof, into a living vertebrate; growing the implanted tumor or portion thereof in the vertebrate for at least a selected time, to generate a tumor characterized by a vasculogenic program; and isolating a selected type of living tissue from the tumor characterized by the vasculogenic program.
In one aspect the invention is a method for inducing host tissue regeneration. The method according to this aspect of the invention includes the steps of implanting an effective amount of an intact tumor, or a nondispersed portion thereof, into a vertebrate host in need of host tissue regeneration; and growing the implanted tumor or portion thereof in the vertebrate host for at least a selected time, to induce host tissue regeneration.
In one embodiment according to this and other aspects of the invention, the implanting is implanting into a site toward which direction regeneration is desired.
In one embodiment according to this and other aspects of the invention, the intact tumor, or the nondispersed portion thereof, is contained within a semipermeable membrane.
In one embodiment according to this and other aspects of the invention, the vertebrate host has vascular disease and the host tissue is artery.
In one embodiment according to this and other aspects of the invention, the vertebrate host has nerve disease and the host tissue is nerve.
In one embodiment according to this and other aspects of the invention, the nerve disease is peripheral nerve disease.
In one embodiment according to this and other aspects of the invention, the nerve disease is spinal cord injury.
In one aspect the invention is a method for inducing host tissue regeneration. The method according to this aspect of the invention includes the step of implanting an effective amount of a tumor product, wherein the tumor product is isolated from a tumor characterized by a vasculogenic program, into a vertebrate host in need of host tissue regeneration, to induce host tissue regeneration.
In one embodiment according to this and other aspects of the invention, the tumor product is selected from the group consisting of cytokines, growth factors, morphogens, angiogenesis factors, and anti-angiogenesis factors.
In one embodiment according to this and other aspects of the invention, the tumor product is a cell.
In one embodiment according to this and other aspects of the invention, the tumor product is contained within a semipermeable membrane.
The invention in one aspect is a method for inducing host tissue regeneration. The method according to this aspect of the invention includes the step of administering an effective amount of a tumor product, wherein the tumor product is isolated from a tumor characterized by a vasculogenic program, to a vertebrate host in need of host tissue regeneration, to induce host tissue regeneration.
In one aspect the invention is a method for identifying a tumor product. The method according to this aspect of the invention includes the steps of providing a tumor characterized by a vasculogenic program; sampling arterial influx to the tumor; sampling venous efflux from the tumor; measuring an amount of a composition in the influx; measuring an amount of the composition in the efflux; and identifying the composition as a tumor product when the amount of the composition in the efflux exceeds the amount of the composition in the influx.
In one aspect the invention is a method for isolating a tumor product. The method according to this aspect of the invention includes the steps of providing a tumor characterized by a vasculogenic program; sampling venous efflux from the tumor, wherein the efflux comprises a tumor product; and isolating the tumor product from the efflux.
In one aspect the invention is a method for analyzing uptake or metabolism of a composition by a tumor. The method according to this aspect of the invention includes the steps of providing a tumor characterized by a vasculogenic program; sampling arterial influx to the tumor; sampling venous efflux from the tumor; measuring an amount of a composition, or a metabolite thereof, in the influx; measuring an amount of the composition, or the metabolite thereof, in the efflux; and comparing the amount of the composition, or the metabolite thereof, in the influx to the amount of the composition, or the metabolite thereof, in the efflux.
In one embodiment according to this aspect of the invention, the composition is a cell.
In one embodiment according to this aspect of the invention, the composition is a drug.
In one embodiment according to this aspect of the invention, the composition or the metabolite thereof in the influx is a drug and wherein the composition or the metabolite thereof in the efflux is a metabolite of the drug.
In one embodiment according to this aspect of the invention, the composition or the metabolite thereof in the influx is a metabolite of a drug and wherein the composition or the metabolite thereof in the efflux is the metabolite of the drug.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing”, “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.



BRIEF DESCRIPTION OF THE DRAWINGS
The figures are illustrative only and are not required for enablement of the invention disclosed herein.
FIGS. 1A, B and C are photographic images showing that implanted tumor elicited large-gauge arteries and veins which linked them to host vasculature. A) A severe combined immunodeficiency (SCID) mouse with a liposarcoma xenograft implanted low on the back was injected with orange latex casting through the left ventricle to identify the arterial network. The tumor, 1.78×1.26 cm at the time of sacrifice, is seen to be well delimited, similar to an organ. A significant artery is seen to connect the tumor to the host. It connects to the aorta at the level of the renal arterial branches. The artery is seen to branch before entering the tumor. Orange color on the mouse surface (including hair) represents latex casting transfer due to handling. B) A vascular conduit as it appears when the skin containing the tumor mass is dissected to the right. The fascia in the vicinity of the conduit is flexible, and the conduit appeared somewhat serpentine in its relaxed position. Here it is shown pulled taut to the right by forceps. The conduit extends from the junction with the tumor (shown by the arrow), leftwards along the back to a point of entry into the abdomen concealed by the skin flap on the left. C) The surgically trimmed clear fibro-adipose sheath, and the distinct artery (red) and vein (dark red) contained within it. This fascial bridge between the abdomen and the fascia abutting the skin appears only in tumor-bearing mice and has no analog in control mice.
FIG. 1D shows angiograms of tumor-bearing and control animals. Angiogram on the left shows the arterial system of a SCID mouse with a liposarcoma xenograft. A main artery supplying the tumor is shown (arrow). It forms a junction with the aorta at the level of the renal arteries. Its gauge is comparable to the femoral artery. Angiogram on the right shows the arterial system of a control SCID mouse.
FIG. 1E) is a bar graph depicting the widths of representative arteries, veins, and nerves in induced and normal tissues. Shown are widths of arteries, veins, and nerves in fascia induced by the tumor, along with normal counterparts at selected sites in control (non-tumor-bearing) animals. Widths were determined in each case by measuring serial sections, and this was repeated for 30 animals (in induced fascial tissue and in the skin and back adjacent to the spine) or for 20 animals (renal artery and vein). Measured structures in the induced tissue include veins, arteries and nerves in the fascial sheath and nerves in the fascia at the tumor interface. Normal structures measured include: 1) nerves in the skin of control animals at the site where tumors were implanted in the test animals; 2) nerves in tissue dorsal to the spine; 3) the renal artery; and 4) the renal vein. Errors are 95% confidence intervals for the mean.
FIG. 2 shows cross-sections of tumor conduit arteries and veins. The artery and vein exhibit properties of mature, functional vasculature. A) Shown is the well-organized, layered structure of the artery in the region of the conduit between the tumor and abdominal wall. The muscular media is highlighted with smooth muscle actin immunostain (reddish brown). An adventitial layer that does not stain is also evident. External to the adventitia is fibro-adipose tissue. Endothelial cells are seen to line the uniform lumen. B) In cross-section is shown the classic, serpentine structure of the elastic lamina in the fully-formed artery, with the smooth muscle actin-labeled muscular media surrounding. A final adventitial layer is displayed. By contrast, the vein is thin-walled, with a relatively large lumen. C) Longitudinal section of artery and vein in a tumor conduit. This section, which passes into, out of, then back into the arterial lumen, shows smooth muscle actin immunostain labeling in the muscular walls of the artery and vein. An accompanying nerve is also present. D) This section follows serially from that in FIG. 2C and is stained for elastin (black) and collagen (red). Three layers in the arterial wall are evident. The internal and external elastic laminae of the artery are well-defined by the elastin stain (black). Perivascular adventitial collagen stains red. In between is the muscular media. E) Cell proliferation in the artery and nerve in the tumor conduit. Ki-67 immunostain shows labeling of endothelium and smooth muscle cells as well as cells in the surrounding connective tissue and within the nerve. Section counterstained with hematoxylin and eosin (H&E) (4×).
FIGS. 3A and B are cross-sections of the tumor conduit neurovascular bundle. A) Neurovascular bundle in the tumor conduit. The localization and orientation of the nerves was demonstrated by immunodetection of axons using an antibody to neurofilament. Section counterstained with H&E. B) Nerve presence in the tumor-conduit interface. Multiple nerve sections are detected by S-100 immunostain (brown). Section counterstained with H&E (20×).
FIG. 3C shows a schematic of the tumor with its induced conduit and a bar graph depicting the quantification of tumor-induced nerve presence. Shown is a schematic of the tumor with its induced conduit, containing a neurovascular bundle embedded in a fibro-adipose support tissue. The conduit extends from the tumor to the abdominal wall ( ̃30 mm—Region 2), traverses the wall (Region 3), and continues on (Region 4) to connect to the aorta near its junction with the renal arteries. Quantified for each region is the ‘nerve presence index’—the average ratio of nerve tissue area to total tissue area measured in each microscope field. In the neurovascular bed at the tumor-conduit interface, the nerve index was highest at 6.98%±1.00, falling off with distance from the tumor to the wall (3.49%±1.52 in Region 2, and 0.39%±0.11 in the abdominal wall). The nerve index then increased slightly in Region 4 (0.65%±0.17). The skin overlying the tumor showed negligibly small nerve detection. Additionally shown are the nerve indices for control, non-tumor-bearing animals (skin: 0.07%±0.05 and abdominal wall: 0.40%±0.20). For statistical accuracy, up to 100 imaged fields (80× magnification) were averaged to obtain the measurements in each region.
FIG. 4 shows tissue cross-sections that depict the identification and localization of cells expressing Sonic hedgehog (Shh) mRNA and protein. A) In situ hybridization with Shh, showing positively-labeled endothelial cells (blue) lining the lumen of the conduit vein (40×). B) Shh protein presence in tumor conduit structures. Shh protein staining in an artery/vein cross-section. Positive labeling (brown) is evident in adipocytes of the fascial bridge and in smooth muscle cells in the muscular media of the artery (40×). C) Shh protein labeling in nerve cross-sections. Strong staining is evident in the epineurium surrounding the nerves. No such labeling is seen in control non-tumor bearing mice. D) Generalized Shh protein labeling in the tumor conduit. A diffuse, granular labeling for Sonic hedgehog protein is seen in mesenchymal areas in the conduit adjacent the neurovascular bundles. This labeling appears to be identifying secreted Shh protein contributing to a morphogenic gradient, similar to what has been observed in embryonic contexts.
FIG. 5 is a diagram that depicts the global pattern of Shh mRNA expression over the neurovascular conduit. In the portion of the tumor conduit extending from the tumor to the abdominal wall ( ̃30 mm), Shh mRNA expression was quantified using digitized densitometric imaging. The tumor with conduit was embedded in paraffin, sectioned longitudinally, and mounted on a microscope slide. Using the successive x-y positions of the midline of the curved conduit for each 400 μm step in the x-position, distance along the conduit from the tumor was calculated. Measured as a function of this distance along the conduit was the ratio of the hybridization signal area detected to the total tissue area in each microscope field. For each x-step, ten adjacent fields were imaged in the y-direction (so as to cover the conduit width) and averaged to arrive at a value at that point along the conduit. The signal starts at 3.6% at the tumor-conduit interface, falling eventually to 0.1% 12 mm from the tumor.



DETAILED DESCRIPTION OF THE INVENTION
The neurovascular system, a hierarchical structure comprised of a network of large and small nerves and vessels, is established during embryogenesis. The neurovascular map in mature animals is essentially determined, although locally perturbable through injury or disease. It has now been demonstrated according to the invention that following tumor implantation there can be a remodeling of the vascular hierarchy of the host, with the tumor acquiring a large-gauge vascular accommodation reminiscent of that to an organ. The confinement of influx to and efflux from the tumor to a coupled artery and vein is likened to that seen in encapsulated organs, e.g. the kidney. Strikingly, nerve induction accompanies the artery and vein, together forming a neurovascular bundle. The ability of a tumor to elicit directed arteriogenesis and neurogenesis in mature animals has ramifications for the exploitation of regenerative potential in the adult setting.
Reasoning that since a tumor consists of a heterogeneous, unstable cell population that is poised to rapidly respond to ambient selection pressures (Kinzler, K. W. et al., Cell 87, 159-170 (1996); Folkman, J. et al., Nature Revs. Molec. Cell Biol. 1, 76-79 (2000)) and to redirect the local environment in a way that meets its own needs, it has been discovered according to the invention that one can channel this capacity to useful biological ends. Inadequate oxygen and nutrient supply is a basic constraint to tumor growth that elicits compensatory responses within the tumor population (e.g., upregulation of angiogenic cytokines) that induce neovascularization in the local environment. Additionally, the tumor's unstable genome contains genetically varied clones, with selection favoring those clones whose chance expressions promote tumor expansion, including more effective nutrient support (Folkman, J. et al., Nature Revs. Molec. Cell Biol. 1, 76-79 (2000); Hanahan, D. et al., Cell 100, 57-70 (2000)). Owing to such dynamics, the tumor in effect represents an efficient “problem solving machine” for those physiological problems whose solutions are tied, or can be made to be tied, to promoting tumor expansion. The underlying process is thus analogous to the Monte Carlo method (Rubinstein, R. Y., Wiley. New York (1981)) employed computationally to discover by stochastics optimum solutions to otherwise intractable dynamical problems. Clearly, tumor-directed arteriogenesis is a solution to the problem of nutrient acquisition, particularly in instances where significant nutrient delivery to a tumor is required while, at the same time, multiple smaller vessels are discouraged. Accordingly, it has now been discovered according to the invention that if a tumor is physically constrained to be more insulated from its surround, the arteriogenesis “solution” is more favored, as is seen in organs.
The fundamental biological constraint that tumors need sufficient vasculature to expand (Folkman, J. et al, Nature Revs. Molec. Cell Biol. 1, 76-79 (2000)) was at the root of the experiments described in the Examples below to go beyond angiogenesis and utilize tumors to promote arteriogenesis. As described in greater detail below, it was surprisingly discovered that not only can developing tumors be used to induce arteriogenesis, but also that the induced arteriogenesis was accompanied by induced neurogenesis.
While there are no previous reports of tumors inducing neurogenesis, and there is no direct rationale for the induction of nerves by tumors, there is sufficient evidence to suggest that neurogenesis and arteriogenesis are closely coupled. In the body plan of all vertebrates the occurrence of large vessels is accompanied by nerves. Pioneering work by Martin and Lewis (Martin, P. et al., Int. J Dev. Biol. 33(3), 379-387 (1989)) concluded that peripheral nerves and blood vessels follow the same route because they respond independently to the same mesenchymal cues. Furthermore, cell components of both nerves and vessels are known to respond to some of the same cytokines, e.g., ephrins/Eph (Shima, D. T. et al., Curr. Opin. Genet. Dev. 10(5), 536-542 (2000)). Platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-beta) are mitogenic for both Schwann cells and for vascular smooth muscle cells. These are two of an increasing list of growth factors that have both neurogenic and angiogenic effects (Fu, S. Y. et al., Molec. Neurobiol. 14(1-2), 67-116 (1997); Nomi, M. et al., Molec. Aspects Med. 23(6), 463-483 (2002)). Following peripheral nerve injury, the angiogenic molecules vascular endothelial growth factor (VEGF), and fibroblast growth factors aFGF and bFGF, promote axon regeneration (Fu, S. Y. et al., Molec. Neurobiol. 14(1-2), 67-116 (1997); Sondell, M. et al., Eur. J Neurosci. 12(12), 4243-4254 (2000); Fujimoto, E. et al., J. Neurocytol. 26(8), 511-528 (1997)). Neuropilin-1, a mediator of neuronal guidance, is also a specific receptor of VEGF165 (Soker, S. et al., Cell 92(6), 735-745 (1998)). More recently, it was reported that sensory nerves determine the patterning of arterial differentiation in developing chick skin, presumably mediated by VEGF (Mukouyama, Y. S. et al., Cell 109(6), 693-705 (2002)).
The embryonic developmental morphogen Sonic hedgehog (Shh) (Lum, L. et al., Science 304, 1755-9 (2004); Chiang, C. et al., Nature 383, 407-13 (1996); Marti, E. et al., A.P., Nature 375, 322-325 (1995); Ingham, P. W. et al., Genes Devel. 15, 3059-87 (2001)) was found to be instrumental in tumor-driven adult neurogenesis and essential for bundle induction.
The invention in certain aspects relates to a method for generating a tumor characterized by a vasculogenic program. As used herein, a vasculogenic program refers to a set of biochemical steps implemented by a given tumor to induce de novo new arteries and veins characterized by the presence of smooth muscle. A described herein, a vasculogenic program can also induce de novo new nerves and/or neurovascular bundles. This vasculogenic program includes for example the expression of appropriate factors to induce and effectuate arteriogenesis and/or neurogenesis. Such factors can include a morphogen, including in particular Sonic hedgehog. Such factors can be cytokines, e.g. ephrins/Eph, platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), vascular endothelial growth factor (VEGF), fibroblast growth factors aFGF and bFGF, and neuropilin-1.
As used- herein, a tumor characterized by a vasculogenic program is a tumor that has the capacity to carry out such a program, i.e., a tumor that can and does promote arteriogenesis and neurogenesis and can effectuate the generation of directed arteries, veins, nerves, and/or neurovascular bundles. A tumor characterized by a vasculogenic program generally exhibits, by its anatomy or by tumor products generated by the tumor, the tumor's ability to induce the formation of arteries, veins, nerves, and/or neurovascular bundles.
As used herein, an artery refers to a tubular vascular structure, other than a capillary, having concentric layers of endothelium, internal elastic lamina, and smooth muscle as recognized by those of skill in the art. Arteries include arterioles, small arteries, muscular arteries, and large elastic arteries. Arteries normally transport blood away from the heart. In one embodiment an artery has an external diameter of at least 1 mm.
As used herein, a vein refers to a tubular vascular structure, other than a capillary, having concentric layers of endothelium and smooth muscle, as recognized by those of skill in the art. Veins include venules, veins of small caliber, veins of medium caliber, and veins of large caliber. Veins normally transport blood toward the heart. In one embodiment a vein has an external diameter of at least 1 mm.
A vasculogenic program includes an arteriogenic program. An arteriogenic program, as used herein, is a type of vasculogenic program that is specifically involved in inducing arteries.
A vasculogenic program is to be distinguished from an angiogenic program. As used herein, an angiogenic program refers to a set of biochemical steps implemented by a given tumor to induce sprouting of capillaries from pre-existing arteries and veins near or adjacent to the tumor, to penetrate into the tumor. The capillaries arising from an angiogenic program are essentially free of smooth muscle and thus are composed essentially of naked endothelial cells.
Various aspects of the invention include the step of implanting an intact tumor, or a nondispersed portion thereof, into a living vertebrate. As used herein, a tumor refers to a mass of abnormally replicating cells of vertebrate origin. Abnormally replicating cells of a tumor are termed tumor cells or, equivalently, cancer cells. A subject having a tumor is a subject in which a detectable tumor is present. Tumors include but are not limited to cancers of the biliary tract; bladder; bone; brain; breast; cervix; colon; endometrium; esophagus; heart; kidney; larynx; liver; lung (e.g., small cell and non-small cell); meninges; oral cavity (including any of lip, tongue, mouth, pharynx); ovary; pancreas; prostate; rectum; skeletal muscle; skin; small intestine; spinal cord; stomach; testis; thyroid; ureter; uterus; as well as choriocarcinoma; intraepithelial neoplasms; leukemias; lymphomas; melanoma; neuroblastoma; retinoblastoma; sarcomas; and other carcinomas and sarcomas. In one embodiment the tumor is a solid tumor that excludes leukemias and other hematologic cancers. Tumors include both primary and metastatic tumors.
An intact tumor can be derived from any suitable source. In one embodiment the intact tumor is derived from a spontaneously occurring tumor in a vertebrate subject. For example, a biopsied or surgically removed tumor obtained from a human subject can be used as an intact tumor. In one embodiment the intact tumor is derived from a tumor cell line. Tumor cell lines are well known in the art and many are available from commercial sources. Example of tumor cell lines include, without limitation, those listed in Table 1, available from American Type Culture Collection (ATCC, Manassas, Va.).







TABLE 1







Exemplary Tumor Cell Lines










ATCC No.
Name
Cancer Type
Tissue Source





CRL-2327
HCC1428
adenocarcinoma
breast


CRL-7850
Hs 588.T
adenocarcinoma
cervix


CCL-249
NCI-H548
adenocarcinoma
colon


CRL-5972
SNU-C1
adenocarcinoma
colon


CRL-7207
Hs 241.T
adenocarcinoma
colon


CRL-7213
Hs 255.T
adenocarcinoma
colon


CRL-7435
Hs 698.T
adenocarcinoma
colon


HTB-78
SW 626
adenocarcinoma
colon; ovary metastasis


CRL-7928
HuTu 80
adenocarcinoma
duodenum


HTB-40
HuTu 80
adenocarcinoma
duodenum


HTB-111
AN3 CA
adenocarcinoma
endometrial cancer; lymph node





metastasis


CRL-7911
A704
adenocarcinoma
kidney


HTB-45
A-704
adenocarcinoma
kidney


HTB-49
SW 839
adenocarcinoma
kidney; clear cell


CRL-5892
NCI-H1755
adenocarcinoma
liver


HTB-52
SK-HEP-1
adenocarcinoma
liver; ascites


CRL-5944
NCI-H2405
adenocarcinoma
lung


CRL-5844
NCI-H838
adenocarcinoma
lung


CRL-5866
NCI-H1373
adenocarcinoma
lung


CRL-5868
NCI-H1395
adenocarcinoma
lung


CRL-5918
NCI-H2073
adenocarcinoma
lung


CRL-5942
NCI-H2347
adenocarcinoma
lung


CRL-7380
Hs 618.T
adenocarcinoma
lung


CRL-5877
NCI-H1573
adenocarcinoma
lung


CRL-5907
NCI-H1944
adenocarcinoma
lung


HTB-57
SK-LU-1
adenocarcinoma
lung


CRL-5850
NCI-H920
adenocarcinoma
lung, lymph node metastasis


CRL-5852
NCI-H969
adenocarcinoma
lung, pleural effusion


CRL-5865
NCI-H1355
adenocarcinoma
lung, pleural effusion


CRL-5872
NCI-H1437
adenocarcinoma
lung, pleural effusion


CRL-5876
NCI-H1568
adenocarcinoma
lung; lymph node metastasis


CRL-5881
NCI-H1623
adenocarcinoma
lung; lymph node metastasis


CRL-5882
NCI-H1648
adenocarcinoma
lung; lymph node metastasis


CRL-5887
NCI-H1693
adenocarcinoma
lung; lymph node metastasis


CRL-5897
NCI-H1819
adenocarcinoma
lung; lymph node metastasis


CRL-5909
NCI-H1993
adenocarcinoma
lung; lymph node metastasis


CRL-5911
NCI-H2009
adenocarcinoma
lung; lymph node metastasis


CRL-5912
NCI-H2023
adenocarcinoma
lung; lymph node metastasis


CRL-5922
NCI-H2087
adenocarcinoma
lung; lymph node metastasis


CRL-5936
NCI-H2250
adenocarcinoma
lung; lymph node metastasis


CRL-5895
NCI-H1792
adenocarcinoma
lung; pleural effusion


CRL-5985
NCI-H2122
adenocarcinoma
lung; pleural effusion


HTB-179
NCI-H676B
adenocarcinoma
lung; pleural effusion


HTB-55
Calu-3
adenocarcinoma
lung; pleural effusion


CRL-5889
NCI-H1703
adenocarcinoma
lung; squamous cell


CRL-2351
AU565
adenocarcinoma
mammary gland; breast


CRL-7222
Hs 274.T
adenocarcinoma
mammary gland; breast


CRL-7226
Hs 280.T
adenocarcinoma
mammary gland; breast


CRL-7227
Hs 281.T
adenocarcinoma
mammary gland; breast


CRL-7245
Hs 343.T
adenocarcinoma
mammary gland; breast


CRL-7253
Hs 362.T
adenocarcinoma
mammary gland; breast


CRL-7477
Hs 739.T
adenocarcinoma
mammary gland; breast


CRL-7480
Hs 741.T
adenocarcinoma
mammary gland; breast


CRL-7647
Hs 902.T
adenocarcinoma
mammary gland; breast


HTB-132
MDA-MB-468
adenocarcinoma
mammary gland; breast


HTB-27
MDA-MB-361
adenocarcinoma
mammary gland; breast


HTB-30
SK-BR-3
adenocarcinoma
mammary gland; breast; malignant





pleural effusion


HTB-128
MDA-MB-415
adenocarcinoma
mammary gland; breast; pleural





effusion


HTB-130
MDA-MB-436
adenocarcinoma
mammary gland; breast; pleural





effusion


HTB-21
CAMA-1
adenocarcinoma
mammary gland; breast; pleural





effusion


HTB-22
MCF7
adenocarcinoma
mammary gland; breast; pleural





effusion


HTB-26
MDA-MB-231
adenocarcinoma
mammary gland; breast; pleural





effusion


HTB-151
Hs 696
adenocarcinoma
metastasis to bone (sacrum)


HTB-147
Hs 700T
adenocarcinoma
metastasis to the pelvis


CRL-10303
MDAH 2774
adenocarcinoma
ovary


HTB-161
NIH:OVCAR-3
adenocarcinoma
ovary


HTB-75
Caov-3
adenocarcinoma
ovary


HTB-76
Caov-4
adenocarcinoma
ovary


HTB-77
SK-OV-3
adenocarcinoma
ovary; ascites


CRL-1687
BxPC-3
adenocarcinoma
pancreas


CRL-1997
HPAF-II
adenocarcinoma
pancreas


CRL-2119
HPAC
adenocarcinoma
pancreas


CRL-2172
SW 1990
adenocarcinoma
pancreas


HTB-79
Capan-1
adenocarcinoma
pancreas


HTB-80
Capan-2
adenocarcinoma
pancreas


CRL-1682
AsPC-1
adenocarcinoma
pancreas; ascites


CRL-1435
PC-3
adenocarcinoma
prostate


CRL-2422
MDA PCa 2b
adenocarcinoma
prostate


CCL-235
SW837
adenocarcinoma
rectum


CRL-1622
KLE
adenocarcinoma
uterus; endometrium


HTB-112
HEC-1-A
adenocarcinoma
uterus; endometrium


HTB-113
HEC-1-B
adenocarcinoma
uterus; endometrium


CRL-7758
TE 206.T
adenocarcinoma
unknown


CRL-5883
NCI-H1650
adenocarcinoma
bronchoalveolar carcinoma lung;





pleural effusion


CRL-5885
NCI-H1666
adenocarcinoma
bronchoalveolar carcinoma lung;





pleural effusion


CRL-5894
NCI-H1781
adenocarcinoma
bronchoalveolar carcinoma lung;





pleural effusion


CRL-2220
CA-HPV-10
adenocarcinoma
prostate


CRL-5800
NCI-H23
adenocarcinoma
non-small cell lung cancer lung


CRL-5810
NCI-H522
adenocarcinoma
non-small cell lung cancer lung


CRL-5870
NCI-H1435
adenocarcinoma
non-small cell lung cancer lung


CRL-5875
NCI-H1563
adenocarcinoma
non-small cell lung cancer lung


CRL-5884
NCI-H1651
adenocarcinoma
non-small cell lung cancer lung


CRL-5891
NCI-H1734
adenocarcinoma
non-small cell lung cancer lung


CRL-5896
NCI-H1793
adenocarcinoma
non-small cell lung cancer lung


CRL-5899
NCI-H1838
adenocarcinoma
non-small cell lung cancer lung


CRL-5908
NCI-H1975
adenocarcinoma
non-small cell lung cancer lung


CRL-5914
NCI-H2030
adenocarcinoma
non-small cell lung cancer lung


CRL-5921
NCI-H2085
adenocarcinoma
non-small cell lung cancer lung


CRL-5935
NCI-H2228
adenocarcinoma
non-small cell lung cancer lung


CRL-5939
NCI-H2291
adenocarcinoma
non-small cell lung cancer lung


CRL-5941
NCI-H2342
adenocarcinoma
non-small cell lung cancer lung


CRL-5834
NCI-H647
adenosquamous carcinoma
lung; pleural effusion; mixed


HTB-178
NCI-H596
adenosquamous carcinoma
lung


CRL-10296
NCI-H295
adrenocortical carcinoma
adrenal gland, cortex


CRL-2170
SW 1573
alveolar cell carcinoma
lung


CRL-1579
C32TG
amelanotic melanoma
skin


CRL-1585
C32
amelanotic melanoma
skin


HTB-137
Hs 695T
amelanotic melanoma
metastasis to lymph node


CRL-9267
W5-6
anaplastic carcinoma
unknown, probably lung


HTB-56
Calu-6
anaplastic carcinoma
unknown, probably lung


CRL-2461
SV7tert
angiomyolipoma
kidney


CRL-1718
CCF-STTG1
astrocytoma
brain


HTB-12
SW 1088
astrocytoma
brain


HTB-13
SW 1783
astrocytoma
brain


CRL-7762
TE 354.T
basal cell carcinoma
skin


CRL-7646
Hs 900.T
benign osteoid osteoma
bone


CRL-7649
Hs 903.T
benign osteoid osteoma
bone


CRL-7672
Hs 919.T
benign osteoid osteoma
bone


CRL-2081
MSTO-211H
biphasic mesothelioma
metastasis to lung


CRL-7588
Hs 853.T
bladder carcinoma
metastasis to lung or bronchus


CRL-2294
BCP-1
body cavity based lymphoma
peripheral blood; B lymphoblast


CRL-5807
NCI-H358
bronchioalveolar carcinoma
lung; bronchiole; alveolus


CRL-5835
NCI-H650
bronchioalveolar carcinoma
lung


CRL-7194
Hs 229.T
bronchogenic adenocarcinoma
lung


HTB-168
ChaGo-K-1
bronchogenic carcinoma
lung; bronchus


CRL-1647
ST486
Burkitt's lymphoma
ascites; B lymphocyte


HTB-62
P3HR-1
Burkitt's lymphoma
ascites; B lymphocyte


CCL-85
EB-3
Burkitt's lymphoma
B lymphocyte


CCL-86
Raji
Burkitt's lymphoma
B lymphocyte


CCL-87
Jiyoye
Burkitt's lymphoma
B lymphocyte


CRL-1432
NAMALWA
Burkitt's lymphoma
B lymphocyte


CRL-1484
HS-Sultan
Burkitt's lymphoma
B lymphocyte


CRL-1648
CA46
Burkitt's lymphoma
B lymphocyte


CRL-2392
GA-10
Burkitt's lymphoma
B lymphocyte


CRL-2393
GA-10
Burkitt's lymphoma
B lymphocyte


CRL-2394
GA-10
Burkitt's lymphoma
B lymphocyte


HTB-61
EB2
Burkitt's lymphoma
B lymphocyte


CRL-7933
P-3J
Burkitt's lymphoma
lymph node; B lymphoblast


CRL-7936
Raji
Burkitt's lymphoma
lymph node; B lymphoblast


CRL-10237
2F7
Burkitt's lymphoma
lymph node; B lymphocyte


CCL-213
Daudi
Burkitt's lymphoma
peripheral blood; B lymphoblast


CCL-214
NC-37
Burkitt's lymphoma
peripheral blood; B lymphoblast


HTB-60
EB1
Burkitt's lymphoma
upper maxilla; B lymphocyte


CRL-1596
Ramos
Burkitt's lymphoma (American)
B lymphocyte


CRL-1923
Ramos
Burkitt's lymphoma (American)
B lymphocyte


CRL-2128
NCI-H295R
carcinoma
adrenal gland, cortex


CRL-1594
C-4I
carcinoma
cervix


CRL-1595
C-4 II
carcinoma
cervix


CRL-7396
Hs 636.T
carcinoma
cervix


CRL-7914
C4II
carcinoma
cervix


CRL-7920
DoTc2 4510
carcinoma
cervix


HTB-31
C-33 A
carcinoma
cervix


HTB-32
HT-3
carcinoma
cervix


CRL-7908
A498
carcinoma
kidney


HTB-44
A-498
carcinoma
kidney


CCL-185
A549
carcinoma
lung


HTB-53
A-427
carcinoma
lung


CRL-5867
NCI-H1385
carcinoma
lymph node; squamous cell


CRL-7316
Hs 540.T
carcinoma
mammary gland; breast


CRL-7336
Hs 566(B).T
carcinoma
mammary gland; breast


CRL-7365
Hs 605.T
carcinoma
mammary gland; breast


CRL-7368
Hs 606
carcinoma
mammary gland; breast


CRL-7721
MB 157
carcinoma
mammary gland; breast;





pleural effusion


CRL-1420
MIA PaCa-2
carcinoma
pancreas


HTB-134
Hs 766T
carcinoma
pancreas


CCL-138
Detroit 562
carcinoma
pharynx


CRL-10995
LNCaP-FGC
carcinoma
prostate


CRL-1740
LNCaP
carcinoma
prostate



clone FGC


CRL-2505
22Rv1
carcinoma
prostate


HTB-81
DU 145
carcinoma
prostate


CRL-5833
NCI-H630
carcinoma
rectum


CRL-7870
Hs 740.T
carcinoma
stomach


CRL-1803
TT
carcinoma
thyroid, medulla


CRL-1472
HT-1376
carcinoma
urinary bladder


CRL-1473
HT-1197
carcinoma
urinary bladder


CRL-7150
Hs 195.T
carcinoma
urinary bladder


CRL-7193
Hs 228.T
carcinoma
urinary bladder


CRL-7833
Hs 172.T
carcinoma
urinary bladder


CRL-7926
HT 1197.T
carcinoma
urinary bladder


CRL-7927
HT 1376.T
carcinoma
urinary bladder


HTB-9
5637
carcinoma
urinary bladder


CRL-1671
RL95-2
carcinoma
uterus; endometrium


HTB-118
SW 962
carcinoma
vulva


CCL-257
NCI-H1688
carcinoma; classic small cell
lung




lung cancer


CRL-5804
NCI-H187
carcinoma; classic small cell
lung




lung cancer


CRL-5808
NCI-H378
carcinoma; classic small cell
lung




lung cancer


CRL-5817
NCI-H889
carcinoma; classic small cell
lung




lung cancer


CRL-5821
NCI-H60
carcinoma; classic small cell
lung; pleural effusion




lung cancer


CRL-5825
NCI-H220
carcinoma; classic small cell
lung; pleural effusion




lung cancer


CRL-5828
NCI-H250
carcinoma; classic small cell
lung




lung cancer


CRL-5832
NCI-N592
carcinoma; classic small cell
lung




lung cancer


CRL-5836
NCI-H711
carcinoma; classic small cell
lung




lung cancer


CRL-5837
NCI-H719
carcinoma; classic small cell
lung




lung cancer


CRL-5840
NCI-H740
carcinoma; classic small cell
lung




lung cancer


CRL-5841
NCI-H748
carcinoma; classic small cell
lung




lung cancer


CRL-5842
NCI-H774
carcinoma; classic small cell
lung; soft tissue




lung cancer


CRL-5846
NCI-H847
carcinoma; classic small cell
lung; pleural effusion




lung cancer


CRL-5849
NCI-H865
carcinoma; classic small cell
lung; pleural effusion




lung cancer


CRL-5854
NCI-H1059
carcinoma; classic small cell
lung




lung cancer


CRL-5855
NCI-H1092
carcinoma; classic small cell
lung




lung cancer


CRL-5856
NCI-H1105
carcinoma; classic small cell
lung




lung cancer


CRL-5861
NCI-H1284
carcinoma; classic small cell
lung




lung cancer


CRL-5862
NCI-H1304
carcinoma; classic small cell
lung; pleural effusion




lung cancer


CRL-5869
NCI-H1417
carcinoma; classic small cell
lung




lung cancer


CRL-5871
NCI-H1436
carcinoma; classic small cell
lung




lung cancer


CRL-5886
NCI-H1672
carcinoma; classic small cell
lung




lung cancer


CRL-5888
NCI-H1694
carcinoma; classic small cell
lung




lung cancer


CRL-5898
NCI-H1836
carcinoma;
lung




classic small cell lung cancer


CRL-5902
NCI-H1876
carcinoma; classic small cell
lung




lung cancer


CRL-5906
NCI-H1930
carcinoma; classic small cell
lung




lung cancer


CRL-5910
NCI-H1994
carcinoma; classic small cell
lung




lung cancer


CRL-5916
NCI-H2059
carcinoma; classic small cell
lung




lung cancer


CRL-5920
NCI-H2081
carcinoma; classic small cell
lung; pleural effusion




lung cancer


CRL-5979
NCI-H1339
carcinoma; classic small cell
lung; pleural effusion




lung cancer


CRL-5824
NCI-H211
carcinoma; small cell
bone marrow




lung cancer


CRL-2049
DMS 79
carcinoma; small cell
lung




lung cancer


CRL-2062
DMS 53
carcinoma; small cell
lung




lung cancer


CRL-2064
DMS 153
carcinoma; small cell
lung




lung cancer


CRL-2066
DMS 114
carcinoma; small cell
lung




lung cancer


CRL-2177
SW 1271
carcinoma; small cell
lung




lung cancer


CRL-2195
SHP-77
carcinoma; small cell
lung; large cell, variant




lung cancer


CRL-5853
NCI-H1048
carcinoma; small cell
lung; pleural effusion




lung cancer


CRL-5858
NCI-H1184
carcinoma; small cell
lung; lymph node metastasis




lung cancer


CRL-5859
NCI-H1238
carcinoma; small cell
lung




lung cancer


CRL-5864
NCI-H1341
carcinoma; small cell
lung




lung cancer


CRL-5874
NCI-H1522
carcinoma; small cell
lung; pleural effusion




lung cancer


CRL-5879
NCI-H1618
carcinoma; small cell
lung




lung cancer


CRL-5901
NCI-H1870
carcinoma; small cell
lung




lung cancer


CRL-5903
NCI-H1882
carcinoma; small cell
lung




lung cancer


CRL-5905
NCI-H1926
carcinoma; small cell
lung; lymph node metastasis




lung cancer


CRL-5913
NCI-H2029
carcinoma; small cell
lung; lymph node metastasis




lung cancer


CRL-5927
NCI-H2141
carcinoma; small cell
lung; lymph node metastasis




lung cancer


CRL-5929
NCI-H2171
carcinoma; small cell
lung; pleural effusion




lung cancer


CRL-5931
NCI-H2195
carcinoma; small cell
lung




lung cancer


CRL-5932
NCI-H2196
carcinoma; small cell
lung




lung cancer


CRL-5933
NCI-H2198
carcinoma; small cell
lung; lymph node metastasis




lung cancer


CRL-5934
NCI-H2227
carcinoma; small cell
lung




lung cancer


CRL-5940
NCI-H2330
carcinoma; small cell
lung; lymph node metastasis




lung cancer


CRL-5976
NCI-H64
carcinoma; small cell
lung




lung cancer


CRL-5978
NCI-H735
carcinoma; small cell
lung




lung cancer


CRL-5982
NCI-H1963
carcinoma; small cell
lung




lung cancer


CRL-5983
NCI-H2107
carcinoma; small cell
lung




lung cancer


CRL-5984
NCI-H2108
carcinoma; small cell
lung




lung cancer


HTB-119
NCI-H69
carcinoma; small cell
lung




lung cancer


HTB-120
NCI-H128
carcinoma; small cell
lung; pleural effusion




lung cancer


HTB-171
NCI-H446
carcinoma; small cell
lung; pleural effusion




lung cancer


HTB-172
NCI-H209
carcinoma; small cell
lung




lung cancer


HTB-173
NCI-H146
carcinoma; small cell
lung; pleural effusion




lung cancer


HTB-175
NCI-H82
carcinoma; small cell
lung; pleural effusion




lung cancer


HTB-180
NCI-H345
carcinoma; small cell
lung




lung cancer


CRL-5813
NCI-H660
carcinoma; small cell
prostate; neuroendocrine




lung cancer;




extrapulmonary origin


HTB-184
NCI-H510A
carcinoma; small cell
lung




lung cancer;




extrapulmonary origin


CRL-11351
H69AR
carcinoma; small cell
lung




lung cancer;




multidrug resistant


CRL-5809
NCI-N417
carcinoma; variant small cell
lung




lung cancer


CRL-5811
NCI-H526
carcinoma; variant small cell
lung




lung cancer


CRL-5823
NCI-H196
carcinoma; variant small cell
lung




lung cancer


CRL-5831
NCI-H524
carcinoma; variant small cell
lung; lymph node metastasis




lung cancer


CRL-5845
NCI-H841
carcinoma; variant small cell
lung; lymph node metastasis




lung cancer


HTB-177
NCI-H460
carcinoma; large cell
lung; pleural effusion




lung cancer


HTB-183
NCI-H661
carcinoma; large cell
lung




lung cancer


CCL-256
NCI-H2126
carcinoma; non-small cell
lung




lung cancer


CRL-5803
NCI-H1299
carcinoma; non-small cell
lung; large cell; neuroendocrine




lung cancer


CRL-5816
NCI-H810
carcinoma; non-small cell
lung; large cell; neuroendocrine




lung cancer


CRL-5818
NCI-H1155
carcinoma; non-small cell
lung; large cell; neuroendocrine




lung cancer


CRL-7891
Hs 819.T
chondrosarcoma
bone


HTB-94
SW 1353
chondrosarcoma
bone


CCL-98
BeWo
choriocarcinoma
placenta


CRL-7394
Hs 630.T
choriocarcinoma
placenta


HTB-144
JAR
choriocarcinoma
placenta


HTB-36
JEG-3
choriocarcinoma
placenta


CRL-1978
ES-2
clear cell carcinoma
ovary


HTB-46
Caki-1
clear cell carcinoma
kidney


HTB-47
Caki-2
clear cell carcinoma
kidney


CCL-251
NCI-H716
colorectal adenocarcinoma
cecum


CCL-252
NCI-H747
colorectal adenocarcinoma
cecum


CCL-253
NCI-H508
colorectal adenocarcinoma
cecum


CCL-254
NCI-H498
colorectal adenocarcinoma
cecum


CCL-218
WiDr
colorectal adenocarcinoma
colon


CCL-220
COLO 320DM
colorectal adenocarcinoma
colon


CCL-220.1
COLO 320HSR
colorectal adenocarcinoma
colon


CCL-221
DLD-1
colorectal adenocarcinoma
colon


CCL-222
COLO 205
colorectal adenocarcinoma
colon


CCL-224
COLO 201
colorectal adenocarcinoma
colon


CCL-225
HCT-15
colorectal adenocarcinoma
colon


CCL-227
SW620
colorectal adenocarcinoma
colon


CCL-228
SW480
colorectal adenocarcinoma
colon


CCL-229
LoVo
colorectal adenocarcinoma
colon


CCL-230
SW403
colorectal adenocarcinoma
colon


CCL-231
SW48
colorectal adenocarcinoma
colon


CCL-233
SW1116
colorectal adenocarcinoma
colon


CCL-237
SW948
colorectal adenocarcinoma
colon


CCL-238
SW1417
colorectal adenocarcinoma
colon


CCL-255
LS123
colorectal adenocarcinoma
colon


CL-187
LS 180
colorectal adenocarcinoma
colon


CL-188
LS 174T
colorectal adenocarcinoma
colon


CRL-2102
C2BBe1
colorectal adenocarcinoma
colon


CRL-7214
Hs 257.T
colorectal adenocarcinoma
colon


CRL-7351
Hs 586.T
colorectal adenocarcinoma
colon


CRL-7352
Hs 587.Int
colorectal adenocarcinoma
colon


HTB-37
Caco-2
colorectal adenocarcinoma
colon


HTB-38
HT-29
colorectal adenocarcinoma
colon


HTB-39
SK-CO-1
colorectal adenocarcinoma
colon


CCL-234
SW1463
colorectal adenocarcinoma
rectum


CRL-7159
Hs 200.T
colorectal adenocarcinoma
rectum


CRL-7184
Hs 219.T
colorectal adenocarcinoma
rectum


CRL-7168
Hs 207.T
colorectal adenocarcinoma
sigmoid colon


CCL-250
SNU-C2B
colorectal carcinoma
cecum


CCL-250.1
SNU-C2A
colorectal carcinoma
cecum


CRL-2134
LS513
colorectal carcinoma
cecum


CRL-2158
LS1034
colorectal carcinoma
cecum


CRL-2159
LS411N
colorectal carcinoma
cecum


CCL-247
HCT 116
colorectal carcinoma
colon


CCL-248
T84
colorectal carcinoma
colon


CRL-7399
Hs 674.T/cc
colorectal carcinoma
rectum


CRL-7456
Hs 722.T
colorectal carcinoma
rectum


CRL-7273
Hs 398.T
condyloma acuminatum
skin; genital wart


CRL-2105
HH
cutaneous T cell lymphoma
peripheral blood; T lymphocyte


CRL-8294
MJ
cutaneous T cell lymphoma;
peripheral blood; T lymphocyte




mycosis fungoides


CRL-7252
Hs 357.T
dermatofibrosarcoma
skin


CRL-7692
Hs 941.T
dermatofibrosarcoma
skin


CRL-7043
Hs 63.T
dermatofibrosarcoma
skin




protuberans


CRL-7233
Hs 295.T
dermatofibrosarcoma
skin




protuberans


HTB-186
Daoy
desmoplastic cerebellar
brain; cerebellum




medulloblastoma


CRL-2260
HT
diffuse mixed lymphoma
ascites; B lymphoblast


CRL-2558
PL45
ductal adenocarcinoma
pancreas


CRL-1918
CFPAC-1
ductal adenocarcinoma;
pancreas


CRL-12420
GI-101A
ductal adenocarcinoma;
mammary gland; breast; duct




infiltrating


CRL-1500
ZR-75-1
ductal carcinoma
mammary gland; breast;





ascites; epithelial


CRL-1504
ZR-75-30
ductal carcinoma
mammary gland; breast;





ascites; epithelial


CRL-1897
UACC-812
ductal carcinoma
mammary gland; breast


CRL-2320
HCC1008
ductal carcinoma
mammary gland; breast


CRL-2338
HCC1954
ductal carcinoma
mammary gland; breast; duct


CRL-7345
Hs 574.T
ductal carcinoma
mammary gland; breast; duct


HTB-121
BT-483
ductal carcinoma
mammary gland; breast


HTB-129
MDA-MB-435S
ductal carcinoma
mammary gland; breast;





pleural effusion


HTB-133
T-47D
ductal carcinoma
mammary gland; breast;





pleural effusion


HTB-20
BT-474
ductal carcinoma
mammary gland; breast


HTB-25
MDA-MB-175
ductal carcinoma
mammary gland; breast;





pleural effusion


CRL-1837
SU.86.86
ductal carcinoma
pancreas


HTB-104
Cates-1B
embryonal carcinoma
testis


CRL-7802
Hs 454.T
eosinophilic granuloma
bone


CRL-1550
Ca Ski
epidermoid carcinoma
cervix


CRL-7932
ME180
epidermoid carcinoma
cervix


HTB-33
ME-180
epidermoid carcinoma
cervix


HTB-34
MS751
epidermoid carcinoma
cervix


CRL-1555
A-431
epidermoid carcinoma
epidermis


CRL-7902
A253
epidermoid carcinoma
epidermis


CCL-199
HLF-a
epidermoid carcinoma
lung


HTB-54
Calu-1
epidermoid carcinoma
lung


CRL-7228
Hs 284.Pe
epidermoid carcinoma
lung; pleural effusion


CRL-7905
A388
epidermoid carcinoma
metastasis to lymph node


CRL-2592
A431NS
epidermoid carcinoma
skin, epidermis


HTB-41
A-253
epidermoid carcinoma
submaxillary salivary gland


CRL-1469
PANC-1
epithelioid carcinoma
pancreas; duct


CRL-2138
VA-ES-BJ
epithelioid carcinoma
metastasis to bone marrow


CRL-7556
Hs 822.T
Ewing's sarcoma
bone


CRL-7598
Hs 863.T
Ewing's sarcoma
bone


HTB-166
RD-ES
Ewing's sarcoma
bone


CRL-7744
TE 115.T
fibromatosis
connective and soft tissue


CRL-7951
HT 1080.T
fibrosarcoma
bone


CRL-7062
Hs 93.T
fibrosarcoma
connective and soft tissue


CRL-7287
Hs 414.T
fibrosarcoma
connective tissue


CRL-7508
Hs 778(A).T
fibrosarcoma
connective tissue


CRL-7509
Hs 778(B).T
fibrosarcoma
connective tissue


CRL-7664
Hs 913(B).T
fibrosarcoma
connective tissue


CRL-7665
Hs 913(C).T
fibrosarcoma
connective tissue


CRL-7824
Hs 15.T
fibrosarcoma
connective tissue


HTB-152
Hs 913T
fibrosarcoma
metastasis to lung


CRL-7666
Hs 913(D).T
fibrosarcoma
metastasis to lung or bronchus


CRL-7668
Hs 913(F).T
fibrosarcoma
metastasis to lung or bronchus


CCL-121
HT-1080
fibrosarcoma
unknown


CRL-7604
Hs 868.T
fibrosarcoma
unknown


HTB-91
SW 684
fibrosarcoma
unknown


TIB-223
GCT
fibrous histiocytoma
metastasis to lung


CRL-7773
TE 615.T
ganglioneuroblastoma
brain


CRL-1739
AGS
gastric adenocarcinoma
stomach


CRL-1863
RF-48
gastric adenocarcinoma
stomach


CRL-1864
RF-1
gastric adenocarcinoma
stomach


CRL-5822
NCI-N87
gastric carcinoma
stomach


HTB-135
Hs 746T
gastric carcinoma
stomach


CRL-5971
NCI-SNU-1
gastric carcinoma
stomach; ascites


CRL-5973
NCI-SNU-5
gastric carcinoma
stomach; ascites


CRL-5974
NCI-SNU-16
gastric carcinoma
stomach; ascites


HTB-103
KATO III
gastric carcinoma
stomach; pleural effusion


CRL-7447
Hs 706.T
giant cell sarcoma
bone


CRL-7473
Hs 737.T
giant cell sarcoma
bone


CRL-7554
Hs 821.T
giant cell sarcoma
bone


CRL-7579
Hs 846.T
giant cell sarcoma
bone


CRL-7617
Hs 883.T
giant cell sarcoma
bone


CRL-7081
Hs 127.T
giant cell sarcoma
connective tissue


CRL-7547
Hs 814.T
giant cell sarcoma
vertebral column


CRL-1620
A172
glioblastoma
brain


CRL-2020
DBTRG-05MG
glioblastoma
brain; glial cell


CRL-7899
A172
glioblastoma
brain; glial cell


HTB-16
U-138 MG
glioblastoma
brain


CRL-1690
T98G
glioblastoma multiforme
brain


HTB-14
U-87 MG
glioblastoma; astrocytoma
brain


HTB-15
U-118 MG
glioblastoma; astrocytoma
brain


HTB-138
Hs 683
glioma
brain


CRL-10741
C3A
hepatoblastoma
liver


CRL-11997
HEP G2/2.2.1
hepatoblastoma;
liver




transfected with a CYP7




minigene/luciferase construct


CRL-2233
SNU-398
hepatocellular carcinoma
liver


CRL-2234
SNU-449
hepatocellular carcinoma
liver


CRL-2235
SNU-182
hepatocellular carcinoma
liver


CRL-2236
SNU-475
hepatocellular carcinoma
liver


HB-8064
Hep 3B2.1-7
hepatocellular carcinoma
liver



(Hep 3B)


HB-8065
Hep G2
hepatocellular carcinoma
liver


CRL-8024
PLC/PRF/5
hepatoma
liver; Alexander cells


CRL-1532
182-PF SK
hereditary adenomatosis
skin


CRL-1533
166-ME SK
hereditary adenomatosis
skin




(Gardner's variant)


CRL-1593.2
U-937
histiocytic lymphoma
macrophage; histiocyte


CRL-2367
TUR
histiocytic lymphoma;
histiocyte




transfected U-937 cells


CRL-7593
Hs 856.T
histiocytoma
connective tissue


CCL-113
RPMI 6666
Hodgkin's disease;
unknown




Hodgkin's lymphoma


CRL-7264
Hs 388.T
Hodgkin's disease;
lymph node




Hodgkin's lymphoma


CRL-7362
Hs 604.T
Hodgkin's disease;
lymph node




Hodgkin's lymphoma


CRL-7488
Hs 751.T
Hodgkin's disease;
lymph node




Hodgkin's lymphoma


HTB-146
Hs 445
Hodgkin's disease;
lymph node




Hodgkin's lymphoma


CRL-7779
TO 175.T
Hodgkin's disease;
skin




Hodgkin's lymphoma


CRL-7373
Hs 611.T
Hodgkin's disease;
spleen




Hodgkin's lymphoma


CRL-7378
Hs 616.T
Hodgkin's disease;
thymus




Hodgkin's lymphoma


CRL-2175
SW 156
hypernephroma
kidney


CCL-244
HCT-8
ileocecal colorectal
colon



(HRT-18)
adenocarcinoma


CRL-7428
Hs 692(A).T
intestinal carcinoma
metastasis to lymph node


CRL-7630
Hs 892.T
keratoacanthoma
skin


CRL-7629
Hs 891.T
kidney carcinoma
metastasis to lymph node


CRL-5878
NCI-H1581
large cell adenocarcinoma
lung


CRL-2262
29SR
large cell immunoblastic
pleural effusion; lymphoblast




lymphoma


CRL-2289
DB
large cell lymphoma
B lymphoblast


CRL-5923
NCI-H2106
large cell neuroendocrine
lung metastasis to lymph node




carcinoma


CRL-7822
Hs 5.T
leiomyosarcoma
connective tissue


HTB-88
SK-LMS-1
leiomyosarcoma
vulva


CRL-10423
JM1
leukemia; lymphoma
pre-B lymphoblast


HTB-92
SW 872
liposarcoma
unknown


CRL-7306
Hs 505.T
lymphocytic lymphoma
lymph node


CRL-7313
Hs 518.T
lymphocytic lymphoma
spleen


CRL-7818
Hs 491.T
lymphocytic lymphoma
lymph node


CRL-7218
Hs 268.T
lymphogranulomatosis
lymph node


CRL-2230
BC-1
lymphoma
B lymphocyte


CRL-2231
BC-2
lymphoma
B lymphocyte


CRL-2277
BC-3
lymphoma
B lymphocyte


CRL-8119
1A2
lymphoma
B lymphocyte


HTB-142
Hs 602
lymphoma
cervical lymph node


CRL-11622
RH9/CB
lymphoma
cutaneous T lymphocyte


CRL-12043
RH9
lymphoma
cutaneous T lymphocyte


HTB-176
H9
lymphoma
cutaneous T lymphocyte


TIB-161
HuT 78
lymphoma
cutaneous T lymphocyte


CRL-7235
Hs 313.T
lymphoma
lymph node


CRL-7507
Hs 777.T
lymphoma
lymph node


CRL-11213
RH9/MSC
lymphoma
T lymphocyte


CRL-8543
H9/HTLV-IIIB
lymphoma
T lymphocyte


CRL-7797
HT 1417
lymphoma
unknown


TIB-162
HuT 102
lymphoma; mycosis fungoides
cutaneous T lymphocyte


CRL-7755
TE 175.T
lymphosarcoma
lymph node


CRL-7641
Hs 898.T
malignant acanthocytosis;
skin




keratoacanthoma


HTB-105
Tera-1
malignant embryonal
embryo




carcinoma


HTB-106
Tera-2
malignant embryonal
embryo




carcinoma


CRL-2365
M059K
malignant glioblastoma;
brain; glial cell




glioma


CRL-2366
M059J
malignant glioblastoma;
brain; glial cell




glioma


CRL-7684
Hs 934.T
malignant melanoma
connective tissue


CRL-7685
Hs 935.T
malignant melanoma
connective tissue


HTB-64
Malme-3M
malignant melanoma
metastasis to lung


HTB-63
HT-144
malignant melanoma
metastasis to subcutaneous tissue


CRL-1424
G-361
malignant melanoma
skin


CRL-1619
A-375
malignant melanoma
skin


CRL-1872
A375.S2
malignant melanoma
skin


CRL-1974
COLO 829
malignant melanoma
skin


CRL-7691
Hs 940.T
malignant melanoma
skin


HTB-65
MeWo
malignant melanoma
skin


HTB-66
RPMI-7951
malignant melanoma
skin


HTB-67
SK-MEL-1
malignant melanoma
skin


HTB-68
SK-MEL-2
malignant melanoma
skin


HTB-69
SK-MEL-3
malignant melanoma
skin


HTB-70
SK-MEL-5
malignant melanoma
skin


HTB-71
SK-MEL-24
malignant melanoma
skin


HTB-72
SK-MEL-28
malignant melanoma
skin


HTB-73
SK-MEL-31
malignant melanoma
skin


CRL-2407
NK-92
malignant non-Hodgkin's
natural killer cell; NK cell




lymphoma


CRL-11732
OV-90
malignant papillary serous
ovary




adenocarcinoma


CRL-1973
NTERA-2 cl.D1
malignant pluripotent
testis




embryonal carcinoma


CRL-8805
TE671
medulloblastoma
brain; cerebellum


HTB-185
D283 Med
medulloblastoma
brain; cerebellum


HTB-187
D341 Med
medulloblastoma
brain; cerebellum


CRL-7724
SH-4
melanoma
lung, pleural effusion


CRL-7426
Hs 688(B).T
melanoma
metastasis to lymph node


CRL-7568
Hs 834.T
melanoma
metastasis to lymph node


CRL-11147
A2058
melanoma
skin


CRL-1675
WM-115
melanoma
skin


CRL-1676
WM-266-4
melanoma
skin


CRL-2500
A7
melanoma
skin


CRL-7299
Hs 432.T
melanoma
skin


CRL-7360
Hs 600.T
melanoma
skin


CRL-7425
Hs 688(A).T
melanoma
skin


CRL-7572
Hs 839.T
melanoma
skin


CRL-7585
Hs 852.T
melanoma
skin


CRL-7637
Hs 895.T
melanoma
skin


CRL-7653
Hs 906(A).T
melanoma
skin


CRL-7654
Hs 906(B).T
melanoma
skin


CRL-7658
Hs 908.Sk
melanoma
skin


CRL-7686
Hs 936.T
melanoma
skin


CRL-7687
Hs 936.T(C1)
melanoma
skin


CRL-7690
Hs 939.T
melanoma
skin


CRL-7898
A101D
melanoma
skin


CRL-7904
A375
melanoma
skin


CRL-9446
CHL-1
melanoma
skin


CRL-9451
CHL-2
melanoma
skin


CRL-9607
HMCB
melanoma
skin


HTB-140
Hs 294T
melanoma
skin


HTB-114
SK-UT-1
mesodermal tumor (mixed)
uterus




consistent with




leiomyosarcoma


HTB-115
SK-UT-1B
mesodermal tumor (mixed);
uterus; endometrium




consistent with




leiomyosarcoma


CRL-5820
NCI-H28
mesothelioma
pleural effusion


CRL-5915
NCI-H2052
mesothelioma
pleural effusion


CRL-5917
NCI-H2066
mixed; small cell lung cancer;
lung




adenocarcinoma;




squamous cell carcinoma


CRL-5938
NCI-H2286
mixed; small cell lung cancer;
lung




adenocarcinoma;




squamous cell carcinoma


CRL-1848
NCI-H292
mucoepidermoid pulmonary
lung




carcinoma


CRL-8644
HuNS1
myeloma
lymphoblast


CCL-127
IMR-32
neuroblastoma
brain; neuroblast


CRL-2137
SK-N-AS
neuroblastoma
brain; neuroblast


CRL-2142
SK-N-FI
neuroblastoma
brain; neuroblast


CRL-2149
SK-N-DZ
neuroblastoma
brain; neuroblast


CRL-2266
SH-SY5Y
neuroblastoma
brain


CRL-2267
BE(2)-M17
neuroblastoma
brain


CRL-2268
BE(2)-C
neuroblastoma
brain


CRL-2270
MC-IXC
neuroblastoma
brain


CRL-2271
SK-N-BE(2)
neuroblastoma
brain


CRL-2273
CHP-212
neuroblastoma
brain


HTB-11
SK-N-SH
neuroblastoma
brain


CRL-5893
NCI-H1770
neuroendocrine carcinoma
lymph node


HTB-10
SK-N-MC
neuroepithelioma
brain


HTB-148
H4
neuroglioma
brain


CRL-7434
Hs 697.Ln
non-caseating granuloma
lymph node


CRL-10236
10C9
non-Hodgkin's lymphoma
lymph node; B lymphocyte


CRL-2261
RL
non-Hodgkin's lymphoma
ascites; B lymphoblast


CRL-2408
NK-92MI
non-Hodgkin's lymphoma,
lymphoblast




malignant; transfected




with human IL-2 cDNA


CRL-2073
NCCIT
nullipotent embryonal
bone




carcinoma; teratocarcinoma


CRL-7609
Hs 871.T
osteoblastoma
bone


CRL-11226
143.98.2
osteosarcoma
bone


CRL-1423
G-292
osteosarcoma
bone


CRL-1427
MG-63
osteosarcoma
bone


CRL-1543
HOS
osteosarcoma
bone


CRL-1544
KHOS/NP
osteosarcoma
bone


CRL-1545
KHOS-240S
osteosarcoma
bone


CRL-1546
KHOS-321H
osteosarcoma
bone


CRL-1547
MNNG/HOS
osteosarcoma
bone


CRL-7005
Hs 3.T
osteosarcoma
bone


CRL-7023
Hs 39.T
osteosarcoma
bone


CRL-7060
Hs 88.T
osteosarcoma
bone; connective tissue


CRL-7134
Hs 184.T
osteosarcoma
bone


CRL-7140
Hs 188.T
osteosarcoma
bone


CRL-7263
Hs 387.T
osteosarcoma
bone


CRL-7444
Hs 704.T
osteosarcoma
bone


CRL-7448
Hs 707(A).T
osteosarcoma
bone


CRL-7471
Hs 735.T
osteosarcoma
bone


CRL-7489
Hs 755(B).T
osteosarcoma
bone


CRL-7511
Hs 781.T
osteosarcoma
bone


CRL-7521
Hs 792(B).T
osteosarcoma
bone


CRL-7537
Hs 805.T
osteosarcoma
bone


CRL-7543
Hs 811.T
osteosarcoma
bone


CRL-7577
Hs 845.T
osteosarcoma
bone


CRL-7595
Hs 860.T
osteosarcoma
bone


CRL-7600
Hs 864.T
osteosarcoma
bone; connective tissue


CRL-7602
Hs 866.T
osteosarcoma
bone


CRL-7606
Hs 870.T
osteosarcoma
bone


CRL-7622
Hs 888.T
osteosarcoma
bone


CRL-7626
Hs 889.T
osteosarcoma
bone


CRL-7628
Hs 890.T
osteosarcoma
bone


CRL-7631
Hs 894(A).T
osteosarcoma
bone


CRL-7632
Hs 894(B).T
osteosarcoma
bone


CRL-7633
Hs 894(C).T
osteosarcoma
bone


CRL-7634
Hs 894(D).T
osteosarcoma
bone


CRL-7642
Hs 899(A).T
osteosarcoma
bone


CRL-7643
Hs 899(B).T
osteosarcoma
bone


CRL-7644
Hs 899(C).T
osteosarcoma
bone


CRL-7645
Hs 899(D)
osteosarcoma
bone


CRL-7765
TE 417.T
osteosarcoma
bone


CRL-7766
TE 418.T
osteosarcoma
bone


CRL-7780
TO 203.T
osteosarcoma
bone


CRL-7783
HT 728.T
osteosarcoma
bone


CRL-7823
Hs 14.T
osteosarcoma
bone


CRL-7939
SaOS
osteosarcoma
bone


CRL-7943
T1-73
osteosarcoma
bone


CRL-8303
143B
osteosarcoma
bone


CRL-8304
143B
osteosarcoma
bone


HTB-85
Saos-2
osteosarcoma
bone


HTB-96
U-2 OS
osteosarcoma
bone


CRL-2098
SJSA-1
osteosarcoma
bone


CRL-7677
Hs 925.T
pagetoid sarcoma
skin


CRL-5819
NCI-H1404
papillary adenocarcinoma
lung


CRL-7573
Hs 840.T
papilloma
pharynx


CCL-155
RPMI 8226
plasmacytoma; myeloma
B lymphocyte


CRL-8033-1
SKO-007
plasmacytoma; myeloma
B lymphocyte


CRL-8033-2
SKO-007
plasmacytoma; myeloma
B lymphocyte


CRL-8083
MC/CAR
plasmacytoma; myeloma
B lymphocyte


CRL-8147
MC/CAR-Z2
plasmacytoma; myeloma
B lymphocyte


TIB-196
U266B1
plasmacytoma; myeloma
B lymphocyte


CRL-9068
NCI-H929
plasmacytoma; myeloma
bone marrow; B lymphocyte


CRL-2237
SNU-387
pleomorphic hepatocellular
liver




carcinoma


CRL-2238
SNU-423
pleomorphic hepatocellular
liver




carcinoma


CRL-5904
NCI-H1915
poorly differentiated
brain




carcinoma


CRL-7753
TE 161.T
possible Burkitt's lymphoma
lymph node


CRL-2335
HCC1806
primary acantholytic
mammary gland; breast




squamous cell carcinoma


CRL-1902
UACC-893
primary ductal carcinoma
mammary gland; breast


CRL-2314
HCC38
primary ductal carcinoma
mammary gland; breast; duct


CRL-2315
HCC70
primary ductal carcinoma
mammary gland; breast; duct


CRL-2316
HCC202
primary ductal carcinoma
mammary gland; breast; duct


CRL-2321
HCC1143
primary ductal carcinoma
mammary gland; breast; duct


CRL-2322
HCC1187
primary ductal carcinoma
mammary gland; breast; duct


CRL-2324
HCC1395
primary ductal carcinoma
mammary gland; breast


CRL-2326
HCC1419
primary ductal carcinoma
mammary gland; breast


CRL-2329
HCC1500
primary ductal carcinoma
mammary gland; breast; duct


CRL-2331
HCC1599
primary ductal carcinoma
mammary gland; breast; duct


CRL-2336
HCC1937
primary ductal carcinoma
mammary gland; breast; duct


CRL-2340
HCC2157
primary ductal carcinoma
mammary gland; breast


CRL-2343
HCC2218
primary ductal carcinoma
mammary gland; breast; duct


CRL-11730
TOV-21G
primary malignant
ovary




adenocarcinoma


CRL-11731
TOV-112D
primary malignant
ovary




adenocarcinoma


CRL-2380
MPanc-96
primary malignant
pancreas




adenocarcinoma


CRL-2330
HCC1569
primary metaplastic
mammary gland; breast




carcinoma


CCL-105
SW-13
primary small cell carcinoma
adrenal gland; cortex


CRL-1231
Sar Nis
pseudoachondroplasia
skin




(autosomal dominant)


CRL-1611
ACHN
renal cell adenocarcinoma
kidney


CRL-1932
786-O
renal celladenocarcinoma
kidney


CRL-1933
769-P
renal celladenocarcinoma
kidney


CRL-1440
G-402
renal leiomyoblastoma
kidney


CRL-7678
Hs 926.T
renal rhabdomyosarcoma
kidney


CRL-7239
Hs 324.T
reticulum cell sarcoma
lymph node


HTB-169
WERI-Rb-1
retinoblastoma
eye; retina


HTB-18
Y79
retinoblastoma
eye; retina


CRL-7713
130T
rhabdomyosarcoma
connective and soft tissue


CRL-7726
T 174
rhabdomyosarcoma
connective and soft tissue


CRL-7763
TE 381.T
rhabdomyosarcoma
connective and soft tissue


CRL-7767
TE 441.T
rhabdomyosarcoma
connective tissue


CRL-7774
TE 617.T
rhabdomyosarcoma
connective tissue


CRL-7862
Hs 729.T
rhabdomyosarcoma
connective tissue


CCL-136
RD
rhabdomyosarcoma
muscle


CRL-1598
A-673
rhabdomyosarcoma
muscle


CRL-2061
SJRH30
rhabdomyosarcoma
muscle


CRL-7752
TE 159.T
rhabdomyosarcoma
unknown


CRL-7900
A204
rhabdomyosarcoma
unknown


HTB-153
Hs 729
rhabdomyosarcoma
unknown


HTB-82
A-204
rhabdomyosarcoma
unknown


CRL-7910
A673
rhabdomyosarcoma or
unknown




undifferentiated carcinoma


CRL-7732
TE 76.T
sacrococcygeal teratoma
bone


CRL-7746
TE 130.T
sacrococcygeal teratoma
bone


HTB-86
SK-ES-1
sarcoma (anaplastic
bone




osteosarcoma or




Ewing's sarcoma)


CRL-7037
Hs 57.T
sarcoma or lymphoma
lung


CRL-7482
Hs 742.T
scirrhous adenocarcinoma
mammary gland; breast


CRL-7800
Hs 444(B).T
seminoma
testis


CRL-7030
Hs 51.T
spindle cell sarcoma
connective and soft tissue


CRL-7085
Hs 132.T
spindle cell sarcoma
connective tissue


CRL-10302
SW756
squamous cell carcinoma
cervix


HTB-35
SiHa
squamous cell carcinoma
cervix


CRL-5928
NCI-H2170
squamous cell carcinoma
lung


HTB-182
NCI-H520
squamous cell carcinoma
lung


HTB-59
SW 900
squamous cell carcinoma
lung


HTB-58
SK-MES-1
squamous cell carcinoma
lung; pleural effusion


CCL-30
RPMI 2650
squamous cell carcinoma
nasal septum; pleural effusion


HTB-43
FaDu
squamous cell carcinoma
pharynx


HTB-107
SW579
squamous cell carcinoma
thyroid


CRL-1623
SCC-15
squamous cell carcinoma
tongue


CRL-1624
SCC-4
squamous cell carcinoma
tongue


CRL-1628
SCC-25
squamous cell carcinoma
tongue


CRL-1629
SCC-9
squamous cell carcinoma
tongue


CRL-2095
CAL 27
squamous cell carcinoma
tongue


HTB-3
SCaBER
squamous cell carcinoma
urinary bladder


HTB-117
SW 954
squamous cell carcinoma
vulva


CRL-5826
NCI-H226
squamous cell carcinoma;
lung; pleural effusion




mesothelioma


CRL-7289
Hs 416.T
squamous papilloma
skin


CRL-7440
Hs 701.T
synovial sarcoma
connective tissue


HTB-93
SW 982
synovial sarcoma
synovium


CRL-1572
PA-1
teratocarcinoma
ovary


CRL-7886
Hs 789.T
transitional cell carcinoma
ureter


CRL-7882
Hs 769.T
transitional cell carcinoma
urethra


CRL-1749
UM-UC-3
transitional cell carcinoma
urinary bladder


CRL-2169
SW 780
transitional cell carcinoma
urinary bladder


HTB-1
J82
transitional cell carcinoma
urinary bladder


HTB-4
T24
transitional cell carcinoma
urinary bladder


HTB-5
TCCSUP
transitional cell carcinoma
urinary bladder


HTB-2
RT4
transitional cell papilloma
urinary bladder


CRL-1649
MC116
undifferentiated lymphoma
ascites; B lymphocyte


CRL-1976
MES-SA
uterine sarcoma
uterus


CRL-2274
MES-SA/MX2
uterine sarcoma
uterus


CRL-1977
MES-SA/Dx5
uterine sarcoma;
uterus




multiple drug resistant


CRL-7102
Hs 156.T
xanthogranuloma
skin









The tumor can but need not necessarily be a tumor of human origin. In one embodiment the tumor is a Lewis lung model.
As used herein, implanting refers to a surgical procedure of placing an intact or nondispersed tumor or other composition in the body of a vertebrate. Implantation can be carried out at any suitable site in the body of the host vertebrate. In one embodiment implantation is subcutaneous, i.e., between the skin and muscle of the vertebrate. In one embodiment implantation is intraperitoneal. Other sites of implantation are contemplated by the invention and specifically include any site toward which it is desired to direct tissue regeneration. The spatial direction of tissue generation is influenced by a gradient of tumor products emanating from the implant.
As used herein, an intact tumor refers to a population of tumor cells having a three-dimensional architecture including extracellular matrix and/or stromal structures generated by the tumor cells as developed when the tumor cells are grown in vivo or in vitro. Intact tumor is to be distinguished from dispersed tumor, the latter referring to any preparation of tumor cells in which the three-dimensional architecture of the intact tumor has been substantially eliminated, e.g., to create a single-cell suspension of tumor cells.
As used herein, a nondispersed portion of an intact tumor refers to a section of an intact tumor having the three-dimensional architecture of the tumor from which the portion is derived. A nondispersed portion of an intact tumor is to be distinguished from a dispersed portion of a tumor, the latter referring to any preparation of tumor cells in which the three-dimensional architecture of a portion of an intact tumor has been substantially eliminated, e.g., to create a single-cell suspension of tumor cells. In one embodiment the nondispersed portion of an intact tumor refers to a segment of intact tumor that has been dissected from the intact tumor. The dissecting in one embodiment involves cutting by any suitable means, e.g., scalpel or scissors. In one embodiment a nondispersed portion of an intact tumor refers to a portion of an intact tumor that is isolated from a region of intact tumor that excludes both outer cortical tissue and central necrotic tissue.
The tumor implant can be a xenograft, an allograft, or an autograft. As used herein the term xenograft refers to a tissue or organ, for example a tumor or a nondispersed piece of a tumor, from a donor of one species, placed into a recipient of another species. As used herein the term allograft refers to a tissue or organ, for example a tumor or a nondispersed piece of a tumor, from an individual donor of one species, placed into another individual recipient of the same species. As used herein the term autograft refers to a tissue or organ, for example a tumor or a nondispersed piece of a tumor, from an individual as donor, placed into the same individual as recipient. Xenografts and allografts may be rejected by an intact immune system of the recipient. Thus in some embodiments the recipient is chosen or manipulated to have an incompetent immune system. In one embodiment the recipient is a SCID mouse.
As used herein, a vertebrate refers to any vertebrate animal, including mammals, fish, birds, reptiles, and amphibians. In one embodiment the vertebrate is a mammal. In one embodiment the mammal is a non-primate mammal, for example, a mouse, rat, rabbit, guinea pig, sheep, pig, goat, or dog. In one embodiment the mammal is a mouse. In one embodiment the mammal is a non-human primate. In one embodiment the mammal is a human. In one embodiment the vertebrate is a fish, e.g., a zebrafish.
Various aspects of the invention include the step of growing the implanted tumor or portion thereof in a vertebrate for at least a selected time. As used herein, growing refers to permitting the implanted tumor or portion thereof to reside in a viable state within the vertebrate host. In one embodiment growing involves an increase in tumor cell population. In one embodiment growing involves an increase in tumor size (e.g., volume). For example, in a typical situation a tumor implant may be 1 mm3 at the time of implantation and at least 1 cm3 after the selected time, a 1000-fold increase in size.
The selected time is any time sufficient to permit establishment of a vasculogenic program and, optionally, at least a desired size. The selected time can range from one day up to one year. In one embodiment at least a selected time is a selected time ranging from one day up to one year. In certain various embodiments the selected time is one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, two months, three months, four months, five months, and six months. In a typical situation the selected time is about one month.
In certain embodiments the implanting and growing steps are repeated serially. A further step of selecting a tumor based on its vasculogenic program, increased vasculogenic program, or reduced angiogenic program, is optionally included after the preceding step of growing, thereby selecting for tumors with the desired phenotype. Each subsequent round of implanting and growing steps are typically performed in a naive vertebrate host, i.e., tumor implanted and grown in host n in round n is implanted and grown in host n+1 in round n+1. Thus a tumor or portion thereof is allowed to grow in a first vertebrate host for the selected amount of time, evaluated for its vasculogenic program, and then used as the tumor or portion thereof implanted into and grown in a second or subsequent vertebrate host. In one embodiment the steps of implanting and growing are performed twice. In one embodiment the steps of implanting and growing are performed three times. In one embodiment the steps of implanting and growing are performed four times. In various embodiments the steps of implanting and growing are performed 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times.
The invention in certain aspects relates to a method for isolating a selected type of living tissue from a tumor. As used herein, a selected type of tissue can be any type of tissue generated or induced by the tumor. In one embodiment the selected type of tissue is an artery. In one embodiment the selected type of tissue is a vein. In one embodiment the selected type of tissue is nerve. In one embodiment the selected type of tissue is a specific type of cell, particularly a stem cell. The selected type of tissue can be isolated from the tumor and then used for any suitable purpose, including a therapeutic purpose.
In one embodiment the isolating can be accomplished in vivo. For example, a neurovascular bundle induced by the tumor can be dissected away from surrounding tissues in a living host, without severing connection between the tumor and the host. The artery and vein of the induced neurovascular bundle may be exposed so that they can be cannulated with a needle or catheter in order to permit sampling of arterial influx to the tumor and venous efflux from the tumor, respectively.
In one embodiment the isolating includes removing the selected tissue from the tumor and from the host. The isolated selected tissue in this embodiment is then available, for example, for use in vivo as a tissue implant at another site in the original host or at any desired site in another host. Alternatively, the isolated selected tissue in this embodiment can be used in vitro for further tissue engineering or characterization, for example in metabolic or pharmacological study.
In one embodiment the isolating includes removing the tumor from the selected tissue, leaving intact the connection between the selected tissue and the host. For example, an artery and a vein included in an induced tissue may be used to create an arteriovenous shunt suitable for use in hemodialysis. In another example, an artery and a vein included in an induced tissue may be used as sites for vascular connection of a kidney allograft.
The invention in certain aspects relates to methods for inducing host tissue regeneration. The methods according to these aspects can employ either tumors or tumor products to induce regeneration of host tissue in a treated vertebrate host.
In various embodiments an intact tumor, a nondispersed portion of an intact tumor, or an effective amount of one or more tumor products is implanted into a vertebrate host in need of host tissue regeneration. In one embodiment the tumor is a tumor characterized by a vasculogenic program, as described herein. As the tumor grows within the host, it induces regeneration of host tissues. Likewise, as the tumor product is released into the host, it induces regeneration of host tissues.
In one embodiment the implanted tumor, nondispersed portion of a tumor, or tumor product is contained within a container bounded at least in part by a semipermeable membrane, the membrane selected to permit transfer of soluble products, such as soluble tumor products, oxygen, nutrients, metabolic waste products, and drugs, between the tumor and the host. The container can be composed completely of semipermeable membrane, or it can be composed partly of impermeable wall and partly of semipermeable membrane. The semipermeable membrane is selected to have a pore size and structure such that transfer of cells cannot occur between the tumor and the host.
In one embodiment the semipermeable membrane is biocompatible, i.e., it does not induce a response by the host. In one embodiment the semipermeable membrane is substantially non-resorbable. In one embodiment the semipermeable membrane is resorbable.
As used herein, a vertebrate host in need of tissue regeneration is a vertebrate having a disease or condition that is characterized by absence or disruption of normal tissue, wherein the absent or disrupted normal tissue may be treated by restoration of normal tissue. The list of diseases and conditions that may benefit from tissue regeneration is extensive and includes, without limitation, cardiovascular disease, coronary insufficiency, myocardial infarction, peripheral vascular disease, peripheral neuropathy, spinal cord injury, stroke, Alzheimer's disease, burns, emphysema, cirrhosis, diabetes mellitus, chronic pancreatitis, amputation.
In one embodiment the vertebrate host has peripheral nerve disease (also termed peripheral neuropathy). Peripheral neuropathy can arise from any of a number of conditions, including ischemia, systemic disease such as diabetes mellitus, infectious disease such as Hansen's disease and Lyme disease, genetic conditions, nerve trauma, and exposure to toxins. Genetically-acquired peripheral neuropathies include Refsum's disease, abetalipoproteinemia, Tangier disease, Krabbe's disease, metachromatic leukodystrophy, Fabry's disease, Dejerine-Sottas syndrome, Charcot-Marie Tooth disease, and others. Exemplary toxic agents which cause neurotoxicities include therapeutic drugs such as antineoplastic agents, various alcohols including ethanol, contaminants in foods, and environmental and industrial pollutants.
In one embodiment the vertebrate host has spinal cord injury. Spinal cord injury includes disruption of spinal cord structure at any location and may result, in certain embodiments, in partial or complete hemiplegia, paraplegia, quadriplegia, and any combination thereof.
In one embodiment the vertebrate host has vascular disease involving an artery. Arterial insufficiency can arise from any of a variety of causes, including atherosclerosis, arteriosclerosis, frostbite, diabetes mellitus, thrombosis, trauma, and vasculitis. The vascular disease is one embodiment can be peripheral vascular disease, e.g., vascular disease of the limbs. The vascular disease in one embodiment involves one or more of the coronary arteries.
In one embodiment the implant is implanted into a site toward which direction the tissue regeneration is desired. Without meaning to be bound to any particular theory or mechanism of action, it is believed that the implanted tumor or tumor products generate a gradient of factors, centered at the location of the implant, along which regeneration of tissue occurs. Thus selection of the location of the implant can be made to best advantage depending on the three-dimensional direction toward which it is desired to regenerate tissue. For example, an implant may be implanted at a site distal to a viable peripheral nerve, which site is in need of peripheral nerve regeneration. As another example, an implant may be positioned at a site distal to adequate coronary flow, which site is at risk of further ischemic injury or insufficiency following completed or threatened myocardial infarction.
As an alternative to implanting a tumor or tumor product, in one aspect of the invention an effective amount of a tumor product is administered to a vertebrate subject in need of tissue regeneration, to induce host tissue regeneration. In one embodiment the tumor product is a tumor product isolated from a tumor characterized by a vasculogenic program, as described herein. The administering may be into a site toward which direction the tissue regeneration is desired.
Various aspects of the invention involve implanting, administering, identifying, or isolating a tumor product. As used herein, a tumor product refers to any composition of tumor origin. Tumor products can include soluble factors and cells originating from the tumor. Thus tumor products do not include regenerated host tissue. In one embodiment the tumor product is a product of a tumor characterized by a vasculogenic program, as described herein. Tumor products include, without limitation, such soluble factors as cytokines, growth factors, morphogens, angiogenesis factors, and anti-angiogenesis factors. A tumor product in one embodiment is a tumor cell. In one embodiment the tumor cell is a stem cell.
As used herein, cytokine refers to any of a number of soluble proteins or glycoproteins that act on immune cells through specific receptors to affect the state of activation and function of the immune cells. Cytokines include interferons, interleukins, tumor necrosis factor, transforming growth factor beta, colony-stimulating factors (CSFs), chemokines, cytokines that stimulate hematopoiesis, as well as others. Interferons specifically include, without limitation, interferon alpha (IFN-α), interferon beta (IFN-β), and interferon gamma (IFN-γ). IFN-α includes a family of about twenty structurally related polypeptides encoded by separate genes. Interleukins specifically include, without limitation, interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, and IL-32. Chemokines are a family of cytokines that stimulate leukocyte movement and migration. Chemokines specifically include, without limitation, so-called C, CC, CXC, and CX3C chemokines, BCA-1, CTACK, CXCL16, ELC, ENA78, eotaxin, fractalkine, GCP-2, Gro-α, Gro-β, Gro-γ, I-309, IP-10, I-TAC, lymphotactin, MCP-1, MCP-2, MCP-3, MCP-4, MDC, MEC, MIG, MIP-1α, MIP-1β, MIP-3α, NAP-2, RANTES, SDF-1, SLC, TARC, and TECK. Colony-stimulating factors specifically include, without limitation, granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte colony-stimulating factor (GM-CSF), and monocyte colony-stimulating factor (M-CSF). Additional cytokines that stimulate hematopoiesis include stem cell factor (c-Kit ligand), erytrhropoietin, and thrombopoietin.
As used herein, growth factor refers to a naturally occurring soluble protein or glycoprotein that stimulates cell division, differentiation, and proliferation. Growth factors are produced by normal cells during embryonic development, tissue growth, and wound healing. Certain growth factors are also considered to be cytokines. Growth factors include, but are not limited to, epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), erythropoietin (EPO), hematopoietic cell growth factor (HCGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), stem cell factors, neurotrophins, transforming growth factor (TGF), and VEGF.
As used herein, morphogen refers to a substance that provides spatial information via a concentration gradient during embryonic development. Morphogens are usually diffusible proteins such as Sonic hedgehog.
Sonic hedgehog (Shh) is a key developmental morphogen in vertebrates, and it plays an essential role in the development of neuronal cells and tissues in both the peripheral and central nervous system(Lum, L. et al., Science 304, 1755-9 (2004)). In addition, it is central in determining dorsal-ventral polarity of the neural tube (Chiang, C. et al., Nature 383, 407-13 (1996); Marti, E. et al., Nature 375, 322-325 (1995); Ingham, P. W. et al., A. P., Genes Devel. 15, 3059-87 (2001)) and the determination of anterior-posterior patterning of limbs. Shh is but one member of the hedgehog protein family, which also includes Indian (Ihh) and Desert (Dhh). Sonic hedgehog signaling has long been considered to be active only during development. However, there are recent reports of continued Shh activity in adult stem cells (Lai, K. et al., Nature Neurosci. 6(1), 21-27 (2003); Ruiz i Altaba et al., Nature Revs. Cancer 2, 361-72 (2002)) and deregulated Shh signaling linked to some specific cancers, e.g. small-cell lung cancer (Watkins, D. N. et al., Nature 422, 313-317 (2003)) and pancreatic adenocarcinomas (Thayer, S. P. et al., Nature 425, 851-856 (2003); di Magliano, M. P. et al., Nature Revs. Cancer 3(12), 903-911 (2003)). Additionally, injection of Shh protein into ischemic limbs of adult mice increased local angiogenesis (Pola, R. et al., Nature Med. 7, 706-711 (2001).
Sonic hedgehog is an important cell signaling molecule expressed during embryonic development. Shh is involved in the patterning of the developing embryonic nervous system, somite and limb. The N-terminal peptide of Shh is released by autoproteolysis and functions through interactions with a multicomponent receptor complex containing the transmembrane proteins Patched (Ptc) and Smoothened (Smo). In the absence of hedgehog protein, Ptc inhibits Smo, a G protein coupled-like receptor. When Shh binds to Ptc, Smo is disinhibited and initiates a signaling cascade that results in activation of a transcription factor, Gli-1. Shh protein is expressed in key embryonic tissues such as the Hensen's node, zone of polarizing activity in the posterior limb bud, notochord, and floor plate of the neural tube. Aberrant activation of the hedgehog pathway has been reported to be associated with certain human cancers, including basal cell carcinoma.
As used herein, Sonic hedgehog antagonist or inhibitor refers to any natural or synthetic substance that decreases, inhibits or abolishes Shh activity, either directly or by affecting any step of the Shh-pathway of signal transduction which involves Patched, Smoothened and Gli. Examples of Shh antagonists include: anti-hedgehog antibodies (5E1), natural products such as the plant alkaloid cyclopamine, small molecule inhibitors such as Cur61414, peptides such as forskolin, the adenylate cyclase activator that functions via protein kinase A to activate the destruction of Ci/Gli (Williams, J. A. et al. Proc Natl Acad Sci USA, 100(8) 4616-4621 (2003)).
As used herein, sonic hedgehog agonist refers to any natural or synthetic substance that activates or stimulate the activity of Shh or of the Shh-signaling pathway. An example of a Shh agonist is Hh-Ag, a synthetic non-peptidyl small molecule (Frank-Kamenetsky, M. et al. J. Biol. 1(2) 10.1-10.19 (2002)).
As used herein, angiogenesis factor refers to a substance secreted by a tumor to induce blood vessel growth (angiogenesis). Angiogenesis is the physiological process involving the formation of new blood vessels from pre-existing vessels, while vasculogenesis is the process of blood vessel formation occurring by a de novo production of endothelial cells. Though similar, the two are different in one aspect: the term angiogenesis denotes the formation of new blood vessels from pre-existing ones, while vasculogenesis is the term used for the formation of new blood vessels when there are no pre-existing ones. Tumors induce blood vessel growth (angiogenesis) by secreting various growth factors (e.g. Vascular Endothelial Growth Factor or VEGF). Growth factors, such as bFGF and VEGF can induce capillary growth into the tumor, supplying required nutrients and allowing for tumor expansion. Thus angiogenesis is a necessary and required step for the transition of tumors from a dormant state to a malignant and perhaps even for the spread of a tumor, or metastasis. Angiogenesis factors are also secreted by macrophages and stimulate neovascularization in healing wounds.
As used herein, anti-angiogenesis factors (or angiogenesis inhibitors) are natural and synthetic substances that inhibit angiogenesis. Examples of natural anti-angiogenesis factors include angiostatin, endostatin, and tumstatin. Angiostatin is a natural anti-angiogenesis factor that is currently in clinical trials for its use in anticancer therapy. It blocks the growth of new blood vessels. Angiostatin is a 57 kDa multimodular fragment of a larger protein, plasminogen. Endostatin is a naturally-occurring chemical in the human body that also serves as an anti-angiogenesis agent. Similar to angiostatins, endostatins interfere with the binding of growth factors (such as VEGF) to angiogenesis factors. An example of a synthetic anti-angiogenesis factor is Avastin®. Avastin® (bevacizumab; Genentech) is a monoclonal antibody directed against an isoform of VEGF. Avastin® interacts with VEGF, thereby inhibiting the binding of VEGF to the receptors that promote angiogenesis.
As used herein, the term stem cell refers to unspecialized human or animal cells that can produce mature specialized body cells and at the same time replicate themselves. Stem cells include embryonic stem cells and adult stem cells. Embryonic stem cells are derived from a blastocyst (the blastula typical of placental mammals), which is very young embryo that contains 200 to 250 cells and is shaped like a hollow sphere. The stem cells themselves are the cells in the blastocyst that ultimately would develop into a person or animal. “Adult” stem cells are derived from the umbilical cord and placenta or from blood, bone marrow, skin, and other tissues. The similar embryonic germ line cells come from a fetus that is 5 to 9 weeks old and are derived from tissue that would have developed into the ovaries or testes. Stem cells could have the capacity to repair or replace damaged body tissues because stem cells are less likely than other foreign cells to be rejected by the immune system when they are implanted in the body. Embryonic stem cells have the capacity to develop into every type of tissue found in an adult. Germ line cells and adult stem cells are widely believed by many to be less versatile, although this view may be rapidly evolving and becoming less restricted as new methods for manipulating adult stem cells are discovered. Stem cells have been used experimentally to form the hematopoietic (blood-making) cells of the bone marrow and heart, blood vessel, muscle, and insulin-producing tissue.
Methods according to the invention for identifying a tumor product and for analyzing uptake or metabolism of a composition by a tumor can be adapted for use in a high throughput format. High throughput screening can include the use of arrays of agents or reagents and, optionally, various robotic devices for sample handling.
A tumor product can be isolated from a tumor using any suitable method. Methods of isolating soluble products from a tissue are well known in the art and can include, without limitation, immunoaffinity chromatography, immunoprecipitation, size exclusion chromatography, high pressure liquid chromatography, and cloning and recombinant expression using a suitable expression vector and expression host cell system. Such methods generally entail no more than routine amounts of experimentation for any given tumor product.
Methods of isolating living cells from a tissue are also well known in the art and can include, without limitation, fluorescence-activated cell sorting, immunobead affinity separation, enzyme-linked immunosorbent assay (ELISA), and the like.
The invention in certain aspects involves sampling arterial influx to a tumor and sampling venous efflux from the tumor. As disclosed herein, the vasculogenic tumor of the invention can resemble an organ with a paired artery and vein serving as the sole or major vascular connection to a host in which it resides. These vascular structures can be sufficiently large as to make it possible to sample contents of the vascular (arterial) influx to the tumor and the vascular (venous) efflux from the tumor. Such sampling is not feasible for tumors having an angiogenic program, for which there are no such large vascular structures supplying and emanating from such tumors.
Arterial influx as used herein refers to a fluid, usually blood, contained or coursing within an artery. Venous efflux as used herein refers to a fluid, usually blood, contained or coursing within a vein.
As used herein, sampling arterial influx to a tumor refers to gaining access to and contacting the blood or other fluid contained or coursing within an artery supplying the tumor. In one embodiment the tumor is a tumor characterized by a vasculogenic program. The sampling can but need not include removing an amount of fluid contained within or flowing through the artery supplying the tumor. Thus sampling can be performed in one embodiment by the introduction of a probe into the lumen of the artery and then acquiring information in situ via the probe. In one embodiment sampling can be performed by introducing a needle or catheter into the lumen of the artery and withdrawing a volume of blood or other fluid contained within or flowing through the artery. The blood or fluid so obtained can then be processed in vitro to obtain information about compositions in the withdrawn sample. The sampling arterial influx can be performed substantially coincident with sampling venous efflux, or it can be performed separate in time from sampling venous efflux.
As used herein, sampling venous efflux to a tumor refers to gaining access to and contacting the blood or other fluid contained or coursing within a vein draining the tumor. In one embodiment the tumor is a tumor characterized by a vasculogenic program. The sampling can but need not include removing an amount of fluid contained within or flowing through the vein draining the tumor. Thus sampling can be performed in one embodiment by the introduction of a probe into the lumen of the vein and then acquiring information in situ via the probe. In one embodiment sampling can be performed by introducing a needle or catheter into the lumen of the vein and withdrawing a volume of blood or other fluid contained within or flowing through the vein. The blood or fluid so obtained can then be processed in vitro to obtain information about compositions in the withdrawn sample. The sampling venous efflux can be performed substantially coincident with sampling arterial influx, or it can be performed separate in time from sampling arterial influx.
In various aspects the methods of the invention include measuring an amount of a composition in the arterial influx and an amount of the composition in the venous efflux. The composition to be measured can be any composition of interest. In various embodiments the composition to be measured can be a tumor product, a cytokine, a growth factor, a morphogen, an angiogenic factor, an anti-angiogenic factor, a hormone, a nutrient, a metabolic waste product, a drug, a drug metabolite, a drug candidate, a test agent, an imaging agent, a labeling agent, a cell, and any combination thereof.
The measuring can be performed using any method suitable for that purpose. The method of measurement will be selected based at least in part on the identity or type of composition to be measured. Those of skill in the art will be able to determine which methods are suited for measuring particular compositions or types of compositions. The methods may involve the use of any one or combination of techniques involving chemical reagents, antibodies, bioassays, mass spectrometry, nuclear magnetic resonance spectroscopy, nucleic acid sequencing, protein sequencing, microarrays, hybridization techniques, and fluorescence-activated cell sorting, to name but a few.
As used herein, measuring an amount refers to measuring an absolute amount or a relative amount. An absolute amount can be an amount expressed in terms of mass, moles, activity, volume, temperature, pressure, and the like. A relative amount can be an amount expressed in terms of concentration, percent, relative activity, and the like.
An aspect of the invention includes the step of identifying a composition as a tumor product when the amount of the composition in the venous efflux exceed the amount of the composition in the arterial influx. The amount of the composition in the venous efflux exceeds the -amount of the composition in the arterial influx when there is a measurable excess amount in the venous efflux as compared to the amount in the arterial influx. In various embodiments the measurable excess represents at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 percent of the influx amount.
In an embodiment the composition to be measured is a drug or a drug metabolite. As used herein, a drug refers to a composition with pharmacological activity. Many drugs are small molecules, i.e., molecules with molecular weight less than or equal to about 1.5 kDa. Not all drugs are small molecules, however, as drugs also include biologics such as antibodies and other macromolecules prepared by recombinant techniques. Drugs include formulated as well as unformulated drugs. Drugs also include agents approved for clinical use as well as agents not currently approved for clinical use. The list of drugs is enormous and includes, without limitation, drugs categorized as abortofacients, analgesics, anesthetics, antibodies, anticholinergics, anticonvulsants, antidiabetic agents, antidotes, antihistamines, anti-infectives, antimalarials, antineoplastics, antiparkinsonian agents, appetite suppressants, biologicals, blood modifiers, bone metabolism regulators, cardiovascular agents, central nervous system stimulants, central nervous system depressants, cholinesterase inhibitors, contraceptives, diuretics, enzymes, gastrointestinal agents, hormones, immunomodulators, immunosuppressives, leukotriene maodulators, mast cell stabilizers, migraine preparations, muscle relaxants, nonsteroidal anti-inflammatory agents, parasympatholytics, parasympathomimetics, prostaglandins, psychotherapeutic agents, sedatives, steroids, sympathomimetics, vaccines, vasodilators, and vitamins.
In one embodiment a drug is an anti-cancer agent. Anti-cancer agents include chemotherapeutic agents, cancer immunotherapeutic agents, and cancer vaccines. Anticancer agents may be used alone or in combination with radiation and/or surgical procedures to treat a tumor or cancer. As used herein, an “anti-cancer agent” refers to an agent which is administered to a subject for the purpose of treating a cancer. As used herein, “treating cancer” includes preventing the development of a cancer, reducing the symptoms of cancer, and/or inhibiting the growth of an established cancer.
The chemotherapeutic agent may be selected from the group consisting of methotrexate, vincristine, adriamycin, cisplatin, non-sugar containing chloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566, RAS farnesyl transferase inhibitor, farnesyl transferase inhibitor, MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470, Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951 f, Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomal doxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, Xeload/Capecitabine, Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid, SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, BMS- 182751/oral platinum, UFT (Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer, Campto/Levamisole, Camptosar/Irinotecan, Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin, Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU 103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD 9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Analog, nitrosoureas, alkylating agents such as melphalan and cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl, Dactinomycin, Daunorubicin HCl, Estramustine phosphate sodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue), Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna, Mitotane (o.p′-DDD), Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA), Azacitidine, Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Pentostatin (2′ deoxycoformycin), Semustine (methyl-CCNU), Teniposide (VM-26), and Vindesine sulfate, but it is not so limited.
The immunotherapeutic agent may be selected from the group consisting of Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1D10 Ab, SMART ABL 364 Ab, and ImmuRAIT-CEA, but it is not so limited.
The cancer vaccine may be selected from the group consisting of EGF, Anti-idiotypic cancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL theratope, BLP25 (MUC-1), liposomal idiotypic vaccine, Melacine, peptide antigen vaccines, toxin/antigen vaccines, MVA-based vaccine, PACIS, BCG vaccine, TA-HPV, TA-CIN, DISC-virus, and ImmuCyst/TheraCys, but it is not so limited.
As used herein, “effective amount” refers to any amount that is necessary or sufficient for achieving or promoting a desired outcome. In some instances an effective amount is a therapeutically effective amount. A therapeutically effective amount is any amount that is necessary or sufficient for promoting or achieving a desired biological response in a subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular agent being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular agent without necessitating undue experimentation.
As used herein, the term “subject” refers to a vertebrate animal. In one embodiment the subject is a mammal. In one embodiment the subject is a human. In other embodiments the subject is a non-human vertebrate animal, including, without limitation, non-human primates, laboratory animals, livestock, domesticated animals, and non-domesticated animals.
As used herein, the term “treat” as used in reference to a disorder, disease, or condition means to intervene in such disorder, disease, or condition so as to prevent or slow the development of, to prevent, slow or halt the progression of, or to eliminate the disorder, disease, or condition.
EXAMPLES
Example 1
Tumor-Induced Arteriogenesis
Intact human liposarcoma pieces, 1 mm3, were implanted subcutaneously in mice. Implantation of a tumor piece, rather than injection of tumor cells, was used to: 1) provide local sequestration rather than interspersion of tumor cells within host tissue; 2) provide a significant isolated source of angiogenic factors to promote homing of the neovasculature; and 3) preserve the important tumor-stromal/tumor-cell architecture necessary to maintain inter-tissue signaling (Hlatky, L. et al., Cancer Res. 54, 6083-6086 (1994); Barcellos-Hoff, M. H. et al, Radiat. Res. 156(5), 618-627 (2001)). The best response was obtained when xenografts were positioned mid-dorsally on the lower back. Well-delimited tumors developed between the skin and muscle. Upon dissection the tumors were easily freed from adjacent tissue with minimal bleeding, attesting to their bounded, organ-like delimitation and vascular accommodation (FIG. 1A). The tumors elicited large-gauge arteries and veins which linked them to the host vasculature through a fascial bridge (FIG. 1C) that has no analog in control mice. The experiments yielded neovasculature (arteries and veins) of >30 mm in length between the tumor and the abdominal wall in the mouse. These vessels tracked within the fibro-adipose fascia (FIG. 1B) lying external to the abdomen, loosening the fascial cohesiveness along the track of the vessels and up to their point of entry into the tumor. The fascial sheath supporting the vascular conduit consisted of connective tissue elements, including fibroblasts, collagen, adipocytes, histiocytes and mast cells. The conduit was easily freed from the adjacent skin and muscle (FIG. 1C). The majority of the xenografts implanted in this manner resulted in well-delimited tumors with substantial artery-vein conduits. Arteriogenesis was also detected in the extension of this artery within the abdominal wall and peritoneal cavity. This was not the case if the tumor was injected as a cell suspension (preserving the full range of cell types present in the tumor). When injected as a suspension, tumors grew poorly, invaded local tissues including the skin, produced a large proportion of smaller extratumoral vessels, and yielded no major artery-vein conduits. Sham surgery, conducted to examine if growth factor production secondary to the implant surgery was sufficient to induce arterialization, failed to induce any vessels of significant size directed to the site. Sham surgeries consisted either of the subcutaneous implantation of a piece of biologically inert clinical-grade implant material (e.g. testicular implant material, Silimed) of similar size (1 mm3) to the tumor implants, or of the simple wounding and suturing of an animal.
Arterial networks in the mice were visualized via angiograms following barium gel injection. For animals with implanted liposarcoma pieces, the arterial conduit to the tumors resembled that to organs (FIG. 1D). It is noted that for the tumor-bearing animal, an artery from the aorta near the renal arterial branch homed to the tumor, extending through the abdominal wall of the animal. The length of the artery between the point of penetration of the abdominal wall and the tumor reached  ̃30 mm. Additionally, the arterial gauges were seen to elaborate to a size comparable to that of the femoral artery (FIG. 1 D,E), as confirmed by dissection (FIG. 1 A). Angiograms of control mice showed no such artery though the abdominal wall. Unlike the classic randomly-oriented and tortuous vasculature induced within tumors, these experiments demonstrated directed, large-gauge arteries and veins induced outside the tumor. Upon reaching the tumor site, noticeable branching into smaller vessels occurs (FIG. 1A). Inside the xenografts a classic tumor vasculature was detected using antibodies to standard endothelial and smooth muscle cell markers, platelet endothelial cell adhesion molecule PECAM-1 (CD-31), CD34 (Hlatky, L. et al., J. Natl. Cancer Inst. 94, 883-893 (2002)) and alpha smooth muscle actin (αSMA).
As confirmed by structural, flow dynamic, and immunohistochemical criteria, the artery and vein of the vascular conduit exhibited properties of mature functional vasculature (FIG. 2A-D). Induced arteriogenesis was demonstrated by the well-developed wall structure of the arteries. In the arterial cross-sections, a characteristic serpentine internal elastic lamina, muscular media, external elastic lamina and adventitia were present, and the wall-thickness-lumen ratio was appropriate, suggesting full viscoelastic and vasomotor functionality (FIG. 2B). Pulsatility and the uni-directional flow in the arteries was established by two-color directional Doppler imaging. Immunohistochemically, the densely αSMA-stained media within the walls of the large vessels was consistent with identification of these vessels as mature arteries and veins (FIG. 2A,B,C). Elastin staining demonstrated the normally prominent internal and external elastic laminae in the arteries and the normally less well-developed elastic layers in the vein (FIG. 2D). Interestingly, despite these impressions of vascular maturity, proliferation data indicated ongoing arteriogenic development. Cell proliferation within the artery and vein was assessed via proliferating cell nuclear antigen (PCNA) and Ki-67 (MIB 1), a nuclear protein expressed in all cycle phases except G0. Unlike normal adult vasculature which is essentially non-proliferating (Hobson, B. et al., J., Br. J. Cancer 49(4), 405-413 (1984); Folkman, J. et al., New York: Cold Spring Harbor Laboratory Press; p. 1-17 (1998)), labeling confirmed proliferative activity in both endothelial and smooth muscle cells of the arteries and veins within the conduits, in addition to proliferative cells in the surrounding connective tissue (FIG. 2E). Cell proliferation was detected to be highest near the tumor. The endothelium of the artery and vein were derived from the host and not from endothelial cells of the implanted tumor. This was determined by implanting tumors in mice transgenically altered with green fluorescent protein, GFP, driven by a Tie-2 promoter to identify host endothelium.
Example 2
Tumor-Induced Neurogenesis
The implanted xenografts in this experiment induced directed nerves. In the fascial bridge connecting the tumor to the abdominal wall, which notably has no analog in the control mouse, nerves were seen to track parallel to the vasculature. The nerves associated with the arteries in a manner analogous to nerve routing in normal development (Martin, P. & Lewis, J. Origins of the neurovascular bundle: interactions between developing nerves and blood vessels in embryonic chick skin. Int. J. Dev. Biol. 33(3), 379-387 (1989); (Mukouyama, Y. S. et al., Cell 109(6), 693-705 (2002)); (FIG. 2C). Nerve localization and orientation was demonstrated by immunodetection of axons using an antibody to neurofilament (FIG. 3A). Enveloping Schwann cells were positively detected via an antibody to S-100 (FIG. 3B). FIG. 2C shows a neurovascular bundle, with nerve, artery and vein marked, as it homes to the xenograft. Labeling with neurofilament and S-100 verified the presence of nerve within the entire conduit. These nerves were often large ( ̃0.12 mm in diameter in paraffin sections—FIG. 1E). Infrequently, nerves were covered by a myofibroblast coat as identified by αSMA staining. Smooth muscle actin staining has been found in contractile myofibroblasts in the perineurium surrounding fascicles containing collections of nerve fibers comprising functional nerves (Chamberlain, L. J. et al., J. Comp. Neurol. 417(4), 415-430 (2000)). The axially-oriented myofibroblasts are believed to create axial tension and align collagen fibers to facilitate the elongation of regenerating axons at a transection site.
At the tumor-conduit interface, the nerves and vessels branched, forming a bed of high vessel and nerve track density (FIG. 3B). Again, close association and parallel orientation of the large neovessels and nerves was observed. To examine the extent of neurogenesis that accompanied tumor growth, nerves along the neurovascular conduit and in the neurovascular bed at the base of the tumor were quantified using immunohistochemistry in combination with digitized imaging. Scoring of peripheral nerves was based on the simultaneous demonstration of axons labeling with neurofilament and Schwann cells labeling with S-100. For the quantification of nerve areas, S-100 labeling was imaged. Ratios of nerve area/tissue area were calculated yielding a “nerve presence index” measure. To capture the dependence of nerve presence with position along the conduit, the entire conduit was scanned, following its extension back to the aorta. The total conduit between the tumor and aorta was divided into four distinct regions: Region-1 (the tumor-conduit interface) directly adjacent to the tumor; Region-2 between the tumor-conduit interface and the abdominal wall ( ̃30 mm); Region-3 within the abdominal wall; and Region-4 between the abdominal wall and the insertion point of the conduit artery into the aorta (FIG. 3C). In Region-1, the nerve index was 6.98%±1.00, in Region-2, 3.49%±1.52, in Region-3, 0.39%±0.11, and in Region-4, 0.65%±0.17. It is noted that considerable nerve presence was induced by the tumor, with 5-10-fold higher nerve presence indicated in the newly-generated portion of the conduit outside the abdominal wall, compared to that visualized within the animal and in the abdominal wall. For control mice without tumors, the nerve index was quantified in the two tissues which lie directly adjacent to the site where tumors would be implanted: 1) the underside of the skin (0.07%±0.05), and 2) the abdominal wall (0.40%±0.20). A 100-fold increase in nerve presence is seen in the interface region around the tumors as compared to the skin in non-tumor bearing mice. The nerve index in the abdominal wall was the same in both the tumor-bearing and control animals (0.39%±0.11 and 0.40%±0.20). Up to 100 imaged fields were averaged to obtain the measurements in each region.
In adult nerves, Schwann cells are quiescent with heterochromatin-rich nuclei. However, in regenerating nerve, Schwann cells proliferate and form columns that serve as scaffolds for regenerating axons by expressing adhesion molecules on the surface plasma membrane and producing various tropic factors for regenerating axons (Evans, G. R., Semin. Surg. Oncol. 19(3), 312-318 (2000)). Proliferation within the nerves induced in this experiment was assessed via the expression of both PCNA and Ki-67. Ki-67, widely used to detect proliferation in tumor and vascular studies, has recently been used as a proliferation marker in adult neurogenesis (Kee, N. et al., J. Neurosci. Meth. 115(1), 97-105 (2002)). Positive Schwann cell labeling was detected with both Ki-67 (FIG. 2E) and PCNA staining, indicating proliferation. Supporting these observations, H&E revealed Schwann cells which appeared plump and active with light chromatin staining, indicative of proliferative status.
Example 3
Sonic Hedgehog Signaling is Instrumental in Tumor-Driven Adult Neurogenesis
This experiment was a study of the role of the embryonic signaling molecule Sonic Hedgehog (Shh) in driving the neurogenic and arteriogenic processes induced by the implanted tumors in these adult mice. Sonic hedgehog, a key developmental morphogen in vertebrates, plays an essential role in the development of neuronal cells and tissues in both the peripheral and central nervous system (Lum, L. et al., Science 304, 1755-9 (2004)). In addition, it is central in determining dorsal-ventral polarity of the neural tube (Chiang, C. et al., Nature 383, 407-13 (1996); Marti, E. et al., Nature 375, 322-325 (1995); Ingham, P. W. et al., Genes Devel. 15, 3059-87 (2001)) and the determination of anterior-posterior patterning of limbs. Sonic hedgehog signaling has long been considered to be active only during development. However, recent insightful reports show continued Shh activity in adult stem cells (Lai, K. et al., Nature Neurosci. 6(1), 21-27 (2003); Ruiz i Altaba et al., Nature Revs. Cancer 2, 361-72 (2002)) and deregulated Shh signaling linked to some specific cancers, e.g. small-cell lung cancer (Watkins, D. N. et al., Nature 422, 313-317 (2003)) and pancreatic adenocarcinomas (Thayer, S. P. et al., Nature 425, 851-856 (2003); di Magliano et al., Nature Revs. Cancer 3(12), 903-911 (2003)). Additionally, injection of Shh protein into ischemic limbs of adult mice increased local angiogenesis (Pola, R. et al., Nature Med. 7, 706-711 (2001)).
To identify and localize cells expressing Shh message and protein, in situ hybridization and immunohistochemistry were used. Significant Shh (both mRNA and protein) was detected in the interface at the base of the tumor and in the nerve, artery and vein of the neurovascular bundles (FIG. 4A-D). Within the artery and vein Shh was detected in both endothelial and smooth muscle cells. In the nerves, Shh protein expression was pronounced in Schwann cells and in the epineurium, which also labeled strongly for CD34, (FIG. 4C). Demonstrating the highest Shh expression were various cell types in the fascial connective tissue surrounding the neurovascular structures within the conduit, including adipocytes and fibroblasts. In response to tumor implantation, cellular Shh expression throughout the whole body of the animals was also examined. Beyond those few locations where Shh is known to be found in adult mice (e.g. in hair follicles), only cells of the induced neurovascular bundle conduit and the surrounding fibro-adipocyte tissue labeled for Shh in tumor-bearing mice. To evaluate the global pattern of Shh mRNA expression over the neurovascular conduit, digitized imaging was used to quantify cellular in situ Shh expression as a function of distance along the conduit. This was accomplished using an automated scanning procedure which detected Shh hybridization signal in a montage of microscope fields covering the entire conduit length. The percentage of tissue per microscope field scoring positive for Shh mRNA decreased markedly as a function of distance from the tumor (FIG. 5). This percentage decreased from 3.6% in the tumor-conduit interface to 0.1% at 12 mm from the tumor, and decreased slowly thereafter. This suggests the signals driving the formation of the neurovascular bundle originated at the tumor site, and that a stable gradient in this signaling was established with distance from the tumor. A stable gradient of this scale is reminiscent of the morphogen gradients found in the developing embryo. Comparing the data of FIG. 5 with that of FIG. 3C, one observes that regions of the highest Shh expression coincide with those having the highest nerve presence index.
To investigate whether Shh developmental pathways played a critical role in the generation of the neurovascular bundles, Shh signaling was blocked in the early stages of tumor growth. Shh acts by inhibiting Patched (Ptc), thereby reducing the normally inhibitory effect of Ptc on Smoothened (Smo). This activates the Shh pathway. Cyclopamine, a plant alkaloid that blocks the action of Smo in the receptor complex and thus the signaling pathway of Shh, was delivered subcutaneously (following the methods of Thayer et al., 2003 (Thayer, S. P. et al., Nature 425, 851-856 (2003)) to tumor-bearing mice 12 days after tumor implantation. Treatment was delivered daily (50 mg/kg) for a period of 5 days. After 4 days of treatment tumor regression was observed. On day 7 following the start of treatment the mice were sacrificed and the tissue examined. Tissue from both treated and untreated tumor-bearing mice were compared. At autopsy, there was no evidence of viable tumor or of a neurovascular bundle after cyclopamine treatment. In contrast, the untreated tumor-bearing animals had established neurovascular bundles and cells in the supporting sheath expressing Shh. Sections of skin and tissue surrounding the tumor implant site after cyclopamine treatment showed only control Shh expressions (e.g. in hair follicles). These findings suggest that Shh signaling is instrumental in the maintenance of the neurogenesis and arteriogenesis process induced by the tumor, and that blocking this signaling abrogates these developmental programs.
Experimental Methods
The following experimental methods were used in the experiments described above.
Tumor Implantation
Primary human liposarcoma tumors were implanted subcutaneously as  ̃1 mm3 pieces in adult (8-10 week old) male and female SCID mice. The resulting tumors were then passed mouse to mouse: grown to  ̃1 cm, excised, and pieces reimplanted through a small incision mid-dorsally low on the back (n=50) of SCID (Massachusetts General Hospital, Boston, Mass.) or nude (Taconic Farms) mice. Animals with tumors 0.5-2 cm in diameter were sacrificed for investigation or prepared for angiograms or latex casting.
Angiograms and Latex Casting
Mice were euthanized (CO2) and sternotomy performed to gain access to the thoracic cavity. A small vent in the right atrium was made, then the left ventricle flushed with 10-15 cc of PBS+heparin (1 ml) until fluid exiting the left atrium is clear. This was followed by perfusion with 4 ml of 5% formalin. Angiograms: 2cc of liquid-coat HD barium sulfate contrast suspension #149705 (Lafayette Pharmaceuticals, Lafayette, Ind.). Liver color was monitored to establish that contrast solution has entered the arteries and arterioles. Angiogram films prepared by routine x-ray. Latex Castings: 3-5 ml of orange Latex (Microfil, Flow Tech., Carver, Mass.) injected until the larger arteries were fully perfused.
Immunohistochemistry
Formalin-fixed, paraffin-embedded, sectioned (5 μm) samples. Slides treated with Proteinase K (Roche Diagnostic, Indianapolis, Ind.) at 37° C. for 15-30 min and blocking buffer (TNT from TSA biotin system kit, NEL 700A, Perkin-Elmer Life Sciences, Boston, MA) for 30 min at 25° C. Primary antibodies used: CD31 (PECAM1) and CD34 (Pharmingen, Bedford, Mass.), Ki-67 (Dako, Carpinteria, Calif.), PCNA (PC10 monoclonal, Dako), α-smooth muscle actin (Sigma), S-1 00 (polyclonal, Signet, Dedham, Mass.), elastin, neurofilament (monoclonal, Signet), and Shh sc-9024 (polyclonal, Santa Cruz Biotech., Santa Cruz, Calif.). Procedure excluding S-100, neurofilament, and elastin: Primary antibodies bind overnight at 4° C. (CD31, CD34, Shh) or 1 hr at 25° C. (PCNA, Ki-67, SMA). The secondary biotinylated antibody (Vector Labs, Burlingame, Calif.) applied for 30-40 min. Signal amplified with biotinyl tyramine amplification reagent (Perkin-Elmer Life Sciences, Boston, Mass.). Peroxidase enzyme added and linked using the ABC peroxidase standard kit (Vector) according to manufacturer's instructions. Visualization accomplished with the Vector Novar (Nova Red) substrate kit. Slides counterstained with hematoxylin. Elastin: Tissue section is overstained with a soluble lake of hematoxylin-ferric chloride-iodine. Differentiation is accomplished by the use of excess solution of ferric chloride to break the tissue-dye complex. S-100 and neurofilament: Signet Ultra-Streptavidin detection system, as per manufacturer's instructions. S-100 visualized with diaminobenzidine (DAB) substrate.
In Situ Hybridization
Samples were fixed in 4% PFA at 4° C. for 10 min, followed by 0.5×SSC for 5 min at 25° C., Proteinase K for 10 min at 37° C., 0.5×SSC for 10 min, 4% PFA for 5 min, 0.5×SSC for 5 min. It was then placed in a prehybridization buffer at 56° C. for 30 min (2 ml SDS 10%, 2 ml SSC 20%, 1 ml tRNA (10 mg/ml), 4 ml dextran sulfate (50%), 10 ml formamide, 1 ml DEPC H2O, 500 μg (25 μg/ml) fish sperm DND. This was followed by treatment with a DIG-labeled riboprobe (1:50) at 55° C. overnight. RNAse A solution (20 mg/ml) was added and slide incubated 30 min at 25° C., incubated in anti DIG overnight at 4° C. (1:500 in blocking buffer), rinsed in buffer 1 for 2×10 min, then in buffer 3 for 5 min, developed in NBT/BCIP at 25° C. Sample counterstained methyl green for 3 min., dehydrated and coverslipped. For the mouse Shh probe, a 1.576 kb fragment was cloned into Eco-R1 site of Bluescript. Antisense—Sal 1 digest/T3 transcript; sense—Pst 1 digest/T7 transcript, 3 μl of 7 μg/μl DNA.
Digitized Imaging
Densitometric imaging was done using a Quantimet 570 computerized imaging facility (Leica), utilizing a Reichert-Jung Polyvar microscope integrated to a 500 Mhz processor-based parallel-tasking computer via a JVC 3-CCD camera. Morphological routines used to remove background “noise”, isolate and measure vascular features, and assess antigen signal ratios. A scan pattern was set up according to the size of tissue sample to be imaged. The computer was calibrated for each magnification so that step sizes would correspond to field dimensions. Masking was done by varying the intensity-detection threshold to define the boundaries of the chromogen to be measured. Shh presence along the tumor conduit was quantified using an automated scanning procedure which detected Shh hybridization signal in 400×400 μm frames forming a montage covering the entire conduit length. Measured data analyzed in Excel.
EQUIVALENTS
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

## Claims

1. A method for identifying a tumor product, comprising
providing a tumor characterized by a vasculogenic program;
sampling arterial influx to the tumor;
sampling venous efflux from the tumor;
measuring an amount of a composition in the influx;
measuring an amount of the composition in the efflux; and
identifying the composition as a tumor product when the amount of the composition in the efflux exceeds the amount of the composition in the influx.

2. The method of claim 1, wherein the tumor product is selected from the group consisting of cytokines, growth factors, morphogens, angiogenesis factors, and anti-angiogenesis factors.

3. The method of claim 1, wherein the tumor product is a cell.

4. A method for isolating a tumor product, comprising
providing an implanted tumor characterized by a vasculogenic program;
sampling venous efflux from the tumor, wherein the efflux comprises a tumor product; and
isolating the tumor product from the efflux.

5. The method of claim 4, wherein the tumor product is selected from the group consisting of cytokines, growth factors, morphogens, angiogenesis factors, and anti-angiogenesis factors.

6. The method of claim 4, wherein the tumor product is a cell.