Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing
Reexamination Certificate
1999-08-04
2001-05-08
Schwartzman, Robert A. (Department: 1635)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
C424S009200, C435S006120, C435S091200
Reexamination Certificate
active
06228345
ABSTRACT:
1. INTRODUCTION
The present invention relates to a novel in vivo assay for quantitating intravasation of cancer cells based on a highly sensitive polymerase chain reaction (PCR) utilized in combination with a chick embryo chorioallantoic membrane (CAM) assay. The assay of the invention provides a method for measuring the metastatic potential of cancer cells. The assay also provides a drug screening assay for identification of agents having anti-metastatic activity. The present invention is based on the discovery that when cancer cells are inoculated onto the CAM of a chick embryo, the ability of cancer cells to invade blood vessels can be measured using a sensitive PCR based assay. In addition, blood vessel intravasation by cancer cells was significantly inhibited when agents such as metalloproteinase inhibitors were co-inoculated with the cancer cells.
2. BACKGROUND OF THE INVENTION
The spread of cancer cells from a primary tumor to a site of metastasis formation involves multiple interactions such as invasion of extracellular matrix, neovascularization, invasion of the blood vessel wall (intravasation), exit from the circulation (extravasation) and establishment of secondary growth. Because cancer cells reach distant sites by disseminating through blood or lymphatic circulation, breaching of the vascular wall is a crucial event in metastasis formation. It is not known how early in disease progression cancer cells acquire the ability to intravasate. However, once established, this pathway appears to remain active as cancer cells can be detected in repeated blood samples of cancer patients.
Escape of cancer cells from the circulation, referred to as extravasation, is thought to be a major rate-limiting step in metastasis, with few cells being able to extravasate. However, highly metastatic cells are believed to extravasate more readily than poorly metastatic cells. A number of studies designed to assess metastasis by measuring extravasation have been performed using a chick embryo choriallantoic membrane (CAM) model system in which a large number of cancer cells are injected directly into the chorioallantoic membrane vein (Koop et al., 1994, Cancer Research 54:4791-4797; Chambers et al., 1997, J. Nat'l Cancer Inst. 84:797-803; Tsuchiya et al., 1993, Cancer Research 53:1397-1402; Shioda et al., 1997, AJP 150:2099-2112; Endo et al., 1990, J. Cancer Res. 81:723-6; Yamamoto et al., 1996, Anticancer Res. 16:413-7; Tsuchiya et al., Int. J. Cancer 56:46-51). However, since cancer cells are injected directly into the blood vessel, such studies fail to assay a tumor cell's ability to invade blood vessel walls (intravasation), a defect that would severely diminish the number of cancer cells delivered into the circulation and reduce the chances of metastatic growth.
While many aspects of cancer dissemination have been extensively studied, very little biochemical information related to the process of intravasation, i.e., the invasion of the blood vessel wall, is available. This may be in part due to the paucity of experimental models capable of mimicking the cellular and molecular interactions required for successful completion of intravasation. Existing models, such as mouse bladder (Poste et al., 1980 Cancer Res. 40:1636-1644) or human amnion denuded of epithelium and re-seeded with endothelial cells (Foltz et al., 1982, In “
Interaction of Platelets and Tumor Cells”,
G. A. Jamieson, (ed.), New York: Alan R. Liss, Inc. pp. 353-371), are used infrequently because they recapitulate poorly the structure of blood vessels and in particular, small vessels such as capillaries and post-capillary venules, where most of the cancer cell invasion is believed to take place. The scarcity of “spontaneously” metastasizing human tumors in experimental animals is another obstacle to the study of intravasation. The reason for the inefficient “spontaneous” metastasis is not understood.
To identify properties that define cells capable of successful intravasation, and to screen for compounds capable of inhibiting metastic growth, it is imperative that quantitative in vivo assays are developed in which intravasation can be measured.
3. SUMMARY OF THE INVENTION
The present invention relates to a novel in vivo assay for quantitating the ability of cancer cells to breach blood vessel walls based on a highly sensitive PCR amplification step used in combination with a chick embryo CAM assay. The assay of the invention provides a method for quantitating the metastatic potential of a cancer cell. The invention further relates to the use of the in vivo assay to screen for agents capable of inhibiting intravasation, and thereby modulating the metastatic potential of cancer cells. The assay of the invention has the advantage of providing a highly sensitive assay system capable of mimicking the in vivo cellular and molecular interactions required for successful completion of intravasation.
The assay of the invention involves the use of a chick embryo CAM model system for measuring intravasation. The assay of the invention comprises the inoculation of tumor cells onto the “upper” chorioallantoic membrane (“upper” CAM) of an artificially created air sac in a chick embryo; and the subsequent detection and quantitation of cancer cells that have entered the vasculature and migrated into the “lower” chorioallantoic membrane (“lower CAM”) of the embryo using PCR amplification of inoculated cell-specific DNA sequences. The presence of cancer cell specific DNA sequences in the lower CAM of the embryo, as detected by PCR, indicates that the inoculated cancer cells are capable of invading intact blood vessel walls, disseminating, and arresting in small blood vessels of the chick embryo tissues such as those located in the lower CAM.
Further, the assay of the invention can be used to screen for agents capable of inhibiting cancer cell intravasation. In assays designed to identify agents with the potential for inhibiting metastasis, a test agent is included with the inoculated cancer cells or injected into the vasculature of the chick embryo. The assay of the invention may also be used to detect phenotypic changes effected by genetic manipulation of cancer cells that results in changes in metastatic potential. The assay can be used in conjunction with new gene discovery methods to test the role of newly discovered genes in control of metastasis.
The invention is based on the observation that when cancer cells are inoculated onto the upper chorioallantoic membrane CAM of the chick embryo, the cells are capable of invading blood vessel walls and migrating to different regions of the embryo as detected by PCR amplification of cancer cell specific DNA sequences. It was further demonstrated that intravasation was inhibited by 90% when the cancer cells were co-inoculated with the metalloproteinase inhibitor, marimastat. In addition, recombinant expression of antisense uPAR in cancer cells inhibited intravasation by 50%-70%.
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Poste, G et al., 1980, Cancer Research 40:1636-1644.
Ossowski, L., 1988,Cell52:321-328.
Chambers, AF et al., 1992,Journal of the National Cancer Institute84:797-803.
Endo, Y et al., 1990,Jpn. J. Cancer Res.81:723-726.
Tsuchiya, Y et al.,Cancer Research53:1397-1402, 1993.
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Koop S., et al, 1996, Proc. Natl. Acad. Sci USA 93:11080-11084.
Yamamoto, H., et al., 1996, Anticancer Research 16:413-418.
Shioda T, et al., 1997, American Journal of Pathology 150:20992112.
Andresen PA et al., Int. J. Cancer 72:1-22, 1997.
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Kim et al. “Requirement for specific proteases in cancer cell intraversation as revealed by a novel semiquantitative PCR-based assay” Cell, vol. 94, p 353-362,
Lacourciere Karen A.
Mount Sinai School of Medicine of New York University
Schwartzman Robert A.
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