Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – The nonhuman animal is a model for human disease
Reexamination Certificate
1997-04-28
2001-05-15
Hauda, Karen M. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
The nonhuman animal is a model for human disease
C435S325000, C435S320100, C424S093210
Reexamination Certificate
active
06232523
ABSTRACT:
TECHNICAL FIELD
The invention relates to the study of tumor progression. Specifically, it concerns model systems for studying the metastasis of tumors in vertebrate systems.
BACKGROUND ART
It has long been recognized that the ability of tumor tissues to metastasize constitutes a major portion of the life-threatening aspects of malignancy. Metastasis is the growth of secondary tumors at sites different from the primary tumor. Thus, despite surgical removal of the primary tumor, it may not be possible to arrest the progress of this condition. An understanding of the mechanism whereby metastasis occurs will be crucial to the development of protocols whereby the growth of secondary tumors can be controlled. In order to understand the mechanism of metastasis, it will be necessary to provide a model which permits identification of small numbers of tumor cells against a background of many host cells so that secondary tumor emboli and micrometastases can be observed over the course of real time.
Others have demonstrated extravasation and initial seeding steps in tumor metastasis in vitro using externally fluorescently labeled tumor cells. Khokha, R. et al.,
Cancer Metastasis Rev
(1995) 14:279-301; Koop, S. et al.,
Cancer Res
(1995) 55:2520-2523. Further, Margolis, L. B. et al.,
In Vitro Cell Dev Biol
(1995) 31:221-226 was able to visualize the migration of externally fluorescently labeled lung tumor cells in host mouse lung in histoculture. In all cases, however, long-term observation was not possible due to the limitation of exogenous fluorescent labels. Retroviral transfer of a green fluorescent protein (GFP) gene has been shown to result in stable transfectants of human cancer cells in vitro. Levy, J. P. et al.,
Nature Biotechnol
(1996) 14:610-614.
Attempts have been made to provide such a model using the &bgr;-galactosidase gene as a marker (Lin, W. C. et al.,
Cancer Res
(1990) 50:2808-2817; Lin, W. C. et al.,
Invasion and Metastasis
(1992) 12:197-209). However, this marker has not proved satisfactory, as fresh or processed tissue cannot be used. The present invention provides a marker which permits visualization of tumor invasion and micrometastasis formation in viable fresh tissue.
DISCLOSURE OF THE INVENTION
The invention provides models which permit the intimate study of formation of metastases from primary tumors in a realistic and real-time setting. By using green fluorescent protein (GFP) as a stable and readily visualized marker, the progression of such metastasis can be modeled and the mechanism elucidated.
Thus, in one aspect, the invention is directed to a method to follow the progression of metastasis of a primary tumor, which method comprises removing fresh organ tissues from a vertebrate subject which has been modified to contain tumor cells that express GFP and observing the excised tissues for the presence of fluorescence.
In another aspect, the invention is directed to a vertebrate subject which has been modified to contain tumor cells expressing GFP.
In still other aspects, the invention is directed to tumor cells modified to produce GFP under control of nonretroviral control elements, to tissues containing metastatic tumors that produce GFP, and to histocultures of tissues which contain such metastasized tumors.
MODES OF CARRYING OUT THE INVENTION
The invention provides model systems for the study of the mechanism of metastasis of tumors generally. Advantage is taken of the visible marker green fluorescence protein (GFP) to label the tumor cells so that their migration and colonization in tissues distal to the tumor can be followed as the migration and colonization progresses.
In general, the model involves modifying a vertebrate, preferably a mammal, so as to contain tumor tissue, wherein the tumor cells have, themselves, been modified to contain an expression system for GFP. Tumors can be formed in such vertebrate systems by administering the transformed cells containing the GFP expression system and permitting these transformed cells to form tumors. Typically such administration is subcutaneous and the tumors are formed as solid masses. The tumors thus formed can be implanted in any suitable host tissue and allowed to progress, metastasize and develop.
Suitable procedures for growing the initial tumor, thus, involve transcutaneous injection of the tumor cells, such as CHO cells, HeLa cells, carcinoma and sarcoma cell lines, and well established cell lines such as the human lung adenocarcinoma line anip 973, and others that may become available in the art. The administered cells will have been modified to contain an expression system for GFP. After administration, solid tumors generally develop, typically at the site of subcutaneous injection. These tumors, which are themselves fluorescent, can then be removed and used for implantation in the model vertebrate.
Techniques for implantation of the solid tumors, now labeled with GFP, into vertebrates include direct implantation by surgical orthotopic implantation (SOI) at the desired site, typically the site from which the tumor cells were derived. Suitable sites include lung, liver, pancreas, stomach, breast, ovary, prostate, bone marrow, brain, and other tissues susceptible to malignancy. Once the solid tumors have been implanted, the vertebrate becomes a model system for studying metastasis. The tumor is thus allowed to progress and develop and the vertebrate is monitored for appearance of the GFP labeled cells at sites distal from the original implantation site. The monitoring can occur either on the whole vertebrate by opening the animal and observing the organs directly with a fluorescent microscope, or the tissues may be excised and examined microscopically. As GFP is visible to the naked eye, no development systems to stain the tissue samples are required. The tissue samples are simply properly processed as fresh samples in slices of suitable size, typically 1 mm thick, and placed under a microscope for examination. Even colonies of less than 10 cells are thus visible. A variety of microscopic visualization techniques is known in the art and any appropriate method can be used.
In addition, the development of the tumor can be studied in vitro in histological culture. Suitable systems for such study include solid supported cultures such as those maintained on collagen gels and the like.
Suitable vertebrate subjects for use as models are preferably mammalian subjects, most preferably convenient laboratory animals such as rabbits, rats, mice, and the like. For closer analogy to human subjects, primates could also be used. Particularly useful are subjects that are particularly susceptible to tumor development, such as subjects with impaired immune systems, typically nude mice or SCID mice. Any appropriate vertebrate subject can be used, the choice being dictated mainly by convenience and similarity to the system of ultimate interest.
The label used to follow the metastasis is green fluorescent protein (GFP). The gene encoding this protein has been cloned from the bioluminescent jellyfish
Aequorea victoria
(Morin, J. et al.,
J Cell Physiol
(1972) 77:313-318). The availability of the gene has made it possible to use GFP as a marker for gene expression. GFP itself is a 283 amino acid protein with a molecular weight of 27 kD. It requires no additional proteins from its native source nor does it require substrates or cofactors available only in its native source in order to fluoresce. (Prasher, D. C. et al.,
Gene
(1992) 111:229-233; Yang, F. et al.,
Nature Biotechnol
(1996) 14:1252-1256; Cody, C. W. et al.,
Biochemistry
(1993) 32:1212-1218.) Mutants of the GFP gene have been found useful to enhance expression and to modify excitation and fluorescence. GFP-S65T (wherein serine at 65 is replaced with threonine) is particularly useful in the invention method and has a single excitation peak at 490 nm. (Heim, R. et al.,
Nature
(1995) 373:663-664). Other mutants have also been disclosed by Delagrade, S. et al.,
Biotechnology
(1995) 13:151-154; Cormack, B. et al.,
Gene
(1996) 173:33-38 and Cra
Chishima Takashi
Tan Yuying
Anticancer Inc.
Beckerleg AnneMarie S.
Hauda Karen M.
Morrison & Foerster / LLP
LandOfFree
Metastasis models using green fluorescent protein (GFP) as a... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Metastasis models using green fluorescent protein (GFP) as a..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Metastasis models using green fluorescent protein (GFP) as a... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2555019