Metastasis models using green fluorescent protein (GFP) as a marker

• US 6,759,038 B2
• Filed: 05/29/2001
• Issued: 07/06/2004
• Est. Priority Date: 04/28/1997
• Status: Expired due to Fees

• Current Assignee: Anticancer Incorporated
• Original Assignee: Anticancer Incorporated
• Inventors: Tan, Yuying, Chishima, Takashi
• Primary Examiner(s): Wehbe′ , Anne M.
• Application Number:
  US09/870,268
• Publication Number:
  US 20020026649A1
• Time in Patent Office: 1,134 Days
• Field of Search: 435/325, 435/320.1, 435/455, 800/8, 800/9, 800/10, 514/44, 424/93.2, 424/93.21
• US Class Current: 424/93.21
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 49/0008 Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
• CPC Class Codes: A61K 49/0047 Green fluorescent protein [GFP]
• CPC Class Codes: A61K 49/0097 Cells, viruses, ghosts, red blood cells, viral vectors, used for imaging or diagnosis in vivo
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae

Abstract

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 is disclosed. The fluorescence can also be monitored by observing the tissues in situ. Vertebrate subjects which contain GFP producing tumors are useful models to study the mechanism of metastasis, as well as to evaluate candidate protocols and drugs. In addition, subjects already harboring tumors can be treated so as to modify the endogenous tumors to contain GFP. This permits clinical applications. Finally, by injecting a contrast dye into a subject harboring a GFP-labeled tumor, angiogenesis in the tumor can be observed directly.

Claims

1. A method to evaluate a candidate protocol or drug for the inhibition of metastasis of a primary tumor which method comprises:

administering said protocol or drug to a subject which is a mouse, rat or rabbit which contains a primary tumor that stably expresses green fluorescent protein (GFP) in cells of said tumor when said tumor metastasizes and monitoring the progression of metastasis by observing the presence, absence or intensity of the fluorescence at various locations in the treated subject;

wherein said subject contains said tumor that expresses GFP and wherein said subject is a genetically immunocompromised mouse, rat or rabbit, or a mouse, rat or rabbit which is syngeneic to said tumor;

monitoring the progression of metastasis in a control, which contains a similar tumor that expresses green fluorescent protein;

wherein said control subject contains said tumor that expresses GFP wherein said control subject is an immunocompromised mouse, rat or rabbit, or a mouse, rat or rabbit which is syngeneic to said tumor; and

comparing the progression of metastasis in said treated subject with the progression of metastasis in said control subject wherein the control subject and treated subject are intact;

whereby a diminution of the progression of metastasis in said treated subject as compared to said control subject identifies the protocol or drug as effective in inhibiting metastasis.

2. The method of claim 1 wherein the progression of metastasis is monitored by fluorescent optical tumor imaging in the intact subject.

3. The method of claim 1 wherein said subject contains said tumor by virtue of surgical orthotopic implantation of said tumor.

4. The method of claim 1 wherein said subject contains said tumor by virtue of injecting cells of a stably transformed tumor cell line which has been transfected with an expression vector containing a first nucleotide sequence encoding green fluorescent protein (GFP) and a second nucleotide sequence encoding a selection marker, both said first and second nucleotide sequences being under control of a viral promoter and wherein said cell line stably effects high level expression of said GFP in the absence of selection agent and maintains a high level expression of GFP when said cell line proliferates through multiple passages of said cell line.

5. A method to monitor metastasis of a primary tumor in a subject which is a mouse, rat or rabbit which contains said primary tumor, and wherein said tumor stably expresses green fluorescent protein (GFP) in cells of said tumor when said tumor metastasizes,

wherein said subject contains said tumor that expresses GFP and wherein said subject is a genetically immunocompromised mouse, rat or rabbit, or a mouse, rat or rabbit which is syngeneic to said tumor;

which method comprises monitoring the progression of metastasis by observing the presence, absence or intensity of the fluorescence as a function of time at various locations in said subject wherein the subject is intact.

6. The method of claim 5 wherein the progression of metastasis is monitored by fluorescent optical tumor imaging in the intact subject.

7. The method of claim 5 wherein said subject contains said tumor by virtue of surgical orthotopic implantation of said tumor.

8. The method of claim 5 wherein said subject contains said tumor by virtue of injecting cells of a stably transformed tumor cell line which has been transfected with an expression vector in containing a first nucleotide sequence encoding green fluorescent protein (GFP) and a second nucleotide sequence encoding a selection marker, both said first and second nucleotide sequences being under control of a viral promoter and wherein said cell line stablely effects high level expression of said GFP in the absence of selection agent and maintains a high level expression of GFP when said cell line proliferates through multiple passages of said cell line.

9. A method to monitor metastasis of a primary tumor in a mammalian subject which contains said primary tumor, and wherein said tumor stably expresses green fluorescent protein (GFP) in cells of said tumor when said tumor metastasizes,

wherein said primary tumor is endogenous to said mammalian subject and expresses said GFP as a result of locally administering a retroviral vector to said subject in the vicinity of said tumor, said retroviral vector containing an expression system for said GFP;

which method comprises monitoring the progression of metastasis by observing the presence, absence or intensity of the fluorescence as a function of time at various locations in said subject.

10. The method of claim 9 wherein the subject is human.

11. The method of claim 9 wherein the progression of metastasis is monitored by excising fresh organ tissues from various locations in said subject.

12. The method of claim 11 wherein said excised tissues are observed by microscopic examination of fresh tissue slices.