Whole Body Imaging Using Portable Observation Systems

Abstract

Methods to image a fluorescent protein in intact subjects using simple portable instruments are described.

Description

EXAMPLE 1

In Vivo Fluorescent Protein Labeling of Tumor Cells

GFP retroviral supernatants are prepared according to Hasegawa, et al. (“In vivo tumor delivery of the green fluorescent protein gene to report future occurrence of metastasis,” Cancer Gene Therapy (2000) 7:1336-1340). Nude mice are prepared with human stomach tumors growing intraperitoneally, also as discussed by Hasegawa, et al. The retroviral supernatants are injected intraperitoneally at days 4 to 10 following implantation of the cancer cells into the mice.

The mice are imaged externally using a GFsP-5 imaging device, which resembles a miner's lamp. Examinations occur every other week so that tumor growth and metastasis formation can be visualized by GFP expression. No normal tissues are transduced by the GFP retrovirus. Two weeks after retroviral GFP delivery, GFP-expressing tumor cells are observed in the gonadal fat, greater omentum, and intestine, indicating that the tumors are efficiently transduced by the GFP gene and can be detected by its expression. Laparotomies are performed to confirm the observations made externally.

EXAMPLE 2

High Throughput External Screening of Subjects with Ovarian Tumor Fragments

Thirty nude mice are implanted with GFP-expressing Chinese hamster ovary tumor fragments (CHO-K1-GFP) of about 1 mm³ to screen potential antitumor compounds for efficacy against the tumor tissue. The nude mice are implanted with tumor fragments into the ovarian serosa of nude mice by surgical orthotopic implantation (SOI) and ovarian tumors develop (See, Chishima, et al., Cancer Res. (1997) 57:2042-2047).

The animals, housed in individual cages, are placed on a rotating table and are passed in front of a GFP-Vid-187 (Biological Laboratory Equipment, Ltd, Budapest, Hungary), which comprises a light source fitted upon a standard digital video camera. The tumors, which are strongly fluorescent, are observable on the video gathered by the camera. Images gathered by the camera are analyzed visually and fed into a computer for further analysis.

Experimental animals receive various candidate compounds while the control animals receive saline. During the study, animals receiving a candidate compound that is efficacious against the tumor cells display less fluorescence than the control animals. Fluorescence in the control animals is observed to spread throughout the peritoneal cavity, including the colon, cecum, small intestine, spleen, and peritoneal wall. GFP fluorescence is used to track tumor spread; numerous micrometastases are detected on the lungs of all control mice and multiple micrometastasis are also detected on the liver, kidney, contralateral ovary, adrenal gland, para-aortic lymph node, and pleural membrane.

Claims

1. A method to visualize a fluorescent protein through the skin of an intact subject, which method comprises applying excitation light to said subject using a portable light source with an attached first filter and observing emission from said protein through a second filter.

2. The method of claim 1, wherein the portable light source is an LED flashlight.

3. The method of claim 1, wherein the second filter is provided as a goggle.

4. The method of claim 1, wherein said fluorescent protein is expressed in tumor cells.

5. The method of claim 4, wherein said tumor cells are orthotopically implanted in an immunocompromised or syngeneic animal.

6. The method of claim 1, wherein the fluorescent protein is expressed by an infectious agent.

7. The method of claim 1, wherein the fluorescent protein is operatively linked to the control system for a gene whose expression is to be studied.

8. The method of claim 1, wherein observation is made of more than one fluorescent protein.

9. A method for monitoring tumor growth in an intact subject comprising: applying excitation light using a portable excitation source with a first filter to a subject comprising tumor cells labeled with fluorescent protein; andobserving the location(s) of said tumor cells in the intact subject using a second filter.

10. The method of claim 9, wherein said applying of excitation light and observing are conducted as a function of time.

11. The method of claim 9, which further comprises treating said subject with a candidate protocol and comparing the location(s) of tumor cells in said subject treated with the protocol with the location(s) of tumor cells in a subject not treated with the protocol.

12. A method for monitoring gene expression in an intact subject which method comprises applying an excitation light from a portable excitation source with a first filter to a subject comprising a nucleotide sequence encoding a fluorescent protein operably linked to control sequences associated with a gene whose expression is to be monitored andobserving the presence or amount of fluorescent protein in the intact subject using a second filter.

13. The method of claim 12, wherein said applying of excitation light and observing are conducted as a function of time.

14. The method of claim 12, which further comprises providing a stimulus to said subject and comparing the level of fluorescence emitted by the subject provided the stimulus to that emitted by a subject not provided with said stimulus.

15. A method to monitor the progress of infection in a subject which method comprises applying excitation light using a portable excitation source with a first filter to a subject comprising an infectious agent labeled with fluorescent protein, andobserving the location(s) of said infectious agent in the intact subject using a second filter.

16. The method of claim 15, wherein said applying of excitation light and observing are conducted as a function of time.

17. The method of claim 15, which further comprises treating said subject with a candidate protocol and comparing the location(s) of infectious agent in said subject treated with said protocol with the location(s) of infectious agent in a subject not treated with the protocol.