Method for predicting the efficacy of anti-cancer drugs

• US 6,448,030 B1
• Filed: 12/05/2000
• Issued: 09/10/2002
• Est. Priority Date: 02/18/2000
• Status: Expired due to Fees

• Current Assignee: University of Nevada Las Vegas
• Original Assignee: University of Nevada Las Vegas
• Inventors: Rust, William L., Huff, Janice L., Plopper, George E.
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US09/730,700
• Publication Number:
  US 20020068316A1
• Time in Patent Office: 644 Days
• Field of Search: 435/29, 435/4, 435/968, 422/186
• US Class Current: 435/29
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive

Abstract

A method for measuring both the anti-migratory and cytotoxic effects of anticancer drugs on cancer tissues measuring the chemosensitivity of human biopsies to therapeutic drugs. Single cell suspension preparations of biopsy tissue is exposed to anticancer drugs and introduced into the first chamber of a migration plate separated from a second chamber by a porous membrane opaque to radiation. A migration stimulus is added to the second chamber. Migrated cells on the side of the membrane facing the second chamber are labeled with a live-cell fluorescent indicator. Non-migrated cells on the side of the membrane facing the first chamber are labeled with a fluorescent indicator of cell death. The emitted fluorescence of both the migrated cells and the non-viable cells is quantified in a fluorescence plate reader. The comparative intensity of fluorescence is an indicator of the susceptibility of the cells to the cytotoxic properties of the drug.

Claims

1. A method for predicting the efficacy of anti-cancer drugs on cells wherein both the cytotoxic and anti-migratory effects of the drug are measured, comprising the steps of:

a. disaggregating a biopsy specimen into individual cells to form a cell suspension;

b. exposing said cells in said suspension to said anti-cancer drug;

c. adding said exposed cells to the first chamber of a migration plate comprising two chambers separated by a porous membrane, said porous membrane being opaque to radiation;

d. placing a chemical agent in the second chamber, said agent being capable of inducing migration of said cells from said first chamber to said second chamber;

e. incubating cells to allow cell migration to occur;

f. labeling cells on the side of the membrane closest to said second chamber with a fluorescent dye, said dye being capable of labeling only live cells;

g. labeling cells on the side of the membrane closest to said first chamber with a fluorescent dye, said dye being capable of labeling only dead cells;

h. stimulating the labeled cells on the side of the membrane closest to said second chamber with electromagnetic radiation of a first wavelength whereby said labeled cells will emit electromagnetic radiation of a second wavelength;

i. measuring the emitted electromagnetic radiation from the side of the radiation opaque membrane closest to said second chamber;

j. stimulating the labeled cells on the side of the membrane closest to said first chamber with electromagnetic radiation of a first wavelength whereby said labeled cells will emit electromagnetic radiation of a second wavelength; and

k. measuring the emitted electromagnetic radiation from the side of the radiation opaque membrane closest to said first chamber, whereby the effect of a drug on said cell migration and said cell death is an indication of the effectiveness of said drug at arresting said cancer tissues.

2. The method according to claim 1, wherein:

said side of the membrane closest to the second chamber is coated with a fixed material capable of inducing migration.

3. The method according to claim 1, wherein:

said fluorescent dye capable of labeling only live cells is metabolically activated.

4. The method according to claim 1, wherein:

said fluorescent dye capable of labeling only live cells is embedded in a material endocytosed only by live cells.

5. The method according to claim 1, wherein:

the steps of stimulating the labeled cells on the side of the membrane closest to said first and said second chamber each further comprises the further step of measuring the electromagnetic radiation with a fluorescent plate reader.

6. A method for testing the efficacy of a drug on cells, in vitro, comprising the steps of:

exposing said cells in said suspension to said drug;

adding said exposed cells to the first chamber of a migration plate comprising two chambers separated by a porous membrane, said porous membrane being opaque to radiation;

placing a chemical agent in the second chamber, said agent being capable of inducing migration of said cells from said first chamber to said second chamber;

labeling cells on the side of the membrane closest to said second chamber with a fluorescent dye, said dye being capable of labeling only live cells;

labeling cells on the side of the membrane closest to said first chamber with a fluorescent dye, said dye being capable of labeling only dead cells;

stimulating the labeled cells on the side of the membrane closest to said second chamber with electromagnetic radiation of a first wavelength whereby said labeled cells will emit electromagnetic radiation of a second wavelength;

measuring the emitted electromagnetic radiation from the side of the radiation opaque membrane closest to said second chamber;

stimulating the labeled cells on the side of the membrane closest to said first chamber with electromagnetic radiation of a first wavelength whereby said labeled cells will emit electromagnetic radiation of a second wavelength; and

measuring the emitted electromagnetic radiation from the side of the radiation opaque membrane closest to said first chamber, whereby the effect of said drug on said cell migration and said cell death is an indication of the effectiveness of said drug.

7. The method according to claim 6, wherein:

the steps of stimulating the labeled cells on the side of the membrane closest to said first and said second chamber each further comprises the further step of measuring the electromagnetic radiation with a fluorescent plate reader.

8. The method according to claim 6, wherein:

said fluorescent dye capable of labeling only live cells is metabolically activated.

9. The method according to claim 6, wherein:

said fluorescent dye capable of labeling only live cells is embedded in a material endocytosed only by live cells.