WO2003038431A1

WO2003038431A1 - Method for screening combinatorial bead library, capturing cells from body fluids, and ligands for cancer cells

Info

Publication number: WO2003038431A1
Application number: PCT/US2001/050865
Authority: WO
Inventors: Derick H. Lau; Kit S. Lam
Original assignee: The Regents Of The University Of California
Priority date: 2001-10-26
Filing date: 2001-10-26
Publication date: 2003-05-08

Abstract

The invention includes a cell-growth-on-bead assay for screening a one-bead-one-compound combinatorial bead library to identify synthetic ligands for cell attachment and growth or proliferation of epithelial cells. Cells are incubated with a compound bead library for 24 to 72-hours, allowing them to attach and grow on the beads. Those beads with cells growing are removed, and the ligand on the bead is identified. Also provided are ligands specific for epithelial cancer cells. The invention also includes a method of capturing epithelial cells from body fluids. In this method, beads are prepared with a known ligand specific for a particular type of cell and incubated for 24 to 72 hours with a sample of the body fluid being tested. Those cells attached to the beads are removed and identified.

Description

METHOD FOR SCREENING COMBINATORIAL BEAD LIBRARY.

CAPTURING CELLS FROM BODY FLUIDS. AND

LIGANDS FOR CANCER CELLS

Derick H. Lau and Kit S. Lam

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to screening methods for one-bead-one-compound

combinatorial libraries and includes a screening assay that uses live cells to identify

synthetic ligands that promote attachment and growth or proliferation of epithelial

cells. Also included are ligands specific for epithelial cancer cells. The invention

also relates to methods for isolating and capturing epithelial cells, including benign

(non-cancerous) and malignant (cancerous) cells, from body fluids.

2. Description of Related Art

One-bead-one-compound combinatorial bead libraries (see Lam, Kit S. et

al. "A new type of synthetic peptide library for identifying ligand-binding

activity. " Nature 354 (1991): 82-84), such as one-bead-one-compound peptide

libraries, are being used to study cell adhesion properties of cancer cells. Using

random peptide bead libraries and suspended cancer cells, peptide ligands that

promote cell attachment have been identified for lymphoma (Park, Steven,¹ Renil

Manat, Brian Vikstrom, Nail Amro, and Kit S. Lam. "Identification of peptide

ligands for «4B1 integrin receptor as potential targeting agents for non-Hodgkin's

lymphoma, " abstract in Peptides: The Wave of the Future, 2nd International Peptide Symposium in conjunction with the 17^th American Peptide Symposium, San

Diego, CA (June 9-14, 2001)) and prostate cancer cell lines (Pennington,

Michael E. , Kit S. Lam and Anne E. Cress. "The use of a combinatorial library

method to isolate human tumor cell adhesion peptides. " Molecular Diversity 2

(1996): 19-28; DeRoock, Ian B., Michael E. Pennington, Thomas C. Sroka, Kit

S. Lam, G. Tim Bowden, Elisabeth L. Bair, and Anne E. Cress. "Synthetic

Peptides Inhibit Adhesion of Human Tumor Cells to Extracellular Matrix

Proteins." Cancer Research 61 (April 15, 2001): 3308-13).

In the existing methods, live cells in suspension are incubated for about one

to four hours with a bead library, and the library is then screened for beads with

peptide ligands that promote cell attachment. This is done by visual selection - the

beads are examined under a dissecting microscope and those beads with attached

cells are removed using a micropipet. Further steps are then performed to confirm

that the removed beads are in fact capable of binding the particular type of cells

tested. Then, the peptides on those beads are sequenced. (See Pennington et al.,

"Use of a combinatorial library method," 19-28.)

In another existing method of testing live cells for peptide ligands that affect

cell growth on culture plates, a bead library is prepared having selectively

cleavable peptides such that a proportion of the peptide on each bead is attached to

the bead by a cleavable linker. When the library is treated with a cleaving agent,

enough of the peptides are released from the beads to cause the biological effect, and the rest of the peptides remain bound to the beads to allow for later sequencing .

Suspended cells are incubated in tissue culture wells with a few beads and with

peptides released from the beads. The effect of the released peptides on the cells

(inhibition or stimulation of cell growth) is determined, and the corresponding

beads are removed. The sequences of the attached peptides are then determined.

(See U. S. pat. no. 5,510,240, issued April 23, 1996 to Lam, Kit S. et al.)

The existing methods, however, are not satisfactory in certain cases. The

methods are difficult to use with epithelial cells, which include the majority of solid

cancer cell cultures, such as lung cancer cells, that exist as adherent cultures rather

than as suspended cells. With incubation periods of only a few hours, these cells

are often only weakly attached to the beads and may easily fall off, rendering the

screening method less accurate because some beads with attached cells are missed.

Also, the existing methods may not detect cell surface receptors that may be altered

by trypsin and/or EDTA. Trypsinization is commonly used to separate tissues or

cell cdltures into a single-cell suspension for testing with a combinatorial library.

The treatment with trypsin may eliminate some, or alter the conformation of, cell

surface receptors. In addition, the existing methods do not select for peptide

ligands that promote cell growth or proliferation, but, rather, for ligands involved

in cell attachment, particularly short-term attachment.

Thus, there is a need for a screening assay that is specific and sensitive,

works well with epithelial cells, can be used to detect cell surface receptors susceptible to trypsin, and selects for ligands that promote not only cell attachment,

but also cell growth or proliferation.

There is also a need for an efficient method of isolating and capturing

epithelial cells, including benign and malignant cells, from body fluids, such as

blood, urine, pleural effusion, pericardial effusion, ascite, and cerebrospinal fluid.

In particular, there is a need for a method of isolating and capturing cancer cells

to assist in diagnosis.

SUMMARY OF THE INVENTION

The present invention is directed to a method for screening a combinatorial

bead library for ligands that promote the attachment and growth or proliferation of

epithelial cells. The method satisfies the need for an assay that is specific and

sensitive, that can be used to detect cell surface receptors susceptible to trypsin,

and that can identify ligands that promote cell growth and proliferation. The

method comprises introducing a suspension of live cells to a combinatorial library

of small molecules, peptides, or other types of molecules, incubating the cells with

the library for about 24 to 72 hours, identifying a solid phase support of the library

with cells growing on the support, isolating the solid phase support, and

determining the chemical structure of the compound attached to that solid phase

support.

The invention also includes ligands specific for cell attachment and growth

or proliferation of epithelial cancer cells, having the chemical structure of cXGXGXXc, in which "c" is D-cysteine; "X" is any L-, D-, unnatural, or

modified amino acid; and "G" is glycine.

The present invention is also directed to a method for isolating and capturing

cells from body fluids and satisfies the need for an efficient method of isolating and

capturing epithelial cells from body fluids. The method comprises introducing a

sample of body fluid to a multiplicity of beads with one or more known ligands

specific for one or more particular types of cell, incubating the body fluid with the

beads for about 24 to 72 hours, identifying a bead with cells growing on the bead,

isolating the bead, and recovering the cells growing on the bead.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a diagram depicting the steps of the cell-growth-on-bead assay.

Fig. 1A shows attached epithelial cells.

Fig. IB shows the detached epithelial cells of Fig. 1A in suspension.

Fig. 1C shows the epithelial cells being mixed with the beads of the bead

library.

Fig. ID shows a top view of three beads, in which two beads have a

monolayer of cells growing on the bead.

Fig. IE shows a top view of the three beads of Fig. ID, after staining, in

which the two beads with a monolayer of cells growing on the bead are stained,

and the one bead without any cells is not stained. Fig. 2 is a diagram depicting the steps of the method for isolating and

capturing epithelial cells from body fluids.

Fig. 2A shows a centrifuge tube containing a sample of body fluid,

containing cells, which has been removed from the human body.

Fig. 2B shows the centrifuge tube containing the body fluid, in which the

cells have been allowed to sediment.

Fig. 2C shows the cells being mixed with beads.

Fig. 2D shows a top view of the beads and the cells, in which some cells are

attached and growing on the beads, and some cells remain unattached.

Fig. 2E shows the beads and the cells after the cells have been removed

from the beads.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One embodiment of the invention includes a method, referred to as the cell-

growth-on-bead assay, for screening a one-bead-one-compound combinatorial bead

library for ligands that promote cell attachment and growth or proliferation.

Ligands that promote cell attachment and growth or proliferation of cancer cells are

also described. Another embodiment of the invention includes a method for

isolating and capturing epithelial cells, such as benign or malignant cells, from

body fluids. This method can be used to aid in the diagnosis of cancer. Cell-Growth-on-Bead Assay

The cell-growth-on-bead assay of the first embodiment includes the

following steps, as shown in Fig. 1. A one-bead-one-compound combinatorial

library is prepared. The library is preferably synthesized using the " split synthesis "

approach described in Lam et al. , "A new type of synthetic peptide library, " 82-84.

The compounds of the library may be small molecules, peptides, or other types of

molecules. An example of a suitable library is a peptide library containing

cXXXXXXc peptides, where "c" is D-cysteine which provides intramolecular

cyclization by disulfide bonding, and "X" is any L, D, unnatural, or modified

amino acid. A suitable solid phase support, such as beads or discs made of

polystyrene, agarose, acrylamide, glass, plastic, or paramagnetic substances, is

used. Polystyrene beads have been found satisfactory. A standard synthetic solid

phase peptide synthesis method, such as fluorenylmethyoxycarbonyl (Fmoc)

chemistry or t-butyloxycarbonyl (Boc) chemistry, is used. For purposes of

illustration, the combinatorial library referred to below is a peptide bead library,

although it is understood that this is only one example of a library that can be used

in this embodiment and that other libraries will also work.

A suspension of live mammalian cells is prepared according to methods

known to those skilled in the art. The cells are preferably epithelial cells and may

be cancerous or non-cancerous. Human cancer cells from a cell line_or derived

from biopsy specimens or body fluid of cancer patients may be used. Fig. 1A shows attached epithelial cells 10. Fig. IB shows the same cells 10 in suspension.

Suspended live cells 10 are mixed with the peptide library in culture

medium, as shown in Fig. 1C, and distributed into culture plates. The ratio of cells

to peptide beads is preferably about 10:1, but can range from about 1:1 to 100:1.

The suspension of cells 10 and beads 12 is mixed gently for sufficient time to

assure contact of beads 12 with suspended cells 10. The culture plates are

incubated in a tissue culture incubator at about 4° C to about 37° C, preferably

37° C, for a period of about 24 to about 72 hours. The suspension of cells 10 and

beads 12 may be kept still or mixed, either continuously or intermittently, during

the incubation period.

After the incubation period, beads 12 are observed under a dissecting

microscope. The presence of an increased number of cells 10 or beads 14 covered

with a monolayer of cells, as shown in Fig. ID, evidences cell growth or

proliferation. These beads 14 (referred to as "positive" beads) are removed from

the culture plates. A tetrazolium dye that stains live, but not dead, cells can be

used to facilitate the identification and removal of the positive beads. If a dye is

used, all of the beads are removed after the incubation period and resuspended in

fresh medium in new culture plates. The dye is added. The plates are then

incubated in a tissue culture incubator at 25° C to 37° C for about one to

four hours. Live cells 10 convert the dye to a colored metabolite, which results in

beads 14 with attached cells appearing colored, allowing them to be easily distinguished from beads 16 without attached cells, which appear colorless, as

shown in Fig. IE. Other dyes that stain live cells can also be used.

After positive beads 14 are removed from the plates, attached cells 10 are

separated from the beads. This can be done with the addition of a chaotrophic

agent, such as 8 M guanidine hydrochloride, or a protease, such as trypsin.

The amino acid sequence of the ligand on each isolated positive bead 14 is

then determined. This is preferably done with an automated protein sequencer,

such as the Procise 494 (Applied Biosy stems, Foster City, CA). Alternatively, the

peptide can be released via a cleavable linker and the amino acid sequence

determined by mass spectroscopy. If the ligand on the bead consists of a small

molecule, mass spectroscopy, and/or encoding strategies can be used.

Using the cell-growth-on-bead assay of the first embodiment, ligands that

promote cell attachment and growth or proliferation have been identified. By

structure/activity relationship studies, ligands identified for epithelial cancer cells,

such as those of lung cancer, have the general structure of cXGXGXXc, where "c"

is D-cysteine; "X" is any L, D, unnatural, or modified amino acid; and "G" is

glycine.

Method of Isolating and Capturing Cells from Body Fluids

The second embodiment of the invention is a method to isolate and capture

benign or malignant epithelial cells from mammalian body fluids as shown in Fig. 2. The method car ^*" e used to capture cancer cells from body fluids, such as

As shown in Fig. 2A, a sample of fluid 20 is removed from the body and

placed in centrifuge tube 22 for about 10 minutes to allow cells 24 to settle to the

bottom, as shown in Fig. 2B. A sample of the sediment containing cells 24 is then

resuspended in culture medium. About 0.1 to 0.5 ml of sediment in about 2 ml of

culture medium works well.

A suitable solid phase support, such as beads or discs made of polystyrene,

agarose, acrylamide, glass, plastic, or paramagnetic substances, is used.

Polystyrene beads have been found to be satisfactory. For purposes of illustration,

beads are used as the solid phase support. Approximately 5,000 to 10,000 beads

containing one or more known ligands specific for one or more particular cell types

are sterilized with ethanol. The ligands may consist of small molecules, peptides,

or other molecules. Generally, one ligand is used; however, more than one ligand

may also be used.

The cell suspension and sterile beads 26 are mixed gently in a cell culture

dish for a few minutes to assure contact of beads 26 with suspended cells 24, as

shown in Fig. 2C. The mixture is incubated in a tissue culture incubator under 5 %

carbon dioxide at about 4° C to about 37° C, preferably 37° C. The dish is

observed under a microscope daily for a period of about 24 to about 72 hours to

check for cell attachment and growth, as shown in Fig. 2D. The medium may be changed if necessary. Typically, a culture can be maintained for up to about

four weeks depending on the rate of cell proliferation on the beads. Cells 28

attached to beads 26 are recovered when the number of cells is sufficient for

counting, morphologic examination, molecular analysis, and further culturing.

The medium is pipetted off the culture dish, and beads 26 with attached

cells 28 are rinsed with fresh medium to remove unattached cells 30, such as red

blood cells, and other cell debris. Beads 26 with attached cells 28 are transferred

to a fresh dish. Attached cells 28 are removed from beads 26 with trypsin and/or

EDTA, as shown in Fig. 2E. Isolated cells 28 are transferred to a centrifuge tube

containing fresh culture medium and concentrated by centrifugation at 500 xg. The

supernatant is removed, and the cells are spread on a glass slide by direct smearing

or by cytospin. Cells on glass slides can be preserved in fixatives such as 95 %

alcohol and then stained with Giemsa or Papanicolou stain for subsequent

morphologic examination.

The concentrated cells can also be fixed in 10% formalin or 4%

paraformaldehyde and embedded in paraffin. The cell block can be sectioned into

5 μ slices and placed on glass slides for morphologic staining and/or

immunohistochemical staining to identify special antigens, for example,

carcinoembryonic antigen (CEA) and estrogen receptor (ER), if necessary to aid

in diagnosis of cancer. In addition, the cells recovered can be analyzed with other cellular or molecular techniques such as laser scanning cytometry, Western

blotting, and microarray.

The cells recovered from the beads may also be cultured for expansion of

cell number or for long-term maintenance. After having been separated from the

beads, cells are pipetted into a 15 ml centrifuge tube. The cell pellet is

resuspended in an appropriate amount of medium in a culture dish or flask. The

cell culture is kept in a tissue culture incubator under 5% carbon dioxide at 37° C.

Cells harvested from the culture can be stored long-term in 90% serum and 10%

DMSO under liquid nitrogen.

Example of Cell-Growth-on-Bead Assay

A one-bead-one-peptide combinatorial library, containing random

cXXXXXXc peptides, was prepared using the "split synthesis" method of Lam et

al. , "A new type of synthetic peptide library, " 82-84. The random peptide library

contained 19⁶ = 4.7 x 10⁷ possible permutations of the formula cXXXXXXc,

where "c" is D-cysteine, and "X" is one of 19 natural L-amino acids.

TentaGel polystyrene beads, with a diameter of 80 μm and with grafted

polyethylene glycol of 0.25 mmol/g, were used as a solid phase support (Rapp

Polymere, Germany). A synthetic solid phase method using

fluorenylmethyoxycarbonyl (Fmoc) chemistry was adapted for synthesizing the

peptide bead library. The non-small-cell lung cancer cell line, A549 (American Type Culture

Collection, Manassas, VA), was used. The cell line was maintained in appropriate

culture media as recommended by American Type Culture Collection. Cells were

grown to confluency in DMEM culture medium supplemented with 10% fetal calf

serum. Attached cells were recovered with trypsin/EDTA, washed, and

resuspended as single cells in culture medium.

About 150,000 peptide beads were mixed with approximately one million

suspended cells in 15 ml of culture medium and distributed into six 3-cm culture

plates. The culture plates were agitated gently at about 100 rpm for about

10 minutes. The culture plates were then incubated in a tissue culture incubator at

37° C for about 24 hours to about 72 hours.

A dissecting microscope was used to examine the beads at about 24, 48, and

72 hours. After about 24 to 72 hours, beads with a monolayer of cells were

observed. Out of a library of about 150,000 beads, about 20 to 30 beads typically

exhibited cell growth.

At the end of the incubation period, all of the beads were removed and

resuspended in fresh medium in a new culture plate. An MTT

([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl] tetrazolium bromide) (Sigma,

St. Louis, MO) dye solution was added to each culture plate to a final concentration

of 0.5 mg/ml. The plates were incubated in a tissue culture incubator at 37° C for

about two hours to allow the purple color to develop. Each purple-colored peptide bead was isolated and removed. The attached cells were separated from the beads

with 8 M guanidine hydrochloride.

The amino acid sequence of each isolated peptide bead was determined using

an automated Procise 494 protein sequencer (Applied Biosy stems, Foster City,

CA). Several consensus peptide sequences were determined, one of which was

cNGRGEQc, where "c" is D-cysteine. This peptide was resynthesized on beads,

which were then rescreened with the A549 cells using the assay of the invention.

Virtually all of the beads with this sequence exhibited cell attachment and growth

on their surfaces.

To test the sensitivity of the assay, blank beads and a linear XXXXXX

peptide bead library of 150,000 beads were each spiked with 10 positive peptide

beads carrying the sequence cNGRGEQc. These libraries were each screened with

the A549 cells. The peptide beads with the sequence of cNGRGEQc were isolated

with a recovery rate of 90% to 100% in two separate experiments.

To test cell type specificity of beads carrying the peptide ligand

cNGRGEQc , cell growth of two other non-small-cell lung cancer cell lines , Calu- 1

and H178, was observed on 70% to 90% of the peptide beads. On the other hand,

cell growth was observed on only 10% of the peptide beads with the non-malignant

bronchoepithelial cell line, HBE-1. Thus, the cNGRGEQc peptide is a ligand

specific for promoting cell attachment and growth of malignant cells of the lung. Through additional secondary library screening and structure/activity

relationship studies, other ligands for epithelial cells have been identified, including

the following, where "c" is D-cysteine: c-DGChgGAN-c, where Chg is a-

cyclohexylglycine; c-NGBpaGQM-c, where Bpa is 4-benzoylphenylalanine;

c-NGAcdtGDBpa-c, where Acdt is 4-amino-4-carboxy-l , l-dioxo-

tetrahydrothiopyran; c-NGTGDG-c; c-NGQGAG-c; c-NGYGSF-c; and

c-NGNleGYG-c, where Nle is norleucine.

Example of Method for Isolating and Capturing Cells from Body Fluid

A sample of human pleural fluid was placed in a centrifuge tube, and the

cells were allowed to settle to the bottom of the tube for 10 minutes. A sample of

about 0.1 to 0.5 ml of the sediment containing the cells was resuspended in about

2 ml of culture medium.

TentaGel polystyrene beads, with a diameter of 80 μ and with grafted

polyethylene glycol of 0.25 mmol/g, were used as a solid phase support (Rapp

Polymere, Germany). Approximately 5,000 to 10,000 beads having the ligand

cNGRGEQc were prepared. The beads were sterilized with 75 % ethanol. The cell

suspension and the sterile beads were mixed gently in a 3-cm cell culture dish for

five minutes. The dish was incubated in a tissue culture incubator under 5%

carbon dioxide at 37° C. The dish was observed daily under a microscope for

about 24 to 72 hours to check for cell attachment and/or growth. Cell growth was

observed at 24 hours. Cells attached to beads were recovered after 72 hours. The medium was pipetted off the culture dish, and the beads were rinsed

with fresh medium to remove unattached cells, such as red blood cells, and other

cell debris. Attached cells were removed from the beads with trypsin/EDTA,

transferred to a centrifuge tube with fresh culture medium, and concentrated by

centrimgation at 500 xg. The supernatant was removed, and the cells were spread

on a glass slide by direct smearing or by cytospin. Cells on glass slides were

preserved in 95 % alcohol and stained with Giemsa or Papanicolou stain. The cells

were identified as malignant adenocarcinoma cells which had a large nucleus,

prominent nucleoli, and mucin in the cytoplasm.

The invention has been described above with reference to the preferred

embodiments. Those skilled in the art may envision other embodiments and

variations of the invention that fall within the scope of the claims.

Claims

CLAIMSWe claim:

1. A method of screening a library of synthetic compounds to identify a ligand,

comprising:

introducing a suspension of live mammalian cells to a combinatorial library,

wherein said library comprises multiple solid phase supports with

synthetic compounds attached thereto;

incubating said cells with said solid phase supports of said library for a

period of about 24 to about 72 hours;

identifying a solid phase support having cells growing thereon;

isolating said identified solid phase support; and

determining the chemical structure of the compound attached to said

identified solid phase support.

2. The method of Claim 1 , wherein said incubating is at a temperature of about

37° C.

3. The method of Claim 1, wherein said cells are epithelial cells.

4. The method of Claim 1, wherein said combinatorial library is a peptide

library.

5. The method of Claim 1 , further comprising, after incubating said cells with

said library, adding a dye that will stain only live cells, and further, incubating said

cells with said library and said dye for a period of about two hours.

6. The method of Claim 1, wherein said solid phase supports are beads.

7. A method of screening a library of peptides and determining the sequence

of a peptide ligand, comprising:

introducing a suspension of live mammalian cells to a one-bead-one-peptide

combinatorial library;

incubating said cells with said beads of said peptide library for a period of

about 24 hours to about 72 hours;

identifying a bead having a peptide ligand for said cells, wherein said bead

has cells growing thereon;

isolating said identified bead; and

determining the amino acid sequence of the peptide ligand attached to said

identified bead.

8. The method of Claim 7, wherein said cells are epithelial cells.

9. A method of identifying a ligand that promotes cell attachment and

proliferation, comprising:

introducing a suspension of live mammalian cells to a combinatorial library,

wherein said library comprises multiple solid phase supports with

synthetic compounds attached thereto;

incubating said cells with said solid phase supports of said library for a

period of about 24 to about 72 hours;

identifying a solid phase support having cells growing thereon; isolating said identified solid phase support; and

determining the chemical structure of the compound attached to said

identified solid phase support.

10. A method for isolating and capturing mammalian cells of a particular type

from body fluid, comprising:

introducing a sample of body fluid to a multiplicity of solid phase supports

having a known ligand specific for a particular type of cell;

incubating said body fluid with said solid phase supports for a period of

about 24 to about 72 hours;

identifying a solid phase support having cells growing thereon;

isolating said solid phase support; and

recovering the cells growing on said isolated solid phase support.

11. The method of Claim 10, wherein said incubating is at a temperature of

about 37° C.

12. The method of Claim 10, wherein said ligand is a peptide specific for a

cancer cell.

13. The method of Claim 10, wherein said ligand is specific for human lung

cancer cells and has the chemical structure of cXGXGXXc, in which "c" is

D-cysteine; "X" is an amino acid selected from the group consisting of L-amino

acids, D-amino acids, unnatural amino acids, and modified amino acids; and "G"

is glycine.

14. The method of Claim 13, wherein said ligand has the chemical structure

cNGRGEQc, in which "c" is D-cysteine.

15. The method of Claim 14, wherein said body fluid is human pleural fluid.

16. A ligand specific for human lung cancer cells, wherein said ligand has the

chemical structure of cXGXGXXc, in which "c" is D-cysteine; "X" is an amino

acid selected from the group consisting of L-amino acids , D-amino acids , unnatural

amino acids, and modified amino acids; and "G" is glycine.