U.S. patent application number 12/168537 was filed with the patent office on 2008-12-04 for methods and tools for detecting collagen degradation.
This patent application is currently assigned to Lonza Walkersville, Inc.. Invention is credited to Dale Greenwalt.
Application Number | 20080299604 12/168537 |
Document ID | / |
Family ID | 35239891 |
Filed Date | 2008-12-04 |
United States Patent
Application |
20080299604 |
Kind Code |
A1 |
Greenwalt; Dale |
December 4, 2008 |
METHODS AND TOOLS FOR DETECTING COLLAGEN DEGRADATION
Abstract
Fluorophore-labeled collagen covalently bound to a cell culture
vessel can be used to assay collagen degradation by a variety of
cell types, including osteoclasts and tumor cells. Such assays
provide a high throughput platform for rapid screening of large
numbers of potential modulators of, for example, tumor metastasis
or osteoclast differentiation and/or function.
Inventors: |
Greenwalt; Dale;
(Walkersville, MD) |
Correspondence
Address: |
BANNER & WITCOFF, LTD.
1100 13th STREET, N.W., SUITE 1200
WASHINGTON
DC
20005-4051
US
|
Assignee: |
Lonza Walkersville, Inc.
Walkersville
MD
|
Family ID: |
35239891 |
Appl. No.: |
12/168537 |
Filed: |
July 7, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10840653 |
May 7, 2004 |
7405037 |
|
|
12168537 |
|
|
|
|
Current U.S.
Class: |
435/34 ;
435/288.7 |
Current CPC
Class: |
G01N 2333/78 20130101;
G01N 33/582 20130101; G01N 33/6887 20130101; G01N 33/5044
20130101 |
Class at
Publication: |
435/34 ;
435/288.7 |
International
Class: |
C12Q 1/04 20060101
C12Q001/04; C12M 1/34 20060101 C12M001/34 |
Claims
1. A cell culture vessel comprising fluorophore-labeled collagen
covalently bound to a culture surface of the cell culture
vessel.
2. The cell culture vessel of claim 1 wherein the fluorophore is a
lanthanide chelate.
3. The cell culture vessel of claim 2 wherein the lanthanide
chelate is a europium chelate.
4. The cell culture vessel of claim 3 wherein the europium chelate
is
Eu.sup.3+--N.sup.1-(p-isothiocyanatobenzyl)diethylenetriamine-N.sup.1,N.s-
up.2,N.sup.3-tetraacetic acid.
5. The cell culture vessel of claim 1 wherein the collagen is type
I.
6. The cell culture vessel of claim 1 wherein the collagen is type
IV.
7. The cell culture vessel of claim 1 wherein the collagen is
mammalian.
8. The cell culture vessel of claim 1 wherein the collagen is
human.
9. The cell culture vessel of claim 1 wherein fluorescence of the
covalently bound collagen is quenched.
10. A cell culture vessel comprising
Eu.sup.3+--N.sup.1-(p-isothiocyanatobenzyl)diethylenetriamine-N.sup.1,N.s-
up.2,N.sup.3-tetraacetic acid-labeled human collagen type I
covalently bound to a culture surface of the cell culture
vessel.
11. A kit for detecting collagen degradation, comprising: (a) a
cell culture vessel comprising fluorophore-labeled collagen
covalently bound to a culture surface of the cell culture vessel;
and (b) instructions for a method comprising steps of: (1)
culturing cells in culture medium on the fluorophore-labeled
collagen, wherein the cells can degrade the collagen or can
differentiate into cells which can degrade the collagen; and (2)
detecting the presence or absence of a fluorescence signal in a
sample of the culture medium, wherein fluorescence signal intensity
reflects the concentration of fluorophore-labeled collagen
fragments in the sample of the culture medium.
12. The kit of claim 11 wherein the fluorophore is a lanthanide
chelate.
13. The kit of claim 12 wherein the lanthanide chelate is a
europium chelate.
14. A kit for detecting collagen degradation, comprising: (a) a
cell culture vessel comprising
Eu.sup.3+-N.sup.1-(p-isothiocyanatobenzyl)diethylenetriamine-N.sup.1,N.su-
p.2,N.sup.3-tetraacetic acid-labeled human collagen type I
covalently bound to a culture surface of the cell culture vessel;
(b) an enhancing solution; and (c) instructions for a method
comprising: (1) culturing cells in culture medium on the
Eu.sup.3+--N.sup.1-(p-isothiocyanatobenzyl)diethylenetriamine-N.sup.1,
N.sup.2,N.sup.3-tetraacetic acid-labeled collagen, wherein the
cells can degrade the collagen or can differentiate into cells
which can degrade the collagen; (2) transferring the sample of the
culture medium from the cell culture vessel to an assay vessel
containing an enhancing solution; and (3) detecting the presence or
absence of a fluorescence signal in a sample of the culture medium,
wherein fluorescence signal intensity reflects the concentration of
Eu.sup.3+--N.sup.1-(p-isothiocyanatobenzyl)diethylenetriamine-N.sup.1,N.s-
up.2,N.sup.3-tetraacetic acid-labeled collagen fragments in the
sample of the culture medium.
15. A kit for detecting collagen degradation, comprising: (a) a
cell culture vessel comprising fluorophore-labeled collagen
covalently bound to a culture surface of the cell culture vessel,
wherein fluorescence of the covalently bound fluorophore-labeled
collagen is quenched; and (b) instructions for a method comprising:
(1) culturing cells in culture medium on the fluorophore-labeled
collagen, wherein the cells can degrade the collagen or can
differentiate into cells which can degrade the collagen; (2)
transferring the sample of the culture medium from the cell culture
vessel to an assay vessel containing an enhancing solution; and (3)
detecting in the cell culture vessel the presence or absence of a
fluorescence signal in a sample of the culture medium, wherein
fluorescence signal intensity reflects the concentration of
fluorophore-labeled collagen fragments in the sample of the culture
medium.
Description
[0001] This application is a division of co-pending Ser. No.
10/840,653 filed May 7, 2004.
FIELD OF THE INVENTION
[0002] The invention relates to methods and tools for detecting
collagen degradation.
BACKGROUND OF THE INVENTION
[0003] Collagen degradation is involved in a variety of processes,
including bone resorption and tumor metastasis. In vitro assays of
the collagen-degrading activity of osteoclasts, for example, are
invaluable to the discovery of drugs for the treatment of
osteoporosis. "TRAP" (tartrate-resistant acid phosphatase) assays
detect acid phosphatase produced by mature osteoclasts via either
histochemical or immunohistochemical staining. Other existing
assays use synthetic calcium phosphate-based matrices as a
surrogate bone substrate to detect osteoclast activity, and culture
dishes coated with calcium phosphate upon which osteoclasts can be
cultured are commercially available.
[0004] Some assays use slices of either dentine or bovine cortical
bone as a surrogate bone substrate. Stearns, Clin Exp Metastasis.
1998 May; 16(4):332-9, discloses an assay in which osteoblasts
first are cultured with .sup.3H-hydroxyproline. The osteoblasts
produce .sup.3H-collagen, which is deposited together with other
matrix proteins on a culture plate surface. The osteoblasts are
removed, and other cells can then be tested for the ability to
degrade the deposited .sup.3H-collagen.
[0005] Tumor cells secrete collagenases which degrade extracellular
matrix components and aid metastasis. Garbisa et al., Cancer Lett.
9, 359-66, 1980; Liotta et al., Cancer Metastasis Reviews 1,
277-88, 1982. Cell-based assays for potential drugs that would
block this process would be useful in identifying anti-metastatic
agents.
[0006] There is a need in the art for sensitive and convenient
cell-based assays of collagen degradation that can be used for
rapid screening of potential therapeutic agents.
BRIEF SUMMARY OF THE INVENTION
[0007] One embodiment of the invention is a method of detecting
collagen degradation. Cells which can degrade collagen or can
differentiate into cells which can degrade collagen are cultured in
culture medium on fluorophore-labeled collagen covalently bound to
a culture surface of a cell culture vessel. The presence or absence
of a fluorescence signal is detected in a sample of the culture
medium. Fluorescence signal intensity reflects the concentration of
fluorophore-labeled collagen fragments in the sample of the culture
medium.
[0008] Another embodiment of the invention is a method of detecting
collagen degradation. Osteoclasts or osteoclast precursors are
cultured in culture medium on fluorophore-labeled collagen
covalently bound to a culture surface of a cell culture vessel. A
sample of the culture medium is transferred from the cell culture
vessel to an assay vessel. The presence or absence of a
fluorescence signal is detected in the assay vessel. Fluorescence
signal intensity reflects the concentration of fluorophore-labeled
collagen fragments in the sample of the culture medium.
[0009] Yet another embodiment of the invention is a cell culture
vessel comprising fluorophore-labeled collagen covalently bound to
a culture surface of the cell culture vessel.
[0010] Still another embodiment of the invention is a cell culture
vessel comprising europium chelate-labeled human collagen type I
covalently bound to a culture surface of the cell culture
vessel.
[0011] Other embodiments of the invention are kits for detecting
collagen degradation. The kits contain cell culture vessels and
instructions for carrying out methods of the invention.
[0012] The invention thus provides methods and tools for carrying
out rapid and sensitive detection of collagen degradation.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1. Graph showing fluorescence resulting from the
release over time of europium chelate-labeled degradation fragments
of collagen from europium chelate-labeled collagen covalently bound
to a maleic anhydride-derivatized polystyrene plate ("OsteoLyse.TM.
plate") on which osteoclast precursors were cultured.
[0014] FIG. 2. Graph showing the release over time of europium
chelate-labeled degradation fragments of collagen on days 7, 8, 9,
and 10 from an OsteoLyse.TM. plate on which osteoclast precursors
were cultured.
[0015] FIG. 3. Graph showing increase of signal-to-noise ratio over
time in an OsteoLyse.TM. assay with and without a change in
medium.
[0016] FIG. 4. Graphs showing effects of adding growth factors at
day 7 [macrophage colony stimulating factor (M-CSF) and soluble
receptor activator of NF-.kappa.B ligand (RANK ligand)] in an
OsteoLyse.TM. Assay. FIG. 4A plots results after one additional day
in culture; FIG. 4B plots results after two additional days in
culture; FIG. 4C plots results after three additional days in
culture.
[0017] FIG. 5. Graph showing effects of calcitonin on
osteoclast-mediated bone matrix degradation in vitro.
[0018] FIG. 6. Graph showing comparison of the TRAP Stain and an
assay of the invention. The upper line denotes TRAP data (day 8
multinucleated TRAP-positive cells/well) while the lower line
represents OsteoLyse.TM. assay data.
[0019] FIG. 7. Graph showing inhibition of in vitro bone matrix
resorption by alendronate.
[0020] FIG. 8. Graph showing alendronate-mediated inhibition of in
vitro bone matrix degradation by primary human osteoclasts as
measured by OsteoLyse.TM. and TRAP assays.
[0021] FIG. 9. Graph showing covalent binding of europium
chelate-labeled collagen to maleic anhydride-derivatized
polystyrene tissue culture plate over time.
[0022] FIG. 10. Graph showing electrostatic collagen adherence to a
Nunclon.TM. .DELTA. tissue culture plate.
[0023] FIG. 11. Graph showing apparent molecular masses of europium
chelate-labeled collagen degradation fragments.
[0024] FIG. 12. Graph showing fluorescence resulting from release
of europium chelate-labeled fragments by collagenase.
[0025] FIG. 13. Graph showing osteoclast-mediated degradation of
FITC-labeled collagen over time.
[0026] FIG. 14. Graph showing fluorescence readings for cell
culture medium samples from osteoclast precursors cultured with
soluble DQ FITC-labeled collagen for 9 days. OCTP, osteoclast
precursors; OC, osteoclasts.
[0027] FIG. 15. Graph showing time course of human collagen type I
degradation by human tumor cell lines.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention provides tools and methods for in
vitro measurement of matrix (e.g., bone or extracellular matrix)
collagen degradation in cell-based assays, including collagen
degradation associated with bone resorption and tumor metastasis.
Assays of the invention provide a high throughput platform for
rapid screening of large numbers of compounds that potentially
modulate, e.g. potentially activate or inhibit, for example, tumor
metastasis or osteoclast differentiation and function. In fact,
assays of the invention require much less time than currently
available assays for examining bone resorption. In addition, data
from such assays correlate very well with more traditional assays
of osteoclast function, such as the TRAP assay (Janckila et al., Am
J. Clin. Pathol. 70 (1):45-55, 1978), although assays of the
invention are much more readily quantifiable and have a greatly
increased throughput.
One- and Two-Step Assay Methods
[0029] Assays of the invention are based on the covalent attachment
of fluorophore-labeled collagen to a cell culture surface of a cell
culture vessel. Cells are seeded onto the covalently-bound collagen
and cultured in a culture medium appropriate for the particular
cell type used. Depending on the cell type, culture condition, or
presence of a test compound, the cells may or may not degrade the
covalently-bound collagen. If degradation of the covalently-bound
collagen occurs, fluorophore-labeled collagen fragments are
released into the culture medium. The intensity of the fluorescence
signal in a sample of the culture medium will reflect the
concentration of fluorophore-labeled collagen fragments in the
sample and, therefore, will reflect the amount of collagen
degradation. A fluorescence signal can be detected either in the
cell culture vessel itself (a "one-step assay") or after transfer
of a sample of the cell culture medium to an assay vessel (a
"two-step assay").
[0030] In some embodiments, osteoclasts or osteoclast precursors
(as defined below) are seeded onto fluorophore-labeled collagen,
preferably type I collagen, which is covalently bound to a culture
surface of a cell culture vessel. If osteoclast precursors are
used, the precursors are allowed to differentiate into
multinucleated osteoclasts before carrying out the assay.
Resorptive activity of the osteoclasts, as reflected by the release
of fluorophore-labeled collagen fragments, can be measured by
sampling the cell culture medium after an appropriate period of
cell culture. Such assays directly measure the release of matrix
metalloproteinases into the resorption lacuna of the osteoclast
(Delaisse et al., Microsc Res Tech. 61:504-13, 2003). Assays of the
invention also can be used, e.g., to screen for potential
modulators (potential inhibitors or potential activators) of
osteoclast function. For such screens, a test compound is added to
a culture of differentiated osteoclasts before carrying out the
assay. Assays of the invention also can be used to screen for
modulators of osteoclast differentiation; in this case, a test
compound is added to a culture of osteoclast precursors but is
removed before the assay is carried out.
[0031] In other embodiments, assays of the invention can be used to
monitor the function and/or differentiation of chondroclasts, which
degrade cartilage types II and X, or other collagen-degrading
cells, such as macrophages. Test compounds can be tested for their
effects on the function and/or differentiation of such cells.
[0032] In still other embodiments, assays of the invention can be
used to monitor collagen degrading activity of tumor cells (e.g.,
primary tumor cells, metastatic tumor cells, or cells of a
metastatic or non-metastatic tumor cell line). To screen for
compounds that may modulate this function, such as potential
inhibitors of tumor metastasis, tumor cells are seeded onto
fluorophore-labeled collagen, preferably type IV collagen, which is
covalently bound to a culture surface of a cell culture vessel.
Components for Use in One- and Two-Step Assays
[0033] Cell Culture Vessel
[0034] Any vessel appropriate for culturing cells can be used as
the cell culture vessel, provided the culture surface of the vessel
(i.e., that portion of the inner surface of the vessel onto which
the cells are seeded) is a material to which collagen can be
covalently attached. Such vessels include, for example,
commercially available tissue culture flasks or 1-, 4-, 6-, 8-,
12-, 24-, 96-, or 384-well plastic tissue culture plates or Petri
dishes. Cell culture vessels can be made from any material which is
appropriate for culturing cells and which can be derivatized to
bind collagen covalently. Such materials include, but are not
limited to, glass, polystyrene, polypropylene, polycarbonate,
copolymers (e.g., ethylene vinylacetate copolymers), polyester, and
the like.
[0035] Collagen
[0036] Any form of recombinant or naturally occurring collagen can
be used in assays of the invention. The collagen is preferably
vertebrate collagen, more preferably mammalian collagen, and even
more preferably human collagen. The collagen is sufficiently intact
and undenatured to permit its covalent binding to a culture surface
of a cell culture vessel and to permit its degradation by enzymes
released by cells cultured on the collagen. Preferably, the
collagen is as intact and undenatured as possible, so as to
increase specificity and sensitivity of assays of the
invention.
[0037] Depending upon the cultured cell type, any type of collagen
can be used (e.g., collagen types I, II, III, IV, V, VI, VII, VIII,
VIX, or X, etc.). When osteoclasts or osteoclast precursors are
used in an assay, type I collagen is preferred. Sources of type I
collagen include rat tail collagen, bovine dermis collagen, human
placental collagen, and kangaroo tail collagen. When tumor cells
are used in an assay, type IV collagen is preferred. Sources of
type IV collagen include human or other mammalian placental
collagen and Engelbreth-Holm-Swarm mouse sarcoma collagen.
[0038] The collagen preparation preferably is as pure as possible
so that detected fluorescence reflects true collagen degradation
and not degradation of impurities. Collagen preparations that are
at least about 90% pure (i.e., at least about 90% by weight of the
protein in the preparation is collagen) are preferred; even more
preferred are collagen preparations that are at least about 91, 92,
93, 94, 95, 96, 97, 98, or 99% pure. Preferably, the collagen is
about 100% pure. "About" as used herein means "plus or minus 5% or
less."
[0039] Fluorophore
[0040] Any fluorophore which can be covalently bound to collagen
and still retain detectability can be used in assays of the
invention. A large variety of such fluorophores are available
including, but are not limited to, Alexa Fluor 350, Alexa Fluor
488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor
647, Alexa Fluor 680, fluorescein isothiocyanate (FITC), Rhodamine
110, Rhodamine 123, Rhodamine 6G, Rhodamine Green, Rhodamine Red,
and Rhodamine B.
[0041] Other suitable fluorophores include quantum dots, i.e.,
semiconductor nanocrystals with size-dependent optical and
electronic properties. Quantum dots demonstrate quantum confinement
effects in their luminescent properties. When quantum dots are
illuminated with a primary energy source, a secondary emission of
energy occurs at a frequency that corresponds to the band gap of
the semiconductor material used in the quantum dot. The band gap
energy of a quantum dot varies with the diameter of the crystal.
See U.S. Pat. No. 6,326,144.
[0042] Highly luminescent semiconductor quantum dots (zinc
sulfide-capped cadmium selenide) have been covalently coupled to
biomolecules for use in ultrasensitive biological detection. U.S.
Pat. No. 6,656,695; Stupp et al., Science 277, 1242-48, 1977; Chan
et al., Science 281, 2016-68, 1998. Compared with conventional
fluorophores, quantum dot nanocrystals have a narrow, tunable,
symmetric emission spectrum and are photochemically stable. Bonadeo
et al., Science 282, 1473-76, 1998. See also Jaiswal et al., Nat.
Biotechnol. 21, 47-51, 2003; Watson et al., Biotechniques 34,
296-300, 2003; Wu et al., Nat. Biotechnol. 21, 41-46, 2003; Kaul et
al., Cell Res. 13, 503-07, 2003.
[0043] Lanthanide Chelates
[0044] Preferred fluorophores are lanthanide chelates (e.g,
.beta.-diketone chelates of cerium, praseodymium, neodymium,
promethium, samarium, europium, gadolinium, terbium, dysprosium,
holmium, erbium, thulium, or ytterbium), which can be used with
time-resolved fluorescence techniques. Lanthanide chelates have a
long fluorescence decay time and a very large Stokes' shift. These
properties permit measurement of fluorescence after subsidence of
the background fluorescence. See Soini and Kojola, Clin. Chem. 29,
65, 1983; Hemmila et al., Anal. Biochem. 137, 335 1984; Lovgren et
al., In: Collins & Hoh, eds., Alternative Immunoassays, Wiley,
Chichester, U.K., p. 203, 1985; Hemmila, Scand. J. Clin. Lab.
Invest. 48, 389, 1988; Mikola et al., Bioconjugate Chem. 6, 235,
1995; Peruski et al., J. Immunol. Methods 263, 35-41, 2002; U.S.
Pat. No. 4,374,120; and U.S. Pat. No. 6,037,185. Suitable
.beta.-diketones are, for example, 2-naphthoyltrifluoroacetone
(2-NTA), 1-naphthoyltrifluoroacetone (1-NTA),
p-methoxybenzoyltrifluoroacetone (MO-BTA),
p-fluorobenzoyltrifluoroacetone (F-BTA), benzoyltrifluoroacetone
(BTA), furoyltrifluoroacetone (FTA), naphthoylfuroylmethane (NFM),
dithenoylmethane (DTM), and dibenzoylmethane (DBM). A preferred
lanthanide chelate is
Eu.sup.3+--N.sup.1-(p-isothiocyanatobenzyl)diethylenetriamine-N.sup.1,N.s-
up.2,N.sup.3-tetraacetic acid (Perkin-Elmer). Lanthanide chelates
are particularly well-suited for use in two-step assays.
[0045] Methods of Covalently Attaching a Fluorophore to
Collagen
[0046] There are a variety of methods known in the art which are
useful for covalently attaching a fluorophore to collagen. For
example, the attachment can be direct via a functional group on the
collagen (e.g., amino, carboxyl and sulflhydryl groups) and a
reactive group on the fluorophore. Free amino groups in the
collagen can be reacted with fluorophores derivatized with
isothiocyanate, maleic anhydride, N-hydroxysuccinimide,
tetrafluorylphenyl and pentafluoryl esters. Free carboxyl groups in
the collagen can be reacted with carbodiimides such as
1-ethyl-3-[dimethylaminopropyl]carbodiimide hydrochloride to create
a reactive moiety that will react with an amine moiety on the
fluorophore. Collagen sulfhydryl groups can be attached to
fluorophores modified with maleimide and iodoacetyl groups,
although such linkages are more susceptible to reduction than
linkages involving free amino groups. The collagen can also be
linked indirectly via an intermediate linker or spacer group, using
chemical groups such as those listed above. Collagen can be
attached by any stable physical or chemical association to a
hydrophilic attachment group of a water-soluble quantum dot
directly or indirectly by any suitable means, such as those
described in U.S. Pat. Nos. 6,468,808 and 6,236,144.
[0047] Covalent Attachment of Fluorophore-Labeled Collagen to a
Cell Culture Vessel
[0048] Fluorophore-labeled collagen can be covalently attached to a
derivatized surface of a cell culture vessel using any method known
in the art. For example, the attachment can be direct via a
reactive group on the collagen (e.g., amino, carboxyl and
sulflhydryl groups) and a chemical entity on the plastic surface.
Free amino groups can be reacted with maleic anhydride,
N-hydroxysuccinimide, tetrafluorylphenyl and pentafluoryl esters,
free carboxyl groups can be reacted with carbodiimides such as
1-ethyl-3-[dimethylaminopropyl]carbodiimide hydrochloride to create
a reactive moiety that can be attached to an amino-modified plastic
surface, and collagen sulfhydryl groups can be attached to a
plastic surface modified with maleimide and iodoacetyl groups. The
collagen can also be linked indirectly via an intermediate linker
or spacer group, using chemical groups such as those listed
above.
[0049] A soluble fluorescein-labeled collagen is commercially
available from Molecular Probes, Inc. ("DQ collagen") and can be
used in one-step assays of the invention. The fluorescein-labeled
collagen is so heavily labeled that internal quenching of the
fluorophore occurs; thus, the fluorescein label can be detected
only when degradation separates some of the fluorescein moieties
sufficiently to avoid quenching. Other fluorophores can also be
used to heavily label collagen and cause internal quenching.
[0050] If DQ collagen (or other similar heavily labeled collagen)
is used, it is preferably not attached to a cell culture vessel
using free amino groups; such attachment vastly reduces the
sensitivity of two-step assays of the invention, probably because
the heavy fluorescein labeling renders the majority of DQ
collagen's free amino groups unavailable for binding to the cell
culture surface of a cell culture vessel. See Example 13. It is
preferred that DQ collagen be attached to a cell culture vessel by
other means (e.g., via stable thioether linkages or via carboxyl
groups).
[0051] Cells and Culture Conditions
[0052] Assays of the invention can be used with any type of cell
which either can degrade collagen or that can differentiate into a
cell which can degrade collagen. For example, cells such as
osteoclasts, osteoclast precursors, or tumor cells can be used.
"Osteoclasts" as used herein includes differentiated osteoclasts as
well as osteoclast-like cell lines. "Osteoclast precursors" as used
herein includes pre-osteoclasts, osteoclast progenitors, and
osteoclast precursor cell lines. "Tumor cells" as used herein
includes primary tumor cells, metastatic tumor cells, or tumor cell
lines (either metastatic or non-metastatic). Purified cell
populations (i.e., populations in which all or a majority of the
cells are the desired type of cell) need not be used.
[0053] Osteoclasts or osteoclast precursors can be, for example,
avian or mammalian. Avian osteoclasts or osteoclast precursors are
disclosed, for example, in Collin-Osdoby et al., Methods Mol Med.
2003; 80:65-88; Collin-Osdoby et al., J Bone Miner Res. 2002
October; 17(10):1859-71. Suitable mammalian osteoclasts or
osteoclast precursors include, but are not limited to, those of
rodents, such as rat (Bushinsky, J Bone Miner Res. 1994 November;
9(11):1839-44) or mouse (Takahashi et al., Methods Mol Med. 2003;
80:129-44); rabbit (Coxon et al., Methods Mol Med. 2003; 80:89-99;
Shimizu et al., Bone Miner. 1989 July; 6(3):261-75); non-human
primates (Povolny & Lee, Exp Hematol. 1993 April; 21(4):532-7;
Takahashi et al., J Bone Miner Res. 1987 August; 2(4):311-7); and
humans (Sabokbar & Athanasou, Methods Mol Med. 2003; 80:101-11;
Benito et al., Cytometry. 2002 Oct. 15; 50(5):261-6).
[0054] A preferred source of human osteoclast precursors are those
available from Cambrex Corporation ("Poietics.TM. Osteoclast
Precursors," product no. 2T-110). See Example 2 for a description
of preferred culture conditions for these cells. Culture medium and
additives for culturing osteoclast precursor cells can also be
obtained from Cambrex Corporation (e.g, PT-8001; PT-8201;
PT-9501).
[0055] Other suitable cells include, but are not limited to, MOCP-5
osteoclast precursors (Chen & Li, J Bone Miner Res. 1998 July;
13(7):1112-23); osteoclast-inductive and osteoclastogenic cell
lines from the H-2K.sup.btsA58 transgenic mouse (Chambers et al.,
Proc. Natl. Acad. Sci. USA 90, 5578-82, 1993); and the immortalized
osteoclast (OCL) precursor cell line derived from mice doubly
transgenic for bcl-XL and large T antigen (Hentunen et al.,
Endocrinology 140, 2954-61, 1999). Pre-osteoclast and
osteoclast-like cell lines also can be used. See, for example,
Fiorelli et al., Proc. Natl. Acad. Sci. USA 92, 2672-76, 1995;
Miyamoto & Suda, Keio J. Med. 52, 1-7, 2003; Arai et al., J.
Exp. Med. 190, 1741-54, 1999; Espinosa et al., J. Cell Sci. 115,
3837-48, 2002; Mbalaviele et al., J. Cell Biol. 141, 1467-76, 1998;
Thomas et al., Endocrinol. 140, 4451-58, 1999; Quinn et al.,
Endocrinol. 139, 4424-27, 1998; Itoh et al., Endocrinol. 142,
3656-62, 2001; and Ragab et al., Am. J. Physiol. Cell Physiol. 283,
C679-C687, 2002. See also MacDonald et al., J Bone Miner Res. 1986
April; 1(2):227-33.
[0056] Primary tumor cells typically are mammalian cells, such as
rat, mouse, guinea pig, rabbit, non-human primate, or human cells.
They can be obtained, for example, from spontaneously arising
tumors in non-human mammals or in humans (e.g., from surgical or
biopsy samples) or from tumors seeded in an experimental non-human
mammalian model. Such tumors include, but are not limited to,
melanomas, non-small cell lung tumors, small cell lung tumors,
renal tumors, colorectal tumors, breast tumors, pancreatic tumors,
gastric tumors, bladder tumors, ovarian tumors, uterine tumors,
lymphoma cells, leukemia cells, and prostate tumors. Metastatic
tumor cells can be obtained, for example, from metastases of
primary tumors.
[0057] Alternatively, mammalian, preferably human, metastatic or
non-metastatic cell lines can be used. Metastatic cell lines
include, e.g., melanoma cell lines A2058, MV3, BLM, SK-MEL-19, Hs
688(A).T, WM-115, and 1F6m; breast cancer cell lines MDA 435, MDA
231, and Hs578T; rhabdomyosarcoma cell line SMF-Ai; prostate tumor
cell line DU145/M, PC-3-M; colorectal adenocarcinoma cell line
SW480; gastric adenocarcinoma cell line RF-1; lung squamous cell
carcinoma cell line KLN 205; and osteosarcoma cell line KHOS.
Non-metastatic cell lines include rhabdomyosarcoma cell line
SMF-Deposit Account No. 19-0733, breast cancer cell lines NM-2C5,
MDA-MB-23 1, or MCF-7, melanoma cell line 530, and prostate cancer
cell line LNCaP.
[0058] The selection of an appropriate culture medium for a given
cell type, as well as other culture conditions such as temperature
and percent CO.sub.2, is well within the skill of those in the art
(see, for example, ANIMAL CELL CULTURE, R. I. Freshney, ed.,
1986).
[0059] Culture times can be varied according to the type of cells
cultured. For example, if osteoclast precursors are used, they
typically are cultured at least 3-4 days before an assay is carried
out so that the cells can differentiate into functional osteoclasts
(see FIG. 1). If osteoclasts or osteoclast-like cell lines are
used, assays can be carried out almost immediately after
seeding.
[0060] Assay Vessel for Use in Two-Step Assays of the Invention
[0061] The assay vessel can be a plastic or glass plate and can be
clear, white, or preferably black. More preferably, an assay vessel
is black-walled with a clear bottom surface. The assay vessel can
have multiple wells (e.g., 96 or 384 wells) to facilitate high
throughput assays.
Detection of Fluorescence Signal
[0062] Detection of the fluorescence signal in either a one-step or
a two-step assay of the invention can be either qualitative or
quantitative. Fluorophore-labeled collagen fragments can be
detected by methods known in the art for detecting the particular
fluorophore used. For example, if the collagen is labeled with
fluorescein, its fluorescence can be detected by use of a
fluorimeter with excitation and emission wavelengths of 485 and 535
nm, respectively. Other fluorophores will have their own unique
excitation and emission maxima, and these are known in the art.
Some types of fluorophores, such as quantum dots, can be imaged by
use of image analysis systems that detect fluorescence.
[0063] Detection of Time-Resolved Fluorescence of Lanthanide
Chelates
[0064] Lanthanide chelates are typically used in two-step assays of
the invention. If desired, fluorescence associated with a
lanthanide chelate can be measured without dissociating the
lanthanide ion from the chelate (see U.S. Pat. No. 4,808,541).
Preferably, however, a low pH enhancement solution is used to
dissociate the lanthanide label from the labeled collagen; free
lanthanide (e.g., Eu.sup.3+, Sm.sup.3+, Tb.sup.3+, Dy.sup.3+) then
forms a stable, fluorescent chelate with components of the
enhancement solution within a protective micelle.
[0065] The enhancement solution can contain a suitable detergent,
such as Triton X-100, and a .beta.-diketone to amplify the
fluorescence after the separation. To further improve the
fluorescence, especially in aqueous solutions, a synergistic
compound such as a Lewis base can be added. Suitable synergistic
compounds include N-heterocyclic compounds (e.g.,
o-phenanthroline), as well as phosphines and phosphine oxides (e.g.
trioctylphosphineoxide) (see U.S. Pat. No. 4,565,790). The EG&G
Wallac DELFIA.RTM. method is particularly useful for measuring
fluorescence associated with a lanthanide chelate. See, e.g., U.S.
Pat. Nos. 5,998,146; 5,859,215; 5,637,509; and 5,457,186.
[0066] One suitable enhancement solution comprises 15 .mu.M
.beta.-naphthoyltrifluoroacetone, 50 .mu.M trioctylphosphine oxide,
0.1% Triton X-100 in phthalatacetate buffer, pH 3.2 (see U.S. Pat.
No. 4,808,541, incorporated herein by reference). DELFIA.RTM.
Enhancement Solution (EG&G Wallac) is preferred, but any acidic
solution sufficient to cause the lanthanide ion to dissociate from
the chelating agent and complex with a second chelating agent, such
as a .beta.-diketone, to form a fluorescent chelate can be
used.
[0067] Fluorescence preferably is detected by a method using time
delay, which reduces or eliminates the contribution of non-specific
background fluorescence to the detected signal. A preferred method
of detection is time-resolved fluorometry, which is especially well
suited for use with fluorescent lanthanide chelates (Soini et al.,
Clin. Chem. 25, 353-61, 1979; U.S. Pat. No. 4,374,120; see also
Example 1, below). Devices suitable for carrying out time-resolved
fluorimetry include a Victor spectrofluorimeter (e.g., Victor or
Victor.sup.2.TM. from EG&G Wallac), SPECTRAmax GEMINI
(Molecular Devices), the LJL-Analyst, and FLUOstar from BMG Lab
Technologies. See also U.S. Pat. No. 6,042,785.
[0068] Test Compounds
[0069] Test compounds to be screened for an ability to modulate
collagen degradation, especially for an ability to inhibit collagen
degradation, can be any pharmacologic agents already known in the
art or can be compounds previously unknown to have any
pharmacological activity. Test substances can be naturally
occurring or synthesized in the laboratory. They can be isolated
from microorganisms, animals, or plants, or can be produced
recombinantly or synthesized by chemical methods known in the
art.
[0070] Test compounds also can be obtained from compound libraries.
Methods of generating combinatorial libraries of test compounds are
known in the art and include, but are not limited to, formation of
"biological libraries," spatially addressable parallel solid phase
or solution phase libraries, synthetic library methods requiring
deconvolution, the "one-bead one-compound" library method, and
synthetic library methods using affinity chromatography selection.
See, e.g., DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909,
1993; Erb et al., Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994;
Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al.,
Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl.
33, 2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33,
2061, 1994; Gallop et al., J. Med. Chem. 37, 1233, 1994; and Lam,
Anticancer Drug Des. 12, 145, 1997.
[0071] Test compounds can be presented to cells, for example, in
solution (Houghten, Biotechniques 13, 412-21, 1992), on beads (Lam,
Nature 354, 82-84, 1991), in plasmids (Cull et al., Proc. Natl.
Acad. Sci. U.S.A. 89, 1865-69, 1992), or in phage (Scott &
Smith, Science 249, 386-90, 1990; Devlin, Science 249, 404-06,
1990; Cwirla et al., Proc. Natl. Acad. Sci. 97, 6378-82, 1990;
Felici, J. Mol. Biol. 222, 301-10, 1991; and U.S. Pat. No.
5,223,409).
Cell Culture Vessels
[0072] The invention also provides cell culture vessels for use in
performing assays of the invention. Cell culture vessels of the
invention are those vessels described above to which
fluorophore-labeled collagen is covalently attached. The physical
form of the cell culture vessel is relatively unimportant as long
as the vessel permits covalent attachment of fluorophore-labeled
collagen and satisfactory culture of collagen-degrading cells.
[0073] Preferred cell culture vessels contain covalently attached
europium chelate-labeled collagen, e.g., collagen labeled with
Eu.sup.3+--N.sup.1-(p-isothiocyanatobenzyl)
diethylenetriamine-N.sup.1,N.sup.2,N.sup.3-tetraacetic acid. More
preferred cell culture vessels are maleic anhydride-derivatized
polystyrene tissue culture plates to which europium chelate-labeled
human type I collagen or europium chelate-labeled type IV collagen
is covalently bound. Other preferred cell culture vessels contain
covalently bound fluorophore-labeled collagen that is internally
quenched and does not fluoresce (such as DQ collagen).
Kits
[0074] The invention also provides kits for carrying out assays of
the invention. The kits can comprise, for example, one or more cell
culture vessels of the invention and instructions for carrying out
one or more embodiments of the assays disclosed herein. The kits
can contain other components, such as assay vessels, reagents for
detecting fluorescence (e.g., an enhancement solution), buffers,
osteoclasts or osteoclast precursors, tumor cells, culture medium
growth factors, and fluorescence standards (e.g., a europium
standard).
[0075] In a preferred embodiment, a kit comprises an enhancement
solution, a maleic anhydride-derivatized polystyrene tissue culture
plate to which europium chelate-labeled human type I collagen is
covalently bound, and instructions for carrying out an
OsteoLyse.TM. assay. In another preferred embodiment, a kit
comprises an enhancement solution, a maleic anhydride-derivatized
polystyrene tissue culture plate to which europium chelate-labeled
type IV collagen is covalently bound, and instructions for carrying
out an assay to detect collagen degradation activity by tumor
cells. The preferred europium chelate for use in such kits is
Eu.sup.3+--N.sup.1-(p-isothiocyanatobenzyl)diethylenetriamine-N.sup.1,N.s-
up.2,N.sup.3-tetraacetic acid. Other kits comprise a cell culture
vessel to which a fluorophore-labeled collagen is covalently
attached and instructions for carrying out a one-step assay, a
two-step assay, or both types of assay. In some kits, fluorescence
of the fluorophore-labeled collagen is internally quenched.
[0076] All patents, patent applications, and references cited in
this disclosure are expressly incorporated herein by reference in
their entireties. The above disclosure generally describes the
present invention. A more complete understanding can be obtained by
reference to the following specific examples, which are provided
for purposes of illustration only and are not intended to limit the
scope of the invention.
EXAMPLE 1
OsteoLyse.TM. Assay Protocol
[0077] This example describes steps in one embodiment of a two-step
assay of the invention, termed an "OsteoLyse.TM. assay."
[0078] Remove a 96-well cell culture plate to which europium
chelate-labeled collagen (e.g., collagen labeled with
Eu.sup.3+--N.sup.1-(p-isothiocyanatobenzyl)diethylenetriamine-N.sup.1,N.s-
up.2,N.sup.3-tetraacetic acid) is covalently bound from 4.degree.
C. storage and let it warm to room temperature. Seed mature
osteoclasts or osteoclast precursors (human or non-human) onto the
cell culture plate in medium containing M-CSF and soluble RANK
ligand. If using Cambrex's primary human osteoclast precursors
(Poietics.TM. Human Osteoclast Precursors), seed the cells at a
density of 10,000 cells/well in osteoclast precursor
differentiation medium (Cambrex product # PT-8001). See Example 2
for a detailed protocol for the culture of primary human osteoclast
precursors (Cambrex product # 2T-110). Precursors cultured in the
absence of soluble RANK ligand can serve as "undifferentiated"
controls.
[0079] Culture the cells for 6 days and then renew the cell culture
medium. Fresh medium preferably contains the same concentrations of
M-CSF and soluble RANK ligand as in the original day 0 medium.
Unused control and differentiation media from day 0 can be frozen
(on day 0) and used for the day 6 medium changes.
[0080] At least two different types of protocols can be used. If
the assay is used to measure the effect of a test compound on
differentiation of the osteoclast precursor, the test compound
typically is added at day 0 and removed at day 6. However, if the
assay is used to measure the effect of a test compound on mature
osteoclast function (i.e., bone matrix collagen degradation), the
test compound typically is added at day 6 with the new medium
addition.
[0081] The cell culture medium can be sampled at any time after the
medium change. Because a very small volume (5 to 10 .mu.l) of
medium is sampled, it is very easy to do time-course studies by
repeatedly sampling the medium on sequential days. Medium volumes
greater than 10 .mu.l are unnecessary and, because an excess of
fluorophore releasing reagent with respect to sample is desired,
actually may lead to inefficient counting of the fluorophore as the
ratio of fluorophore releasing reagent to sample decreases.
[0082] Prior to sampling the cell culture medium, remove the
fluorophore releasing reagent (DELFIA.RTM. Enhancement Solution,
available as "Fluorophore Releasing Reagent" from Cambrex Corp.)
from 4.degree. C. storage and let it warm to room temperature--do
not warm this reagent in a water bath. Place 200 .mu.l of the
fluorophore releasing reagent in each well of a black/black-wall
96-well assay vessel; black-walled low background plates are
recommended.
[0083] Transfer 10 .mu.l of cell culture medium to the wells of the
assay vessel containing fluorophore releasing reagent using a
separate pipette tip for each new cell culture medium. Briefly mix
the samples in the assay vessel.
[0084] Determine the fluorescence of each well of the assay vessel
in a time-resolved fluorescence fluorimeter (e.g., a Wallac Victor,
with excitation at 340 nm and emission at 615 nm) over a 400
.mu.second time period after an initial delay of 400
.mu.seconds.
[0085] If the amount of collagen degraded as a percentage of the
total available collagen is to be calculated, determine the total
amount of intact collagen per well by placing 200 .mu.l of
fluorophore releasing reagent in each of three unused wells of the
culture plate. Mix the contents of the wells and then transfer 1
.mu.l per well to corresponding wells in an assay vessel containing
200 .mu.l of fluorophore releasing reagent per well as described in
paragraph [64], above. Determine the fluorescence of each well of
the assay vessel in a time-resolved fluorescence fluorimeter and
multiply the result by 200 to calculate the total amount of intact
collagen per well.
EXAMPLE 2
Culture of Human Osteoclast Precursors
[0086] This example provides detailed instructions for culturing
Poietics.TM. Human Osteoclast Precursors for either one- or
two-step assays of the invention.
[0087] Preparation of Media
[0088] Use pre-warmed (37.degree. C.) supplemented medium for
culturing osteoclast precursors. Decontaminate the external
surfaces of a 100 ml bottle of Osteoclast Precursor Basal Medium
(Cambrex product no. PT-8201) with 70% v/v ethanol or isopropanol.
Make up Osteoclast Precursor Growth Medium by adding fetal bovine
serum (FBS), L-glutamine, penicillin and streptomycin "SingleQuots"
to the bottle of Osteoclast Precursor Basal Medium; the final
concentrations of the supplements will be 10%, 2 mM, 100 units/ml
and 100 .mu.g/ml respectively.
[0089] Thawing of Cells/Initiation of Culture Process
[0090] Warm 100 ml of Osteoclast Precursor Growth Medium in a
37.degree. C. water bath. Quickly but completely thaw the vial of
frozen cells in a 37.degree. C. water bath. Wipe the outside of the
vial with 70% ethanol. Aseptically transfer the cell suspension to
a 50 ml conical tube. Rinse the cryovial with 1 ml of Osteoclast
Precursor Growth Medium. Add the rinse dropwise to the cells while
gently swirling the tube (.apprxeq.1 minute).
[0091] Slowly add additional medium drop-wise to the cells until
the total volume is 5 ml, while gently swirling after each addition
of several drops of medium (.about.3 minutes). Slowly bring the
volume up to 40 ml by adding 1 to 2 ml volumes of medium drop wise,
while gently swirling after each addition of medium (.about.10
minutes).
[0092] Centrifuge the cell suspension at 200.times.g at room
temperature for 15 minutes. Carefully remove by pipette and save
most of the wash, leaving approximately 3 ml behind so the cell
pellet is not disturbed. Gently resuspend the cell pellet in the
remaining medium and transfer to a 15 ml conical tube.
[0093] Rinse the 50 ml conical tube with 2 ml of Osteoclast
Precursor Growth Medium and add drop-wise to the cells in the 15 ml
conical tube. Slowly bring the volume up to 10 ml by adding 1 to 2
ml volumes of Osteoclast Precursor Growth Medium drop-wise while
gently swirling after each addition of medium.
[0094] Centrifuge the cell suspension at 200.times.g at room
temperature for 15 minutes. Carefully remove by pipette all but 1
ml of the wash. Gently resuspend the cell pellet in the remaining
medium and count (e.g, using a hemocytometer). When washing the
cells, do not attempt to remove too much of the wash. Leave a
minimum of 1 ml of wash at the bottom of the tube. If the final
cell count is low, some of the pellet may have been removed with
the wash.
[0095] Dilute 20 .mu.l of the cell suspension in 20 .mu.l of 0.4%
Trypan Blue and do a cell count and determine % viability. Recovery
should be greater than 90%. If the cell count is lower than
expected, centrifuge the previously saved wash at a higher speed,
count, and combine if necessary.
[0096] Maintenance and Osteoclast Precursor Differentiation
Procedure
[0097] Primary human osteoclast precursors cannot be "passaged."
They can be differentiated, but in the absence of specific
differentiation signals, the cells will senesce. As a negative
control, some cells can be cultured in the absence of soluble RANK
ligand. While the precursors will expand in number, no functional
differentiated osteoclasts will develop in controls without soluble
RANK ligand.
[0098] If the precursors are not to be treated with test
samples
[0099] To prepare Osteoclast Differentiation Medium (Cambrex
product no. PT-8201), add the entire contents of the M-CSF
SingleQuot to 30 ml of Osteoclast Precursor Growth medium--the
final concentration will be 33 ng/ml. The vial of M-CSF may have to
be centrifuged at very low speed to recover the entire content of
the vial. Remove 1 ml of the M-CSF supplemented medium for the
culture of undifferentiated control cells.
[0100] Add 1.0 ml of the medium containing M-CSF to a vial of
lyophilized soluble RANK ligand. Cap the vial, mix and remove the
contents and add the RANK ligand SingleQuot to the remaining 29 ml
of the M-CSF supplemented medium. The final concentration of
soluble RANK ligand will be 66 ng/ml. Add osteoclast precursors to
the control and Differentiation Medium at a concentration of 50,000
cells/ml. Seed 10,000 osteoclast precursors/well at 0.2
ml/well.
[0101] If the precursors are to be treated with test samples
[0102] To prepare Osteoclast Differentiation Medium, add the entire
contents of the M-CSF SingleQuot to 15 ml of Osteoclast Precursor
Growth medium--the final concentration will be 33 ng/ml upon
addition of 0.1 ml of test sample. The vial of M-CSF may have to be
centrifuged at very low speed to recover the entire content of the
vial. Remove 0.5 ml of M-CSF supplemented medium for the culture of
undifferentiated control cells.
[0103] Add 1.0 ml of the medium containing M-CSF to the vial of
lyophilized soluble RANK ligand. Cap the vial, mix and remove the
contents and add the RANK ligand SingleQuot to the remaining 14.5
ml of the M-CSF supplemented medium. The final concentration of
soluble RANK ligand will be 66 ng/ml upon addition of 0.1 ml of
test sample.
[0104] Add osteoclast precursors to the control and Differentiation
Medium at a concentration of 100,000 cells/ml. Seed 10,000
Osteoclast Precursors/well at 0.1 ml/well.
[0105] Set up a 24-well dilution plate with appropriate volumes of
Osteoclast Precursor Growth Medium/well and make serial dilutions
of the test sample(s) to be assayed. Add 0.1 ml of each different
concentration of test sample to the wells of osteoclast precursors.
Each assay should be done in triplicate.
[0106] Control wells can be set up which contain 1) no added test
sample and 2) solvent only if the test samples were dissolved in
solvents such as DMSO, ethanol, etc.
[0107] Cell Culture
[0108] Incubate the cells at 37.degree. C. in a humidified
atmosphere of 5% CO.sub.2.
[0109] Day 7 osteoclasts can be identified by phase microscopy as
unusually large multinucleate cells. The majority of each well's
bottom surface should be covered by such cells. The culture can be
continued for an additional week, with or without feeding, during
which time the osteoclasts will continue to increase in size.
[0110] To document osteoclast differentiation, cultures can be
stained for the .alpha..sub.v.beta..sub.3 integrin complex or for
tartrate-resistant acid phosphatase (TRAP).
EXAMPLE 3
Release of Fluorescent Collagen Peptides Over Time in an
OsteoLyse.TM. Assay
[0111] Primary human osteoclast precursors were seeded onto
europium chelate-labeled collagen
(Eu.sup.3+--N.sup.1-(p-isothiocyanatobenzyl)diethylenetriamine-N.sup.1N.s-
up.2,N.sup.3-tetraacetic acid; Perkin-Elmer) covalently bound to a
maleic anhydride-derivatized polystyrene plate (Pierce
Reacti-Bind.TM.) at 10,000 cells/well and cultured in medium
containing M-CSF and soluble RANK ligand as described in Example 2.
Samples of culture medium (10 .mu.l) were removed every 24 hours
and counted in 200 .mu.l Fluorophore Releasing Reagent (DELFIA.RTM.
Enhancement Solution) in a time-resolved fluorescence-capable plate
reader.
[0112] The release of fluorescent collagen peptides by
differentiating primary human osteoclasts occurs between the third
and fourth days of culture. See FIG. 1.
[0113] The fluorescence of the medium samples diluted in the wells
with Fluorophore Releasing Reagent is directly proportional to the
resorptive activity of the mature osteoclast. The fluorescent
read-out of the OsteoLyse.TM. assay is proportional to cell number
and the degree of osteoclast differentiation. See FIG. 2 for
documentation that the accumulation of collagen fragments is
directly related to duration of cell culture.
[0114] The release of collagen degradation fragments was
substantially linear with time and the signal-to-background ratio,
which also increased with time, was as high as 38 after 10 days of
culture. The coefficient of variation of the OsteoLyse.TM. assay
was <20% and the Z' value ranged from 0.5 to 0.7.
EXAMPLE 4
Release of Fluorescent Collagen Peptides After a Change of Medium
in an OsteoLyse.TM. Assay
[0115] Primary human osteoclast precursors were seeded onto
europium chelate-labeled collagen covalently bound to a maleic
anhydride-derivatized polystyrene plate (Pierce Reacti-Bind.TM.) at
10,000 cells/well and cultured in medium containing soluble RANK
ligand. The medium was changed after day 6.
[0116] Samples of culture medium (10 .mu.l) were removed and
counted in 200 .mu.l Fluorophore Releasing Reagent (DELFIA.RTM.
Enhancement Solution) in a time-resolved fluorescence-capable
plate-reader, after 7, 8, 9 and 10 days of total cell culture
time.
[0117] The signal-to-noise (S:N) ratios of the OsteoLyse.TM. assay
increases with time of incubation and after a day 6 change of
medium. These results are shown in FIG. 3. Left bar of each pair of
bars=no change of medium; right bar of each pair of bars=with
medium change.
EXAMPLE 5
Dependence of Osteoclast Collagen-Degrading Activity on M-CSF and
Soluble RANK Ligand in an OsteoLyse.TM. Assay
[0118] Primary human osteoclast precursors were seeded onto
europium chelate-labeled collagen covalently bound to a maleic
anhydride-derivatized polystyrene plate (Pierce Reacti-Bind.TM.) at
10,000 cells/well and cultured in differentiation medium. After 7
days of culture, the medium with and without various combinations
of M-CSF and soluble RANK ligand, was renewed. Samples of culture
medium (10 .mu.l) were removed and counted after an additional 1
(FIG. 4A), 2 (FIG. 4B) and 3 (FIG. 4C) days. "2 GF"=M-CSF (33
ng/ml) and RANK ligand (66 ng/ml); "0 GF"=neither M-CSF nor RANK
ligand.
[0119] This example demonstrates that the collagen-degrading
activity of differentiated (day 6) primary human osteoclasts is
dependent upon the presence of both M-CSF and soluble RANK
ligand.
EXAMPLE 6
Inhibition of Bone Matrix Resorption by Calcitonin in an
OsteoLyse.TM. Assay
[0120] Primary human osteoclast precursors were seeded onto
europium chelate-labeled collagen covalently bound to a maleic
anhydride-derivatized polystyrene plate (Pierce Reacti-Bind.TM.) at
10,000 cells/well and cultured in differentiation medium containing
(1) no calcitonin, (2) calcitonin added only at day 5, or (3)
calcitonin added on both days 0 and 5. Ten .mu.l samples of culture
media were counted after a total of 6 days. Measurement of the in
vitro inhibition of bone resorption by alendronate was also assayed
similarly in an OsteoLyse.TM. assay and gave an IC.sub.50 value of
approximately 2 .mu.M.
[0121] The results are shown in FIG. 5. Treatment of human
osteoclasts with 1 nM calcitonin for 24 hours (day 5 of culture)
inhibited bone matrix degradation by 88%. Treatment of the cells
with calcitonin after prior exposure to calcitonin on day 0 had
little effect on the resorptive activity of the osteoclasts.
Calcitonin added at day 0 effectively resulted in the osteoclasts
becoming refractory to calcitonin added on day 5.
EXAMPLE 7
Comparison of TRAP and OsteoLyse.TM. Assays
[0122] Primary human osteoclast precursors were seeded onto
europium chelate-labeled collagen covalently bound to a maleic
anhydride-derivatized polystyrene plate at 10,000 cells/well and
cultured in medium containing soluble RANK ligand with and without
interferon .gamma.. After 9 days, cell culture media were assayed
for fluorescent collagen peptides as described in Example 1. Cells
in the plate were subsequently stained for TRAP (Sigma #
386-A).
[0123] The results are shown in FIG. 6. Data are expressed as
percent inhibition relative to controls not treated with interferon
.gamma.. Upper curve, results of TRAP assay. Lower curve, results
of OsteoLyse.TM. assay. Data from these two assays gave nearly
identical results with IC.sub.50 values of approximately 0.1
ng/ml.
EXAMPLE 8
Comparison of TRAP and OsteoLyse.TM. Assays in Determinations of
Alendronate-Mediated Inhibition of Osteoclast Function
[0124] Primary human osteoclast precursors were seeded onto
europium chelate-labeled collagen covalently bound to a maleic
anhydride-derivatized polystyrene plate at 10,000 cells/well and
cultured in medium containing M-CSF only or in medium containing
both M-CSF and soluble RANK ligand with and without alendronate (10
.mu.M).
[0125] The media were renewed after 7 days. After an additional 24
hours, the cell culture media were sampled as described in Example
1. The results are shown in FIG. 7.
[0126] In another experiment, osteoclast precursors were cultured
in differentiation medium with and without different concentrations
of alendronate. After 7 days the media were renewed and, after an
additional 3 days, the cell culture media were sampled as described
in Example 1. Cells in the plate were subsequently stained for TRAP
(Sigma # 386-A).
[0127] The results are shown in FIG. 8. Alendronate did not inhibit
the differentiation of the osteoclast precursors as measured by the
presence of multinucleated TRAP-positive osteoclasts.
EXAMPLE 9
Covalent Binding of Europium Chelate-Labeled Collagen to Maleic
Anhydride-Derivatized Polystyrene Tissue Culture Plate Over
Time
[0128] Europium chelate-labeled human type I collagen was placed in
the wells of a maleic anhydride-derivatized polystyrene tissue
culture plate (50 .mu.l/well) and incubated at 37.degree. C. At
various times, wells were aspirated and washed with detergent and 1
M sodium chloride to remove unbound collagen. Fluorophore Releasing
Reagent (100 .mu.l/well) was placed in each well and 1 .mu.l/well
was then withdrawn and diluted in 200 .mu.l Fluorophore Releasing
Reagent in a 96-well assay vessel. The fluorescence of each well
was then determined. The results are shown in FIG. 9.
EXAMPLE 10
Release Of Non-Covalently Bound Europium Chelate-Labeled Collagen
Over Time
[0129] Europium chelate-labeled human type I collagen (at 55
.mu.g/ml) was placed in the wells of a non-derivatized 96-well
tissue culture plate (50 .mu.l/well). The plate was then incubated
at 37.degree. C. for 2.5 hours. Excess collagen was removed and the
wells were air-dried overnight. Each well was then rinsed with cell
culture medium (200 .mu.l/well) five times, filled with 200 .mu.l
medium and incubated at 37.degree. C. for 24 hours. The medium was
then sampled (5 .mu.l) and each well rinsed 4 times with 200 .mu.l
medium. Each rinse was saved and sampled (5 .mu.l).
[0130] The wells were then filled with medium (200 .mu.l) and
incubated for an additional 96 and 120 hours, after which the
medium was sampled. The wells were then emptied and filled with 200
.mu.l Fluorophore Releasing Reagent (DELFIA.RTM. Enhancement
Solution), 5 .mu.l of which was then sampled. Each 5 .mu.l sample
was diluted in 200 .mu.l Fluorophore Releasing Reagent and
counted.
[0131] The data show that less than 5% of the total europium
chelate-labeled collagen remained attached to the plastic surface
of the culture plate after 5 days of incubation. The results are
shown in FIG. 10.
EXAMPLE 11
Demonstration of Apparent Molecular Masses of Europium
Chelate-Labeled Collagen Degradation Fragments
[0132] Osteoclast precursors were cultured on europium
chelate-labeled collagen covalently bound to a maleic
anhydride-derivatized polystyrene plate and differentiated as
described in Example 2. After 7 days of culture, the medium was
renewed and the culture continued. After 24 hours, culture media
were combined, mixed with 100 .mu.l of protein standards and loaded
onto a 16 mm.times.600 mm Sephacryl-300 HD gel filtration column.
The column was eluted with phosphate-buffered saline at 0.5
ml/minute. Three 150 ml fractions were collected, and 10 .mu.l
samples of each were counted in Fluorophore-Releasing Reagent
(DELFIA.RTM. Enhancement Solution). The elution profile of the
protein standards was determined by the absorbance of the fractions
at 405 nm.
[0133] The results show that europium chelate-labeled degradation
fragments of collagen have apparent molecular masses of less than
12,000 daltons. See FIG. 11.
EXAMPLE 12
Release Of Fluorophore-Labeled Collagen Fragments by
Collagenase
[0134] Collagenase (type I, Worthington) was made up in DMEM cell
culture medium and added to the wells of a maleic
anhydride-derivatized polystyrene plate to which europium-labeled
collagen (100 .mu.l/well at 1 unit/ml) was covalently bound and
incubated at 37.degree. C. After 10, 20 and 30 minutes, 5 .mu.l of
the enzyme solution was added to 200 .mu.l of Fluorophore-Releasing
Reagent (DELFIA.RTM. Enhancement Solution) and counted.
[0135] The results show that collagenase degradation of the
covalently-bound collagen substrate released fluorophore-labeled
fragments into the cell culture medium. See FIG. 12.
EXAMPLE 13
Osteoclast-Mediated Degradation of FITC-Labeled Collagen
[0136] FITC-labeled bovine type I collagen (DQ collagen, Molecular
Probes, Inc.) was covalently bound to the surface of a maleic
anhydride-derivatized 96-well tissue culture plate. Osteoclast
precursors were cultured, with and without differentiation, in the
plate as described in Example 2. Interferon .gamma. (1 ng/ml) was
added to some wells. At days 5, 7 and 9 of culture, 10 .mu.l
samples of each well were collected and added to 200 .mu.l
phosphate-buffered saline and counted in a fluorimeter (485/535
nm).
[0137] The results are shown in FIG. 13. There was a statistical
difference between the undifferentiated and differentiated cells.
Interferon .gamma. completely inhibited osteoclast-mediated
collagen degradation. However, the absolute number of RFU was very
low and the S:N ratio was close to 1. A likely explanation for this
unsuitability is that the heavy fluorescein labeling renders the DQ
collagen's free amino groups unavailable for binding to the cell
culture surface of a cell culture vessel.
EXAMPLE 14
Unsuitability of Soluble DQ Collagen in a Cell-Based Collagen
Degradation Assay
[0138] Osteoclast precursors were cultured with and without
differentiation in a plastic 96-well tissue culture plate as
described in Example 2. At day 0, soluble DQ FITC-labeled collagen
(Molecular Probes, Inc.) was added to the cultures (1
.mu.g/well).
[0139] After 9 days of culture, samples of the media (10 .mu.l)
were added to 200 .mu.l phosphate-buffered saline and counted in a
fluorimeter (485/535 nm).
[0140] The results are shown in FIG. 14. There was no statistically
significant difference in the relative fluorescence units (RFU)
between differentiated and undifferentiated cells. These results
demonstrate that soluble DQ collagen is not suitable for use in
cell-based collagen degradation assays of the invention. A likely
explanation for this unsuitability is that fluorescently labeled
collagen substrate must be attached to the cell culture surface of
the cell culture vessel. A mature osteoclast forms a "resorption
lacuna" or "bay" at the basal side of the cell, which is separated
from the cell culture medium and into which proteolytic enzymes are
secreted. Collagen in the supernatant would not be available to
these enzymes.
EXAMPLE 15
Osteoclast-Mediated Degradation of DQ Collagen in a One-Step
Assay
[0141] Primary human osteoclast precursors (Cambrex product #
2T-110) are seeded onto DQ collagen covalently bound via stable
thioester linkages to a maleimide-derivatized polystyrene plate
(Pierce Reacti-Bind.TM.) at 10,000 cells per well and cultured in
medium containing M-CSF and soluble RANK ligand, as described in
Example 2.
[0142] After 7 days of culture, the tissue culture plate is read in
a fluorimeter (485/535 nm). The fluorescence read-out is due solely
to fluorophore-labeled collagen fragments. This is because
fluorescence of the covalently bound DQ collagen is quenched due to
the heavy labeling density (i.e., the close proximity of one
fluorescein molecule to another). Thus, the fluorescence signal in
the cell culture medium in the wells of the tissue culture plate is
directly proportional to the resorptive activity of mature
osteoclasts.
EXAMPLE 16
Assay of Collagen I Degradation by Metastatic Tumor Cells
[0143] Two cancer cell lines (HT-1080, which has been shown to be
highly metastatic, and BT-474, which has been shown to be less
metastatic) were seeded onto europium chelate-labeled collagen
covalently bound to a maleic anhydride-derivatized polystyrene
plate at 10,000 cells/well and cultured at 37.degree. C. Every 24
hours, 10 .mu.l of the cell culture medium were collected and
counted in a 96-well plate containing 200 .mu.l of
Fluorophore-Releasing Reagent (DELFIA.RTM. Enhancement Solution).
The results are shown in FIG. 15.
[0144] While the invention has been described in conjunction with
specific embodiments, the description and examples above are
intended to illustrate, but not limit the scope of the invention.
This application is intended to cover those changes and
substitutions that may be made by those skilled in the art without
departing from the spirit and the scope of the invention. Other
aspects, advantages and modifications will be apparent to those
skilled in the art to which the invention pertains, and these
aspects and modifications are within the scope of the invention,
which is limited only by the appended claims.
* * * * *