U.S. patent application number 14/391750 was filed with the patent office on 2015-03-19 for methods and compositions for cancer diagnosis.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University, Univeristy of Washington through its Center for Commreicalization. Invention is credited to Teri Brentnall, Sheng Pang, Juergen Karl Willmann.
Application Number | 20150079003 14/391750 |
Document ID | / |
Family ID | 48325963 |
Filed Date | 2015-03-19 |
United States Patent
Application |
20150079003 |
Kind Code |
A1 |
Brentnall; Teri ; et
al. |
March 19, 2015 |
Methods and Compositions for Cancer Diagnosis
Abstract
The present invention provides reagents and methods for
detecting cancer precancerous lesions in a patient.
Inventors: |
Brentnall; Teri; (Seattle,
WA) ; Willmann; Juergen Karl; (Stanford, CA) ;
Pang; Sheng; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Univeristy of Washington through its Center for
Commreicalization
The Board of Trustees of the Leland Stanford Junior
University |
Seattle
Palo ALto |
WA
CA |
US
US |
|
|
Family ID: |
48325963 |
Appl. No.: |
14/391750 |
Filed: |
May 1, 2013 |
PCT Filed: |
May 1, 2013 |
PCT NO: |
PCT/US13/39070 |
371 Date: |
October 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61641210 |
May 1, 2012 |
|
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61763598 |
Feb 12, 2013 |
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Current U.S.
Class: |
424/9.2 ;
424/9.1; 424/9.52 |
Current CPC
Class: |
A61K 49/223 20130101;
G01N 33/57438 20130101; A61K 49/221 20130101 |
Class at
Publication: |
424/9.2 ;
424/9.1; 424/9.52 |
International
Class: |
A61K 49/22 20060101
A61K049/22 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with U.S. government support under
grant number R21 5R21CA139279-02 awarded by the National Institutes
of Health. The U.S. Government has certain rights in the invention.
Claims
1. A method for detecting pancreatic cancer in a patient, or for
determining a risk for pancreatic cancer development in a patient
comprising one of: (a) (i) administering a detectable Thy-1 binding
molecule to a patient at risk of having or developing pancreatic
cancer, under conditions suitable to promote binding complex
formation between the Thy-1 binding molecule and Thy-1 present in
pancreatic tumor neovasculature or a precancerous lesion; and (ii)
detecting the presence or absence of the binding complexes; wherein
the presence of an increased number of the binding complexes
compared to control is indicative of the presence of pancreatic
cancer in the patient or indicates a risk of pancreatic cancer
development in the patient, or (b) (i) administering a detectable
binding molecule to a patient at risk of having or developing
pancreatic cancer, wherein the binding molecule is selected from
the group consisting of (a) MMRN1 binding molecules, (b) MRC2
binding molecules, (c) NRP1 binding molecules, and/or (d) VCAM1
binding molecules, under conditions suitable to promote binding
complex formation between the binding molecule and a binding
molecule target present in pancreatic tumor neovasculature or a
recancerous lesion; and (ii) detecting the presence or absence of
the binding complexes; wherein the presence of an increased number
of the binding complexes compared to control is indicative of the
presence of pancreatic cancer in the patient or indicates a risk of
pancreatic cancer development in the patient.
2. The method of claim 1, wherein the molecule is selected from the
group consisting of anti-human Thy-1 antibodies and a protein
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOS:1-20.
3. The method of claim 1, wherein the binding molecule is attached
to the surface of a microbubble.
4. The method of claim 1, wherein the detecting is carried out by
ultrasound molecular imaging.
5. The method of claim 1, wherein the presence of the binding
complexes is indicative of the presence of precancerous lesions,
and thus indicative of a risk of pancreatic cancer development in
the patient.
6. The method of claim 1, wherein the presence of the binding
complexes is indicative of the presence of pancreatic ductal
adenocarcinoma in the patient.
7. (canceled)
8. A method for determining efficacy of pancreatic cancer therapy
in a patient, comprising one of: (a) (i) administering a detectable
Thy-1 binding molecule to a patient undergoing or who has
previously undergone pancreatic cancer therapy, under conditions
suitable to promote binding of the Thy-1 binding molecule to Thy-1
in the pancreatic tumor neovasculature to form a binding complex;
and (ii) detecting the presence or absence of binding complexes;
wherein the presence or absence of binding complexes is indicative
of the efficacy of the anti-cancer therapy in the patient; or (b)
(i) administering a detectable binding molecule selected from the
group consisting of (i) MMRN1 binding molecules, (ii) MRC2 binding
molecules, (iii) NRP1 binding molecules, and (iv) VCAM1 binding
molecules, to a patient undergoing or who has previously undergone
pancreatic cancer therapy, under conditions suitable to promote
binding complex formation between the binding molecule and its
target present in pancreatic tumor neovasculature to form a binding
complex; and (ii) detecting the presence or absence of the binding
complexes; wherein the presence or absence of binding complexes is
indicative of the efficacy of the anti-cancer therapy in the
patient.
9. (canceled)
10. A composition, comprising: (a) a first microbubble; and (b) a
plurality of Thy-1 binding molecules attached to a surface of the
first microbubble.
11. The composition of claim 10, wherein the Thy-I binding
molecules are selected from the group consisting of anti-human
Thy-1 antibodies and a protein comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS:1-20.
12. The composition of claim 10, wherein the first microbubble
further comprises a plurality of other binding molecules attached
to a surface of the first microbubble, wherein the other binding
molecules are selected from the group consisting of (a) VEGFR2
binding molecules, (b) MMRN1 binding molecules, (c) MRC2 binding
molecules, (d) NRP1 binding molecules, and/or (e) VCAM1 binding
molecules.
13. The composition of claim 10, wherein the composition further
comprises a second microbubble, wherein the second microbubble has
attached to its surface a plurality of other binding molecules,
wherein the other binding molecules are selected from the group
consisting of (a) VEGFR2 binding molecules, (b) MMRN1 binding
molecules, (c) MRC2 binding molecules, (d) NRP1 binding molecules,
and/or (e) VCAM1 binding molecules.
14. The composition of claim 10, wherein the composition comprises
at least 10.sup.7 first microbubbles and/or second
microbubbles.
15. The composition of claim 10, further comprising one or more
anti-cancer therapeutics on or in the first microbubble and/or the
second microbubble.
16. (canceled)
17. A method for detecting cancer in a patient, or for determining
a risk for cancer development in a patient comprising: (a)
administering the composition of claim 10 to a patient at risk of
having or developing cancer, under conditions suitable to promote
binding complex formation between the binding molecules and targets
of the binding molecules present in neovasculature of the tumor or
a precancerous lesion; and (b) detecting a presence or absence of
the binding complexes using ultrasound molecular imaging; wherein
the presence of an increased number of the binding complexes
compared to control is indicative of the presence of cancer in the
patient or indicates a risk of cancer development in the
patient.
18. The method of claim 17, wherein the binding complex comprises a
complex between the binding molecule and binding molecule target
present in neovasculature of a tumor or a precancerous lesion.
19. The method of claim 17, wherein the presence of the binding
complexes is indicative of the presence of a precancerous lesion,
and thus indicative of a risk of cancer development in the
patient.
20. The method of claim 17, wherein the presence of the binding
complexes is indicative of the presence of cancer in the patient.
Description
CROSS-REFERENCE
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. Nos. 61/641,210, filed May 1, 2012 and 61/763,598
filed Feb. 12, 2013, incorporated by reference herein in their
entirety
BACKGROUND
[0003] Pancreatic cancer is the fourth leading cause of
cancer-related death in both women and men in the USA with an
estimated 36,000 deaths in 2012 and 43,000 new diagnoses. Survival
from pancreas cancer is stage dependent and currently the disease
is most frequently detected at advanced tumor stages. Patients
diagnosed with advanced pancreatic cancer have a median survival
time of less than one year and are considered incurable at the time
of diagnosis. Populations who are at elevated risk of pancreatic
cancer include: adult-onset diabetics have a 1:300 lifetime risk,
individuals who inherit a genetic predisposition to the disease
(such as BRCA2 carriers) and individuals who have 2 or more family
members with the disease.
For these moderate-to-high risk groups, methods for surveillance
are currently limited. Therefore, novel, early
detection/surveillance assays that are non-invasive, inexpensive
and accurate are critically needed in order to reduce mortality
from pancreatic cancer.
SUMMARY OF THE INVENTION
[0004] In one aspect, the present invention provides methods for
detecting pancreatic cancer in a patient, or for determining a risk
for pancreatic cancer development in a patient comprising:
[0005] (a) administering a detectable Thy-1 binding molecule to a
patient at risk of having or developing pancreatic cancer, under
conditions suitable to promote binding complex formation between
the Thy-1 binding molecule and Thy-1 present in pancreatic tumor
neovasculature or a precancerous lesion; and
[0006] (b) detecting the presence or absence of the binding
complexes; wherein the presence of an increased number of the
binding complexes compared to control is indicative of the presence
of pancreatic cancer in the patient or indicates a risk of
pancreatic cancer development in the patient.
[0007] In another aspect, the invention provides methods for
detecting pancreatic cancer in a patient, or for determining a risk
for pancreatic cancer development in a patient comprising:
[0008] (a) administering a detectable binding molecule to a patient
at risk of having or developing pancreatic cancer, wherein the
binding molecule is selected from the group consisting of (a) MMRN1
binding molecules, (b) MRC2 binding molecules, (c) NRP1 binding
molecules, and/or (d) VCAM1 binding molecules, under conditions
suitable to promote binding complex formation between the binding
molecule and a binding molecule target present in pancreatic tumor
neovasculature or a precancerous lesion; and (b) detecting the
presence or absence of the binding complexes; wherein the presence
of an increased number of the binding complexes compared to control
is indicative of the presence of pancreatic cancer in the patient
or indicates a risk of pancreatic cancer development in the
patient.
[0009] In a further aspect, the invention provides methods for
determining efficacy of pancreatic cancer therapy in a patient,
comprising:
[0010] (a) administering a detectable Thy-1 binding molecule to a
patient undergoing or who has previously undergone pancreatic
cancer therapy, under conditions suitable to promote binding of the
Thy-1 binding molecule to Thy-1 in the pancreatic tumor
neovasculature to form a binding complex; and (b) detecting the
presence or absence of binding complexes; wherein the presence or
absence of binding complexes is indicative of the efficacy of the
anti-cancer therapy in the patient.
[0011] In a further aspect, the present invention provides methods
for determining efficacy of pancreatic cancer therapy in a patient,
comprising:
[0012] (a) administering a detectable binding molecule selected
from the group consisting of (i) MMRN1 binding molecules, (ii) MRC2
binding molecules, (iii) NRP1 binding molecules, and (iv) VCAM1
binding molecules, to a patient undergoing or who has previously
undergone pancreatic cancer therapy, under conditions suitable to
promote binding complex formation between the binding molecule and
its target present in pancreatic tumor neovasculature to form a
binding complex; and (b) detecting the presence or absence of the
binding complexes; wherein the presence or absence of binding
complexes is indicative of the efficacy of the anti-cancer therapy
in the patient.
[0013] In another aspect, the invention provides compositions,
comprising:
[0014] (a) a first microbubble; and
[0015] (b) a plurality of Thy-1 binding molecules attached to a
surface of the first microbubble.
[0016] In one embodiment the first microbubble may further comprise
a plurality of other binding molecules attached to a surface of the
first microbubble, wherein the other binding molecules are selected
from the group consisting of (a) VEGFR2 binding molecules, (b)
MMRN1 binding molecules, (c) MRC2 binding molecules, (d) NRP1
binding molecules, and/or (e) VCAM1 binding molecules. In another
embodiment, the compositions may further comprise a second
microbubble, wherein the second microbubble has attached to its
surface a plurality of other binding molecules, wherein the other
binding molecules are selected from the group consisting of (a)
VEGFR2 binding molecules, (b) MMRN1 binding molecules, (c) MRC2
binding molecules, (d) NRP1 binding molecules, and/or (e) VCAM1
binding molecules.
[0017] In another aspect, the invention provides compositions,
comprising
[0018] a) a first microbubble; and
[0019] (b) a plurality of binding molecules attached to a surface
of the first microbubble, wherein the binding molecules attached to
the surface of the first microbubble are selected from the group
consisting of (a) MMRN1 binding molecules, (b) MRC2 binding
molecules, (c) NRP1 binding molecules, and/or (d) VCAM1 binding
molecules.
[0020] In a further aspect, the invention provides methods for
detecting cancer in a patient, or for determining a risk for cancer
development in a patient comprising:
[0021] (a) administering the composition of any embodiment of the
invention to a patient at risk of having or developing cancer,
under conditions suitable to promote binding complex formation
between the binding molecules and targets of the binding molecules
present in neovasculature of the tumor or a precancerous lesion;
and
[0022] (b) detecting a presence or absence of the binding complexes
using ultrasound molecular imaging; wherein the presence of an
increased number of the binding complexes compared to control is
indicative of the presence of cancer in the patient or indicates a
risk of cancer development in the patient.
DESCRIPTION OF THE FIGURES
[0023] FIG. 1. Overview of study design, from identification of
novel human PDAC-neovasculature-associated imaging biomarker Thy1,
to target validation, creation of human Thy1-targeted ultrasound
contrast agent, generation of a novel orthotopic PDAC xenograft
model expressing human Thy1 on its neovasculature, and in vivo
testing of imaging properties of Thy1-targeted ultrasound contrast
agent.
[0024] FIG. 2. Immunohistochemistry (IHC) analysis of Thy1 staining
in human pancreatic tissue samples. Examples of normal pancreas (A)
and primary chronic pancreatitis (B) with no Thy1 staining on
vasculature. (C). Example of positive Thy1-staining on vessels
associated with PDAC. D. Summary of IHC scores on Thy1 stained
tissues from normal pancreas (normal), chronic pancreatitis (CP),
and pancreatic cancer (PC).
[0025] FIG. 3. Evaluation of Thy1 expression on vascular
endothelial cells. (A) Stably transfected cells and wild-type cells
were assessed for human Thy1 expression by immunofluorescence
staining Clone 1 showed strong Thy1 staining, clone 2 showed low
staining, and wild-type cells showed no Thy1 staining. (B) Human
Thy1 expression levels on different cell types were quantitatively
assessed by FACS analysis. Histogram overlay of signals from cells
with different levels of Thy1 expression is shown. (C) Mean
fluorescence intensity values are shown in bar graph. Error bars
are .+-.standard deviations.
[0026] FIG. 4. Dynamic cell culture binding assay of microbubbles
(MB) in a parallel plate flow chamber setting. Phase-contrast
bright-field micrographs show binding of MBThy1 and MBControl
(white spheres, arrowheads) to different cell types; binding could
be substantially blocked by incubation of cells beforehand with an
anti-human Thy1 antibody (quantitative date in Table 5).
[0027] FIG. 5. In vivo ultrasound molecular imaging of orthotopic
PDAC xenografts in mice and corresponding ex vivo
immunofluorescence analysis. Transverse ultrasound images obtained
in contrast mode following intravenous injection of MBThy1 and
MBControl show strong imaging signal in human Thy1-positive tumor
and background signal in both types of control tumors (scale bar=5
mm; scale is shown for molecular ultrasound imaging signal in
arbitrary units, a.u.). Note low imaging signal following MBControl
in all tumor types (circles, region of interest). Corresponding
immunofluorescence micrographs of merged double-stained sections
(murine CD31, red; human thy1, green) confirm human Thy1 expression
on neovasculature in Thy1-positive tumors (yellow) while both
negative control tumors did not show human Thy1 staining on the
neovasculature (scale bar=50 .mu.m).
DETAILED DESCRIPTION OF THE INVENTION
[0028] All references cited are herein incorporated by reference in
their entirety. Within this application, unless otherwise stated,
the techniques utilized may be found in any of several well-known
references such as: Molecular Cloning: A Laboratory Manual
(Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene
Expression Technology (Methods in Enzymology, Vol. 185, edited by
D. Goeddel, 1991. Academic Press, San Diego, Calif.), "Guide to
Protein Purification" in Methods in Enzymology (M. P. Deutshcer,
ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to
Methods and Applications (Innis, et al. 1990. Academic Press, San
Diego, Calif.), Culture of Animal Cells: A Manual of Basic
Technique, 2.sup.nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York,
N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E.
J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion
1998 Catalog (Ambion, Austin, Tex.).
[0029] As used herein, the singular forms "a", "an" and "the"
include plural referents unless the context clearly dictates
otherwise. "And" as used herein is interchangeably used with "or"
unless expressly stated otherwise.
[0030] As used herein, the term "about" means+1-5% of the stated
value.
[0031] As used herein, the amino acid residues are abbreviated as
follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp;
D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E),
glutamine (Gln; Q), glycine (Gly; G), histidine (His; H),
isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine
(Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser;
S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and
valine (Val; V).
[0032] All embodiments of any aspect of the invention can be used
in combination, unless the context clearly dictates otherwise.
[0033] Unless the context clearly requires otherwise, throughout
the description and the claims, the words `comprise`, `comprising`,
and the like are to be construed in an inclusive sense as opposed
to an exclusive or exhaustive sense; that is to say, in the sense
of "including, but not limited to". Words using the singular or
plural number also include the plural and singular number,
respectively. Additionally, the words "herein," "above," and
"below" and words of similar import, when used in this application,
shall refer to this application as a whole and not to any
particular portions of the application.
[0034] The description of embodiments of the disclosure is not
intended to be exhaustive or to limit the disclosure to the precise
form disclosed. While the specific embodiments of, and examples
for, the disclosure are described herein for illustrative purposes,
various equivalent modifications are possible within the scope of
the disclosure, as those skilled in the relevant art will
recognize.
[0035] In a first aspect, the present invention provides novel
compositions, comprising
[0036] (a) a first microbubble; and
[0037] (b) a plurality of Thy-1 binding molecules attached to a
surface of the first microbubble.
[0038] The inventors have discovered that Thy-1 is present in newly
created micro-blood vessels in cancer, but is not present in normal
blood vessels. Thy-1 specific binding molecules are shown herein
for the first time to stain cancer-related blood vessels in
pre-cancer as well as cancer. Thus, the compositions of the
invention find wide use as, for example, imaging agents to enhance
early stage cancer detection, as well as pre-cancerous states in
individuals at risk for developing cancer, including but not
limited to pancreatic cancer.
[0039] Human Thy-1 or CD90 (Cluster of Differentiation 90) is a
25-37 kDa N-glycosylated, glycophosphatidylinositol (GPI) anchored
conserved cell surface protein. Its amino acid sequence is provided
in SEQ ID NO:1.
[0040] As used herein, "microbubbles" refer to micron-sized
contrast agents composed of a shell and a gas core, as is well
known to those of skill in the art. Microbubbles are commercially
available from a number of sources. The shell may be formed from
any suitable material, including but not limited to albumin,
polysaccharides (such as galactose), lipids (such as
phospholipids), polymers and combinations thereof. Any suitable gas
core can be used in the microbubbles of the invention, including
but not limited to air, octafluoropropane, perfluorocarbon, sulfur
hexafluoride or nitrogen. The gas core determines the echogenecity
of the microbubble. When gas bubbles are caught in an ultrasound
frequency field, they compress, oscillate, and reflect a
characteristic echo, this generates the strong and unique sonogram
in contrast-enhanced ultrasound. Gas cores can be composed of air,
or heavy gases like octafluoropropane, perfluorocarbon, sulfur
hexafluoride or nitrogen. Heavy gases are less water-soluble so
they are less likely to leak out from the microbubble to impair
echogenecity. The average diameter of the microbubble can be
between 1 .mu.m and 25 .mu.m. In general, the microbubbles have a
diameter of about 1 .mu.m and about 10 .mu.m on average, and more
preferably between about 1 .mu.m and 5 .mu.m, 1 .mu.m and 4 .mu.m,
1 .mu.m and 3 .mu.m, 1 .mu.m and about 2 .mu.m, 2 .mu.m and 5
.mu.m, 2 .mu.m and 4 .mu.m, 2 .mu.m and 3 .mu.m, 3 .mu.m and 5
.mu.m, 3 .mu.m and 4 .mu.m, or about as 1 .mu.m, 2 .mu.m, 2.5
.mu.m, 3 .mu.m, 3.5 .mu.m or 4 .mu.m on average. OPTISON.RTM..
(made by GE Healthcare) was the first microbubble approved by Food
and Drug Administration (FDA), and has an albumin shell and
octafluoropropane (C.sub.3F.sub.8) gas core. The second
FDA-approved microbubble, LEVOVIST.RTM., (made by Schering AG), has
a palmitic acid/galactose shell and an air core. Other examples of
microbubble include, but are not limited to ALBUNEX.RTM. (made by
Molecular Biosystems), SONOVUE.RTM. (made by Bracco Diagnostics,
Inc.), SONOZOID.RTM. (made by Schering AG), SONOVIST.RTM. (made by
Schering AG), and DEFINITY.RTM. (made by DuPont Pharmaceuticals).
ALBUNEX.RTM. has an albumin shell and an air core. SONOVUE.RTM. and
contains a sulfur hexafluoride (SF.sub.6) gas core that is
stabilized in aqueous dispersion of a monolayer of phospholipids.
SONOZOID.RTM. is another microbubble preparation containing a
perfluorocarbon gas core and a lipid shell. DEFINITY.RTM. is
another FDA-approved microbubble that contains a lipid shell and an
octafluoropropane (C.sub.3F.sub.8) gas core. In one exemplary
embodiment, microbubbles of the present invention comprise a lipid
shell and perfluorocarbon gas core of between about 1 .mu.m and
about 5 .mu.m, 1 .mu.m and about 4 .mu.m diameter, 1 .mu.m and
about 3 .mu.m, or 1 .mu.m and about 2 .mu.m, on average.
[0041] The microbubbles of the invention can be used, for example,
as a contrast agent for ultrasound imaging. Microbubbles have a
high degree of echogenicity (i.e.: the ability of an object to
reflect ultrasound waves). The echogenicity difference between the
gas in the microbubbles and the soft tissue surroundings of the
body is large. Thus, ultrasonic imaging using microbubble contrast
agents enhances the ultrasound backscatter, or reflection of the
ultrasound waves, to produce a unique sonogram with increased
contrast due to the high echogenicity difference.
[0042] The microbubbles can be functionalized in any suitable
manner for binding of the Thy-1 binding molecules (or other binding
molecules, as described herein). Such techniques are well known to
those of skill in the art, such as those for functionalizing the
surface of a microbubble to permit binding of a protein. In one
embodiment, the microbubble surface if functionalized to permit
direct attachment of the binding agent to the microbubble surface.
In another embodiment, the microbubble surface is functionalized to
permit indirect attachment of the binding molecule to the
microbubble surface. In one non-limiting embodiment of indirect
binding, the microbubble surface can be coated with streptavidin,
to which biotinylated binding molecules can be bound. Any other
suitable binding pair can be similarly used, as will be apparent to
those of skill in the art.
[0043] Following administration to the patient (such as by
intravenous injection), the targeted microbubbles accumulate at
tissue sites that over-express Thy-1 (or other markers discussed
herein), causing a local increase in the ultrasound imaging signal.
Due to their small size, the microbubbles stay predominantly within
the vascular compartment after intravenous injection. Thus, the
microbubbles can be used, for example, to exclusively detect
vascular endothelial cell associated molecular markers that are
present in early stage cancers or precancerous lesions.
[0044] A "Thy-1 binding molecule" is any molecular entity capable
of selectively binding to human Thy-1. Exemplary specific binding
agents include, but are not limited to, Thy-1 itself (SEQ ID NO:1),
anti-Thy-1 antibodies or fragments thereof, aptamers selective for
Thy-1, the I-domain (SEQ ID NO:3) of .alpha.X (CD11c) integrin
domain, or proteins comprising this I domain, including but not
limited to .alpha.X (SEQ ID NO:2, noting that AA 1-19 constitutes
the signal peptide, and thus can be deleted), and .alpha.X-.beta.2
integrin; the I-domain (SEQ ID NO:5) of .alpha.M integrin domain,
or proteins comprising this I domain, including but not limited to
.alpha.M (SEQ ID NO:4) and .alpha.M-.beta.2 integrin (see Choi et
al., Biochemical and Biophysical Research Communications; 07/2005;
331(2):557-61. DOI:10.1016/j.bbrc.2005.04.006); integrin
.alpha.v-.beta.3 (CD51/CD61), Mac-1 (CD11b/CD18), .beta.3-integrin
(SEQ ID NO:6) or the I-domain (SEQ ID NO:7) of 33-integrin (see
Leyton et al., Current Biology, Volume 11, Issue 13, 1028-1038, 10
Jul. 2001), adhesion G-protein coupled receptor CD97 (SEQ ID NO:8)
(see Wandel et al, The Journal of Immunology Feb. 1, 2012 vol. 188
no. 3 1442-1450), and a protein selected from the group consisting
of SEQ ID NOS:9-20 as listed in Table 1.
TABLE-US-00001 TABLE 1 Other Interacting proteins for THY1 ITGAV
Integrin alpha-V (SEQ ID NO: 9) FYN Tyrosine protein kinase-Fyn
(SEQ ID NO: 10) LCK Tyrosine protein kinase -Lck (SEQ ID NO: 11)
ITGAM Integrin alpha-M(SEQ ID NO: 12) ITGB3 Integrin Beta-3 (SEQ ID
NO: 13) YWHAB 14-3-3 protein beta/alpha (SEQ ID NO: 14) APP amyloid
beta A4 protein(SEQ ID NO: 15) ITGB2 Integrin beta-2 (SEQ ID NO:
16) STAT6 Signal transducer and activator of transcription 6 (SEQ
ID NO: 17) ARHGAP35 Rho GTPase activating protein 35 (SEQ ID NO:
18) ARHGAP5 Rho GTPase activating protein 5 (SEQ ID NO: 19) BOAT
Brother of ataxin-1 (SEQ ID NO: 20)
[0045] In another embodiment, the first microbubble further
comprises a plurality of other binding molecules attached to a
surface of the first microbubble, wherein the other binding
molecules are selected from the group consisting of (a) VEGFR2
binding molecules, (b) multimerin-1 (MMRN1) binding molecules, (c)
mannose receptor type 2 (MRC2) binding molecules, (d) neuropilin 1
(NRP1) binding molecules, and/or (e) vascular cell adhesion
molecule 1 (VCAM1) binding molecules.
[0046] As disclosed in the examples that follow, the inventors have
identified MMRN1 (SEQ ID NO:21), MRC2 (SEQ ID NO:55), NRP1 (SEQ ID
NO:57), and VCAM1 (SEQ ID NO:90) as additional vascular endothelial
cell markers that can be used in identifying tumor neovasculature,
and which thus can be used in combination with Thy-1 (as can
VEGFR2) as markers for early stage cancer and precancerous lesion
detection. In one embodiment, the first microbubble, such as a
population of first microbubbles, would each comprise both Thy-1
binding molecules and "other" binding molecules for one or more of
VEGFR2, MMRN1, MRC2, NRP1, and VCAM1. In a preferred embodiment,
the first population of microbubbles contains other binding
molecules for 1, 2, 3, or all 4 of MMRN1, MRC2, NRP1, and
VCAM1.
[0047] In another embodiment, the composition further comprises a
second microbubble, wherein the second microbubble has attached to
its surface a plurality of other binding molecules, wherein the
other binding molecules are selected from the group consisting of
(a) VEGFR2 binding molecules, (b) MMRN1 binding molecules, (c) MRC2
binding molecules, (d) NRP1 binding molecules, and/or (e) VCAM1
binding molecules. In this embodiment, composition would comprise
different subsets of microbubbles: a first microbubble (such as a
population of first microbubbles) that comprises Thy-1 binding
molecules, and at least a second microbubble (such as a population
of second microbubbles) that includes binding molecules for one or
more of VEGFR2, MMRN1, MRC2, NRP1, and VCAM1. In one embodiment,
the second population of microbubbles may each comprise VEGFR2,
MMRN1, MRC2, NRP1, and VCAM1. In another embodiment, the second
population of microbubbles may each comprise other binding
molecules for only one of VEGFR2, MMRN1, MRC2, NRP1, and VCAM1. In
another embodiment, the second population of microbubbles may
comprise a second population of microbubbles that each have VEGFR2
binding molecules, a third population of microbubbles that each
have MMRN binding molecules, a fourth population of binding
molecules that each have MRC2 binding molecules, a fifth population
of microbubbles that each have NRP binding molecules, and a sixth
population of microbubbles that each have VCAM1 binding molecules.
In a further embodiment, the second population of microbubbles may
comprise a second population that each have binding molecules for
two of MMRN1, MRC2, NRP1, and VCAM1, and a third population of
microbubbles comprising binding molecules for the other two of
MMRN1, MRC2, NRP1, and VCAM1. Many variations are possible that use
all or a subset of the recited binding molecules, as will be
understood by those of skill in the art based on the teachings
herein. In a preferred embodiment, the other binding molecules are
binding molecules for one or more of MMRN1, MRC2, NRP1, and
VCAM1.
[0048] In embodiments where at least a first and second population
of microbubbles are present in the composition, the different
populations of microbubbles (i.e.: first microbubble, second
microbubble, etc.) can be prepared so as to be distinguishable from
each other, though this is not a requirement for use of multiple
populations of microbubbles in the methods of the invention. Any
suitable means to distinguish the microbubbles can be used,
including but not limited to, differentially labeling each
population with a separate detectable label (fluorescent,
radioactive, etc.) and using different sized microbubbles for each
different microbubble population.
[0049] Multimerin-1 (MMR) is a soluble human protein found in
platelets and in the endothelium of blood vessels. It is composed
of linked subunits to form large, variably sized homomultimer. MMR1
has a number of identified ligands. A "MMR1 binding molecule" is
any molecular entity capable of selectively binding to human MMR1.
Exemplary specific binding molecules include, but are not limited
to, MMR1 itself (SEQ ID NO:21), anti-MMR1 antibodies, and a protein
selected from the group consisting of SEQ ID NOS: 22 to 54 (see
Table 2 below).
TABLE-US-00002 TABLE 2 Interacting proteins for MMR1 F5 Coagulation
factor V (SEQ ID NO; 22) APC Adenomatous polyposis coli protein
(SEQ ID NO; 23) ALB albumin (SEQ ID NO; 24) APP amyloid beta (A4)
precursor protein (SEQ ID NO; 25) EGF epidermal growth factor (SEQ
ID NO; 26) F8 Coagulation factor 8 (SEQ ID NO; 27) FN1 fibronectin
1 (SEQ ID NO; 28) KNG1 Kininogen 1 (SEQ ID NO; 29) PF4 Platelet
factor 4 (SEQ ID NO; 30) PLG plasminogen (SEQ ID NO; 31) PPBP
pro-platelet basic protein (SEQ ID NO; 32) SERPINE1 serpin
peptidase inhibitor, clade E (SEQ ID NO; 33) SPARC secreted protein
acidic cysteine-rich (SEQ ID NO; 34) SRGN serglycin (SEQ ID NO; 35)
TGFB1 transforming growth factor, beta 1 (SEQ ID NO; 36) TGFB3
transforming growth factor, beta 3 (SEQ ID NO; 37) A2M
alpha-2-macroglobulin (SEQ ID NO; 38) ACTN1 actinin, alpha 1 (SEQ
ID NO; 39) ACTN2 actinin, alpha 2 (SEQ ID NO; 40) ACTN4 actinin,
alpha 4 (SEQ ID NO; 41) ALDOA aldolase A (SEQ ID NO; 42) CFD
complement factor D (SEQ ID NO; 43) CLU clusterin (SEQ ID NO; 44)
F13A1 coagulation factor 13, A1 polypeptide (SEQ ID NO; 45) FIGF
c-fos induced growth factor (SEQ ID NO; 46) GAS6 growth
arrest-specific 6 (SEQ ID NO; 47) SERPINA1 serpin peptidase
inhibitor, clade A (SEQ ID NO; 48) SERPINF2 serpin peptidase
inhibitor, clade F (SEQ ID NO; 49) SERPING1serapin peptidase
inhibitor, clade G (SEQ ID NO; 50) TGFB2 transforming growth
factor, beta 2 (SEQ ID NO; 51) TIMP1 TIMP metallopeptidase
inhibitor 1 (SEQ ID NO; 52) VEGFC vascular endothelial growth
factor C (SEQ ID NO; 53) CDKN2A cyclin-dependent kinase inhibitor
2A (SEQ ID NO; 54)
[0050] A "MRC2 binding molecule" is any molecular entity capable of
selectively binding to human MRC2. Exemplary specific binding
molecules include, but are not limited to, MRC2 itself (SEQ ID
NO:55), anti-MRC2 antibodies, and the NDEL1 protein comprising the
amino acid SEQ ID NO: 56, an identified ligand for MRC2.
[0051] A "NRP1 binding molecule" is any molecular entity capable of
selectively binding to human NRP 1. Exemplary specific binding
molecules include, but are not limited to, NRP 1 itself (SEQ ID
NO:57), anti-NRP1 antibodies, and a protein selected from the group
consisting of SEQ ID NOS:58-89, identified ligands for NRP11 (see
table below).
TABLE-US-00003 TABLE 3 Interacting proteins for NRP1 VEGFA Vascular
endothelial growth factor A (SEQ ID NO: 58) KDR Vascular
endothelial growth factor receptor 2 (SEQ ID NO: 59) SEMA3A
Semaphorin-3A (SEQ ID NO: 60) SEMA3F Semaphorin-3F (SEQ ID NO: 61)
VEGFB Vascular endothelial growth factor B (SEQ ID NO: 62) FGF4
fibroblast growth factor 4 (SEQ ID NO: 63) FLT1 vascular
endothelial growth factor receptor 1 (SEQ ID NO: 64) NRP2
Neuropilin-2 (SEQ ID NO: 65) PGF placenta growth factor (SEQ ID NO:
66) SRRM1 serine/arginine repetitive matrix protein 1 (SEQ ID NO:
67) GIPC1 PDZ domain-containing protein GIPC1 (SEQ ID NO: 68)
SEMA3C semaphorin-3C (SEQ ID NO: 69) FGF1 fibroblast growth factor
1 (SEQ ID NO: 70) FGF2 fibroblast growth factor 2 (SEQ ID NO: 71)
FGF7 fibroblast growth factor 7 (SEQ ID NO: 72) FGFR1 fibroblast
growth factor receptor 1 (SEQ ID NO: 73) PTK2 Focal adhesion kinase
1 (SEQ ID NO: 74) SEMA3B semaphoring-3B (SEQ ID NO: 75) FGFBP1
fibroblast growth factor-binding protein 1 (SEQ ID NO: 76) SQSTM1
sequestosome-1 (SEQ ID NO: 77) IGHG1 immunoglobulin heavy constant
gamma 1 (SEQ ID NO: 78) IGKC immunoglobulin kappa constant (SEQ ID
NO: 79) L1CAM L1 cell adhesion molecule (SEQ ID NO: 80) SEMA3D
semaphorin 3D (SEQ ID NO: 81) SEMA3G semaphorin 3G (SEQ ID NO: 82)
PLXNA1 plexin A1 (SEQ ID NO: 83) PLXNA2 plexin A2 (SEQ ID NO: 84)
SEMA3E semaphoring 3E (SEQ ID NO: 85) ITGA5 integrin, alpha 5 (SEQ
ID NO: 86) ATN1 atrophin 1(SEQ ID NO: 87) GFI1B growth factor
independent 1B transcription repressor (SEQ ID NO: 88) (SEQ ID NO:
89)
[0052] A "VCAM1 binding molecule" is any molecular entity capable
of selectively binding to human VCAM 1. Exemplary specific binding
molecules include, but are not limited to, VCAM1 itself (SEQ ID
NO:90), anti-VCAM1 antibodies, and a protein selected from the
group consisting of SEQ ID NOS: 91 to 130, identified ligands for
VCAM1 (see table below).
TABLE-US-00004 TABLE 4 40 Interacting proteins for VCAM1 ITGB7
integrin beta-7 (SEQ ID NO: 91) IL13 interleukin-13 (SEQ ID NO: 92)
EZR ezrin (SEQ ID NO: 93) MSN moesin (SEQ ID NO: 94) CCL17 c-c
motif chemokine 17 (SEQ ID NO: 95) CTSG cathepsin G (SEQ ID NO: 96)
ELANE neutrophil; elastase (SEQ ID NO: 97) ITGAD integrin alpha-D
(SEQ ID NO: 98) ITGB1 integrin beta-1 (SEQ ID NO: 99) CCL22 C-C
motif chemokine 22 (SEQ ID NO: 100) IRF1 interferon regulatory
factor 1 (SEQ ID NO: 101) NFKB1 nuclear factor NF-kappa-B p105
subunit (SEQ ID NO: 102) LGALS3 Galectin-3 (SEQ ID NO: 103) RELB
transcription factor RelB (SEQ ID NO: 104) RDX radixin (SEQ ID NO:
105) RELA transcription factor p65 (SEQ ID NO: 106) CYBA cytochrome
b-245, alpha polypeptide (SEQ ID NO: 107) CYBB cytochrome b-245,
beta polypeptide (SEQ ID NO: 108) ITGA4 integrin, alpha 4 (SEQ ID
NO: 109) ITGA9 integrin, alpha 9 (SEQ ID NO: 110) NCF2 neutrophil
cytosolic factor 2 (SEQ ID NO: 111) NOX1 NADPH oxidase 1 (SEQ ID
NO: 112) PRKCA protein kinase C, alpha (SEQ ID NO: 113) PRKCH
protein kinase C, eta (SEQ ID NO: 114) SRC v-src sarcoma (SEQ ID
NO: 115) NOX3 NADPH oxidase 3 (SEQ ID NO: 116) PRKCD protein kinase
C, delta (SEQ ID NO: 117) PRKCE protein kinase C, epsilon (SEQ ID
NO: 118) PRKCG protein kinase c, gamma (SEQ ID NO: 119) PRKCQ
protein kinase C, theta (SEQ ID NO: 120) PIK3CA
phosphatidylinosital-4,5-bisphosphate 3-kinase, catalytic subunit
alpha (SEQ ID NO: 121) PIK3CB phosphatidylinosital-4,5-bisphosphate
3-kinase, catalytic subunit beta (SEQ ID NO: 122) PIK3CD
phosphatidylinosital-4,5-bisphosphate 3-kinase, catalytic subunit
delta (SEQ ID NO: 123) PIK3CG phosphatidylinosital-4,5-bisphosphate
3-kinase, catalytic subunit gamma (SEQ ID NO: 124) PIK3R1
phosphoinositide-3-kinase, regulatory subunit 1 (alpha) (SEQ ID NO:
125) PIK3R2 phosphoinositide-3-kinase, regulatory subunit 2 (beta)
(SEQ ID NO: 126) PIK3R3 phosphoinositide-3-kinase, regulatory
subunit 3 (gamma) (SEQ ID NO: 127) PIK3R5
phosphoinositide-3-kinase, regulatory subunit 5 (SEQ ID NO: 128)
GRCh37 (SEQ ID NO: 129) GRCh37 (SEQ ID NO: 130)
[0053] A "VEGFR2 binding molecule" is any molecular entity capable
of selectively binding to human VEGFR2. Exemplary specific binding
molecules include, but are not limited to, VEGFR2 itself (SEQ ID
NO:131), and anti-VEGFR2 antibodies.
[0054] In a second aspect, the present invention provides
compositions, comprising
[0055] a) a first microbubble; and
[0056] (b) a plurality of binding molecules attached to a surface
of the first microbubble, wherein the binding molecules attached to
the surface of the first microbubble are selected from the group
consisting of (a) MMRN1 binding molecules, (b) MRC2 binding
molecules, (c) NRP1 binding molecules, and/or (d) VCAM1 binding
molecules.
[0057] The inventors have discovered that each of MMRN1, MRC2,
NRP1, and VCAM1 are present in newly created micro-blood vessels in
cancer, but is not present in normal blood vessels. Thy-1 specific
binding molecules are shown herein for the first time to stain
cancer-related blood vessels in pre-cancer as well as cancer. Thus,
the compositions of the invention find wide use as, for example,
imaging agents to enhance early stage cancer detection, as well as
pre-cancerous states in individuals at risk for developing cancer,
including but not limited to pancreatic cancer
[0058] All definitions and embodiments of the first aspect of the
invention apply equally to this second embodiment.
[0059] In all of these embodiments, antibodies that can be used as
binding molecules mean an immunoglobulin molecule immunologically
reactive with the recited target, and includes polyclonal and
monoclonal antibodies. Various isotypes of antibodies exist, for
example IgG1, IgG2, IgG3, IgG4, and other Ig, e.g., IgM, IgA, IgE
isotypes. The term also includes genetically engineered forms such
as chimeric antibodies (e.g., humanized murine antibodies) and
heteroconjugate antibodies (e.g., bispecific antibodies), and fully
humanized antibodies. As used throughout the application, the term
"antibody" includes fragments with antigen-binding capability
(e.g., Fab', F(ab').sub.2, Fab, Fv and rIgG, as are well known in
the art. The term also refers to recombinant single chain Fv
fragments (scFv). The term antibody also includes bivalent or
bispecific molecules, diabodies, triabodies, and tetrabodies, as
are known in the art. Antibodies for many if not all of the
proteins disclosed herein are commercially available.
[0060] In all of these embodiments of the microbubbles of the
invention, the binding molecules on the surface of the microbubbles
can be at any density that is effective for targeting of the
microbubble to target-containing neovasculature in a target tumor
or precancerous lesion. In one embodiment, the average number of
binding molecules per square micrometer of the microbubble surface
in a microbubble population is at least 1,000/cm.sup.2; in various
further embodiments, it is at least 2,000/cm.sup.2, 3,000/cm.sup.2,
4,000/cm.sup.2, 5,000/cm.sup.2, 6,000/cm.sup.2, 7,000/cm.sup.2,
7,500/cm.sup.2, or 7,600/cm.sup.2.
[0061] The compositions may comprise any number of microbubbles in
the composition that can be detected once targeted to the
target-containing neovasculature in a target tumor or precancerous
lesion. In one embodiment that can be combined with any other
embodiment herein, the compositions comprise at least 10.sup.6
microbubbles (i.e.: combined total of all microbubbles, whether all
first microbubbles, or a combination of first and second
microbubbles. In various further embodiments, the compositions
comprises at least 2.times.10.sup.6, 3.times.10.sup.6
5.times.10.sup.6, 7.5.times.10.sup.6, 10.sup.7, 2.times.10.sup.7,
3.times.10.sup.7, 5.times.10.sup.7, 7.5.times.10.sup.7, 10.sup.8,
2.times.10.sup.8, 3.times.10.sup.8, 5.times.10.sup.8,
7.5.times.10.sup.8, or at least 10.sup.9 microbubbles.
[0062] Since the microbubbles of the invention target tumor or
precancerous lesion neovasculature upon administration, they can
also be used as a drug delivery device. Thus, in another
embodiment, the composition of any embodiment or combination of
embodiments of the present invention further comprises one or more
anti-cancer therapeutics on or in the first microbubble and/or the
second microbubble. In this embodiment, any suitable anti-cancer
therapeutic can be loaded onto or into the microbubbles, including
but not limited to alkylating agents such as busulfan, cis-platin,
mitomycin C, and carboplatin; antimitotic agents such as
colchicine, vinblastine, paclitaxel, and docetaxel; topo I
inhibitors such as camptothecin and topotecan; topo II inhibitors
such as doxorubicin and etoposide; RNA/DNA antimetabolites such as
5-azacytidine, 5-fluorouracil and methotrexate; DNA antimetabolites
such as 5-fluoro-2'-deoxy-uridine, ara-C, hydroxyurea and
thioguanine; antibodies such as Herceptin.RTM.. and Rituxan.RTM.,
melphalan, chlorambucil, cyclophosamide, ifosfamide, vincristine,
mitoguazone, epirubicin, aclarubicin, bleomycin, mitoxantrone,
elliptinium, fludarabine, octreotide, retinoic acid, tamoxifen and
alanosine.
[0063] Techniques for loading compounds onto or into microbubbles
are well known in the art; any such suitable technique can be used
to produce the microbubbles of this embodiment of the
invention.
[0064] In a third aspect, the present invention provides methods
for detecting cancer in a patient, or for determining a risk for
cancer development in a patient comprising:
[0065] (a) administering the composition of any embodiment or
combination of embodiments of the first or second aspect of the
invention to a patient at risk of having or developing cancer,
under conditions suitable to promote binding complex formation
between the binding molecules and targets of the binding molecules
present in neovasculature of the tumor or a precancerous lesion;
and
[0066] (b) detecting a presence or absence of the binding complexes
using ultrasound molecular imaging; wherein the presence of an
increased number of the binding complexes compared to control is
indicative of the presence of cancer in the patient or indicates a
risk of cancer development in the patient.
[0067] As discussed above, the inventors have found that the
microbubbles of the present invention can be used as a general tool
for detection of tumor neovasculature, such as in early stage
tumors and precancerous lesions. As used herein, "neovasculature"
means the vasculature of tumors.
[0068] The methods according to this aspect of the invention can be
used with any patient at risk of having or developing cancer. The
patient may be any mammal, such as a human. As used herein the term
"cancer" is intended to mean any cellular malignancy whose unique
trait is the loss of normal controls which results in unregulated
growth, lack of differentiation and ability to invade local tissues
and metastasize. Cancer can develop in any tissue of any organ.
More specifically, cancer is intended to include, without
limitation, prostate cancer, leukemia, hormone dependent cancers,
breast cancer, colon cancer, epidermal cancer, liver cancer,
esophageal cancer, stomach cancer, hepatic carcinoma, melanoma,
epidermoid carcinoma, pancreatic cancer, brain malignancies (such
as neuroblastoma, glioblastoma, glioma, medulloblastoma,
astrocytoma, acoustic neuroma, oligodendroglioma and meningioma),
lung cancer (such as small cell lung and non-small cell lung
cancer) ovarian adenocarcinoma, bladder cancer, and renal
cancer.
[0069] As disclosed herein, the inventors have discovered that
Thy-1 and the other recited markers are expressed in pancreatic
neovasculature but not in normal pancreatic vasculature, and thus
can be used to detect cancer. The inventors have also demonstrated
use of the microbubbles of the invention to selectively target
tumor neovasculature. The methods of this aspect of the invention
thus provide early cancer detection, thus allowing earlier clinical
intervention in treatment and substantially improved clinical
outcome. Thus, the methods may further comprise treating the
patient based on the presence of an increased number of binding
complexes compared to control.
[0070] Any suitable control can be used, including but not limited
to comparison to a number of binding complexes identified in a
subject or population of subjects known to not have cancer. Any
amount of Thy-1 or the other markers above control levels may
indicate presence of cancer in the patient or indicates a risk of
cancer development in the patient. In various embodiments, the
increase is binding complexes is at least 10% above control, and
preferably at least 25%, 50%, 100%, or more above control.
[0071] The methods can be used with patients that are at risk of
having cancer, based on symptoms they presently have. The methods
can also be used with patients who are at risk of developing
cancer. Such risk factors include, but are not limited to, a family
history of cancer and genetic disorders indicating a propensity to
develop cancer.
[0072] The microbubbles can be administered by any suitable
technique, including but not limited to parenterally,
transmucosally (orally, nasally, or rectally) or transdermally.
Parenteral administration includes, but is not limited to,
intravenous, intra-arteriole, intramuscular, intradermal,
subcutaneous, intraperitoneal, intraventricular, and intracranial
administration. In one embodiment administration of the
microbubbles is parenteral, via intravenous injection. Following
administration to the patient (such as by intravenous injection),
the targeted microbubbles accumulate at tumor sites that
over-express Thy-1 or other marker, causing a local increase in the
ultrasound imaging signal. In a preferred embodiment, microbubbles
with an average diameter of between about 1 .mu.m and 5 .mu.m, 1
.mu.m and 4 .mu.m, 1 .mu.m and 3 .mu.m, 1 .mu.m and about 2 .mu.m,
2 .mu.m and 5 .mu.m, 2 .mu.m and 4 .mu.m, 2 .mu.m and 3 .mu.m, 3
.mu.m and 5 .mu.m, 3 .mu.m and 4 .mu.m, or about as 1 .mu.m, 2
.mu.m, 2.5 .mu.m, 3 .mu.m, 3.5 .mu.m or 4 .mu.m on average are
intravenously injected. Due to their small size, the microbubbles
stay predominantly within the vascular compartment after
intravenous injection. Thus, the microbubbles can be used, for
example, to exclusively detect vascular endothelial cell associated
molecular markers that are present in early stage cancers or
precancerous lesions.
[0073] The "conditions suitable to promote binding complex
formation" used in the methods of the invention will depend on the
means by which the binding molecule is labeled, the type of assay
(i.e.: in vitro or in vivo), and all other relevant factors, and
can be determined by one of skill in the art based on the teachings
herein).
[0074] Specifics of methods for performing the ultrasound molecular
imaging disclosed herein are well known to those of skill in the
art; exemplary embodiments are disclosed in the examples that
follow.
[0075] In a fourth aspect, the present invention provides methods
for detecting pancreatic cancer in a patient, or for determining a
risk for pancreatic cancer development in a patient comprising:
[0076] (a) administering a detectable Thy-1 binding molecule to a
patient at risk of having or developing pancreatic cancer, under
conditions suitable to promote binding complex formation between
the Thy-1 binding molecule and Thy-1 present in pancreatic tumor
neovasculature or a precancerous lesion; and
[0077] (b) detecting the presence or absence of the binding
complexes; wherein the presence of an increased number of the
binding complexes compared to control is indicative of the presence
of pancreatic cancer in the patient or indicates a risk of
pancreatic cancer development in the patient.
[0078] In a fifth aspect, the present invention provides methods
for detecting pancreatic cancer in a patient, or for determining a
risk for pancreatic cancer development in a patient comprising:
[0079] (a) administering a detectable binding molecule selected
from the group consisting of (i) MMRN1 binding molecules, (ii) MRC2
binding molecules, (iii) NRP1 binding molecules, and (iv) VAM1
binding molecules, to a patient at risk of having or developing
pancreatic cancer, under conditions suitable to promote binding
complex formation between the binding molecule and its target
present in pancreatic tumor neovasculature or a precancerous
lesion; and
[0080] (b) detecting the presence or absence of the binding
complexes; wherein the presence of an increased number of the
binding complexes compared to control is indicative of the presence
of pancreatic cancer in the patient or indicates a risk of
pancreatic cancer development in the patient.
[0081] As disclosed herein, the inventors have discovered that
Thy-1 and the other recited markers are expressed in pancreatic
neovasculature but not in normal pancreatic vasculature, and thus
can be used to detect pancreatic cancer. The methods of the
invention provide early detection of pancreatic cancer or
precancerous lesions, thus allowing earlier clinical intervention
in treatment and substantially improved clinical outcome. Thus, the
methods may further comprise treating the patient based on the
presence of an increased number of binding complexes compared to
control.
[0082] Any suitable control can be used, including but not limited
to comparison to a number of binding complexes identified in a
subject or population of subjects known to not have pancreatic
cancer or precancerous lesions. Any amount of Thy-1 or the other
markers above control levels may indicate presence of pancreatic
cancer in the patient or indicates a risk of pancreatic cancer
development in the patient. In various embodiments, the increase is
binding complexes is at least 10% above control, and preferably at
least 25%, 50%, 100%, or more above control.
[0083] The methods can be used with patients that are at risk of
having pancreatic cancer, based on symptoms they presently have,
including but not limited to abdominal pain, lower back pain,
heartburn, significant weight loss, Trousseau sign, pulmonary
embolisms, new onset of diabetes in elderly individuals, and
jaundice. The methods can also be used with patients who are at
risk of developing pancreatic cancer. Such risk factors include,
but are not limited to, a family history of pancreatic cancer;
genetic disorders including but not limited to autosomal recessive
ataxia-telangiectasia, autosomal dominantly inherited mutations in
the BRCA2 gene and/or PALB2 gene, Peutz-Jeghers syndrome due to
mutations in the STK11 tumor suppressor gene, hereditary
non-polyposis colon cancer (Lynch syndrome), familial adenomatous
polyposis, familial atypical multiple mole melanoma-pancreatic
cancer syndrome (FAMMM-PC) due to mutations in the CDKN2A tumor
suppressor gene; cigarette smoking, age 60 or above, and
obesity.
[0084] According the methods of this aspect of the invention, the
Thy-1 or other binding molecule is detectable. Any suitable
technique for making the binding molecule detectable can be used,
including wherein the detectable Thy-1 binding molecule comprises a
detectable imaging agent selected from the group consisting of a
radioactive agent (e.g., radioiodine (125I, 131I); technetium;
yttrium; 35S or 3H) or other radioisotope or radiopharmaceutical; a
contrast agent (e.g., gadolinium; manganese; barium sulfate; an
iodinated or noniodinated agent; an ionic agent or nonionic agent);
a magnetic agent or a paramagnetic agent (e.g., gadolinium,
iron-oxide chelate); liposomes (e.g., carrying radioactive agents,
contrast agents, or other imaging agents); nanoparticles; a
positron emitting isotope for PET scanner, MRI contrast agents, and
ultrasound agents (e.g., microbubble). In one embodiment, the Thy-1
or other binding molecule is present on the surface of a
microbubble, including but not limited to the Thy-1 or other
binding molecule containing microbubbles of the invention disclosed
herein.
[0085] The binding molecules can be administered by any suitable
technique, including but not limited to parenterally,
transmucosally (orally, nasally, or rectally) or transdermally.
Parenteral administration includes, but is not limited to,
intravenous, intra-arteriole, intramuscular, intradermal,
subcutaneous, intraperitoneal, intraventricular, and intracranial
administration. In one embodiment administration of the binding
molecules is parenteral, via intravenous injection. This embodiment
is particularly preferred when the binding molecules are present on
microbubbles, such as the microbubbles of the present invention.
Following administration to the patient (such as by intravenous
injection), the targeted microbubbles accumulate at pancreatic
sites that over-express Thy-1 or other marker, causing a local
increase in the ultrasound imaging signal. In a preferred
embodiment, microbubbles with an average diameter of between about
1 .mu.m and 5 .mu.m, 1 .mu.m and 4 .mu.m, 1 .mu.m and 3 .mu.m, 1
.mu.m and about 2 .mu.m, 2 .mu.m and 5 .mu.m, 2 .mu.m and 4 .mu.m,
2 .mu.m and 3 .mu.m, 3 .mu.m and 5 .mu.m, 3 .mu.m and 4 .mu.m, or
about as 1 .mu.m, 2 .mu.m, 2.5 .mu.m, 3 .mu.m, 3.5 .mu.m or 4 .mu.m
on average are intravenously injected. Due to their small size, the
microbubbles stay predominantly within the vascular compartment
after intravenous injection. Thus, the microbubbles can be used,
for example, to exclusively detect vascular endothelial cell
associated molecular markers that are present in early stage
cancers or precancerous lesions.
[0086] The "conditions suitable to promote binding complex
formation" used in the methods of the invention will depend on the
means by which the binding molecule is labeled, the type of assay
(i.e.: in vitro or in vivo), and all other relevant factors, and
can be determined by one of skill in the art based on the teachings
herein.
[0087] Detection of binding molecule binding complexes with present
in pancreatic neovasculature can be by any suitable means, and will
depend at least in part on the means by which the binding molecule
is made detectable. In one embodiment, pancreatic biopsies can be
obtained and detectably labeled binding molecules can be contacted
to a biopsy sample under conditions suitable to promote binding
complex formation to target in the biopsy sample, and binding
complexes can be detected via immunohistochemistry or other
suitable technique. In another embodiment, the detection means is
non-invasive, meaning that detection of the binding molecules does
not require obtaining any type of sample (blood, tissue, bone,
urine, or saliva) from the patient. Methods for non-invasive
detection of binding molecules include magnetic resonance imaging
(MRI), positron-emission tomography (PET), single photon emission
tomography (SPECT) and ultrasound imaging, including,
high-intensity focused ultrasound (HIFU) and contrast-enhanced
ultrasound (CEUS).
[0088] In a preferred embodiment, binding molecules are present on
the surface of microbubbles and are administered to a patient under
conditions suitable to promote binding complex formation between
target in the pancreas and the binding molecules. Detection then
comprises using non-invasive contrast-enhanced ultrasound, to
determine the presence or absence of the binding complexes, wherein
the binding complex is only detectable in the presence of target
and wherein the presence of binding complexes is indicative of the
presence of pancreatic cancer, or a risk of pancreatic cancer in
the patient. Non-invasive contrast-enhanced ultrasound (as well as
other non-invasive detection means) can be performed so as to
localize detection to the pancreas. Specifics of methods for
performing the non-invasive detection techniques disclosed herein
are well known to those of skill in the art.
[0089] In one embodiment, an increased number of the binding
complexes compared to control is indicative of the presence of
pancreatic cancer in the patient. In this embodiment, the results
can be used to help direct patient treatment, including but not
limited to aggressive chemotherapy and/or radiation treatments
and/or surgical resection of the tumor. In one embodiment, the
pancreatic cancer is pancreatic ductal adenocarcinoma. Median
survival of patients with pancreatic ductal adenocarcinoma (PDAC)
is less than one year; and thus the earlier detection provided by
the methods of the present invention allows earlier surgical
resection, which offers the best hope for longer patient
survival.
[0090] In one embodiment, the presence of the binding complexes is
indicative of the presence of precancerous lesions, and thus
indicative of a risk of pancreatic cancer development in the
patient. Pre-cancerous lesions are associated with a significantly
increased risk of cancer. Non-limiting examples include cervical
squamous intraepithelial lesion, ductal carcinoma in situ, Bowen's
disease, colon polyps, prostatic intraepithelial neoplasia, and
pancreatic intraepithelial neoplasia. In a further embodiment, the
pre-cancerous cells are pre-cancerous pancreatic cells, including
but not limited to pancreatic intraepithelial neoplasia form
(PanIN) 1A-B, PanIN 2, and PanIN 3. These terms as defined as
follows:
[0091] PanIN-1A: Pyloric gland metaplasia, goblet cell metaplasia,
mucinous hypertrophy, flat duct lesion without atypia, mucinous
ductal hyperplasia, simple hyperplasia, mucinous cell hyperplasia,
flat ductal hyperplasia, non-papillary epithelial hypertrophy.
[0092] PanIN-1B: Papillary hyperplasia, papillary duct lesion
without atypia, and ductal hyperplasia.
[0093] PanIN-2: Atypical hyperplasia, papillary duct lesion with
atypia, low-grade dysplasia, and some cases of moderate dysplasia.
Mucous metaplasia and pyloric gland metaplasia commonly involve
small branch ducts or extend into lobules surrounding PanIN in
ducts. Such involvement has been called adenomatoid or adenomatous
hyperplasia, especially when the change dominates involvement of
ductal epithelium. It is regarded as part of the spectrum of
PanIN-1.
[0094] PanIN-3: Carcinoma in situ, intraductal carcinoma,
high-grade dysplasia, severe dysplasia, and some cases of moderate
dysplasia.
[0095] In this embodiment, the results can be used to help direct
patient treatment, including but not limited to aggressive
chemotherapy and/or radiation treatments to limit development of
the lesion into a tumor.
[0096] In one embodiment, an increased number of binding complexes
compared to control, wherein the binding complexes are multifocal
(i.e.: scattered throughout the pancreas) indicates the presence of
a precancerous lesion in the patient. In another embodiment, an
increased number of binding complexes compared to control, wherein
the binding complexes are focused in the pancreas (i.e.:
predominately in a single location within the pancreas) indicates
the presence of pancreatic cancer, such as PDAC.
[0097] In a sixth aspect the present invention provides methods for
determining efficacy of pancreatic cancer therapy in a patient,
comprising:
[0098] (a) administering a detectable Thy-1 binding molecule to a
patient undergoing or who has previously undergone pancreatic
cancer therapy, under conditions suitable to promote binding of the
Thy-1 binding molecule to Thy-1 in the pancreatic tumor
neovasculature to form a binding complex; and
[0099] (b) detecting the presence or absence of binding complexes;
wherein the presence or absence of binding complexes is indicative
of the efficacy of the anti-cancer therapy in the patient.
[0100] In a seventh aspect, the present invention provides methods
for determining efficacy of pancreatic cancer therapy in a patient,
comprising:
[0101] (a) administering a detectable binding molecule selected
from the group consisting of (i) MMRN1 binding molecules. (ii) MRC2
binding molecules, (iii) NRP1 binding molecules, and (iv) VCAM1
binding molecules, to a patient undergoing or who has previously
undergone pancreatic cancer therapy, under conditions suitable to
promote binding complex formation between the binding molecule and
its target present in pancreatic tumor neovasculature to form a
binding complex; and
[0102] (b) detecting the presence or absence of the binding
complexes; wherein the presence or absence of binding complexes is
indicative of the efficacy of the anti-cancer therapy in the
patient.
[0103] All embodiments and combinations of embodiments of the first
through fifth aspects of the invention can be used in these sixth
and seventh aspects. The methods of these aspects of the invention
can be used, for example, to assess the growth, regression, or
metastasis of the pancreatic tumor, and thus whether a patient
being treated for pancreatic cancer is benefitting from therapy, or
for monitoring patients who have completed treatment for recurrence
of cancer.
[0104] According the methods of this aspect of the invention, the
Thy-1 or other binding molecule is detectable. Any suitable
technique for making the binding molecule detectable can be used,
including wherein the detectable Thy-1 binding molecule comprises a
detectable imaging agent selected from the group consisting of a
radioactive agent (e.g., radioiodine (125I, 131I); technetium;
yttrium; 35S or 3H) or other radioisotope or radiopharmaceutical; a
contrast agent (e.g., gadolinium; manganese; barium sulfate; an
iodinated or noniodinated agent; an ionic agent or nonionic agent);
a magnetic agent or a paramagnetic agent (e.g., gadolinium,
iron-oxide chelate); liposomes (e.g., carrying radioactive agents,
contrast agents, or other imaging agents); nanoparticles; a
positron emitting isotope for PET scanner, MRI contrast agents, and
ultrasound agents (e.g., microbubble). In one embodiment, the Thy-1
or other binding molecule is present on the surface of a
microbubble, including but not limited to the Thy-1 or other
binding molecule containing microbubbles of the invention disclosed
herein.
[0105] The binding molecules can be administered by any suitable
technique, including but not limited to parenterally,
transmucosally (orally, nasally, or rectally) or transdermally.
Parenteral administration includes, but is not limited to,
intravenous, intra-arteriole, intramuscular, intradermal,
subcutaneous, intraperitoneal, intraventricular, and intracranial
administration. In one embodiment administration of the binding
molecules is parenteral, via intravenous injection. This embodiment
is particularly preferred when the binding molecules are present on
microbubbles, such as the microbubbles of the present invention.
Following administration to the patient (such as by intravenous
injection), the targeted microbubbles accumulate at pancreatic
sites that over-express Thy-1 or other marker, causing a local
increase in the ultrasound imaging signal. In a preferred
embodiment, microbubbles with an average diameter of between about
1 .mu.m and 5 .mu.m, 1 .mu.m and 4 .mu.m, 1 .mu.m and 3 .mu.m, 1
.mu.m and about 2 .mu.m, 2 .mu.m and 5 .mu.m, 2 .mu.m and 4 .mu.m,
2 .mu.m and 3 .mu.m, 3 .mu.m and 5 .mu.m, 3 .mu.m and 4 .mu.m, or
about as 1 .mu.m, 2 .mu.m, 2.5 .mu.m, 3 .mu.m, 3.5 .mu.m or 4 .mu.m
on average are intravenously injected. Due to their small size, the
microbubbles stay predominantly within the vascular compartment
after intravenous injection. Thus, the microbubbles can be used,
for example, to exclusively detect vascular endothelial cell
associated molecular markers that are present in early stage
cancers or precancerous lesions.
[0106] The "conditions suitable to promote binding complex
formation" used in the methods of the invention will depend on the
means by which the binding molecule is labeled, the type of assay
(i.e.: in vitro or in vivo), and all other relevant factors, and
can be determined by one of skill in the art based on the teachings
herein).
[0107] Detection of binding molecule binding complexes with present
in pancreatic neovasculature can be by any suitable means, and will
depend at least in part on the means by which the binding molecule
is made detectable. In one embodiment, pancreatic biopsies can be
obtained and detectably labeled binding molecules can be contacted
to a biopsy sample under conditions suitable to promote binding
complex formation to target in the biopsy sample, and binding
complexes can be detected via immunohistochemistry or other
suitable technique. In another embodiment, the detection means is
non-invasive, meaning that detection of the binding molecules does
not require obtaining any type of sample (blood, tissue, bone,
urine, or saliva) from the patient. Methods for non-invasive
detection of binding molecules include magnetic resonance imaging
(MRI), positron-emission tomography (PET), single photon emission
tomography (SPECT) and ultrasound imaging, including,
high-intensity focused ultrasound (HIFU) and contrast-enhanced
ultrasound (CEUS).
[0108] In a preferred embodiment, binding molecules are present on
the surface of microbubbles and are administered to a patient under
conditions suitable to promote binding complex formation between
target in the pancreas and the binding molecules. Detection then
comprises using non-invasive contrast-enhanced ultrasound, to
determine the presence or absence of the binding complexes, wherein
the binding complex is only detectable in the presence of target
and wherein the presence of binding complexes is indicative of the
presence of pancreatic cancer, or a risk of pancreatic cancer in
the patient. Non-invasive contrast-enhanced ultrasound (as well as
other non-invasive detection means) can be performed so as to
localize detection to the pancreas. Specifics of methods for
performing the non-invasive detection techniques disclosed herein
are well known to those of skill in the art.
Example 1
Ultrasonic Molecular Imaging of Thymocyte Differentiation Antigen 1
for Pancreatic Ductal Adenocarcinoma Detection
[0109] Background and Aims:
[0110] Median survival of patients with pancreatic ductal
adenocarcinoma (PDAC) is less than one year; earlier detection that
allows surgical resection offers the best hope for longer survival.
Ultrasound molecular imaging has the potential to detect
neoangiogenesis markers in cancer at the molecular level with high
sensitivity. We sought to identify a novel molecular marker of
neoangiogenesis in PDAC and assess its potential as an imaging
target for PDAC detection using ultrasound.
[0111] Methods:
[0112] Proteomic analysis followed by immunohistochemistry was
performed on tissues from patients with PDAC, chronic pancreatitis,
and normal pancreas to identify molecular markers differentially
expressed on the neovasculature of PDAC. Thymocyte
[0113] Differentiation Antigen 1 (Thy1) was identified as a novel
neoangiogenesis biomarker of PDAC. To assess its utility for in
vivo imaging, binding of a human Thy1-targeted ultrasound contrast
agent was tested in cell culture in a flow chamber assay and in
vivo in a novel orthotopic PDAC xenograft model in mice expressing
human Thy1 on its neovasculature.
[0114] Results:
[0115] Vascular Thy1 expression was significantly higher in PDAC
compared to chronic pancreatitis (P=0.007) and normal tissue
(P<0.0001). Thy1-targeted ultrasound contrast agent showed
significantly increased attachment to Thy1-positive cells compared
to negative control cells (P=0.008). In vivo, Thy1-targeted
ultrasound molecular imaging signal significantly increased in
human Thy1-positive PDAC xenografts.
[0116] Conclusion:
[0117] Our study represents the discovery and in vivo validation of
a neoangiogenesis target, Thy1, for ultrasound molecular imaging of
PDAC. The development of a specific imaging agent along with the
identification of Thy1 as a novel biomarker provides potential
clinical utility for the diagnosis and management of human
PDAC.
INTRODUCTION
[0118] Pancreatic ductal adenocarcinoma (PDAC) is the fourth
leading cause of cancer death in both women and men in the USA (1).
The American Cancer Society estimated 43,920 new diagnoses of PDAC
and 37,390 deaths from this cancer in 2012 in the USA. Survival
from pancreas cancer is stage dependent. Because the disease is
most frequently detected at advanced tumor stages, patients
diagnosed with PDAC have a median survival of less than one year
and only 5% of patients survive five years after diagnosis (2, 3).
Unfortunately, current chemotherapy and radiotherapy approaches
offer only moderate survival benefits and surgery for localized
disease is only possible in 15-20% of patients at the time of
diagnosis (4, 5). Therefore, earlier detection of PDAC that allows
potentially curable surgical resection offers our best hope to
improve patient survival (6). One potential strategy for earlier
detection of cancer, including PDAC, involves screening moderate
and high risk patients with a highly accurate and inexpensive blood
biomarker test (or combination of biomarkers) followed by a
second-level, imaging-based test to confirm a positive biomarker
result and anatomically localize the cancer (7). Serum CA-19-9,
currently the only clinically used blood biomarker, lacks the
sensitivity and, more importantly, the specificity needed to detect
early-stage PDAC; active research to discover more accurate
blood-based or saliva-based biomarkers is underway (8). Imaging
tests are also limited in accuracy for early detection of
pancreatic cancer. Currently used imaging techniques, including
abdominal computed tomography (CT), magnetic resonance imaging and
cholangiopancreatography (MRI/MRCP), retrograde
cholangiopancreatography (ERCP), as well as trans-abdominal and
endoscopic ultrasound (EUS), are often unreliable and non-specific
in detecting early stage PDAC. This is particularly true regarding
the detection of non-cystic precursor lesions and small size (<1
cm) foci of cancer (9, 10). Therefore, development and testing of
novel imaging approaches for earlier detection of PDAC is
critically needed. The vasculature of cancer, termed
neovasculature, differs from normal blood vessels at the molecular
and protein level (11). Molecularly-targeted contrast-enhanced
ultrasound (henceforth ultrasound molecular imaging) is a promising
new imaging technique with the potential to detect molecular
markers overexpressed in the neovasculature of cancer (12, 13) and
potentially increase the sensitivity and specificity of ultrasound
in detecting early cancer. For ultrasound molecular imaging,
micro-sized gas-filled contrast agents (microbubbles; MB) that are
modified to bind to molecular markers are intravenously injected.
After a few minutes, the MB accumulate at tissue sites that
over-express the molecular target, causing a local increase in the
ultrasound imaging signal and enhancing the ability to see small
lesions (14). Since contrast MB are several microns in size, they
remain in the intravascular space and can thus be used to
exclusively detect and visualize molecular markers over-expressed
on the neovasculature of precursor lesions or early stage cancer.
However, similar to the blood biomarker research mentioned above,
the challenge for ultrasound molecular imaging is to discover and
validate imaging targets that are differentially expressed on the
vasculature of PDAC versus normal pancreatic tissue and benign
diseases, such as chronic pancreatitis, to maximize diagnostic
accuracy in early cancer detection (13).
[0119] In this study, we exemplify a proof-of-principle approach
from discovery to in vivo validation of thymocyte differentiation
antigen 1 (Thy1) as a promising new ultrasound molecular imaging
target in PDAC. We report on the discovery and validation of Thy1
in human tissue samples as a target for PDAC neovasculature
imaging, and describe the cell culture and in vivo testing of
Thy1-targeted contrast MB for ultrasound molecular imaging in a
novel orthotopic mouse PDAC model expressing human Thy1 on its
neovasculature.
Materials and Methods
[0120] The overall experimental set up of this study is summarized
in FIG. 1.
Thy1 Target Identification
[0121] Proteomic analysis was performed on whole tissues from
patients with PDAC (n=5), chronic pancreatitis (n=5), and normal
pancreas from donors (n=10) using a LTQ-Orbitrap.TM. hybrid mass
spectrometer (Thermo Fisher Scientific, Waltham, Mass.) coupled
with a nano-flow HPLC (Eksigent Technologies, Dublin, Calif.) as
previously described 16. A total of 118 proteins were
over-expressed by a factor of 2.0 or more in PDAC compared to
normal pancreas. These were searched against literature reports to
identify proteins associated with tumor neovasculature. To create
an ultrasound molecular imaging approach using contrast MB
targeting the luminal site of PDAC-associated tumor vascular
endothelial cells, putative neovascular proteins were further
triaged based on 1) the highest expression in cancer; 2) lack of or
low expression in chronic pancreatitis tissue; 3) membrane
association of the protein; and 4) assessment of protein expression
in normal tissues using the Human Protein Atlas and/or published
literature in PubMed, with lack of expression in normal organs
being preferred. The highest rated candidate was the membrane
protein Thy1 (17). Selection of Thy1 was further supported by its
association with tumor vascular endothelium as previously described
(18, 19).
Validation of Thy1 Expression in Human Pancreatic Tissues
[0122] Samples used in these studies were collected with Human
Subjects approval at the University of Washington and Stanford
University. Furthermore, a pancreatic tissue microarray was
purchased from USBioMax.TM. (Rockville, Md.) Immunohistochemical
(IHC) analysis of Thy1 expression was performed in pancreatic
tissue obtained from 4 normal patients; 15 primary chronic
pancreatitis tissues (defined as chronic pancreatitis not
associated with PDAC); 21 PDAC; and in a commercial tissue
microarray with 24 normal pancreatic tissues and 175 PDAC cases.
Consecutive tissue sections were stained for the vascular
endothelial cell marker CD31 and for Thy1 using standard techniques
(Supplementary Materials and Methods). All slides were reviewed and
graded by a pathologist, experienced in pancreatic pathology.
Vascular endothelial cell staining of Thy1 was scored with a
semi-quantitative IHC score from 0 to 3+ as previously described
20. In brief, cases with Thy1-staining of less than 5%, 5-32%,
33-67%, and greater than 67% of CD31 positive vessels were scored
as 0, 1+, 2+, and 3+, respectively.
Human Thy1-Expressing Vascular Endothelial Cells
[0123] Murine vascular endothelial (MS1) cells stably expressing
human Thy1 on the cell surface were generated using standard
protocols (Supplementary Materials and Methods). Stably-transfected
cells (selected by incubation with 5 .mu.g/ml puromycin; Sigma, St.
Louis, Mo.) were confirmed for Thy1 expression by flow cytometry
analysis and by immunofluorescence staining using standard
techniques (Supplementary Materials and Methods). For subsequent
flow chamber experiments (see below), two clones with high (clone
1) and low (clone 2) human Thy1 expression were selected. Clone 1
was also used for the generation of a novel human PDAC xenograft
model in mice expressing human Thy1 on its neovasculature (see
below).
Preparation of Microbubble Contrast Agents
[0124] Human Thy1-targeted (MBThy1) and control (MBControl)
contrast MB were prepared by attaching anti-human Thy1 antibody or
isotype-matched control IgG antibody onto the surface of
perfluorocarbon-containing, lipid-shelled MB as described
previously 21 (Supplementary Materials and Methods). Flow cytometry
analysis (incubation of targeted MB with Fluorescein-conjugated
anti-biotin antibody; Jackson ImmunoResearch, 1:200) showed that
the average number of attached antibodies per square micrometer of
the MB surface was approximately 7,600 for both MB types.
Flow Chamber Experiments
[0125] MBThy1 binding to clones 1 and 2, as well as to wild-type
negative control vascular endothelial cells was assessed in cell
culture experiments under flow shear stress conditions, simulating
flow in tumor capillaries, using a previously described protocol
(22, 23) (Supplementary Materials and Methods).
Human Thy1-expressing and Control Orthotopic Pancreatic Ductal
Adenocarcinoma Xenografts in Mice
[0126] The Administrative Panel on Laboratory Animal Care of
Stanford University approved all procedures using laboratory
animals. Human AsPC1 pancreatic ductal adenocarcinoma cells (ATCC,
Manassas, Va.) were cultured to 70-80% confluency in Dulbecco's
modified Eagle's medium (DMEM; supplemented with 10% (vol/vol)
fetal bovine serum; penicillin (100 U/mL); and streptomycin (100
.mu.g/mL); Invitrogen) at 37.degree. C. in 5% CO2 and 95% air
atmosphere before trypsinization. After midline laparotomy, the
pancreas of the mice was exposed, and AsPC1 cells along with clone
1 cells at 1:5 ratio (total of 6.times.10.sup.6 cells, dissolved in
25 .mu.l of Matrigel.TM. containing epidermal growth factor (0.7
ng/mL), insulin-like growth factor (16 ng/ml), and transforming
growth factor-beta (2.3 ng/ml); BD Biosciences, San Jose, Calif.)
co-injected into the body or tail of the pancreas in 25 female nude
mice (6-8 weeks old; Charles River, Wilmington, Mass.) into the
body or tail of the pancreas. In negative control animals (n=14),
AsPC1 cells alone (n=7) or combined with wild-type cells (n=7) were
co-injected at the above-mentioned ratio. The abdomen was then
closed by layers. Orthotopic xenografts were allowed to grow
between 7 and 21 days to yield a spectrum of different tumor sizes
with volumes ranging between 100 and 1304 mm.sup.3 (mean, 403
mm.sup.3) as measured by B-mode ultrasound and using the formula
for a prolate ellipsoid
(.pi./6.times.length.times.width.times.height).
In Vivo Ultrasound Molecular Imaging of Pancreatic Ductal
Adenocarcinoma Xenografts
[0127] All ultrasound molecular imaging was performed using a
dedicated small animal ultrasound machine; the technical
specifications are detailed in the Supplementary Materials and
Methods. In all PDAC xenografts, intra-animal comparisons of
ultrasound imaging signals were performed following injection of
both MBThy1 and MBControl in a randomized order during the same
imaging session. Four minutes following tail vein injection of
either MBThy1 or MBcontrol (5.times.10.sup.7; bolus injected within
2 seconds through a tail vein catheter), 250 imaging frames were
acquired of each tumor within 25 seconds (22, 23). This was
followed by a one-second destruction pulse to destroy all MB in the
field-of-view (destruction pulse of 3.7 MPa; transmit power, 100%;
mechanical index, 0.63). Nine seconds after the destruction pulse,
another 250 imaging frames were acquired to capture the influx of
freely circulating MB. After a waiting time of at least 30 minutes
to allow MB to clear from previous injection (24, 25), this imaging
sequence was repeated following intravenous injection of
5.times.107 MB of the second MB type of either MBThy1 or MBControl,
with the transducer maintained at the same anatomical location.
Furthermore, an in vivo competition assay with anti-human Thy1
antibodies was performed using mouse anti-human Thy1 antibody (100
.mu.g; eBioscience; San Diego, Calif.) as described previously (22,
23).
Imaging Analysis of In Vivo Ultrasound Molecular Imaging
[0128] Ultrasound molecular imaging was analyzed offline using
dedicated software with motion compensation capabilities (VevoCQ,
Visualsonics; Toronto, Canada). The imaging signal (expressed in
arbitrary units, a.u.) from attached MB was defined as the
difference between pre- and post-destruction imaging signals as
described 23. Regions of interest were draws over the different
tumors and over adjacent normal pancreas tissue by one reader,
blinded to the types of MB (MBThy1 vs. MBControl) and tumor
(positive vs. control tumors).
Ex Vivo Analysis of Pancreatic Ductal Adenocarcinoma Xenografts
[0129] To confirm expression of human Thy1 on vascular endothelial
cells of the tumor neovasculature in mice, all tumors were excised
after ultrasound imaging and immunostained for human Thy1 and mouse
CD31 according to standard protocols (Supplementary Materials and
Methods); AsPC1 tumors were also stained for murine Thy1.
Statistical Analysis
[0130] All continuous measurements were expressed as
means.+-.standard deviation. Differences in the Thy1 expression
levels between groups of patient samples were tested for
statistical significance using the Mann-Whitney test. Empirical
receiver operating characteristic (ROC) curves were used to
determine the sensitivity and specificity of Thy1 in separating
PDAC from non-cancer controls. The two-sample Wilcoxon test was
used for pair-wise comparisons of measurements in flow chamber
experiments. For comparison between MBThy1 vs. MBControl in the
same tumor and for competition assays, the one-sample paired
Wilcoxon test was used. The two-sample t-test was used for
comparisons in multiple groups (Thy1-positive and two control tumor
types). For assessing associations between Thy1-expression levels
and cell attachment, Spearman's correlation coefficient was
estimated and the corresponding P-value was obtained based on
Fisher's transformation. Statistical analyses of Thy1 IHC scores
were performed using GraphPad Prism (La Jolla, Calif.); the
remainder of the statistical analyses was performed with R 2.10.1
with a significance level of 0.05.
Results
[0131] Validation of Thy1 Expression in Human Pancreatic Tissues To
verify the expression of Thy1 in PDAC-associated neovasculature in
humans, IHC was performed on pancreatic tissue samples from normal
pancreas (n=28), primary chronic pancreatitis (n=15) and PDAC
patients (n=199). The expression of Thy1 was essentially restricted
to the neovasculature associated with PDAC, with occasional
staining of the peritumoral stromal compartment. There was no
expression within the neoplastic epithelium in most of the cases;
therefore, only vascular staining (guided by CD31 staining) was
evaluated. The expression of Thy1 was significantly increased in
the PDAC patients (score, 2.1.+-.0.1) and was minimal in normal
controls (0.5.+-.0.1; P<0.0001) and primary chronic pancreatitis
(0.6.+-.0.15; P=0.007) (FIGS. 2A-C). Considering IHC scores of 2+
or 3+ as positive staining, 81% of PDAC cases stained positive for
Thy1 while normal pancreas and chronic pancreatitis cases were
positive in 11% and 7%, respectively. ROC analysis indicated that
Thy1 neovascular immunostaining could distinguish PDAC from normal
and chronic pancreatitis tissues with 90% specificity and 81%
sensitivity [area under curve (AUC) value=0.89].
Microbubble Binding in Flow Chamber Experiments
[0132] Mouse neovascular endothelial cells do not express human
Thy1, thus to recapitulate the protein expression of Thy1 in human
PDAC neovasculature, we created two mouse vascular endothelial cell
lines with different expression levels of human Thy1 (clone 1 and
clone 2) and confirmed the Thy1 expression levels by fluorescence
microscopy (FIG. 3A), and FACS analysis (FIG. 3B, C). Table 5
summarizes binding of MBThy1 and MBControl to clone 1 (high human
Thy1 expression), clone 2 (low human Thy1 expression) and to
wild-type vascular endothelial cells (no Thy1 expression) in flow
chamber experiments. Binding of MBThy1 to both clone 1 (P=0.008)
and clone 2 (P=0.01) was significantly higher than binding to
wild-type endothelial cells (FIG. 4). Blocking of Thy1 receptors
with anti-Thy1 antibodies resulted in significantly decreased
MBThy1 attachment (P=0.03), confirming binding specificity of
MBThy1 to human Thy1. In contrast, compared to MBThy1, MBControl
only demonstrated background attachment to all three cell types
without statistically significant difference of MBControl
attachment among the three different cell types (P>0.5). The
number of attached MBThy1 highly correlated with the expression
levels of Thy1 on vascular endothelial cells (rho=0.92,
P<0.001).
TABLE-US-00005 TABLE 5 Binding of Microbubbles to Vascular
Endothelial (MS1) Cells with Different Expression Levels of Human
Thy1. MB.sub.Thy1 MB.sub.Thy1 MB.sub.Control after blocking High
Thy1 expression (clone 1) 4.4 .+-. 2.0 0.8 .+-. 0.3 0.7 .+-. 0.3
Low Thy1 expression (clone 2) 1.8 .+-. 0.5 0.9 .+-. 0.5 NA No Thy1
expression (wild-type) 0.6 .+-. 0.4 0.6 .+-. 0.2 NA Data are means
.+-. standard deviation of attached microbubbles (MB) per cell.
Note blocking was performed by incubating cells with anti-human
Thy1 antibody.
In Vivo Ultrasound Molecular Imaging in Pancreatic Ductal
Adenocarcinoma Xenografts in Mice
[0133] To prove binding of MBThy1 to human Thy1 in vivo, we
developed a novel orthotopic PDAC xenograft mouse model by
co-injecting AsPC1 cells with mouse vascular endothelial cells
expressing human Thy1. As negative control tumors, either AsPC1
cells only or AsPC1 cells mixed with human Thy1-negative
endothelial cells were injected orthotopically into the pancreas in
mice. Growth rates among the different tumor types were not
significantly different (P>0.05) and tumors over a broad size
spectrum between 100 and 1304 mm3 were images. Imaging enhancement
of Thy1-positive tumors (n=25) was significantly increased
(P<0.001) following injection of MBThy1 compared to MBControl
(FIG. 5; Table 6). Control xenografts (n=14) and normal pancreas
tissue adjacent to the three different tumor types (n=39) showed
significantly lower imaging signal using MBThy1 (Table 6). In vivo
competition assay (n=7) using antihuman Thy1 antibodies resulted in
a significant decrease (P=0.008) of human Thy1-targeted imaging
signal in Thy1-positive xenografts, which further confirmed binding
specificity of MBThy1 to human Thy1 in vivo.
TABLE-US-00006 TABLE 6 Summary of Ultrasound Molecular Imaging
Signals in Three Different Types of Orthotopic Pancreatic Ductal
Adenocarcinoma Xenografts and Control Pancreas Tissue in Mice using
Human Thy1- targeted Microbubbles (MB.sub.Thy1) and Control
Microbubbles (MB.sub.Control). P (MB.sub.Thy1 vs. MB.sub.Thy1
MB.sub.Control MB.sub.Control) Thy1-positive tumors (n = 25) 7.7
.+-. 2.3 1.4 .+-. 2.2 <0.001 Thy1-negative control tumors 1.9
.+-. 1.8 1.9 .+-. 1.7 1.0 (n = 7) AsPC1 only control tumors 1.7
.+-. 1.6 1.4 .+-. 0.6 0.68 (n = 7) Adjacent normal pancreas tissue
1.7 .+-. 1.6 1.6 .+-. 2.2 0.37 (n = 39) P (Thy1-postive vs. Thy1-
0.001 0.54 negative tumors) P (Thy1-positive vs. AsPC1 <0.001
0.06 only tumors) P (Thy1-negative vs. AsPC1 0.82 0.56 only tumors)
P (Thy1-positive vs. normal <0.001 0.18 pancreas) P
(Thy1-negative vs. normal 0.81 0.74 pancreas) P (AsPC1 tumors vs.
normal 0.98 0.69 pancreas) Data are means .+-. standard deviation
of ultrasound molecular imaging signal in arbitrary units. AsPC1 is
a human pancreatic ductal adenocarcinoma cell line.
[0134] After imaging, xenografts were excised and stained for human
Thy1 and mouse
[0135] CD31. Merged immunofluorescent images confirmed
overexpression of human Thy1 on the neovasculature in Thy1-positive
xenografts while there was no human or mouse Thy1 expression
observed in Thy1-negative and AsPC1-only xenografts.
Discussion
[0136] There is a great need for inexpensive and highly accurate
imaging of lethal cancers such as PDAC. Earlier detection of cancer
is limited, in part, by the availability and cost of the diagnostic
test. Ultrasound fulfills many prerequisites for becoming a
promising imaging tool for early cancer detection: It is
noninvasive and inexpensive compared to other imaging modalities;
does not use ionizing irradiation; has a very high spatial and
temporal (real-time exam) resolution (26-28); provides deep tissue
penetration (e.g., compared to optical based approaches); and is
routinely available in almost all clinical imaging departments
worldwide. Several morphological imaging criteria of pre-invasive
or early PDAC have been described on endoscopic ultrasound,
including parenchymal heterogeneity, echogenic foci, and
hypoechogenic nodules (29). However, these findings are very
subtle, require interpretation by expert endosonographers, and are
often nonspecific compared to chronic pancreatitis, and therefore
must be interpreted with caution (29). Indeed, in a recent study
the inter-observer agreement of (17) expert endosonographers
interpreting endoscopic US scans in high risk patients for PDAC was
only fair to poor and did not improve even on consensus
interpretations (30). Adding molecular imaging capabilities to
future clinical ultrasound scans may substantially increase the
diagnostic accuracy of ultrasound imaging in detecting early
neoplastic pancreatic lesions at little additional cost to the
procedure (12). However, this requires identification of molecular
imaging targets that allow differentiation of normal from
precancerous or malignant pancreatic tissue. Furthermore, these
molecular markers must be cancer-specific enough to allow
differentiation of malignant from benign lesions to minimize
false-positive results, thereby decreasing the number of
unnecessary biopsies or surgical interventions.
[0137] The goal of this proof-of-principle study was to discover
and validate a PDAC associated protein that can be used as an
imaging target for ultrasound molecular imaging of the pancreas and
that may increase diagnostic accuracy of ultrasound in earlier
detection of PDAC. Since most current ultrasound contrast agents
remain exclusively within the vascular compartment, molecular
imaging targets for ultrasound need to be expressed on the luminal
site of vascular endothelial cells of the pancreatic
neovasculature. Through quantitative proteomic analysis and several
prioritizing steps, we identified Thy1, a membrane protein, as a
promising new tissue marker for ultrasound molecular imaging
expressed on PDAC-associated neovasculature.
[0138] Thy1, also known as cluster of differentiation 90 (CD90), is
a cell-surface glycoprotein that belongs to the immunoglobulin-like
supergene family (31). While Thy1 was originally described as a
marker for thymocyte differentiation in mice (32), it was later
found to be expressed on various other tissues 15 and up-regulated
on the surface of newly formed blood vessels (33). Our
immunohistochemical validation study demonstrated positive Thy1
expression on the neovasculature of 81% of human pancreatic
adenocarcinoma samples while there was minimal Thy1 staining in
normal pancreatic tissue. Chronic pancreatitis samples showed
marginally higher Thy1 staining than normal pancreas, but
significantly lower Thy1 staining than PDAC. Some Thy1 staining in
chronic pancreatitis cases is expected because Thy1 expression had
been associated with inflammatory tissues in previous studies. For
example, in rat models of inflammation including a renal ischemia
model following renal artery ligation, and in a balloon injury
model of the carotid arteries, Thy1 has been shown to be
overexpressed on angiogenic vessels (37).
[0139] In that study, vascular microvessel exposure with necrosis
factor-.alpha. and interleukin-1.beta. stimulated cellular Thy1
expression, suggesting that inflammatory cytokines may increase
Thy1 expression (37). Chronic pancreatitis is characterized by
fibroinflammatory changes to the pancreatic tissue with variable
extents of superimposed acute inflammatory changes depending on the
etiology and the stage of the disease (38). Such active
inflammation may have stimulated Thy1 expression in some of our
chronic pancreatic cases. After identification and validation of
Thy1 as a promising molecular imaging target for PDAC, we designed
a new contrast MB targeted to human Thy1. In flow chamber
experiments that simulated shear stress flow in tumor capillaries,
we confirmed binding specificity of MBThy1 to Thy1-positive
vascular endothelial cells and showed good correlation between the
number of cell-attaching contrast MB and the number of Thy1
receptors expressed on the cell surface of vascular endothelial
cells as assessed by flow cytometry. To further test the
performance of MBThy1 to image human Thy1 expression in vivo, an
animal model expressing human Thy1 on the tumor vasculature was
needed. Since in animal xenograft models the tumor vasculature from
sprouting angiogenic vessels is derived from the host (e.g., murine
vessels in xenografts in mice), we expected that human Thy1 would
not be expressed on tumor angiogenic vessels after injection of
human PDAC cells into the mice Immunohistochemical analysis of
several subcutaneous human PDAC xenografts (using human AsPC1,
CaPan2, and PANC1 pancreatic adenocarcinoma cells) confirmed no
human or murine Thy1 expression on the tumor vasculature. This
necessitated the design of a novel mouse PDAC model with tumor
vessels expressing the human Thy1 receptor on the vascular cell
surface. Embedding spheroids containing human umbilical vein
endothelial cells in a Matrigel-fibrin matrix has been previously
shown to generate a functional three-dimensional human vascular
network in severe combined immunodeficiency mice (39, 40). We
modified this approach by directly co-injecting mouse vascular
endothelial MS1 cells stably expressing human Thy1 along with human
PDAC cells embedded in a Matrigel matrix enriched with growth
factors. MS 1 cells are murine pancreatic microvasculature cells
transduced with a temperature-sensitive simian virus 40 large T
antigen (41). When injected into immunocompromised mice, these
cells form benign, maximum 2-mm3 hemangiomas (41). In our study,
coinjection of AsPC1 cells along with MS1 cells resulted in
neovasculature with Thy1-expressing MS1 cells aligning along newly
forming tumor vessels. This was confirmed by ex vivo
immunofluorescence staining of Thy1-positive tumors which showed
co-localization of Thy1 with the vascular endothelial cell marker
CD31. Negative control tumors had no Thy1 staining. To the best of
our knowledge, this mouse model is the first to show that a human
neoangiogenesis receptor can be expressed on the neovasculature of
a murine tumor model by using a straight forward co-injection
technique of modified vascular endothelial and tumor cells along
with tissue matrix and growth factors. In the future, this new
mouse model may provide versatility for in vivo testing of
neoangiogenesis-targeted ultrasound contrast agents in mice by
simply replacing the human Thy1 gene by any other human-specific
receptor gene. In our study, we confirmed feasibility of in vivo
human Thy1 visualization with ultrasound imaging in this new animal
model. In orthotopically implanted Thy1-positive tumors in the
pancreas, injection of MBThy1 resulted in an about 4-5.5-fold
higher imaging signal compared to control conditions. While the
goal of our study was not to determine the smallest detectable size
of PDAC, Thy1 expressing tumors down to a size of 100 mm.sup.3
could be visualized with ultrasound molecular imaging.
[0140] In conclusion, our proof-of-principle study illustrates the
development and testing process of neovascular protein target
discovery and validation in human pancreatic tissues to testing of
in vivo ultrasound molecular imaging properties of new human
Thy1-targeted microbubble in a novel orthotopic human PDAC model in
mice that expresses human Thy1 on angiogenic vessels.
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Supplemental Material and Methods
Immunohistochemistry of Thy1 and CD31 Expression
[0184] Immunohistochemistry was performed on standard serial
sections of paraffin-embedded pancreatic tissue slices and
microarrays. Briefly, prior to incubation with the primary
antibody, sections were pre-treated with CC1 antigen retrieval
solution (Ventana, Tucson, Ariz.). Thereafter, primary antibody to
human CD31 (no dilution; Ventana Medical Systems, catalog 760-4378)
and human Thy1 (1:100 dilution; Novus Biologicals, catalog
NBP1-42068) was applied at 37.degree. C. for 16 min followed by
counter staining by Hematoxylin. Labeling was performed on an ES
automatic immunohistochemical stainer (Ventana Medical Systems).
Slides were imaged using Aperio Imagescope.
Murine Vascular Endothelial Cells Stably Expressing Human Thy1
[0185] The human Thy1 DNA sequence
(gi|224589802:c119294246-119288655) was first optimized for
mammalian codon usage as described.sup.1 and then synthesized into
double-stranded DNA. Restriction sites, XhoI (5') and Nhe-I (3')
were added onto the amplified Thy1 double-stranded DNA using
primers. The flanked Thy1 sequence was then cloned into
pCR-BluntII-Topo.TM. vector (Invitrogen, Carlsbad, Calif.),
amplified by colony PCR (one-step inactivation of chromosomal genes
in Escherichia coli K-12 using PCR products), and verified by
sequencing. Flanked (Xho-I (5') and Nhe-I (3')) Thy1 was then
subcloned into pcDNA3.1 (Invitrogen), modified by substitution of
CMV for an ubiquitin promoter and neomycin for puromycin resistance
selection gene. Thy1-pcDNA was amplified in Escherichia coli and
isolated using the Pure-Yield.TM. Maxi Prep Kit (Promega, Madison,
Wis.) according to manufacturer instructions.
[0186] Murine pancreatic vascular endothelial cells [MS 1; ATCC,
Manassas, Va.; cultured in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% fetal bovine serum, penicillin (100
U/mL), and streptomycin (100 .mu.g/mL; Invitrogen)] were
transfected with 10 .mu.g of Thy1-pcDNA3.1 ubi/puro-plasmid or with
empty control vector (no human Thy1 gene; henceforth control
wild-type cells) using Lipofectamine 2000 (Invitrogen) according to
manufacturer's instructions.
Flow Cytometry and Immunofluorescence Staining of Murine Vascular
Endothelial Cells
[0187] Flow Cytometry:
[0188] One million live cells of both transfected and wild-type
cells were washed in phosphate buffered serum (PBS) and incubated
with mouse anti-human Thy1 primary antibody (eBioscience; at 1:100)
for 30 min at room temperature. This was followed by incubation
with anti-mouse PE-antibodies (Jackson ImmunoResearch, 1:200) for
15 min on ice. Expression levels of the Thy1 receptors, on the cell
surface of Thy1-expressing and wild-type MS1 cells, were analyzed
with a FACSCalibur.TM. (Becton Dickinson, San Jose, Calif.) and the
geometric mean fluorescence intensity was determined using Flow Jo
software (Stanford University, Stanford, Calif.).
[0189] Immunofluorescence Staining:
[0190] To further confirm human Thy1 expression on vascular
endothelial MS1 cells, immunofluorescence staining of the cells was
performed using standard techniques. In brief, wild-type and Thy-1
expressing cells were grown on cover slips under standard
conditions in DMEM complete growth media for 24 hours; after the
media was removed, cells were washed in PBS and fixed in 4%
paraformaldehyde in PBS solution for 30 min at room temperature.
Cells were then washed in PBS, and 1% bovine serum albumin (BSA)
blocking solution was applied for one hour. Primary antibody
(rabbit anti-human Thy1, Sigma, 1:100) incubation was performed
overnight at 4'C. After washing in PBS, secondary antibody
(anti-rabbit FITC, Jackson Immunolaboratories, 1:500) was added for
one hour at room temperature. Cells were then washed in PBS,
counterstained with 4'',6-diamidino-2-phenylindole (DAPI), mounted
onto glass slides with anti-fade solution and imaged with an
Olympus IX81 system.
Preparation of Microbubble Contrast Agents
[0191] Perfluorocarbon-filled, lipid-shelled, streptavidin-coated
contrast microbubbles (MicroMarker.TM., VisualSonics, Toronto,
Canada) were reconstituted in 1 mL sterile saline (0.9% sodium
chloride) according to the manufacturer's protocol. The mean
diameter of the microbubbles as assessed by a cell counter and
sizer (Multisizer.TM. III Coulter Counter; Beckman Coulter,
Fullerton, Ca) was 1.5.+-.0.1 .mu.m (range, 1-2 .mu.m). Targeted MB
were prepared by mixing 6 .mu.g of biotinylated antibodies to
5.times.10.sup.7 streptavidin-coated MB for 10 min at room
temperature. Two different types of targeted MB were prepared:
Microbubbles targeted to human Thy1 (MB.sub.Thy1) using
biotinylated mouse anti-human Thy1 monoclonal antibodies
(eBiosciences; San Diego, Calif.); and, control MB
(MB.sub.Control), targeted with an isotype matched control IgG
antibody (eBioscience, San Diego, Calif.); non-bound antibodies
were removed by washing in PBS.
Flow Chamber Experiments
[0192] The three cell types (clone 1 and 2 as well as the wild-type
negative control vascular endothelial cells) were grown on
neutral-charged glass microscope slides triple-coated with
Sigmacote.RTM. (Sigma, Mo.) for 48 hours, then mounted on a
parallel plate flow chamber (GlycoTech Corporation, Rockville,
Md.). A syringe infusion/withdrawal pump (Genie Plus.TM., Kent
Scientific Corporation, Torrington, Conn.) was used to pass
solutions over the cells at a flow rate of 0.6 ml/min,
corresponding to a wall shear stress rate of 100 sec.sup.-1 similar
to that in capillaries.sup.2. Solutions were passed over cells in
the following order: 1) PBS for 2 min; 2) 5.times.10.sup.7 of
either MB.sub.Thy1 or MB.sub.Control in PBS for 4 min; and 3)
2-minute washing with PBS. At least six random fields of view of
these slides were immediately imaged with a phase-contrast
brightfield microscope (200.times.; Axiovert 25; Carl Zeiss,
Thornwood, N.Y.) to assess the number of MB attached to different
cell types (as directly visualized on phase-contrast microscopy).
Cell blocking studies to confirm binding specificity of MB.sub.nyi
to human Thy1 were performed by pre-incubating Thy1-positive cells
with mouse anti-human Thy1 monoclonal antibody (eBiosciences, San
Diego, Calif.; 30 .mu.g/ml growth medium) for 30 min at 37.degree.
C. All experiments performed under different conditions were
performed in triplicates.
Small Animal Ultrasound Molecular Imaging Settings and Protocol
[0193] All mice were anesthetized with 2% isoflurane in room air (2
L/min) during scanning. Ultrasound molecular imaging was performed
using a dedicated small animal ultrasound machine (Vevo 2100;
VisualSonics, Toronto, Canada). Images were acquired in a
transverse plane with a high-frequency transducer (MS250; center
frequency of 21 MHz; lateral and axial resolution of 165 .mu.m and
75 .mu.m, respectively; focal length, 10 mm; transmit power, 4%;
mechanical index, 0.2; dynamic range, 40 dB). The acoustic focus
was centered at the level of the PDAC xenografts with the imaging
plane aligned in the center of the tumor. The same imaging setting
was used in all imaging sessions.
Immunofluorescence Staining of Pancreatic Cancer Xenografts
[0194] Prior to tumor excision, mice were perfused in vivo with 4%
paraformaldehyde at a rate of 4 mL/min for 5 minutes. Mice were
then sacrificed and the tumors excised, fixed in 4%
paraformaldehyde at 4.degree. C. for 24 hours and transferred into
30% sucrose solution at 4.degree. C. for another 24 hours. Tumors
were cryosectioned (slice thickness of 10 .mu.m) and sections were
analyzed from the center of the tumor that approximated the
corresponding imaging plane from ultrasound imaging. Incubation in
5% normal goat serum in PBS for 30 min was performed to block
nonspecific proteins. Sections were simultaneously incubated for 12
hours (4.degree. C.) with 1:100 each of primary antibodies: rabbit
anti-human Thy1 antibody (Sigma) and rat anti-mouse CD31 (BD
Pharmingen). Secondary antibodies (goat anti-rabbit Alexa.TM. Fluor
488 antibody and donkey anti-rat Alexa.TM. Fluor 594 antibody;
Invitrogen) were simultaneously applied at 1:600 dilutions in PBS
for 30 minutes at room temperature to confirm Thy1 expression on
the tumor neovasculature. Fluorescent images were acquired using
Metamorph.TM. software (Universal Imaging Corp., West Chester, Pa.)
and a Zeiss Axioscope.TM. microscope (Axiophot, Carl Weiss AG,
Thornwood, N.Y.) attached to a digital camera (AxioCam.TM. MRc,
Bernried, Germany).
REFERENCES
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Example 2
Additional Pancreatic Cancer-Associated Vascular Endothelial Cell
Markers as Imaging Targets of Pancreatic Cancer Associated
Angiogenesis
[0198] We have identified additional high priority candidate
markers through our proteomic profiling, as disclosed in Example 1.
These candidates include multimerin 1 (MMRN1), mannose receptor
type C2 (MRC2), neuropilin (NRP1), and VCAM1.
TABLE-US-00007 TABLE 7 Additional Four Vascular Endothelial Cell
Marker Candidates for Specific Aim 3. Candidates Location
Endothelial MMRN1 (multimerin 1) Membrane & Y Extracellular
MRC2 (mannose Membrane Y receptor type C2) Also called endo180 NRP1
(neuropilin) Membrane & Y Extracellular VCAM1 Membrane
Cytokine-activated endothelium
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150079003A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150079003A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References