U.S. patent application number 15/573498 was filed with the patent office on 2018-04-19 for tmem active test and uses thereof in diagnosis, prognosis and treatment of tumors.
This patent application is currently assigned to ALBERT EINSTEIN COLLEGE OF MEDICINE, INC.. The applicant listed for this patent is ALBERT EINSTEIN COLLEGE OF MEDICINE, INC.. Invention is credited to John S. Condeelis, Allison S. Harney.
Application Number | 20180106811 15/573498 |
Document ID | / |
Family ID | 57393661 |
Filed Date | 2018-04-19 |
United States Patent
Application |
20180106811 |
Kind Code |
A1 |
Condeelis; John S. ; et
al. |
April 19, 2018 |
TMEM ACTIVE TEST AND USES THEREOF IN DIAGNOSIS, PROGNOSIS AND
TREATMENT OF TUMORS
Abstract
Disclosed are kits and methods for detecting the presence of
tumor sites that are active in tumor cell dissemination and uses
thereof for determining the risk of tumor cells undergoing
hematogenous metastasis, for assessing the prognosis of a subject
undergoing treatment for a localized tumor, for determining a
course of treatment for a localized tumor, and for identifying
agents to treat or prevent hematogenous metastasis.
Inventors: |
Condeelis; John S.; (Bronx,
NY) ; Harney; Allison S.; (Bronx, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALBERT EINSTEIN COLLEGE OF MEDICINE, INC. |
Bronx |
NY |
US |
|
|
Assignee: |
ALBERT EINSTEIN COLLEGE OF
MEDICINE, INC.
Bronx
NY
|
Family ID: |
57393661 |
Appl. No.: |
15/573498 |
Filed: |
May 24, 2016 |
PCT Filed: |
May 24, 2016 |
PCT NO: |
PCT/US2016/033862 |
371 Date: |
November 13, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62166730 |
May 27, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/574 20130101;
G01N 2800/50 20130101; G01N 33/57415 20130101; G01N 33/57484
20130101; C07K 2317/76 20130101; C07K 16/22 20130101; G01N 2800/52
20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C07K 16/22 20060101 C07K016/22 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
number CA100324 awarded by the National Institutes of Health and
grant number W81XWH-13-1-0010 awarded by the Department of Defense
Breast Cancer Research Program. The government has certain rights
in this invention.
Claims
1. A method of determining the presence of one or more sites that
are active in tumor cell dissemination in a subject, the method
comprising treating a tumor sample from the subject to detect Tie2,
VEGFA, CD68, CD31, and VE-Cadherin and/or ZO-1, wherein the
presence of CD68 indicates the presence of a macrophage and wherein
the presence of CD31 indicates the presence of an endothelial cell,
and analyzing levels of Tie2, VEGFA, and VE-Cadherin and/or ZO-1,
wherein Tie2.sup.Hi/VEGFA.sup.Hi peri-vascular macrophages
associated with low levels of VE-Cadherin and/or ZO-1 endothelial
staining indicate the presence of sites that are active in tumor
cell dissemination (TMEM.sup.Active sites), and wherein
Tie2.sup.Hi/VEGFA.sup.Hi peri-vascular macrophages associated with
high levels of VE-Cadherin and/or ZO-1 endothelial staining
indicate that TMEM sites are inactive in tumor cell
dissemination.
2. A method for determining the risk of tumor cells undergoing
hematogenous metastasis comprising assaying a tumor sample from a
subject for the presence of TMEM.sup.Active sites by the method
according to claim 1, wherein the risk of tumor cells undergoing
hematogenous metastasis increases with the presence of
TMEM.sup.Active sites.
3. A method for determining a course of treatment for a tumor in a
subject comprising assaying a tumor sample from the subject for the
presence of TMEM.sup.Active sites by the method according to claim
1, wherein the presence of TMEM.sup.Active sites indicates that the
subject should be treated for a metastatic tumor or wherein a lack
of TMEM.sup.Active sites indicates that the subject does not need
to be treated for a metastatic tumor.
4. A method for assessing the efficacy of an anti-cancer therapy in
inhibiting tumor cell dissemination and metastasis in a subject
comprising assaying a tumor sample from the subject for the
presence of TMEM.sup.Active sites by the method according to claim
1, wherein a reduction of TMEM.sup.Active sites in the subject
undergoing anti-cancer therapy indicates that the anti-cancer
therapy is effective and wherein a lack of reduction of
TMEM.sup.Active sites in the subject undergoing anti-cancer therapy
indicates that the anti-cancer therapy may not be effective.
5. The method of claim 4, wherein the anti-cancer therapy comprises
administration to the subject of a drug that inhibits TMEM
function.
6. The method of claim 4, wherein the anti-cancer therapy comprises
administration of a Tie2 kinase inhibitor to the subject.
7. A method of preventing or reducing tumor cell dissemination and
metastasis in a subject comprising: a) receiving an identification
of the subject as having tumor sites that are active in tumor cell
dissemination by the method according to claim 1; and b)
administering an anti-cancer therapy to the subject identified as
having tumor sites that are active in tumor cell dissemination.
8. The method of claim 7, wherein the anti-cancer therapy comprises
administration to the subject of a drug that inhibits TMEM
function.
9. The method of claim 7, wherein the anti-cancer therapy comprises
administration of a Tie2 kinase inhibitor to the subject.
10. A method for identifying an agent to treat or prevent
hematogenous metastasis, the method comprising contacting a tumor
sample with the agent, assaying the tumor sample for the presence
of TMEM.sup.Active sites by the method according to claim 1, and
analyzing whether or not the agent reduces the number of
TMEM.sup.Active sites, wherein an agent that reduces the number of
TMEM.sup.Active sites is a candidate agent for treating or
preventing hematogenous metastasis.
11. The method of claim 1, wherein the tumor is a breast, pancreas,
prostate, colon, brain, liver, lung, head or neck tumor.
12. The method of claim 11, wherein the tumor is a breast
tumor.
13. A kit for detecting the presence of tumor sites that are active
in tumor cell dissemination, the kit comprising reagents to detect
one or more of Tie2, VEGFA, CD68, CD31, and VE-Cadherin and/or
ZO-1.
14. The kit of claim 13, further comprising instructions for a
procedure to detect the presence of tumor sites that are active in
tumor cell dissemination.
15. The kit of claim 13, wherein the tumor is a breast, pancreas,
prostate, colon, brain, liver, lung, head or neck tumor.
16. The kit of claim 15, wherein the tumor is a breast tumor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 62/166,730, filed on May 27, 2015, the
contents of which are herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] Various publications are referred to in parentheses
throughout this application. Full citations for these references
may be found at the end of the specification immediately preceding
the claims. The disclosures of these publications are hereby
incorporated by reference in their entireties into the subject
application to more fully describe the art to which the subject
application pertains.
[0004] For almost two decades tumor vasculature has been described
as abnormal with increased vascular permeability (1, 2). Vascular
endothelial growth factor A (VEGFA) is known to promote vascular
permeability, and inhibition of VEGFA results in the normalization
of tumor vasculature and a decrease in permeability (3, 4). Due to
the significant effects of VEGFA on tumor angiogenesis and vascular
permeability, inhibitors of VEGF signaling have become an important
research focus in the development of anti-tumor therapies.
[0005] Tumor-associated macrophages (TAMs) have been implicated in
tumor progression, angiogenesis and metastasis (5, 6). A
subpopulation of perivascular TAMs that have features of
pro-tumorigenic macrophages, promoting tumor angiogensis and
metastasis, has been identified as Tie2-expressing macrophages
(TEMs) (7). Perivascular macrophages are also an essential
component of the microanatomical sites termed "tumor
microenvironment of metastasis" (TMEM) that consist of a TAM in
direct contact with a Mammalian enabled (Mena) over-expressing
tumor cell and endothelial cell (8, 28). TMEM have been associated
with tumor cell intravasation (9, 10) and TMEM density predicts
distant metastatic recurrence in breast cancer patients
independently of other clinical prognostic indicators (8, 11, 28).
However, the mechanistic link between perivascular macrophages and
tumor cell intravasation remained unclear. Further,
hyperpermeability in tumor vasculature is not uniform, but rather
is spatially and temporally heterogeneous (12). In a VEGFA
overexpression model inducing vascular permeability, the presence
of macrophages at vascular branch points was observed at hotspots
of vascular permeability (4). Although hyperpermeability of tumor
vasculature is widely accepted, a mechanistic understanding of the
heterogeneity of vascular permeability, the contribution of TAMs,
and the link with tumor cell intravasation has not been
described.
[0006] There is a need for reliable methodologies to predict the
risk for metastatic disease in cancer patients in order both to
administer proper treatment to patients whose tumors have a high
risk of metastasizing and to avoid unnecessary administration of
chemotherapy to patients whose tumor had a negligible risk of
metastasizing. The present invention addresses this need.
SUMMARY OF THE INVENTION
[0007] Provided are methods of determining the presence of one or
more sites that are active in tumor cell dissemination in a
subject, the methods comprising
[0008] treating a tumor sample from the subject to detect Tie2,
VEGFA, CD68, and VE-Cadherin and/or ZO-1, wherein the presence of
CD68 indicates the presence of a macrophage, and
[0009] detecting levels of Tie2, VEGFA, and VE-Cadherin and/or
ZO-1,
[0010] wherein Tie2.sup.Hi/VEGFA.sup.Hi pen-vascular macrophages
associated with low levels of VE-Cadherin and/or ZO-1 endothelial
staining indicate the presence of sites that are active in tumor
cell dissemination (TMEM.sup.Active sites), and
[0011] wherein Tie2.sup.Hi/VEGFA.sup.Hi pen-vascular macrophages
associated with high levels of VE-Cadherin and/or ZO-1 endothelial
staining indicate that there are no active sites of tumor cell
dissemination.
[0012] Also provided are methods for determining the risk of tumor
cells undergoing hematogenous metastasis comprising determining
whether or not a tumor sample from a subject contains
TMEM.sup.Active sites, wherein the risk of tumor cells undergoing
hematogenous metastasis increases with the presence of
TMEM.sup.Active sites.
[0013] Still further provided are methods for determining a course
of treatment for a tumor in a subject, the method comprising
determining whether or not a tumor sample from a subject contains
TMEM.sup.Active sites, wherein the presence of TMEM.sup.Active
sites indicates that the subject should be treated for a metastatic
tumor or wherein a lack of TMEM.sup.Active sites indicates that the
subject does not need to be treated for a metastatic tumor.
[0014] Also provided is a method for assessing the efficacy of an
anti-cancer therapy in inhibiting tumor cell dissemination and
metastasis in a subject comprising assaying a tumor sample from the
subject for the presence of TMEM.sup.Active sites by the method
disclosed herein, wherein a reduction of TMEM.sup.Active sites in
the subject undergoing anti-cancer therapy indicates that the
anti-cancer therapy is effective and wherein a lack of reduction of
TMEM.sup.Active sites in the subject undergoing anti-cancer therapy
indicates that the anti-cancer therapy may not be effective.
[0015] A method of preventing or reducing tumor cell dissemination
and metastasis in a subject is provided, where the method
comprises:
[0016] a) receiving an identification of the subject as having
tumor sites that are active in tumor cell dissemination by the
method disclosed herein; and
[0017] b) administering an anti-cancer therapy to the subject
identified as having tumor sites that are active in tumor cell
dissemination.
[0018] Still further provided are methods for identifying agents to
treat or prevent hematogenous metastasis, the methods comprising
contacting tumor samples with the agent and analyzing whether or
not the agent reduces the number of TMEM.sup.Active sites, wherein
an agent that reduces the number of TMEM.sup.Active sites is a
candidate agent for treating or preventing hematogenous
metastasis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1A-1M. Transient, local blood vessel permeability
events accompany intravasation, at TMEM. (A) Time 0' in the left
panel indicating TMEM (white box) from time-lapse IVM. Macrophages
(M), Tumor cells (TC) and blood vessels (155 kDa Dextran-TMR).
Single time point of tumor cell and macrophage streaming towards
non-migratory TMEM (asterisk). Streams and TMEM are in different
focal planes. Scale bar, 50 .mu.m. (B) 3D reconstruction of
time-lapse IVM from (A) of TC and macrophage streaming towards TMEM
(asterisk). Scale bar, 20 .mu.m. (C) 3D reconstruction of TC
intravasation (arrowhead) at TMEM (luminal surface of the
endothelium dashed white line). (D) IVM time-lapse of tumor cell
intravasation at TMEM. TC arrives at TMEM (arrowhead) and undergoes
transendothelial migration (arrow) while TMEM macrophage and TC
remain relatively immobile. Scale bar=10 .mu.m. (E) Schematic
summary diagram of panels A-D where TC (T2) and macrophage (M2)
stream towards non-migratory TMEM (box, T1 and M1), where the TC
(T2) undergoes transendothelial migration. (F) IVM time-lapse of
155 kDa dextran-TMR extravasation and tumor cell intravasation.
TMEM (white box). Blood vessel permeability sites (arrowheads) and
intravasating TC (dotted line, 9'). Clearance of dextran and
decrease of CTC at 30'. Scale bar, 50 .mu.m. At 9' and 30' TMEM
tumor cells and macrophages are added in false color to increase
visibility after bleaching. (G) Isolated 155 kDa dextran-TMR
channel from F. Arrows mark dextran extravasation (white). Dashed
line indicates the luminal side of the endothelium. (H) Isolated
tumor cell channel from (F). Arrowhead marks site of intravasating
TC (dashed line) at TMEM. White dashed line marks the luminal
surface of the endothelium. Box indicates the region adjacent to
TMEM with elevated CTC. (I) Single time point of tumor cell
intravasation (dashed line) by time-lapse IVM. Scale bar, 50 .mu.m.
(J) 3D reconstruction of time-lapse IVM from I of tumor cell
intravasation at TMEM. Transmigrating tumor cells (individually
numbered, dashed white lines) are isolated from other cell types
for clarity with time in minutes from start (J0') to end of
transmigration (J3'). The luminal endothelial surface is outlined
in a dashed line. Extravascular dextran at TMEM indicated with an
arrowhead. (K) Frequency of blood vessel permeability events in the
presence of TMEM or away from TMEM in 100 .mu.m windows (n=16, **,
P=0.0034). (L) Frequency of tumor cell intravasation events in the
presence of TMEM or away from TMEM in 100 .mu.m windows (n=16, **,
P=0.0012). (M) Quantification of extravascular dextran intensity
and CTC area at TMEM over time from F. (.circle-solid.)
Extravascular dextran, (.box-solid.) CTC.
[0020] FIG. 2A-2N. Macrophage depletion reduces vascular
permeability and tumor cell intravasation. (A) Time lapse imaging
demonstrates that laser-induced damage to the endothelium creates a
hole allowing for extravasation of 155 kDa dextran-TMR. The
location of the hole is marked by a white dot (2 .mu.m) and an
arrowhead. 155 kDa dextran-TMR extravasates and increases over time
up to 60' filling the field of view and not clearing from the
tissue (n=4). Scale bar, 50 .mu.m. (B) 155 kDa dextran-TMR is
injected by tail vein i.v. catheter followed by 8 .mu.g of
VEGFA.sub.165 at 0'. VEGFA.sub.165 induces blood vessel
permeability in all of the blood vessels in the field of view. Peak
extravascular dextran is observed at 20' followed by clearance by
60' after resealing of vascular junctions (n=4). Scale bar, 50
.mu.m. (C) Spontaneous vascular permeability at TMEM is both
transient and local. Local peak extravasation of 155 kDa
dextran-TMR occurs after 20' (arrowhead) and clears within 60'
(n=11). Scale bar, 50 .mu.m. (D) Quantification of total
extravascular 155 kDa dextran-TMR area after laser-induced damage,
i.v. injection of VEGFA.sub.165 or spontaneous permeability at TMEM
from individual animals represented in A, B and C. Peak of 155 kDa
dextran-TMR area in spontaneous permeability at TMEM indicated with
an arrowhead. (.circle-solid.) laser damage, (.box-solid.)
intravenous VEGFA.sub.165 and (.tangle-solidup.) spontaneous
vascular permeability at TMEM. (E) Quantification of average
relative intensity of extravascular 155 kDa dextran-TMR after
(.circle-solid.) laser damage (n=4), (.box-solid.) intravenous
VEGFA.sup.1.sub.65 n=4 and (.tangle-solidup.) spontaneous
permeability (n=11). (F) Table of parameters from curve fitting to
an Exponentially Modified Gaussian function using data from (E).
(G) Immunofluorescence imaging of tumor sections stained for TMEM.
Vasculature (CD31), tumor cells (Mena) and macrophages (CD68) and
DAPI. TMEM are outlined in a white box. Scale bar, 20 .mu.m. H-N
are changes in parameter shown after removal of macrophages with
the agent B/B. (H) Quantification of total CD68+ macrophages in
tumor tissue (****, P<0.0001), (I) in TMEM (***, P=0.0003). (J)
Immunfluorescence imaging of tumor sections stained for vasculature
(CD31), 155 kDa dextran-TMR and DAPI, ZO-1 or VE-Cadherin as
indicated. Scale bar, 50 .mu.m. (K) Quantification of extravascular
155 kDa dextran-TMR (vehicle n=7, B/B homodimerizer n=8; ***,
P=0.0009) (L) circulating tumor cells (***, P=0.0007) (M) Vascular
ZO-1 (**, P=0.006) and (N) Vascular VE-Cadherin (* P=0.02).
[0021] FIG. 3A-3K. Inhibition of VEGFA from Tie2.sup.Hi/VEGF.sup.Hi
TMEM macrophages reduces blood vessel permeability and tumor cell
intravasation. (A) Immunofluorescence imaging of TMEM. Macrophages
(CD68), blood vessels (CD31), tumor cells (Mena), and DAPI. TMEM in
white box (right panel). Scale bar, 15 .mu.m. (B)
Immunofluorescence imaging of VEGFA.sup.Hi macrophages in TMEM in
sequential sections. Scale bar, 10 .mu.m. Tumor cell, spotted line;
macrophages, solid line; and blood vessels, dashed line. Left
panel: Macrophages (CD68), tumor cells (Mena), blood vessels
(CD31), and DAPI. Sequential section (center panel): VEGFA, Tie2,
blood vessels (CD31), and DAPI. Schematic representation (right
panel) of protein expression in TMEM; tumor cells with Mena.sup.Hi
endothelial cells CD31 and macrophages CD68, VEGFA.sup.Hi and
Tie2.sup.Hi M, macrophage; TC, tumor cell; and EC, endothelial
cell. (C) Immunofluorescence images of Tie2, VEGF and CD31. Lines
indicate regions of intensity profiling of VEGF intensity for CD31
(EC), macrophage (M) and tumor tissue (TC). Scale bar, 25 .mu.m.
(D) Fluorescence intensity profile of VEGF from (C) of macrophage,
endothelial cell and tumor tissue. (E) Immunofluorescence imaging
of sequential PyMT tumor sections for TEM markers. VEGFA.sup.Hi
TMEM macrophages express F4/80, MRC1, CD11b and CD68 as indicated
by an arrowhead in sequential sections. CD31+ endothelium is
indicated by an arrowhead. (F) VEGFA.sup.Hi TMEM macrophages
express Tie2, MRC1, and CD68 but not CD11c as indicated by an
arrowhead. CD31+/Tie2+ endothelium is indicated by an arrowhead.
Scale bar, 25 .mu.m. (G) Immunfluorescence imaging of tumor
sections after blocking VEGFA with anti-VEGFA blocking antibody
(B20-4.1.1). Tumors are stained for vasculature (CD31), 155 kDa
dextran-TMR and DAPI, ZO-1 or VE-Cadherin as indicated. Scale bar,
50 .mu.m. (H) Quantification of extravascular 155 kDa dextran-TMR
and (n=10; **, P=0.0015) (I) circulating tumor cells (*, P=0.0497),
(J) Vascular ZO-1 (**, P=0.005) and (K) Vascular VE-Cadherin (*,
P=0.0463).
[0022] FIG. 4A-4M. Macrophage-specific ablation of Vegfa in PyMT
implant tumors (Vegfa.sup.flox;Csflr-Cre) compared to control
(Vegfa.sup.flox) blocks blood vessel permeability and tumor cell
intravasation. (A) Immunfluorescence of tumor sections stained for
vasculature (CD31), 155 kDa dextran-TMR and DAPI, ZO-1 or
VE-Cadherin as indicated. Scale bar, 50 .mu.m. (B) Quantification
of extravascular 155 kDa dextran-TMR and (Vegfa.sup.flox n=5,
Vegfa.sup.flox;Csflr-Cre n=3; **; P=0.0029) (C) circulating tumor
cells (*, P=0.0177) (D) Vascular ZO-1 (**, P=0.0054) and (E)
Vascular VE-Cadherin (*, P=0.0457). (F, H) Immunofluorescence of
tumor sections stained for the presence of vascular junction
proteins at TMEM macrophages. Tumor sections are stained for
VE-cadherin, CD31 and VEGFA. Sequential sections are stained for
CD31, Tie2 and CD68. (F) Control tumors (Vegfa.sup.flox) or (H)
after ablation of Vefga (Vegfa.sup.flox;Csflr-Cre). CD68+
macrophage in TMEM outlined in white box, adjacent endothelium in
TMEM in box. Merged signal of CD31 and Tie2 (left) or CD31 and
VE-Cadherin (right). Decreased VE-Cadherin at TMEM (F, right) seen
as decreased VE-Cadherin. Scale bar, 15 .mu.m. (G, I)
Quantification of the relative intensity of VEGFA or vascular
junction proteins (ratio of VE-cadherin to CD31 in blood vessels)
in F, H at TMEM or away from TMEM in (G) control tumors
(Vegfa.sup.flox) or (I) after ablation of Vegfa in
Vegfa.sup.flox;Csflr-Cre tumors along 25 .mu.m lengths of blood
vessel (n=3). (.circle-solid.) Relative fluorescence intensity of
VE-Cadherin/CD31, (.box-solid.) Relative VEGFA intensity. Dashed
line indicates the presence of a CD68+ macrophage. (J)
Quantification of average pixel intensity of VE-Cadherin/CD31
immunofluorescence staining in 25 .mu.m lengths of blood vessel at
TMEM or away from TMEM in the presence of VEGFA.sup.Hi macrophages
(Vegfa.sup.flox, n=3) or after macrophage-specific ablation of
Vegfa (Vegfa.sup.flox;Csflr-Cre, n=3) from data in G and I.
Post-ANOVA comparisons with significant difference indicated (*,
**). (K) Cartoon summarizing TMEM macrophage-mediated induction of
blood vessel permeability promotes tumor cell intravasation. TMEM
assemble with close association between the non-migratoryTMEM TC
(T1) and Tie2.sup.Hi/VEGFA.sup.Hi macrophage (bM1) on blood
vessels. VEGFA destabilizes vascular junctions resulting in
vascular permeability and TC (T2) intravasation. (L) Human breast
cancer tumor sections stained for the presence of vascular junction
proteins at TMEM macrophages. Tumor sections are stained for TMEM;
Mean, CD68 and CD31 by IHC and for VE-cadherin, Tie2 and VEGFA
stained by immunofluorescence in sequential sections. TMEM outlined
in black box in IHC and white box in immunofluorescence. Scale bar,
15 .mu.m. (M) Quantification of normalized average pixel intensity
of VE-Cadherin staining in vasculature at Tie2Hi/VEGFAHi
macrophages of TMEM or away from TMEM in (n=23 at TMEM, n=24 away
from TMEM in 5 individual patient samples, ***, P=0.0001).
[0023] FIG. 5A-5E. Tumor cell intravasation and transient blood
vessel permeability occur exclusively at TMEM. (A) Intravital
imaging microscopy time-lapse of tumor cell intravasation at TMEM.
TMEM is composed of a tumor cell (TC), macrophage (M) and
endothelial cell (EC) in direct contact. Another tumor cell
(arrowhead) from the stream behind appears adjacent to TMEM and
undergoes transendothelial migration (arrow). Scale bar=10 .mu.m.
(B) 3D reconstruction of TMEM from live tumor time lapse. Tumor
cells approaching the blood vessel in a stream are indicated by
arrowheads. The TMEM macrophage (M), tumor cell (TC) and their
associated blood vessel endothelial cell (EC) are indicated with
arrows. The TMEM-associated tumor cell (TC) does not move while the
streaming tumor cells (arrowheads) approach and intravasate at
TMEM. Scale bar=5 .mu.m. (C) Frequency of blood vessel permeability
events in the presence of TMEM or absence of TMEM per imaging
field. (D) Frequency of tumor cell intravasation events in the
presence of TMEM or absence of TMEM per imaging field. (E-H)
Macrophage depletion in PyMT tumors in the MAIFA (macrophage
fas-induces apoptosis) mouse model reduces blood vessel
permeability and tumor cell intravasation. (E) Quantification of
extravascular 155 kDa dextran-TMR (vehicle n=7, B/B
homodimerizer-induced removal of macrophages, n=8; ***, P=0.0009)
(F) circulating tumor cells (***, P=0.0007) (G) Vascular ZO-1 (**,
P=0.006) and (H) Vascular VE-Cadherin (* P=0.02).
[0024] FIG. 6A-6B. TMEM macrophages are
VEGF.sup.Hi/Tie2.sup.Hi/CD68+. (A) Immunofluorescence imaging of
TMEM. Macrophages (CD68), blood vessels (CD31), tumor cells (Mena),
and DAPI. TMEM in white box (right panel). (B) Immunofluorescence
imaging of VEGF.sup.Hi macrophages in TMEM in sequential sections.
Tumor cell, spotted line; macrophages, solid line; and blood
vessels, dashed line. Left panel: Macrophages (CD68), tumor cells
(Mena), blood vessels (CD31), and DAPI. Sequential section (center
panel): VEGFA, Tie2, blood vessels (CD31), and DAPI. Schematic
representation (right panel) of protein expression in TMEM; tumor
cells with Mena.sup.Hi, endothelial cells CD31 and macrophages
CD68, VEGFA.sup.Hi and Tie2.sup.Hi. M, macrophage; TC, tumor cell;
and EC, endothelial cell.
[0025] FIG. 7A-7D. Macrophage-specific ablation of Vegfa in PyMT
implant tumors blocks blood vessel permeability and tumor cell
intravasation. Quantification in the presence (Vegfa.sup.flox n=5)
or absence (Vegfa.sup.flox;Csflr-Cre n=3) of Vegfa in macrophages
of (A) extravascular 155 kDa dextran-TMR in (**; P=0.0029) (B)
circulating tumor cells (*, P=0.0177) (C) Vascular ZO-1 (**,
P=0.0054) and (D) Vascular VE-Cadherin (*, P=0.0457).
[0026] FIG. 8A-8E. Inhibition of VEGFA reduces blood vessel
permeability and tumor cell intravasation. Quantification in tumor
sections after treatment with anti-VEGFA blocking antibody
(B20-4.1.1) or IgG control antibody of (A) extravascular 155 kDa
dextran-TMR and (n=10; **, P=0.0015) (B) circulating tumor cells
(*, P=0.0497), (C) Vascular ZO-1 (**, P=0.005) and (D) Vascular
VE-Cadherin (*, P=0.0463). (E) Schematic diagram of TMEM activation
leading to blood vessel permeability and tumor cell intravasation.
At TMEM a tumor cell and macrophage interact with the endothelium.
When Tie2.sup.Hi macrophage of TMEM signals with elevated VEGF,
endothelial cell junction remodeling occurs through VEGF signaling.
Vascular junctions are degraded leading to decreased VE-Caderhin
and ZO-1 in CD31+ endothelial cells resulting in increased vascular
leakiness. Increased vascular permeability supports tumor cell
intravasation at TMEM sites.
[0027] FIG. 9A-9B. Blood vessel permeability and tumor cell
intravasation are linked and occur only at TMEM sites. (A)
Frequency of blood vessel permeability events in the presence of
TMEM or away from TMEM (n=16, **, P=0.0034). (B) Frequency of tumor
cell intravasation events in the presence of TMEM or away from TMEM
(n=16, **, P=0.0012).
[0028] FIG. 10. Quantification of normalized average pixel
intensity of VE-Cadherin staining in vasculature at
Tie2.sup.Hi/VEGFA.sup.Hi macrophages of TMEM or away from TMEM
(n=23 at TMEM, n=24 away from TMEM) in 5 individual patient samples
(***, P=0.0001).
[0029] FIG. 11A-11B. Rebastinib, an inhibitor of Tie2 macrophage
function in TMEM, inhibits TMEM activity. (A) Quantification of
TMEM density in 10 40.times. fields (not significant). (B)
Quantification of vascular permeability activity of tumor TMEM
using vascular permeability marker IV-dextran/ZO-1 staining
intensity (n=7 and 9; *, P=0.0177).
[0030] FIG. 12A-12B. Knockout of the VEGF gene (csfl-cre) in Tie2Hi
macrophages blocks TMEM macrophage function. Quantification of (A)
Vascular ZO-1 (**, P=0.0054) and (B) Vascular VE-Cadherin (*,
P=0.0457), normalized to blood vessel area (ZO-1 or VE-Cad staining
intensity/anti-CD31 staining intensity).
DETAILED DESCRIPTION OF THE INVENTION
[0031] Provided is a method of determining the presence of one or
more sites that are active in tumor cell dissemination in a
subject, the method comprising
[0032] treating a tumor sample from the subject to detect Tie2,
VEGFA, CD68, CD31, and VE-Cadherin and/or ZO-1, wherein the
presence of CD68 indicates the presence of a macrophage and wherein
the presence of CD31 indicates the presence of an endothelial cell,
and
[0033] analyzing levels of Tie2, VEGFA, and VE-Cadherin and/or
ZO-1,
[0034] wherein Tie2.sup.Hi/VEGFA.sup.Hi peri-vascular macrophages
associated with low levels of VE-Cadherin and/or ZO-1 endothelial
staining indicate the presence of sites that are active in tumor
cell dissemination (TMEM.sup.Active sites), and
[0035] wherein Tie2.sup.Hi/VEGFA.sup.Hi peri-vascular macrophages
associated with high levels of VE-Cadherin and/or ZO-1 endothelial
staining indicate that there are no sites that are active in tumor
cell dissemination.
[0036] Tie2.sup.Hi/VEGFA.sup.Hi CD68+ cells in direct contact with
a blood vessel identify the presence of a TMEM site (see
Experimental Details below). TMEM.sup.Active sites are TMEM sites
that are active in tumor cell dissemination.
[0037] Also provided is a method for determining the risk of tumor
cells undergoing hematogenous metastasis comprising assaying a
tumor sample from a subject for the presence of TMEM.sup.Active
sites by the method disclosed herein, wherein the risk of tumor
cells undergoing hematogenous metastasis increases with the
presence of TMEM.sup.Active sites.
[0038] Still further provided is method for determining a course of
treatment for a tumor in a subject comprising assaying a tumor
sample from the subject for the presence of TMEM.sup.Active sites
by the method disclosed herein, wherein the presence of
TMEM.sup.Active sites indicates that the subject should be treated
for a metastatic tumor or wherein a lack of TMEM.sup.Active sites
indicates that the subject does not need to be treated for a
metastatic tumor.
[0039] Also provided is a method for assessing the efficacy of an
anti-cancer therapy in inhibiting tumor cell dissemination and
metastasis in a subject comprising assaying a tumor sample from the
subject for the presence of TMEM.sup.Active sites by the method
disclosed herein, wherein a reduction of TMEM.sup.Active sites in
the subject undergoing anti-cancer therapy indicates that the
anti-cancer therapy is effective and wherein a lack of reduction of
TMEM.sup.Active sites in the subject undergoing anti-cancer therapy
indicates that the anti-cancer therapy may not be effective.
[0040] A method of preventing or reducing tumor cell dissemination
and metastasis in a subject is provided, where the method
comprises:
[0041] a) receiving an identification of the subject as having
tumor sites that are active in tumor cell dissemination by the
method disclosed herein; and
[0042] b) administering an anti-cancer therapy to the subject
identified as having tumor sites that are active in tumor cell
dissemination.
[0043] Preferably, the anti-cancer therapy comprises administration
to the subject of a drug that inhibits TMEM function. The
anti-cancer therapy can comprise administration of a Tie2 kinase
inhibitor to the subject. Rebastinib is an example of a Tie2 kinase
inhibitor (32, 33). FIG. 11 illustrates that rebastinib, an
inhibitor of Tie2 macrophage function in TMEM, inhibits TMEM
activity.
[0044] Still further provided is a method for identifying an agent
to treat or prevent hematogenous metastasis, the method comprising
contacting a tumor sample with the agent, assaying the tumor sample
for the presence of TMEM.sup.Active sites by the method disclosed
herein, and analyzing whether or not the agent reduces the number
of TMEM.sup.Active sites, wherein an agent that reduces the number
of TMEM.sup.Active sites is a candidate agent for treating or
preventing hematogenous metastasis. In different embodiments, the
tumor is contacted with the agent in vivo or ex vivo.
[0045] Also provided is a kit for detecting the presence of tumor
sites that are active in tumor cell dissemination, the kit
comprising reagents to detect one or more of Tie2, VEGFA, CD68,
CD31, and VE-Cadherin and/or ZO-1. The reagent can be, for example,
an antibody, an antibody fragment, a peptide or an aptamer.
Antibody fragments include, but are not limited to, F(ab').sub.2
and Fab' fragments and single chain antibodies. The kit can further
comprise instructions for a procedure to detect the presence of
tumor sites that are active in tumor cell dissemination.
[0046] In any of the methods or kits disclosed herein, the tumor
can be, for example, a secretory epithelial tumor. The tumor can
be, for example, a prostate, pancreas, colon, brain, liver, lung,
head or neck tumor, or in particular a breast tumor.
[0047] The present invention is illustrated in the following
Experimental Details section, which is set forth to aid in the
understanding of the invention, and should not be construed to
limit in any way the scope of the invention as defined in the
claims that follow thereafter.
EXPERIMENTAL DETAILS
Introduction and Overview
[0048] Dissemination of tumor cells from the primary tumor is an
essential step in metastasis. Direct contact between a macrophage,
tumor and endothelial cell [Tumor MicroEnvironment of Metastasis
(TMEM)], correlates with metastasis. As disclosed herein, it is
shown using intravital high-resolution two-photon microscopy, that
transient vascular permeability and tumor cell intravasation occur
simultaneously and exclusively at TMEM. The hyperpermeable nature
of tumor vasculature has been described as spatially and temporally
heterogeneous. Using real-time imaging it was observed that
vascular permeability is transient, restricted to TMEM sites, and
required for tumor cell dissemination. VEGFA signaling from
Tie2.sup.Hi TMEM macrophages causes local loss of vascular
junctions, resulting in transient vascular permeability and tumor
cell intravasation, demonstrating a role for TMEM within the
primary mammary tumor. These data provide insight into the
mechanism of tumor cell intravasation and vascular permeability in
breast cancer, and explain the prognostic value of TMEM density as
a predictor of distant metastatic recurrence in patients.
[0049] Tumor vasculature is abnormal with increased vascular
permeability. VEGFA signaling from Tie2.sup.Hi TMEM macrophages
results in transient permeability and tumor cell intravasation at
tumor blood vessels proximal to TMEM, explaining the previously
unresolved heterogeneity in vascular permeability. These data
provide evidence for the mechanism underlying the association of
TMEM with distant metastatic tumor recurrence in mouse models and
human breast cancer patients, offering a rationale for the
development of therapeutic approaches targeting TMEM formation and
function.
Materials and Methods
[0050] Summary Tumor cell intravasation at sites of transient
vascular permeability associated with TMEM was studied using mouse
mammary tumor virus--polyoma middle T antigen (MMTV-PyMT)
autochthonous and implanted models of human patient-derived mammary
carcinoma. Single cell resolution of cell activity at TMEM in live
animals was achieved using extended time-lapse imaging on a
custom-built two-laser multiphoton microscope. To investigate the
role of macrophages in tumor cell intravasation and blood vessel
permeability, the MAFIA mouse model was used to deplete
macrophages. Observation of extravascular dextran, vascular
junction proteins and protein expression in tissue was performed
using immunofluorescence microscopy. Further investigation of the
mechanism of macrophage-mediated tumor cell intravasation and blood
vessel permeability at TMEM utilized anti-mouse-VEGFA inhibitory
antibody and the ablation of Vegfa expression in monocytes using a
myeloid-specific (Csflr promoter), tamoxifen-inducible Cre
expressing mouse strain was crossed with Vegfa.sup.flox/flox mice
with gene ablation induced with tamoxifen.
[0051] Tumor staging in PyMT. Early carcinoma and late carcinoma
tumors were characterized by a pathologist according to previously
characterized features (13). Briefly, early carcinoma tumors were
used from PyMT mice 7-9 weeks old that are characterized by
distended acinar structures with focal stromal invasion, high
density of leukocytic infiltration, and increased cytological
atypia and late carcinoma tumors from mice 12-14 weeks old that are
characterized by solid sheets of epithelial cells with little or no
remaining acinar structures visible (13).
[0052] Immunofluorescence Image Analysis.
[0053] To measure vascular junctions and extravascular dextran, the
CD31 channel (blood vessel), dextran and vascular junction (ZO-1 or
VE-Cadherin) were each thresholded to just above background based
upon intensity. Thresholding was verified by eye. A binary mask of
the blood vessels was created to define the boundaries of the
signal inside blood vessels. Structures smaller than 100 px.sup.2
were excluded as debris, and holes were filled. The extravascular
dextran area was isolated by subtracting the blood vessel mask from
the dextran mask. The remaining extravascular dextran area, and
blood vessel area were then measured. To measure vascular junction
area the vascular junction image was thresholded to just above
background in the blood vessel using the blood vessel mask and a
binary mask was made of the vascular junction area. The area of
vascular junctions and extravascular dextran was normalized to the
area of blood vessels in each image.
[0054] Vascular VE-Cadherin adjacent to TMEM was quantified by the
mean VE-Cadherin staining intensity in CD31+ vasculature adjacent
to CD68+/Tie2.sup.Hi/VEGFA.sup.Hi macrophages, CD68+ macrophages or
in the absence of macrophages in sequential tissue sections. Four
different fields (of 2.times.2 40.times. fields with 15% overlap)
were acquired per mouse. To measure vascular VE-Cadherin, the CD31
channel (blood vessel), VEGFA and VE-Cadherin were each thresholded
to just above background based upon intensity. Thresholding was
verified by eye. A binary mask of the blood vessels was created to
define the boundaries of the signal inside blood vessels. A box
(ROI) was moved along the vasculature in 0.5 .mu.m lengths of
vasculature in a sliding window fashion as defined by a freehand
drawn line. Average pixel intensity was measured in each of the
CD31, VE-Cadherin and VEGFA channels for each ROI and moved along
another 0.5 .mu.m lengths of vasculature. Measurements were
repeated until the end of the length (25 .mu.m) of the line drawn.
This method was adapted for use in measuring NG-2 staining
intensity as a measure of pericyte coverage of vasculature.
Pericyte coverate adjacent to TMEM was quantified by the mean NG-2
staining intensity in CD31+ vasculature adjacent to
CD68+/CD206+/VEGFA.sup.Hi macrophages, CD68+ macrophages or in the
absence of macrophages in sequential tissue sections. Four
different fields (of 2.times.2 40.times. fields with 15% overlap)
were acquired per mouse. To measure perivascular NG-2, and the CD31
channel (blood vessel), were thresholded to just above background
based upon intensity. Thresholding was verified by eye. A box (ROI)
was moved along the vasculature in 0.5 .mu.m lengths of vasculature
in a sliding window fashion as defined by a freehand drawn line.
Average pixel intensity was measured in each of the CD31 and NG-2
channels for each ROI and moved along another 0.5 .mu.m lengths of
vasculature. Measurements were repeated until the end of the length
(25 .mu.m) of the line drawn. Average pixel intensity in NG-2 was
determined for each length of vasculature measured and averaged for
each animal.
[0055] To measure vascular VE-cadherin in human samples, the
vasculature was outlined by CD31 staining in the IHC section. The
ROI was applied to the VE-cadherin fluorescence channel and average
signal intensity measured. Regions of vasculature adjacent to
CD68/Tie2.sup.Hi/VEGFA.sup.Hi macrophages or away from TMEM are
measured (n=23 at TMEM, n=24 away from TMEM) in 5 independent
patient samples.
[0056] Intravital Imaging.
[0057] Z-stacks of up to 50 .mu.m of depth were acquired with a 2
.mu.m slice interval for up to 4 h. Three time frames were acquired
after the injection of 155 kDa TMR-dextran before the
administration of 1.5 mg of 10 kDa fluorescein-dextran or 8 .mu.g
(0.2 mg/kg) of VEGFA.sub.165 peptide (PeproTech) by the tail vein
catheter to induce systemic vascular permeability as previously
described (29, 30). For laser-induced damage, the laser was held at
a position on the endothelium at 200 mW for 2 s (generating 400 mJ)
after injection of 155 kDa-dextran-TMR. Extended time-lapse images
were acquired.
[0058] Intravital microscopy image analysis. All images were
acquired as 16-bit TIFF images and all quantitative analysis was
performed on the raw 16-bit TIFF images. As previously described,
image channels were balanced and subtracted to isolate the CFP
signal (31). An average intensity Z-projection was made for all
channels. The ImageJ plug-in StackReg was used to register the
images over time. The average intensity Z-projection is used for
dextran analysis to determine quantitatively the mean dextran
intensity within a volume of interest. The first image in the
time-lapse sequence (t=0 min) was used to define the boundaries of
the vasculature. Circular ROIs were placed adjacent to the
vasculature in the tumor tissue at sites of spontaneous, transient
vascular permeability. Average fluorescence intensity of dextran
channel was measured in the ROI for each Z-projection time frame of
the time-lapse sequence. Fluorescence intensity values were
normalized to the maximum fluorescence intensity for each
individual vascular permeability event. To determine the kinetics
of transient vascular permeability (FIG. 2E), individual
permeability events were aligned with 0 min determined as the first
frame where dextran intensity increases above background.
Individual permeability events were averaged to determine the
average kinetics. For VEGFA.sub.165 injection and laser damage
experiments t=0 min is the start of image acquisition for all
animals. To determine the total dextran area (FIG. 2D) a binary
mask was made from the blood vessel signal within the first frame
of the time-lapse sequence. The blood vessel mask was applied to
all subsequent channels and subtracted from the dextran channel to
measure only extravascular dextran. The dextran channel was
intensity thresholded to just above background and verified by eye
and the total area was measured. Maximum intensity Z-projection was
made for the tumor cell channel to best highlight the cells and
suppress background. To measure circulating tumor cells, time-lapse
stacks were cropped to an area over the blood vessel immediately
downstream of TMEM sites with extravasation of vascular probes. The
blood vessel and tumor cell channels were intensity thresholded to
just above background and verified by eye. A binary mask was made
from the blood vessel signal within the first frame of the
time-lapse sequence. This image was used to define intact blood
vessels and determine the boundaries of what is intra- and
extravascular for the rest of the time-lapse sequence. Structures
smaller than 100 .mu.m.sup.2 were excluded as debris, and all holes
were filled. The blood vessel mask was applied to the tumor cell
channel which was then thresholded just above background to detect
all circulating tumor cells. The area in the tumor cell channel was
measured as the area of circulating tumor cells which was used as a
surrogate measure of the number for circulating tumor cells as the
area increases with number. The area is a conservative measurement
of circulating tumor cells as it increases directly with the cell
number and any inaccuracy would be cause by spatial overlap in
cells that would result in reducing the total area of circulating
tumor cells measured. The area of circulating tumor cells was
measured in each image in the time-lapse sequence. The area of
circulating tumor cells was normalized to the frame with the
maximum area of circulating tumor cells. A single optical plane is
presented in the figures unless otherwise described. For
three-dimensional reconstructions, data was imported into Imaris
software (BitPlane) for surface rendering.
[0059] Sliding window measurement of tumor cell intravasation and
vascular permeability. In this measurement a 100 .mu.m sized boxes,
size chosen based on the size of a TMEM, are placed along the
vessels consecutively in a FOV. Each box is then interrogated for
the presence of TMEM, tumor cell intravasation and vascular
permeability events. Time-lapse sequences of z-stacks are
interrogated to examine vasculature in 3D.
Results
[0060] TMEM-Associated Tumor Cells and Macrophages are Stationary
in TMEM Structures.
[0061] To examine the functional role of TMEM in tumor cell
dissemination, the spontaneous autochthonous mouse mammary cancer
model was used where the mouse mammary tumor virus long terminal
repeat drives the polyoma middle T antigen (MMTV-PyMT), in which
tumors exhibit histology similar to human luminal breast cancer,
and progress to metastasis (13). Immunohistochemistry (IHC)
revealed that TMEM structures in mouse tumors have the same
microanatomical structure as identified in humans (11). TMEM
density increases with tumor progression with elevated TMEM scores
in late carcinoma (LC) as compared to early carcinoma (EC) as seen
by IHC though total perivascular macrophage (including macrophages
not associated with tumor cells) density is not significantly
different (13). High-resolution imaging demonstrates that in TMEM
structures, tumor cells and macrophages extend protrusions but are
relatively non-migratory and stay in direct contact over time.
[0062] Vascular Permeability and Tumor Cell Intravasation Occur
Concurrently at TMEM.
[0063] To directly observe TMEM function in vivo, extended
time-lapse intravital microscopy (IVM) with high spatial and
temporal resolution was used. To visualize blood flow, vessels were
labeled with a high molecular weight compound (155 kDa dextran or
quantum dots) (1, 14) (FIG. 1). In LC, transient, local blood
vessel permeability was observed at TMEM sites by the extravasation
of quantum dots or 155 kDa dextran-tetramethylrhodamine (TMR) (FIG.
1F, G, K, 2C). In PyMT LC, tumor cell intravasation occurs at TMEM
sites concurrently with transient permeability (FIG. 1M). Migratory
tumor cells and macrophages stream towards TMEM at sites with
vascular permeability whereupon tumor cells undergo
transendothelial migration at TMEM (FIG. 1A-F, H-J).
Transendothelial crossing of tumor cells is visualized by the
hourglass shape of tumor cells as they are partially in the vessel
lumen and partially in the tissue (FIG. 1C, F, H-J). During
transendothelial migration of tumor cells, the TMEM tumor cell and
macrophage neither migrate nor intravasate indicating that tumor
cells entering the blood vessel at TMEM are supplied by the
migratory stream of cells (FIGS. 1A, B and D). The stationary
phenotype of these cells is consistent with previous results
showing macrophage contact-initiated invadopodium formation
uniquely in the TMEM tumor cell (9) and that perivascular
invadopodium-containing tumor cells are relatively non-motile in
vivo (15).
[0064] The peak of extravascular dextran intensity and the
appearance of circulating tumor cells coincide temporally and
spatially (FIGS. 1F-H, J, and M) demonstrating a direct link
between localized blood vessel permeability and tumor cell
intravasation at TMEM. The coincidence of spontaneous, transient
vascular permeability with tumor cell intravasation at TMEM also
has been observed in a patient-derived xenograft model of
triple-negative breast cancer, TN1.
[0065] To confirm that TMEM is associated with transient vascular
permeability and tumor cell intravasation a 100 .mu.m window, the
approximate width of a TMEM site, was consecutively slid along all
blood vessels (window measurement) to quantify the frequency of
tumor cell intravasation and vascular permeability events in the
presence or absence of TMEM. Vascular permeability and tumor cell
intravasation occur exclusively within the 100 .mu.m window when it
contains a TMEM, but never when the 100 .mu.m window does not
contain a TMEM in PyMT (FIGS. 1K and L). Similar results were
observed in the human TN1 model highlighting the importance of TMEM
in transient vascular permeability and tumor cell
intravasation.
[0066] Vascular Permeability at TMEM is a Highly Localized and
Transient Event.
[0067] Tumor vasculature has been previously described as abnormal
with increased vascular permeability, which has been attributed to
larger vascular intercellular openings (1, 12, 16). However,
vascular permeability is not spatially or temporally uniform, with
hotspots at vascular branch points (4, 12). Here it is demonstrated
that vascular permeability is transient, occurs exclusively at TMEM
sites, and is temporally heterogeneous, explaining the previously
unresolved heterogeneity in vascular permeability (FIGS. 1K, 2C).
Events of spontaneous, local vascular permeability and tumor cell
intravasation at TMEM occur predominantly at vascular branch
points, consistent with previous reports of vascular permeability.
If tumor blood vessels were uniformly leaky high-molecular weight
vascular probes would extravasate immediately and continuously
after injection. While the high-molecular weight probe, 155 kDa
dextran-TMR, remains in the vasculature in the absence of transient
TMEM-associated permeability events for the duration of the
time-lapse imaging, a low molecular weight dextran, 10 kDa
dextran-fluorescein isothiocynate (FITC), below the molecular
cutoff size of the endothelium (1, 14) leaks from blood vessels and
clears from the vascular space.
[0068] Further, transient permeability events are distinct from
mechanical damage to the endothelium. After creating a 2 .mu.m hole
in the endothelium with a laser, 155 kDa dextran-TMR extravasates
continuously, filling the field of view (FIG. 2A). By contrast,
VEGFA-mediated permeability is transient (12). Intravenous
injection of VEGFA.sub.165, the soluble isoform of VEGFA with
properties of native VEGF (17), results in vascular permeability
with peak intensity of extravascular dextran at 20 min (FIG. 2B).
Spontaneous vascular permeability at TMEM follows similar kinetics
to VEGFA.sub.165-mediated permeability with peak intensity of
extravascular dextran at 20 min but is restricted to individual
TMEM sites (FIG. 2C). The curves obtained for average intensity of
extravascular 155 kDa dextran-TMR after laser damage, VEGFA.sub.165
and spontaneous permeability were fit to an exponentially modified
Gaussian function (FIGS. 2E and F). While the curve for laser
damage does not have a clearance term as dextran continues to
extravasate for the entire time-lapse, both the VEGFA.sub.165 and
spontaneous curves have similar extravasation and clearance rates.
A significant difference between VEGFA.sub.165 and spontaneous
TMEM-mediated permeability is that permeability at TMEM is highly
local, while VEGFA.sub.165 results in dextran extravasation from
all blood vessels within a field of view (FOV). Thus, the area of
extravascular 155 kDa dextran-TMR from local TMEM-mediated
permeability is markedly less than permeability from VEGFA.sub.165
or laser-induced damage (FIG. 2D) further emphasizing the local
nature of TMEM-mediated vascular permeability.
[0069] TMEM-Associated Macrophages are Essential for Vascular
Permeability and Tumor Cell Intravasation.
[0070] To determine if TMEM macrophages regulate vascular
permeability and tumor cell intravasation, macrophages were
depleted in the mammary tumor using the previously characterized
mouse model, MAFIA (macrophage fas-induced apoptosis) (18, 19) with
orthotopic MMTV-PyMT tumor implants. Depletion of macrophages is
systemic, including the mammary tumor, thus resulting in a
depletion of TAM and TMEM by 67% and 72% respectively (FIG. 2G-I).
When macrophages are depleted, extravascular dextran decreases, as
does the number of circulating tumor cells (FIGS. 2J, K and L).
These data demonstrate that macrophages are essential for vascular
permeability and tumor cell intravasation at TMEM.
[0071] Since blood vessel permeability observed by IVM is
restricted to TMEM, it was examined if vascular junction protein
localization was altered in the absence of macrophages, reflecting
a requirement for macrophage-dependent signaling events to induce
vascular permeability. Staining for vascular junction proteins ZO-1
and VE-Cadherin increased in the tumor vasculature after depletion
of macrophages in MAFIA mouse tumors (FIGS. 2J, M and N) indicating
that macrophages are involved in vascular junction disassembly
during vascular permeability events at TMEM.
[0072] Tie2-Expressing Macrophages are Localized in TMEM
Structures.
[0073] In PyMT mammary carcinoma, a subpopulation of TAMs has been
identified as Tie2.sup.Hi perivascular macrophages (7, 20, 21).
Tie2-expressing macrophages (TEMs) have been shown to upregulate
the Tie2 tyrosine kinase receptor by 100 fold after recruitment to
the tumor (22). TEMs have features of pro-tumorigenic macrophages
and promote tumor angiogenesis (7). TEMs are further characterized
as MRC1+/CD11b+/F4/80+/CD11c- and are associated with CD31+ tumor
blood vessels (20). Thus, it was determined if Tie2-expressing
macrophages are located in TMEM. Immunofluorescence of TMEM markers
Mena (tumor cells), CD31 (endothelial cells) and CD68 (macrophage)
(FIG. 3A) compared to Tie2, VEGFA and CD31 in sequential tissue
sections demonstrates that Tie2.sup.Hi/VEGFA.sup.Hi macrophages are
enriched in TMEM structures (FIG. 3B). VEGFA is elevated in
Tie2.sup.Hi macrophages, as compared to the adjacent endothelial
cells and surrounding tumor tissue (FIGS. 3C and D). Further, 100%
of Tie2.sup.Hi/VEGFA.sup.Hi TMEM-associated macrophages express the
TEM markers MRC1, CD11b and F4/80 while lacking CD11c (FIG. 3E,
F).
[0074] Inhibition of VEGFA signaling reduces vascular permeability
and tumor cell intravasation. To investigate the importance of
VEGFA in TMEM function, VEGFA binding to VEGF receptors was blocked
using a neutralizing antibody (B20-4.1.1), which resulted in a
decrease in extravascular dextran and circulating tumor cells
(FIGS. 3G, H and I). Binding of VEGFA to VEGR2 leads to junction
disassembly (23). Vascular ZO-1 and VE-Cadherin staining increased
during VEGFA inhibition suggesting an increase in integrity of
endothelial adherens and tight junctions from reduced
bioavailability of VEGFA, including VEGFA from TMEM (FIGS. 3G, J
and K).
[0075] VEGFA Signaling from Tie2.sup.Hi/VEGFA.sup.Hi TMEM
Macrophages Mediates Vascular Permeability and Tumor Cell
Intravasation.
[0076] To determine if the subpopulation of
Tie2.sup.Hi/VEGFA.sup.Hi macrophages in TMEM are an essential
source of VEGFA in the tumor microenvironment required for
transient vascular permeability at TMEM and tumor cell
intravasation, VEGFA was selectively ablated in monocytes and
macrophages using the Vegfaflox/flox; Csflr-Mer-iCre-Mer transgenic
mouse depletion model of Vegfa that targets myeloid cells
expressing Csflr, including both Ly6C.sup.Hi and Ly6C.sup.Lo
populations, including the TEM population (24). Macrophage-specific
depletion of VEGFA reduced transient vascular permeability, and
circulating tumor cells, while restoring vascular junctions (FIGS.
4A, B, C, D and E). Immunofluorescence of sequential sections
demonstrates that blood vessels adjacent to
CD68+/Tie2.sup.Hi/VEGFA.sup.Hi TMEM macrophage have significantly
reduced vascular VE-Cadherin/CD31 relative intensity compared to
regions of vasculature away from TMEM sites in Vegfa.sup.flox
tumors (FIGS. 4 F and G). Further, when VEGFA has been ablated in
Vegfa.sup.flox; Csflr-Cre tumors VE-Cadherin/CD31 relative staining
intensity is the same along the tumor vasculature as in regions
away from TMEM (FIGS. 4H and I). Therefore, vascular junction
integrity, as measured by VE-Cadherin/CD31 relative staining
intensity, is only significantly reduced in regions of vasculature
adjacent to VEGFA.sup.Hi TMEM macrophages in TMEM (FIG. 4J).
Further, pericyte coverage of the vasculature is reduced in regions
of VEGFA.sup.Hi TEMs in TMEM as compared to regions away from
VEGFA.sup.Hi TMEM structures. A decrease in pericyte coverage of
vasculature has been correlated with increased metastasis and
vascular permeability (25).
[0077] To establish the relevance of Tie2.sup.Hi/VEGFA.sup.Hi
macrophages in TMEM structures in mediating vascular permeability
and tumor cell dissemination in metastastic breast cancer, vascular
junction staining was measured in human breast cancer patient
samples. Staining of sequential sections demonstrates that blood
vessels adjacent to Tie2.sup.Hi/VEGFA.sup.Hi macrophages in TMEM
have significantly reduced vascular VE-Cadherin fluorescence
intensity compared to regions of vasculature away from TMEM (FIG.
4L, M).
[0078] Together these data establish that the
Tie2.sup.Hi/VEGFA.sup.Hi TMEM macrophages interact with endothelial
cells through VEGFA signaling to mediate local, transient blood
vessel permeability demonstrating the mechanism underlying the
clinically-demonstrated association of TMEM density with metastatic
recurrence of breast cancer.
DISCUSSION
[0079] Although the abnormality and permeability of tumor
vasculature has been well characterized, the mechanism leading to
spatial and temporal heterogeneity in permeability has not been
resolved. The use of high-resolution multiphoton microscopy has
allowed for the study of vascular permeability and tumor cell
dissemination in mammary carcinoma at unprecedented spatial and
temporal resolution. The present data show that in the PyMT
authochthonous mouse mammary carcinoma and human patient-derived
xenograft TN1 models, that vascular permeability is dynamic,
localized, and restricted to TMEM. These data are consistent with
previous findings that hyperpermeability of tumor vasculature is
heterogeneous and often in the presence of perivascular macrophages
(4), but further explains the observed heterogeneity and that tumor
cell intravasation occurs at sites of vascular permeability.
[0080] The sites of dynamic tumor vascular permeability have been
identified at sites of VEGFA.sup.Hi perivascular macrophages at
TMEM. The clinical significance of TMEM density in predicting
metastatic risk has been recently expanded to a large cohort of
patients, further emphasizing the importance of TMEM in breast
cancer metastasis (26). These data demonstrate that
Tie2.sup.Hi/VEGFA.sup.Hi perivascular macrophages in TMEM share the
characteristics of the pro-angiogenic and pro-metastatic
Tie2-expressing macrophages (7).
[0081] Mechanistically, macrophage/tumor cell streams migrate to
TMEM sites through the EGFR/CSF-1R paracrine loop (27). Elevated
expression of VEGFA in the Tie2.sup.Hi TMEM macrophage results in
transient permeability of tumor blood vessels proximal to TMEM that
occurs by disassembling endothelial cell junctions. The
simultaneous attraction of migratory tumor cells and transient
blood vessel permeability results in a concurrent spike in tumor
cell intravasation with vascular permeability at TMEM sites (FIG.
4K). These data, together with the clinical association of TMEM
with distant metastatic tumor recurrence in human breast cancer
patients explain why TMEM density can predict metastasis and argues
for the development of therapeutic approaches targeted against both
TMEM formation and function. TMEM.sup.Active Test
[0082] Hematogenous dissemination of tumor cells from the primary
tumor is an essential step in metastasis and is unrelated to growth
potential. The sites of tumor cell dissemination are called TMEM
which is defined as the direct contact between a macrophage, tumor
cell and endothelial cell (9, 28). The sum of TMEM number in ten
40.times. fields predicts the risk of distant recurrence in breast
cancer patients (11, 28). Intravital high-resolution two-photon
microscopy of live mammary tumors shows that vascular leakiness and
tumor cell intravasation occur exclusively at TMEM (FIG. 5A-D).
This, dissemination of tumor cells from solid tumors such as breast
tumors occurs only at TMEM (FIG. 9A,B). Ablation of TMEM
macrophages blocks TMEM-associated vascular leakiness and
intravasation, demonstrating an essential role of macrophages in
TMEM function (FIG. 5E-H).
[0083] Macrophages in TMEM are Tie2.sup.Hi/VEGFA.sup.Hi (FIG. 6).
Tie2.sup.Hi/VEGFA.sup.Hi pen-vascular macrophages are the type of
macrophage that is found in TMEM. The presence of a
Tie2.sup.Hi/VEGFA.sup.Hi macrophage in contact with a blood vessel
indicates a site of TMEM. TMEM can be identified as
Tie2.sup.Hi/VEGFA.sup.Hi CD68+ cells in direct contact with a blood
vessel.
[0084] VEGFA signaling from Tie2.sup.Hi/VEGFA.sup.Hi
TMEM-associated macrophages causes local loss of vascular
endothelial cell junctions (ZO-1 and VE-Cadherin decrease),
resulting in transient endothelial permeability and tumor cell
intravasation (FIG. 7). This discovery demonstrates a way to assess
the activity status of TMEM in disseminating tumor cells and to
investigate the efficacy of drug intervention in vascular leakiness
and associated tumor cell intravasation (FIG. 8).
[0085] Hence, the simultaneous staining of Tie2, VEGFA, CD68, CD31
and VE-Cadherin and/or ZO-1 in (e.g., Formalin-Fixed,
Paraffin-Embedded (FFPE)) tumor tissue provides a test to assess
the activity status of TMEM (TMEM.sup.Active) in a patient and the
efficacy of dissemination inhibitor drugs that inhibit TMEM
activity. Tie2.sup.Hi/VEGFA.sup.Hi pen-vascular macrophages
associated with low levels of VE-Cadherin and ZO-1 endothelial
staining indicate TMEM sites that are active in tumor cell
dissemination while Tie2.sup.Hi/VEGFA.sup.Hi pen-vascular
macrophages associated with high levels of VE-Cadherin and ZO-1
endothelial staining indicate TMEM sites that are inactive in tumor
cell dissemination (FIGS. 5E-H, 7A-D and 8).
[0086] When TMEM is active the endothelial cell junctions between
blood vessel endothelial cells in contact with TMEM are disrupted
leading to a loss of VE-Cadherin and ZO-1 endothelial staining,
which is correlated with tumor cell intravasation and
dissemination. When TMEM are inactive the level of VE-Cadherin and
ZO-1 endothelial staining at TMEM will be higher than in active
TMEM and identical to the level of staining observed in blood
vessels that are not associated with TMEM in neighboring tissue
(FIG. 10). Hence, the TMEM.sup.Active assay is inherently
quantitative and always has an intrinsic control in the same tissue
section (areas away from TMEM=no TMEM in FIG. 10).
[0087] The relative activity of TMEM can be quantified in (e.g.,
FFPE) tissue sections in several ways, e.g.:
[0088] 1. as the ratio of IV-dextran (or other IV contrast agent)
that has leaked from the blood vessel into the tissue at the time
of Formaldehyde Fixation after IV injection of contrast agent in
preparation for FFPE (measure of local vascular permeability)
divided by ZO-1 or VE-Cad staining intensity at TMEM (FIG. 11).
Higher dextran/ZO-1=more TMEM activity;
[0089] 2. as the ratio of ZO-1 or VE-Cad staining intensity at TMEM
per area of blood vessel (CD31 staining area) (FIG. 12). Higher
VE-Cad or ZO-1/CD31=less TMEM activity;
[0090] 3. as the absolute intensity of ZO-1 or VE-Cad staining
intensity at TMEM sites (FIG. 10). Higher VE-Cad staining=less TMEM
activity.
[0091] The activity status of TMEM (TMEM.sup.Active) in patient
tissue samples can be used to assess the efficacy of dissemination
inhibitor drugs that inhibit TMEM activity. For example, observe
the effects of the following dissemination inhibitors on
TMEM.sup.Active using the quantitation methods for TMEM activity
described in #1-3 above as follows:
[0092] a. Rebastinib, the Tie2 inhibitor which blocks VEGF.sup.Hi
TMEM macrophage function, (FIG. 11);
[0093] b. Knockout of the VEGF gene (Csflr-cre) in macrophages
which blocks TMEM macrophage function (FIG. 12).
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