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US20180106811A1 - Tmem active test and uses thereof in diagnosis, prognosis and treatment of tumors - Google Patents

Tmem active test and uses thereof in diagnosis, prognosis and treatment of tumors Download PDF

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US20180106811A1
US20180106811A1 US15/573,498 US201615573498A US2018106811A1 US 20180106811 A1 US20180106811 A1 US 20180106811A1 US 201615573498 A US201615573498 A US 201615573498A US 2018106811 A1 US2018106811 A1 US 2018106811A1
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tmem
tumor
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vegfa
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John S. Condeelis
Allison S. Harney
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Albert Einstein College of Medicine
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G01N2033/57403—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast

Definitions

  • VEGFA Vascular endothelial growth factor A
  • Tumor-associated macrophages have been implicated in tumor progression, angiogenesis and metastasis ( 5 , 6 ).
  • 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 ).
  • the mechanistic link between perivascular macrophages and tumor cell intravasation remained unclear.
  • hyperpermeability in tumor vasculature is not uniform, but rather is spatially and temporally heterogeneous ( 12 ).
  • the presence of macrophages at vascular branch points was observed at hotspots of vascular permeability ( 4 ).
  • 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.
  • determining the presence of one or more sites that are active in tumor cell dissemination in a subject comprising
  • Tie2 Hi /VEGFA 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 Active sites), and
  • Tie2 Hi /VEGFA 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.
  • 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 Active sites, wherein the risk of tumor cells undergoing hematogenous metastasis increases with the presence of TMEM Active sites.
  • Still further provided are methods for determining a course of treatment for a tumor in a subject comprising determining whether or not a tumor sample from a subject contains TMEM Active sites, wherein the presence of TMEM Active sites indicates that the subject should be treated for a metastatic tumor or wherein a lack of TMEM Active sites indicates that the subject does not need to be treated for a metastatic tumor.
  • 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 Active sites by the method disclosed herein, wherein a reduction of TMEM 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 Active sites in the subject undergoing anti-cancer therapy indicates that the anti-cancer therapy may not be effective.
  • a method of preventing or reducing tumor cell dissemination and metastasis in a subject comprises:
  • 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 Active sites, wherein an agent that reduces the number of TMEM Active sites is a candidate agent for treating or preventing hematogenous metastasis.
  • 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 ⁇ m.
  • B 3D reconstruction of time-lapse IVM from (A) of TC and macrophage streaming towards TMEM (asterisk). Scale bar, 20 ⁇ m.
  • C 3D reconstruction of TC intravasation (arrowhead) at TMEM (luminal surface of the endothelium dashed white line).
  • 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.
  • FIG. 1 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 ⁇ 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.
  • M Quantification of extravascular dextran intensity and CTC area at TMEM over time from F.
  • ⁇ Extravascular dextran, ( ⁇ ) CTC.
  • FIG. 2A-2N Macrophage depletion reduces vascular permeability and tumor cell intravasation.
  • B 155 kDa dextran-TMR is injected by tail vein i.v. catheter followed by 8 ⁇ g of VEGFA 165 at 0′.
  • D Quantification of total extravascular 155 kDa dextran-TMR area after laser-induced damage, i.v.
  • VEGFA 165 or spontaneous permeability at TMEM injection of VEGFA 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.
  • ⁇ laser damage
  • F Table of parameters from curve fitting to an Exponentially Modified Gaussian function using data from (E).
  • TMEM 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 ⁇ 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),
  • J Immunfluorescence imaging of tumor sections stained for vasculature (CD31), 155 kDa dextran-TMR and DAPI, ZO-1 or VE-Cadherin as indicated.
  • FIG. 3A-3K Inhibition of VEGFA from Tie2 Hi /VEGF 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 ⁇ m.
  • B Immunofluorescence imaging of VEGFA Hi macrophages in TMEM in sequential sections. Scale bar, 10 ⁇ m. Tumor cell, spotted line; macrophages, solid line; and blood vessels, dashed line.
  • FIG. 1 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 Hi endothelial cells CD31 and macrophages CD68, VEGFA Hi and Tie2 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 ⁇ m.
  • VEGFA 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 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 ⁇ m.
  • FIG. 1 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 ⁇ m.
  • FIG. 4A-4M Macrophage-specific ablation of Vegfa in PyMT implant tumors (Vegfa flox ;Csflr-Cre) compared to control (Vegfa flox ) blocks blood vessel permeability and tumor cell intravasation.
  • Vegfa flox compared to control (Vegfa 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 ⁇ m.
  • ⁇ Relative fluorescence intensity of VE-Cadherin/CD31
  • ⁇ Relative VEGFA intensity. Dashed line indicates the presence of a CD68+ macrophage.
  • TMEM assemble with close association between the non-migratoryTMEM TC (T1) and Tie2 Hi /VEGFA Hi macrophage (bM1) on blood vessels. VEGFA destabilizes vascular junctions resulting in vascular permeability and TC (T2) intravasation.
  • T1 non-migratoryTMEM TC
  • bM1 Tie2 Hi /VEGFA Hi macrophage
  • T2 vascular permeability
  • 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 ⁇ m.
  • 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.
  • B 3D reconstruction of TMEM from live tumor time lapse. Tumor cells approaching the blood vessel in a stream are indicated by arrowheads.
  • TMEM macrophage M
  • tumor cell TC
  • EC blood vessel endothelial cell
  • 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.
  • FIG. 6A-6B TMEM macrophages are VEGF Hi /Tie2 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 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.
  • TMEM When Tie2 Hi macrophage of TMEM signals with elevated VEGF, endothelial cell junction remodeling occurs through VEGF signaling. VEGF signaling. VEcular 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.
  • FIG. 9A-9B Blood vessel permeability and tumor cell intravasation are linked and occur only at TMEM sites.
  • FIG. 11A-11B Rebastinib, an inhibitor of Tie2 macrophage function in TMEM, inhibits TMEM activity.
  • A Quantification of TMEM density in 10 40 ⁇ fields (not significant).
  • a method of determining the presence of one or more sites that are active in tumor cell dissemination in a subject comprising
  • 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
  • Tie2 Hi /VEGFA 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 Active sites), and
  • Tie2 Hi /VEGFA 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.
  • TMEM Active sites are TMEM sites that are active in tumor cell dissemination.
  • 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 Active sites by the method disclosed herein, wherein the risk of tumor cells undergoing hematogenous metastasis increases with the presence of TMEM Active sites.
  • 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 Active sites by the method disclosed herein, wherein the presence of TMEM Active sites indicates that the subject should be treated for a metastatic tumor or wherein a lack of TMEM Active sites indicates that the subject does not need to be treated for a metastatic tumor.
  • 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 Active sites by the method disclosed herein, wherein a reduction of TMEM 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 Active sites in the subject undergoing anti-cancer therapy indicates that the anti-cancer therapy may not be effective.
  • a method of preventing or reducing tumor cell dissemination and metastasis in a subject comprises:
  • 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.
  • Still further provided is a method for identifying an agent to treat or prevent hematogenous metastasis comprising contacting a tumor sample with the agent, assaying the tumor sample for the presence of TMEM Active sites by the method disclosed herein, and analyzing whether or not the agent reduces the number of TMEM Active sites, wherein an agent that reduces the number of TMEM Active sites is a candidate agent for treating or preventing hematogenous metastasis.
  • the tumor is contacted with the agent in vivo or ex vivo.
  • kits for detecting the presence of tumor sites that are active in tumor cell dissemination 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′) 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.
  • 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.
  • TMEM Tumor MicroEnvironment of Metastasis
  • VEGFA signaling from Tie2 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.
  • Tumor vasculature is abnormal with increased vascular permeability.
  • VEGFA signaling from Tie2 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.
  • TMEM 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.
  • TMEM tumor cell intravasation and blood vessel permeability
  • the CD31 channel blood vessel
  • dextran and vascular junction ZO-1 or VE-Cadherin
  • ZO-1 or VE-Cadherin vascular junction
  • ZO-1 or VE-Cadherin vascular junction
  • VE-Cadherin vascular junction
  • a binary mask of the blood vessels was created to define the boundaries of the signal inside blood vessels. Structures smaller than 100 px 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.
  • Vascular VE-Cadherin adjacent to TMEM was quantified by the mean VE-Cadherin staining intensity in CD31+ vasculature adjacent to CD68+/Tie2 Hi /VEGFA Hi macrophages, CD68+ macrophages or in the absence of macrophages in sequential tissue sections. Four different fields (of 2 ⁇ 2 40 ⁇ 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 ⁇ 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 ⁇ m lengths of vasculature. Measurements were repeated until the end of the length (25 ⁇ 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 Hi macrophages, CD68+ macrophages or in the absence of macrophages in sequential tissue sections.
  • Four different fields (of 2 ⁇ 2 40 ⁇ fields with 15% overlap) were acquired per mouse.
  • a box (ROI) was moved along the vasculature in 0.5 ⁇ 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 ⁇ m lengths of vasculature. Measurements were repeated until the end of the length (25 ⁇ m) of the line drawn. Average pixel intensity in NG-2 was determined for each length of vasculature measured and averaged for each animal.
  • Z-stacks of up to 50 ⁇ m of depth were acquired with a 2 ⁇ 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 ⁇ g (0.2 mg/kg) of VEGFA 165 peptide (PeproTech) by the tail vein catheter to induce systemic vascular permeability as previously described ( 29 , 30 ).
  • 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.
  • 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.
  • 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.
  • 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.
  • Sliding window measurement of tumor cell intravasation and vascular permeability In this measurement a 100 ⁇ 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.
  • TMEM-Associated Tumor Cells and Macrophages are Stationary in TMEM Structures.
  • TMEM 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.
  • TMEM extended time-lapse intravital microscopy
  • IVAM intravital microscopy
  • vessels were labeled with a high molecular weight compound (155 kDa dextran or quantum dots) ( 1 , 14 ) ( FIG. 1 ).
  • 155 kDa dextran or quantum dots 155 kDa dextran or quantum dots
  • FIG. 1F 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, 2 C).
  • TMR dextran-tetramethylrhodamine
  • TMR dextran-tetramethylrhodamine
  • FIG. 1M tumor cell intravasation occurs at TMEM sites concurrently with transient permeability
  • 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).
  • 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).
  • FIGS. 1F-H , J, and M 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.
  • TMEM transient vascular permeability and tumor cell intravasation
  • a 100 ⁇ 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 ⁇ m window when it contains a TMEM, but never when the 100 ⁇ 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.
  • Vascular Permeability at TMEM is a Highly Localized and Transient Event.
  • Tumor vasculature has been previously described as abnormal with increased vascular permeability, which has been attributed to larger vascular intercellular openings ( 1 , 12 , 16 ).
  • vascular permeability is not spatially or temporally uniform, with hotspots at vascular branch points ( 4 , 12 ).
  • 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.
  • transient permeability events are distinct from mechanical damage to the endothelium.
  • 155 kDa dextran-TMR extravasates continuously, filling the field of view ( FIG. 2A ).
  • VEGFA-mediated permeability is transient ( 12 ).
  • Intravenous injection of VEGFA 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 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 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 165 and spontaneous curves have similar extravasation and clearance rates.
  • VEGFA 165 A significant difference between VEGFA 165 and spontaneous TMEM-mediated permeability is that permeability at TMEM is highly local, while VEGFA 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 165 or laser-induced damage ( FIG. 2D ) further emphasizing the local nature of TMEM-mediated vascular permeability.
  • TMEM-Associated Macrophages are Essential for Vascular Permeability and Tumor Cell Intravasation.
  • 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.
  • 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.
  • Tie2-Expressing Macrophages are Localized in TMEM Structures.
  • Tie2 Hi perivascular macrophages 7 , 20 , 21 .
  • Tie2-expressing macrophages 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.
  • FIG. 3A Immunofluorescence of TMEM markers Mena (tumor cells), CD31 (endothelial cells) and CD68 (macrophage)
  • FIG. 3B VEGFA and CD31 in sequential tissue sections demonstrates that Tie2 Hi /VEGFA Hi macrophages are enriched in TMEM structures
  • FIGS. 3C and D VEGFA is elevated in Tie2 Hi macrophages, as compared to the adjacent endothelial cells and surrounding tumor tissue.
  • 100% of Tie2 Hi /VEGFA Hi TMEM-associated macrophages express the TEM markers MRC1, CD11b and F4/80 while lacking CD11c ( FIG. 3E , F).
  • 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).
  • VEGFA Signaling from Tie2 Hi /VEGFA Hi TMEM Macrophages Mediates Vascular Permeability 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 Hi and Ly6C Lo populations, including the TEM population ( 24 ).
  • FIGS. 4A , B, C, D and E 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 Hi /VEGFA Hi TMEM macrophage have significantly reduced vascular VE-Cadherin/CD31 relative intensity compared to regions of vasculature away from TMEM sites in Vegfa flox tumors ( FIGS. 4 F and G).
  • 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 Hi TMEM macrophages in TMEM ( FIG. 4J ). Further, pericyte coverage of the vasculature is reduced in regions of VEGFA Hi TEMs in TMEM as compared to regions away from VEGFA Hi TMEM structures. A decrease in pericyte coverage of vasculature has been correlated with increased metastasis and vascular permeability ( 25 ).
  • vascular junction staining was measured in human breast cancer patient samples. Staining of sequential sections demonstrates that blood vessels adjacent to Tie2 Hi /VEGFA Hi macrophages in TMEM have significantly reduced vascular VE-Cadherin fluorescence intensity compared to regions of vasculature away from TMEM ( FIG. 4L , M).
  • TMEM tumor cell dissemination
  • 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 ⁇ 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 ).
  • Macrophages in TMEM are Tie2 Hi /VEGFA Hi ( FIG. 6 ).
  • Tie2 Hi /VEGFA Hi pen-vascular macrophages are the type of macrophage that is found in TMEM.
  • the presence of a Tie2 Hi /VEGFA Hi macrophage in contact with a blood vessel indicates a site of TMEM.
  • TMEM can be identified as Tie2 Hi /VEGFA Hi CD68+ cells in direct contact with a blood vessel.
  • VEGFA signaling from Tie2 Hi /VEGFA 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 ).
  • ZO-1 and VE-Cadherin decrease vascular endothelial cell junctions
  • VE-Cadherin decrease
  • 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 Active ) in a patient and the efficacy of dissemination inhibitor drugs that inhibit TMEM activity.
  • TMEM Active TMEM Active
  • dissemination inhibitor drugs that inhibit TMEM activity.
  • Tie2 Hi /VEGFA 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 Hi /VEGFA 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 , 7 A-D and 8 ).
  • TMEM 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.
  • TMEM 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 ).
  • TMEM relative activity of TMEM can be quantified in (e.g., FFPE) tissue sections in several ways, e.g.:
  • TMEM Active The activity status of TMEM (TMEM 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 Active using the quantitation methods for TMEM activity described in #1-3 above as follows:
  • Rebastinib the Tie2 inhibitor which blocks VEGF Hi TMEM macrophage function, ( FIG. 11 );

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KR20200012227A (ko) * 2018-07-26 2020-02-05 부산대학교 산학협력단 Zo-1을 포함하는 혈관 질환 진단용 바이오 마커 조성물
KR102129761B1 (ko) 2018-07-26 2020-07-03 부산대학교 산학협력단 Zo-1을 포함하는 혈관 질환 진단용 바이오 마커 조성물
CN113466453A (zh) * 2021-05-13 2021-10-01 天津市肿瘤医院（天津医科大学肿瘤医院） Hcc组织切片中巨噬细胞包绕肿瘤细胞簇结构及其作为肿瘤标志物的用途和识别方法

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