Detection of Activation of Endothelial Cells as Surrogate Marker for Angiogenesis

Abstract

Methods, compositions and kits are provided for assessing angiogenesis through sensitive, direct detection of activation of endothelial cells at molecular levels. In general, activation of endothelial cells is detected by measuring the levels of cellular components and their protein complexes participating in a specific angiogenesis signaling pathway in endothelial cells. The methods can be used for assessing status of diseases associated with undesirable angiogenesis, such as the likelihood of developing the disease, presence or absence of the disease, prognosis of the disease and the likelihood of response or resistance to a particular anti-angiogenic therapy. The methods can also be used to guide the design of effective therapeutic regimens targeting a specific angiogenic signaling pathway, as well as in conjunction with therapeutic intervention of diseases or conditions associated with undesirable angiogenesis.

1 . A method for detecting activation of endothelial cells in a test sample, comprising:
 measuring the level of a protein complex in the endothelial cells in a test sample, wherein the protein complex is formed between a first cellular component and a second cellular component that are cellular components in an angiogenesis signaling pathway; and   wherein a difference in the level of the protein complex relative to the level of the protein complex in a reference sample detects activation of endothelial cells in the test sample.   
     
     
         2 . The method of  claim 1 , wherein the first cellular component and the second cellular component are each independently selected from the group consisting of VEGFR, Nrp, heparin sulphate, VE-cadherin, Tie, VEGF, P1GF, PDGFR, EphA, EphB, Flt, FGFR, Stat, BAD, RSK, P13K, FAK, Src, P70S6K, SHC, SHC, Akt, Erk, JNK, P38, and MEK. 
     
     
         3 . The method of  claim 1 , wherein the said protein complex is selected from the group consisting of VEGFR1 homodimers, VEGFR2 homodimers, VEGFR1-VEGFR2 heterodimers, VEGFR2-VEGFR3 heterodimers, VEGFR2-SHC complexes, and VEGFR3-SHC complexes. 
     
     
         4 . The method of  claim 1 , wherein the test sample contains circulating endothelial cells or circulating endothelial cell progenitors. 
     
     
         5 . The method of  claim 4 , further comprising isolating circulating endothelial cells or circulating endothelial cell progenitors. 
     
     
         6 . The method of  claim 5 , wherein circulating endothelial cells or circulating endothelial cell progenitors are isolated by immunomagnetic isolation. 
     
     
         7 . The method of  claim 1 , wherein the test sample contains tumor endothelium. 
     
     
         8 . The method of  claim 1 , wherein the test sample is a blood sample. 
     
     
         9 . The method of  claim 1 , wherein the test sample is a fixed tissue sample. 
     
     
         10 . The method of  claim 1 , wherein the step of measuring the level of the protein complex in the endothelial cells comprises:
 mixing (i) the test sample; (ii) a cleaving probe, which is capable of binding the first cellular component and has a cleavage-inducing moiety with an effective proximity; and (iii) one or more binding compounds, wherein each of the binding compounds is capable of binding the first or second cellular component and wherein each of the one or more binding compounds has one or more molecular tags each attached thereto by a cleavable linkage; wherein cleavage of the cleavable linkage(s) within the effective proximity of the cleaving-inducing moiety of the cleaving probe releases the molecular tag(s), wherein detecting the released molecular tag(s) provides a measurement of the protein complex.   
     
     
         11 . The method of  claim 10 , wherein the first or second cellular component is a cell surface receptor. 
     
     
         12 . The method of  claim 11 , wherein the cell surface receptor is selected from the group consisting of VEGFR, Tie, PDGFR, and FGFR. 
     
     
         13 . The method of  claim 11 , wherein the cell surface receptor is VEGFR2 or Tie-2. 
     
     
         14 . The method of  claim 10 , wherein the molecular tag(s) attached to different binding compounds each have a different separation characteristics. 
     
     
         15 . The method of  claim 14 , further comprising separating the released molecular tag(s). 
     
     
         16 . The method of  claim 10 , wherein said step of mixing includes generating an active species by said cleavage-inducing moiety, wherein the active species cleaves said cleavable linkages within said effective proximity. 
     
     
         17 . The method of  claim 10 , further comprising measuring the level of an effector protein in the angiogenesis signaling pathway that has a post-translational modification site in the endothelial cells in the test sample. 
     
     
         18 . The method of  claim 17 , wherein the effector protein is selected from the group consisting of Stat, BAD, RSK, P13K, FAK, Src, P70S6K, SHC, Akt, Erk, JNK, P38, and MEK. 
     
     
         19 . The method of  claim 1 , wherein the test sample is obtained from an individual who is suspected of having a disease associated with undesirable angiogenesis, and wherein detecting activation of endothelial cells in the test sample indicates that the individual has the disease. 
     
     
         20 . A method for screening patients to determine the likelihood that a patient will respond to treatment by an anti-angiogenic agent, comprising:
 measuring the level of a protein complex in the endothelial cells in a test sample from a patient, wherein the protein complex is formed between a first cellular component and a second cellular component that are cellular components in an angiogenesis signaling pathway;   wherein an increase in the level of the protein complex in the test sample from the patient relative to a reference level characteristic of normal endothelial cells indicates that the likelihood that the patient will respond to treatment by an anti-angiogenic agent.   
     
     
         21 . A method for determining whether a patient will respond to treatment by an anti-angiogenic agent, comprising:
 measuring the level of a protein complex in the endothelial cells in a test sample from a patient who has been treated with an anti-angiogenic agent, wherein the protein complex is formed between a first cellular component and a second cellular component that are cellular components in an angiogenesis signaling pathway;   wherein a decrease in the level of the protein complex in the vascular (or CEC/CECP) endothelial cells from the test sample relative to a reference level of the protein complex in endothelial cells in the patient prior to the treatment, indicates that the patient is likely to respond to the treatment by the anti-angiogenic agent.   
     
     
         22 . A method for determining whether a patient has developed resistance to treatment of an anti-angiogenic agent, comprising:
 measuring the level of a protein complex in the endothelial cells in a test sample from a patient who has been treated with an anti-angiogenic agent, wherein the protein complex is formed between a first cellular component and a second cellular component that are cellular components in an angiogenesis signaling pathway;   wherein an increase in the level of the protein complex in the endothelial cells from the test sample relative to a reference level of the protein complex in the endothelial cells in the patient prior to treatment, indicates that the patient has likely developed resistance to the treatment of the anti-angiogenic agent.   
     
     
         23 . A method for detecting activation of endothelial cells in a test sample, comprising:
 measuring in a test sample the levels of two or more different cellular components that participate in one or more angiogenesis signaling pathways;   wherein a difference in the levels of the two or more different cellular components relative to reference levels of the two or more different cellular components, indicates activation of endothelial cells in a test sample.   
     
     
         24 . The method of  claim 23 , wherein the two or more cellular component are selected from the group consisting of VEGFR, Nrp, heparin sulphate, VE-cadherin, Tie, VEGF, P1GF, PDGFR, EphA, EphB, Flt, FGFR, Stat, BAD, RSK, P13K, FAK, Src, P70S6K, SHC, SHC, Akt, Erk, JNK, P38, and MEK. 
     
     
         25 . The method of  claim 23 , wherein the test sample contains circulating endothelial cells or circulating endothelial cell progenitors. 
     
     
         26 . The method of  claim 23 , wherein the test sample contains tumor endothelium. 
     
     
         27 . The method of  claim 23 , wherein the test sample is a blood sample. 
     
     
         28 . The method of  claim 23 , wherein the test sample is a fixed tissue sample. 
     
     
         29 . The method of  claim 23 , wherein the step of measuring the levels of two or more different cellular components in the endothelial cells includes
 mixing (i) the test sample; (ii) a cleaving probe, which is capable of binding one of the two or more cellular components and has a cleavage-inducing moiety with an effective proximity; and (iii) one or more binding compounds, wherein each of the two or more cellular components is bound by at least one member of the one or more binding compounds, and wherein each of the binding compounds has one or more molecular tags each attached thereto by a cleavable linkage; wherein cleavage of the cleavable linkage(s) within the effective proximity of the cleaving-inducing moiety of the cleaving probe releases the molecular tag(s), wherein detecting the released molecular tag(s) provides a measurement of the levels of two or more different cellular components in the endothelial cells.   
     
     
         30 . The method of  claim 29 , wherein at least one of the two or more cellular components is a cell surface receptor. 
     
     
         31 . The method of  claim 30 , wherein the cell surface receptor is selected from the group consisting of VEGFR, Tie, PDGFR, and FGFR. 
     
     
         32 . The method of  claim 30 , wherein the cell surface receptor is VEGFR2 or Tie-2. 
     
     
         33 . The method of  claim 30 , wherein the molecular tag(s) attached to different binding compounds each have a different separation characteristics. 
     
     
         34 . The method of  claim 33 , further comprising separating the released molecular tag(s). 
     
     
         35 . The method of  claim 30 , wherein said step of mixing includes generating an active species by said cleavage-inducing moiety, wherein the active species cleaves said cleavable linkages within said effective proximity. 
     
     
         36 . The method of  claim 30 , further comprising measuring the level of an effector protein in the angiogenesis signaling pathway that has a post-translational modification site in the endothelial cells in the test sample. 
     
     
         37 . The method of  claim 36 , wherein the effector protein is selected from the group consisting of Stat, BAD, RSK, P13K, FAK, Src, P70S6K, SHC, Akt, Erk, JNK, P38, and MEK.