TUMOR CELL-DERIVED MICROVESICLES
20220187300 · 2022-06-16
Inventors
- Janusz RAK (Montreal, CA)
- Khalid AL-NEDAWI (St-Lambert, CA)
- Brian MEEHAN (Montreal, CA)
- Abhijit GUHA (Toronto, CA)
Cpc classification
G01N2333/912
PHYSICS
G01N33/57484
PHYSICS
A61K45/06
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to a method for diagnosis of cancer and for monitoring the progression of cancer and/or the therapeutic efficacy of an anticancer treatment in a sample of a subject by detecting oncogenic and cancer related proteins in microvesicles, and to the use of an agent blocking exchange of microvesicles for treating cancer.
Claims
1. A method of detecting the presence of an oncogenic protein or an MV-associated protein in a subject, comprising: collecting a sample from the subject; isolating microvesicles from the sample; and detecting the presence of the oncogenic protein or the MV-associated protein in the microvesicles.
2. A method for diagnosing or determining prognosis of a cancer in a subject, comprising: detecting the presence of an oncogenic protein or an MV-associated protein in microvesicles in accordance with the method of claim 1, wherein the presence of the oncogenic protein or the MV-associated protein in the sample is indicative that the subject may have cancer.
3. A method for monitoring progression of a cancer or therapeutic efficacy of an anti-cancer treatment in a subject, comprising: a) collecting a first sample from a subject having cancer at a first timepoint, isolating microvesicles from the first sample, and measuring an oncogenic protein or an MV-associated protein in the microvesicles obtained from the first sample; and b) collecting a second sample from the subject having cancer at a second timepoint. the second timepoint occurring after the first timepoint. isolating microvesicles from the second sample. and measuring the oncogenic protein or the MV-associated protein in the microvesicles obtained from the second sample; wherein a change in the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the second sample compared to the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the first sample is indicative of progression of the cancer, or a reduction or no change in the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the second sample compared to the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the first sample indicates therapeutic efficacy of the anti-cancer treatment when the first timepoint occurs before the subject has received an anti-cancer treatment. And the second timepoint occurs after the subject has received the anti-cancer treatment; and wherein said sample is a bodily fluid selected from the group consisting of blood, lymph, urine, cerebrospinal fluid, ascites, saliva, lavage, semen, glandular secretions, exudate, contents of cysts and feces.
4. The method according to any one of claims 1-3, wherein said oncogenic or MV-associated protein is selected from the group consisting of EGFRvIII, EGFR, HER-2, HER-3, HER-4-, MET, cKit, PDGFR, Wnt, beta-catenin, K-ras, H-ras, N-ras, Raf, N-myc, c-myc, IGFR, PI3K, Akt, BRCA1, BRCA2, PTEN, FGFR3, EphB2, ROR1, EphA2, EphA4, receptors of cancer associated cells selected from the group consisting of VEGFR-2, VEGFR-1, Tie-2, TEM-1 and CD276, and proteins listed in Tables 1 to 4.
5. The method according to claim 1, further comprising the step of measuring the phosphorylation state of the oncogenic protein.
6. The method according to claim 3, wherein: an increase in the amount of the oncogenic protein in the microvesicles obtained from the second sample compared to the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the first sample indicates that the cancer has progressed or continued to proliferate; a reduction in the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the second sample, or a change in the composition of the microvesicles obtained from the second sample, compared to the amount of the oncogenic or MV-associated protein in the microvesicles, or the composition of the microvesicles, obtained from the first sample indicates that the cancer has regressed; and no change in the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the second sample compared to the amount of the oncogenic or MV-associated protein in the microvesicles obtained from the first sample indicates the cancer has not progressed.
7. The method according to claim 1, wherein at least two oncogenic or MV-associated proteins are detected in the microvesicles.
8. The method according to claim 1, wherein the microvesicles are isolated by ultracentrifugation, immunoprecipitation, affinity purification or microfiltration.
9. The method according to claim 1, wherein the presence of the oncogenic or MV-associated protein in the microvesicles is detected or measured by immunoblot, immunoprecipitation, ELISA, RIA, flow cytometry, electron microscopy or mass spectrometry.
10. The method according to claim 1, wherein the oncogenic or MV-associated protein in the microvesicles is detected or measured by ELISA with wells coated with Annexin V.
11. The method according to claim 3, wherein the anti-cancer treatment is surgery, radiology, chemotherapy, a targeted cancer treatment, or a combination thereof.
12. The method according to claim 2, 3 or 11, wherein said cancer is selected from the group consisting of breast cancer, glioma, brain cancer, lung cancer, pancreatic cancer, skin cancer, prostate cancer and colorectal cancer.
13-17. (canceled)
18. The method according to claim 2, wherein more than one oncogenic or MV-associated protein is detected in the microvesicles, and the combination of oncogenic or MV-associated proteins detected in the microvesicles is diagnostic of the cancer type.
19. The method according to claim 18, wherein the phosphorylation state of the oncogenic or MV-associated proteins is determined.
20-23. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, an embodiment or embodiments thereof, and in which:
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0067] In accordance with the present invention, there is provided a method of detecting the presence of an oncogenic protein in a subject, comprising collecting a sample from the subject, isolating microvesicles (MVs) from the sample, and detecting the presence of the oncogenic protein in the microvesicles.
[0068] There is also provided herein a method for diagnosing cancer in a sample of a subject by detecting oncogenic proteins in microvesicles.
[0069] In an embodiment, cancer is detected by analyzing microvesicles in a sample, such as a bodily fluid, such as blood, urine, cerebrospinal fluid, lymph, ascites, saliva, lavage, semen, and glandular secretions, as well as feces, exudate, contents of cysts and other sources.
[0070] In another embodiment, a method for prognosis of cancer, by detecting oncogenic proteins in microvesicles, is provided.
[0071] In yet another embodiment, a method for monitoring progression of cancer and/or response to treatment is provided.
[0072] Cancer refers herein to a cluster of cancer cells showing over proliferation by non-coordination of the growth and proliferation of cells due to the loss of the differentiation ability of cells.
[0073] The term “cancer” includes but is not limited to, breast cancer, large intestinal cancer, lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal or colorectal cancer, anal cancer, colon cancer (generally considered the same entity as colorectal and large intestinal cancer), fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vulval cancer, squamous cell carcinoma, vaginal carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, glioma, astrocytoma, glioblastoma multiforme, primary CNS lymphoma, bone marrow tumor, brain stem nerve gliomas, pituitary adenoma, uveal melanoma (also known as intraocular melanoma), testicular cancer, oral cancer, pharyngeal cancer or a combination thereof. In an embodiment, the cancer is a brain tumor, e.g. glioma. In another embodiment, the cancer expresses certain oncoproteins, e.g. HER-2, HER-3 etc. The term “cancer” also includes pediatric cancers, including pediatric neoplasms, including leukemia, neuroblastoma, retinoblastoma, glioma, rhabdomyoblastoma, sarcoma and other malignancies.
[0074] Non-limiting examples of oncogenic proteins which can be detected using the methods of the invention are as follows: (i) membrane-associated oncoproteins derived from cancer cells such as EGFRvIII in glioma, EGFR in squamous cell carcinoma, glioma, lung cancer, or bladder cancer, breast cancer mutant (e.g. Iressa sensitive, mutant or non-expressed tumor suppressor proteins BRCA1 and/or BRCA2), EGFR in lung cancer. HER-2 in breast and ovarian carcinoma, MET in various metastatic and invasive cancers, Kit in gastro-intestinal stromal tumors, PDGFR in glioma, Wnt in various tumors, various phosphatases; (ii) combinatorial clusters of transforming receptors such as EGFR/HER-2 in breast cancer, HER-2/HER-3 in various tumors; (ill) membrane-associated cytoplasmic molecules with transforming properties such as K-ras in colorectal, pancreatic and lung cancer, PTEN (lack of or inactivated) in glioma and prostate cancer, (iv) signaling complexes that could be present (and active) in lipid rafts and microvesicles such as PI3K/Akt, Raf/MEK/MAPK; and (v) tumor related endothelial receptor related to tumor angiogenesis and antiangiogenesis such as VEGFR-2, VEGFR-1, Tie-2 and TEMs (e.g. TEM-1, CD276). These proteins may be detected alone or in combination.
[0075] Other non-limiting examples of oncogenic proteins include EGFRvIII, EGFR, HER-2, HER-3, HER-4, MET, cKit, PDGFR, Wnt, beta-catenin, K-ras, H-ras, N-ras, Raf, N-myc, c-myc, IGFR, IGFR, PI3K, and Akt; tumor suppressor proteins such as BRCA1, BRCA2 and PTEN; cancer-related host receptors and microvesicle-associated molecules, e.g. those involved in angiogenesis such as VEGFR-2, VEGFR-1, Tie-2, TEM-1 and CD276. It is contemplated that all oncogenic proteins, tumor suppressor proteins, host-cell related receptors and microvesicle-associated molecules may be used, alone or in combination, in the methods, compositions and kits of the present invention. It is further contemplated that any oncogenic protein, and any combination of oncogenic proteins, which is determined to be mechanistically, diagnostically, prognostically or therapeutically important for cancer, may be used in the methods, compositions and kits of the present invention.
[0076] The invention described herein is based, at least in part, on the novel and unexpected observation that EGFRvII oncoprotein can be emitted and shared between glioma cells via intercellular transfer of the activated receptor that occurs as cargo of membrane-derived microvesicles released from cells producing the mutant protein. Indeed, EGFRvII stimulates the formation of lipid-raft related microvesicles, to which it becomes incorporated.
[0077] Microvesicles containing EGFRvIII oncoprotein are released to conditioned media or blood of tumor bearing mice and can merge with the plasma membranes of tumor cells lacking this receptor. Such transfer of EGFRvIII triggers the activation of downstream signaling pathways (MAPK and Akt), progression-related changes in gene expression (VEGF, BdxL, p27) and manifestation of exacerbated cellular transformation, notably altered morphology and increased soft agar colony formation efficiency. These observations point to the role of membrane microvesicles in horizontal propagation of transforming proteins between different subsets of cancer cells and suggest that the transforming impact of membrane-associated oncoproteins may extend beyond the cells harboring the corresponding mutant genes.
[0078] Activated cells of various types are known to produce and shed into their surroundings membrane microvesicles, also known as microparticles, ectosomes, or argosomes; in the case where such vesicles originate from the lysosomal pathway, they are often referred to as exosomes. The biological role of these structures is poorly understood, but may include secretory processes, immunomodulation, coagulation and intercellular communication (Janowska-Wieczorek et al., 2005, Int. J Cancer, 20: 752-760).
[0079] Microvesicles may vary in the mechanism of their generation, size and composition, but often (especially ectosomes) contain material associated with membrane lipid rafts, including functional transmembrane proteins. For instance, procoagulant tissue factor (TF) can be released in this fashion from inflammatory cells and, importantly, becomes subsequently incorporated into membranes of platelets, endothelium and other cells where it exerts its biological effects. As used herein, the term “microvesicles” includes microvesicles, microparticles, ectosomes, argosomes, exosomes, tumor vesicles and all other vesicular bodies released from cells.
[0080] Cancer cells lacking the p53 tumor suppressor gene may in some instances mimic this process by releasing altered amounts of TF-containing (Yu et al., 2005, Blood, 105: 1734-1741), or secretory (Yu et al., 2006, Cancer Res, 66: 4795-47801) microvesicles to blood and the pericellular milieu.
[0081] Oncogenic receptors often reside within the regions of the plasma membrane, from which microvesicles originate in cancer cells (e.g. lipid rafts). It is disclosed herein that the oncogenic receptors can themselves become included in the microvesicle cargo. This is of particular interest for example in malignant brain tumors (gliomas) where activation of membrane associated EGFR represents a major transforming event, and in nearly 30% of cases with glioblastoma multiforme (GBM) expression of the EGFRvIII oncogenic mutant is readily detectable.
[0082] In order to explore this phenomenon further, the production of microvesicles by cultured U373 glioma cells lacking the activated EGFR and their counterparts, engineered to express EGFRvIII (U373vIII cells) was examined. Interestingly, the presence of the EGFRvIII oncogene in the latter cell line resulted in formation of multiple vesicular protrusions on the cell surface, an effect that was accompanied by an increase in recovery of protein from the microvesicular fraction of the culture media (see
[0083] In a particular embodiment, proteins enriched in microvesicles, such as EGFRvIII, HER-2, and MET, can be detected by various techniques known in a the art. For example, lysates of microvesicles can be analyzed by immunoblotting using antibodies such as anti-EGFRvIII or anti-EGFR. Concentration of the microvesicles by centrifugation is necessary, but also provides a considerable quantitative and qualitative advantage over the analysis of the whole plasma. This is because microvesicle isolation can improve the sensitivity of detection of certain molecules, e.g. EGFRvIII (due to their enrichment in microvesicles), increase the specificity (as microvesicles are not random collections of plasma membrane molecules), protect the cargo from proteolysis, dephosphorylation or degradation (owing to the microvesicle membrane), and broaden the scope of the analysis (owing to the presence of unique and diagnostically informative combinations of proteins in microvesicle cargo). In this regard, the sensitivity of microvesicle analysis can be increased by switching from ultracentrifugation to microfiltration, the latter of which may simplify and improve the recovery of microvesicles. Another technique to detect microvesicular proteins is immunoprecipitation of microvesicle-related material from magnetic beads coated with e.g. Annexin V (as the MVs express large amounts of phosphatidyl serine) or an antibody binding an oncogenic protein that is expressed on the surface of the MV, such as anti-EGFRvIII antibody.
[0084] Further, an ELISA assay based on two antibodies (e.g. 2×anti-EGFRvIII or anti-EGFRvIII+anti-EGFR) or a radioimmune assay (RIA) based on two antibodies (e.g. 2×anti-EGFRvIII or anti-EGFRvIII+anti-EGFR) can also be used. In addition, ELISA based on binding of microvesicles to surfaces coated with Annexin V (as e.g. in commercial TF assays) or with EGFRvIII/EGFR antibodies could be used in conjunction with a detection component based on the anti-EGFRvIII antibody. Other techniques that can be used include flow cytometry, where microvesicles are captured by beads coated with e.g. Annexin V, or antibodies specific to molecules (proteins, carbohydrates and other) present on their surfaces, or with non-antibody affinity reagents (receptors, aptamers, lectins, nucleic acid sequences, and so on), and stained with e.g. anti-EGFRvIII antibody, and mass spectrometry, where EGFR is detected in the proteome of microvesicle preparations. All such reagents and capture methods could also be used to isolate microvesicles using affinity purification columns and multiplex platforms. It is contemplated that standard techniques known in the art for preparation of microvesicles and for detection of proteins can be used in the methods described herein.
[0085] The present invention is based at least in part, on the observation that abundant expression of EGFRvIII protein is detected in lysates not only of U373vIII cells themselves, but also in their derived microvesicles, demonstrating that the intact oncoprotein is released in this fashion into the circulation. Although the parental U373 cells did release detectable quantities of flotilin-1 containing microvesicles, they contained only trace amounts of wild type EGFR (wtEGFR) and no EGFRvIII. These results were validated against EGFR-negative endothelial cells (HUVEC) and A431 cells expressing only wtEGFR, as well as their respective microvesicle preparations (
[0086] Heterogenous EGFRvIII expression in human glioma suggests that different tumor cell subsets could shed EGFRvIII-containing microvesicles into the common intercellular space. Since microvesicles can readily fuse with cellular membranes via a phosphatidylserine-dependent mechanism, it is here demonstrated that oncogenic EGFRvIII can be transferred in this manner from more aggressive to indolent glioma cells. EGFRvIII-negative U373 cells were, therefore, incubated with preparations of microvesicles obtained from either their U373vIII counterparts harboring EGFRvII, or from U373vIII-GFP cells engineered to express a green fluorescent protein (GFP)-tagged EGFRvIII oncogene (EGFRvIII-GFP). Interestingly, this resulted in an extensive uptake of the microvesicular content by U373 cell, as demonstrated by their de novo surface expression of the EGFRvIII antigen and GFP fluorescence, respectively (
[0087] The apparent intercellular microvesicle-mediated transfer of the ostensibly intact EGFRvIII receptor raises the question, as to the signaling consequences (if any) of this event for the ‘acceptor’ (U373) cells. To address this question, U373 cells 24 hours after their exposure to EGFRvIII-containing microvesicles were examined for activation of the MAPK and Akt cascades, both known to mediate transforming effects downstream of this oncogene. Indeed, incorporation of EGFRvIII into the U373 plasma membrane resulted in a consistent increase in Erk1/2 phosphorylation. This event was. dependent on the transfer of active EGFRvIII, as U373-derived microvesicles, containing no EGFRvIII were ineffective. Moreover, the irreversible blockade of this receptor by preincubation of U373vIII-derived microvesicles with pan-ErbB inhibitor (CI-1033) markedly reduced Erk1/2 phosphorylation (
[0088] The transforming effects of EGFRvIII-dependent pathways are ultimately mediated by deregulation of several genes responsible for tumor growth, survival and angiogenesis. With regard to the latter, it was noted that U373 cells exposed to U373vIII-derived microvesicles exhibited a marked (2-3 fold) increase in production of vascular endothelial growth factor (VEGF). a potent mediator of brain tumor angiogenesis and a known EGFR target. EGFRvIII activity was essential for this effect as U373-derived microvesicles (devoid of EGFRvIII), or those from U373vIII, but preincubated with CI-1033 were unable to induce this release of VEGF (
[0089] While VEGF upregulation often heralds activation of oncogenic pathways. cellular transformation downstream of EGFRvII is mediated by changes in expression of genes directly involved in cellular proliferation and survival. In this regard. U373 cells treated with EGFRvIII-containing microvesicles revealed an increase in expression of the antiapoptotic protein BdxL and decrease in levels of p27/Kip1 cyclin dependent kinase inhibitor, both known EGFR targets (
[0090] The functional consequences of the aforementioned repertoire of molecular responses evoked by incorporation of EGFRvIII-containing microvesicles can lead to a higher degree of cellular transformation, as demonstrated by more spindle morphology of U373 cells exposed to this material (
[0091] It is well recognized that in human GBMs, only a small sub-population of tumor cells harbor the primary genetic mutation leading to EGFRvIII expression, though there is increased growth of the entire tumor. In this regard, it is disclosed herein that EGFRvIII expression provokes formation of cellular microvesicles, to which this transmembrane protein becomes incorporated and shed to the pericellular micromilieu (
[0092] Confirming that in human GBMs, only a small sub-population of tumor cells harbor the primary genetic mutation leading to EGFRvIII expression, both wild type EGFR and EGFRvIII bands were detected at the expected sizes and resolved using a standard SDS-PAGE protocol in microvesicles collected from human plasma of patients with GBM (
[0093] It is encompassed that similar microvesicular transfer can also involve other transforming, mutant, upregulated, or otherwise activated membrane-associated oncogenic tyrosine kinases (e.g. HER-2, wtEGFR, cKit or MET) and proteins operative in a variety of human tumors. Host cells (e.g. endothelium) may also be targets of oncogene-containing microvesicles. In one aspect, the tumor promoting functions (e.g. angiogenesis) of a host cell could be exacerbated by microvesicular transfer. Conversely, tumor-associated host cells are often profoundly altered or even contain overt cytogenetic alterations (Akino et al Am. J. Pathol. 2009), and their derived microvesicles (e.g. containing tumor endothelial markers (TEMs) of endothelial cells) could possess diagnostic, prognostic and predictive value.
[0094] It is also encompassed herein that agents capable of blocking exchange of microvesicles between cells (e.g. annexin V derivatives) may be useful as therapeutic agents, e.g. to Inhibit cancer spreading and growth by inhibiting the fusion of microvesicles with cells. In an embodiment, methods for treating cancer are provided comprising administration of a microvesicle exchange blocking agent, e.g. annexin V and/or derivatives thereof, to a subject in need thereof. It is contemplated that any agent that could be used for blocking microvesicle, microparticle, ectosome or exosome transfer can be used in the methods of the inventions described herein. Other non-limiting examples of such agents include agents blocking P-selectin or its ligand, PSGL.
[0095] In another aspect, the invention provides methods of diagnosing cancer by allowing detection of multiple oncogenic proteins and/or multiple phosphorylation sites within them by performing the analysis in microvesicles. For example, a cancer could be characterized by determining whether it carries EGFRvIII. HER-2, wtEGFR, cKit or MET, alone or in combination, by analyzing the protein composition of the microvesicles. The number of oncogenic protein-containing microvesicles found in a bodily fluid can also be used as a way of determining the aggressiveness of a tumor, i.e. its tendency to spread or metastasize. The methods of the invention can thus aid in diagnosis and/or prognosis. In one embodiment, diagnosis and/or prognosis of breast cancer can be determined by detecting the presence of EGFR and/or HER-2. In another embodiment, diagnosis and/or prognosis of tumors is determined by detecting the presence of HER-2 and/or HER-3.
[0096] In another embodiment, the invention provides methods of monitoring the progression of a cancer and/or monitoring the effectiveness of a treatment or therapeutic regimen. For example, the size and the nature of a tumor can be followed by monitoring the amount and composition of an oncogenic protein. or proteins, e.g. EGFRvII, HER-2, HER-3, cKit or MET, released in microvesicles. It would be expected, for example, that a larger tumor would include more cells and therefore release more microvesicles than a smaller one. This could be used to monitor therapy by providing a means to measure a change in size of a tumor, which may either shrink, grow, or stay the same. Such methods would be valuable in evaluating the effectiveness of a therapy in a patient population as a whole, or in an individual patient. It is also contemplated that the progression of a cancer and/or the response to treatment can be monitored by measuring a combination of oncogenic proteins found in microvesicles. In an embodiment, EGFR and HER-2 can be measured in combination, for example in breast cancer, thereby providing indication as to genetic status (e.g. genotype) and progression (or recurrence) of malignancy, in some aspects irrespectively of the actual tumor size. In other embodiments, HER-2 and HER-3 or HER-2 and EGFR can be measured in combination, for example. Furthermore, as microvesicles may contain intact oncoproteins, in another embodiment the phosphorylation status of the oncoproteins can be determined to monitor or measure the efficacy of targeted treatments. For instance, monitoring the phosphorylation status of the EGFR/HER-2 combination in microvesicles derived from breast cancer could indicate the efficacy of a HER-2-directed drug such as Herceptin®, an EGFR-directed drug such as Tarceva®, or similar anti-cancer treatments, alone or in combination. In another aspect. the molecular environment surrounding an oncogenic protein in the microvesicles, e.g. other molecules, the entire proteome, or the phosphoproteome, may be used to monitor the progression of cancer and/or efficiacy of an anti-cancer treatment. For example, the presence or absence or phosphorylation status of PTEN in the microvesicles may be indicative of progression or cancer and/or efficacy of an anti-cancer treatment.
[0097] In a further aspect, the invention provides methods of monitoring the progression of a cancer and/or monitoring the efficacy of an anti-cancer treatment or therapeutic regimen. It is contemplated that any anti-cancer treatment or therapeutic regimen known in the art could be used in the methods described herein. Non-limiting examples of treatments and therapeutic regimens encompassed herein include surgery, radiology, chemotherapy, and administration of targeted cancer therapies and treatments, which interfere with specific mechanisms involved in carcinogenesis and tumour growth. Non-limiting examples of targeted cancer therapies include therapies that inhibit tyrosine kinase associated targets (such as Iressa®, CI-1033, Tarceva® and Gleevec®), inhibitors of extracellular receptor binding sites for hormones, cytokines, and growth factors (Herceptin®, Erbitux®), proteasome inhibitors (Velcade®) and stimulators of apoptosis (Genasense®). Such targeted therapies can be achieved via small molecules, monoclonal antibodies. antisense, siRNA, aptamers and gene therapy. A subject may also receive a combination of treatments or therapeutic regimens. Any other treatment or therapeutic regimen known in the art can be used in the methods described herein, alone or in combination with other treatments or therapeutic regimens.
[0098] In another aspect, the invention provides methods of diagnosing cancer by allowing detection of multiple phosphorylated oncogenic proteins and/or multiple phosphorylation sites within them by performing the analysis in microvesicles. In another embodiment, the invention provides methods of monitoring the progression of a cancer and/or monitoring the effectiveness of a treatment or therapeutic regiment by measuring the phosphorylation state of an oncogenic protein in the microvesicles.
[0099] Receptor tyrosine kinases (RTKs), such as EGFR, contain an extracellular ligand binding domain connected to a cytoplasmic domain by a single transmembrane helix. The cytoplasmic domain contains a conserved protein tyrosine kinase core and additional regulatory sequences that are subject to autophosphorylation and phosphorylation by heterologous protein kinases. Men a ligand binds to the extracellular domain of an RTK, dimerisation of the RTK with other adjacent RTKs is triggered. Dimerisation leads to a rapid activation of the proteins' cytoplasmic kinase domains, the first substrate for these domains being the receptor itself. As a result the activated receptor becomes autophosphorylated on multiple specific intracellular tyrosine residues. The phosphorylation of specific tyrosine residues within the activated receptor creates binding sites for Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain containing proteins. Specific proteins containing these domains include Src and phospholipase Cy, and the phosphorylation and activation of these two proteins upon receptor binding leads to the initiation of signal transduction pathways. Other proteins that interact with the activated receptor act as adaptor proteins and have no intrinsic enzymatic activity of their own. These adaptor proteins link RTK activation to downstream signal transduction pathways, such as the MAP kinase signalling cascade. The activity of virtually all RTKs can be enhanced, even in the absence of ligand binding, by treatment of cells with protein tyrosine phosphatase inhibitors. Thus, the persistent activation of a RTK, or an oncogenic receptor, that triggers abnormal expression of genes involved in cell proliferation, survival and angiogenesis, is positively regulated by one or several phosphotyrosine sites in the activation loop.
[0100] Phosphorylation of RTKs can be measured using a number of methods. Non-limiting examples of such methods include phosphospecific antibodies. phosphoantibody arrays, staining with antibodies against phosphotyrosine residues, and direct kinase assays with phosphorylatable substrates. Another way to determine the phosphorylation status of multiple receptors on microvesicles could be to assess their total phosphoproteome using mass spectrometer (MS) related methods. It is contemplated that standard techniques known in the art for measuring and detecting phosphorylated proteins (also referred to as phospho-proteins) and the phosphorylation state of a protein can be used in the methods described herein.
[0101] We further report herein that oncogenic transformation may impact a cancer cell in such a way as to cause a change in the wider spectrum of MV-associated proteins, the linkages of which to the triggering oncogene may or may not be mechanistically obvious, but may be meaningful for use as biomarkers. Thus we report herein the identification of cancer-associated proteins present in circulating MVs of mice harbouring subcutaneous human tumour xenografts or in MVs from the culture medium of human tumour cell fines. These proteins represent biomarkers which can be used, for example, to monitor progression of a tumour or response to anti-tumour therapy. The biomarkers can also be used for diagnosis and prognosis of a tumour in a subject. We demonstrate herein that many proteins related to malignancy. such as oncogenes, tumour suppressors, receptor tyrosine kinases, and mediators of cellular signaling are detected in MVs derived from several types of human cancer cells. Examples of such cancer-associated proteins and phosphoproteins found in tumour-derived MVs which can be used as biomarkers are given in Tables 1-4 below.
[0102] It is noted that the cancer-associated proteins and phosphoproteins found in MVs include both known oncoproteins such as HER-2 and unexpected proteins such as VEGFR2 or Tie 2, which are angiogenic receptors usually found on epithelial cells. Thus, analysis of the protein content of tumour-derived MVs as demonstrated herein will aid the characterization, prognosis and diagnosis of tumours, including the complexities of their microenvironment and host stroma. Determining the protein content of tumour-derived MVs may also lead to the potential identification of new therapeutic targets. For example, the human VEGFR3 phosphoprotein was identified in MVs from mice harbouring PANC-1 xenografts, suggesting that the cancer cell associated VEGFR3 phosphoprotein may be a hitherto unappreciated biomarker and/or therapeutic target for at least a subset of pancreatic cancers (see Table 4). In addition, the combination of proteins present in MVs may be characteristic of the tumour from which the MVs are derived (see Table 4, for example). Thus the protein or phosphoprotein profile, or a subset thereof, of the MVs may be used in the methods provided herein, e.g. for monitoring anti-tumour therapy or for diagnosis or prognosis of a tumour.
[0103] As many of the proteins identified herein are phosphoproteins which are known to be activated or inactivated by phosphorylation, the activation or functional state of the cancer-associated proteins can be assessed by monitoring their phosphorylation status. Thus the phosphorylation status of MV-associated proteins, alone or in combination, can also be used in the methods provided herein, e.g. for monitoring anti-tumour therapy or for diagnosis or prognosis of a tumour.
[0104] It should be understood that the MV-associated proteins and phosphoproteins identified herein, and combinations thereof, can be used in the methods and kits of the invention.
[0105] In an aspect, there is provided herein a microvesicle-based blood test, wherein MVs are isolated from a sample of blood from a subject and assayed for MV-associated proteins, or assayed to assess the phosphorylation status of MV-associated proteins. Such methods are demonstrated below, for example in MVs expressing EGFRvIII and wild type EGFR, in mice bearing a xenograft of a human tumour cell line, and in glioblastoma (GBM) patients. We have demonstrated the feasibility of this approach in numerous cancer cell types (such as breast cancer, glioma, brain cancer, lung cancer, pancreatic cancer, skin cancer, prostate cancer and colorectal cancer).
[0106] It is noted that another potential advantage of using circulating MVs rather than a biopsy specimen is the problem of tumor heterogeneity: we have observed on one occasion that a tumor biopsy sample was negative for EGFRvIII, whereas the corresponding blood MV sample was positive (see Example 3). Thus, by obtaining a tissue biopsy sample that is not representative of the overall tumor phenotype/genotype. the result is misleading, whereas the blood sample is representative of the entire tumor, and thus more reliable.
[0107] The present invention will be more readily understood by referring to the following examples, which are given to illustrate the invention rather than to limit its scope.
Example 1
Cell Culture and Isolation of Microvesicles (MVs)
[0108] U373 (human astrocytoma) cells, their stable variant U373vIII expressing Tet-off regulated EGFRvIII or EGFRvIII fused at C-terminus to green fluorescent protein (pEGFPN1) cassette (U373vIII-GFP) and A431 are maintained as described previously (Viloria-Petit et al Am. J. Pathology, 1997, 6:1523-1530; Yu et al., 2005, Blood, 105: 1734-1741) in medium containing microvesicle-depleted fetal bovine serum FBS. HUVEC cells are maintained in EGM-2 (Cambrex Bioscience, Walkesville, Md., USA). Microvesicles are collected from conditioned media or mouse plasma, as previously described (Yu & Rak, J. Thromb. Haemost, 2004, 2:2065-67; AI-Nedawi et al., 2005, Arterioscler. Thromb. Vasc. Biol., 25: 1744-1749). Briefly, media are subjected to two successive centrifugations at 300 g and 12000 g to eliminate cells and debris. Microvesicles are pelleted by ultracentrifugation for 2 hours at 100 000 g and quantified by protein content and analyzed for EGFR or EGFRvIII content. For scanning electron microscopy (SEM) the cells are grown on cover slips, fixed with 2.5% gluteraldehyde, stained with 1% OsO4, covered with gold and visualized using the JEOL 840A instrument. For in vivo analyses tumors are generated by injection of 1-10×10.sup.6 U373vIII or U373 cells into immunodeficient (SCID) mice (Charles River, Canada). In some cases mice are treated daily with the pan-ErbB inhibitor CI-1033 as indicated. Blood is collected from tumor bearing, or control mice by cardiac puncture into heparinized syringes. Platelet-free plasma is used to prepare microvesicles.
[0109] Flow cytometry (FACS) is employed to detect EGFRvIII, or EGFRvIII-GFP on the surface of viable not permeabilized cells and is carried out either with cells that expressed these receptors endogenously, or with those that have acquired such expression upon transfer of microvesicles. Typically, U373 cells are treated, with microvesicles (MVs) obtained from U373vIII or U373vIII-GFP cells for 24 hours. The cells are then detached using 2 mM EDTA (ethylenediaminetetraacetic acid) to obtain a single-cell suspension the aliquots of which (1.5×10.sup.6/sample) are washed in phosphate-buffered saline (PBS) with 1% FBS and 0.1% sodium azide. The cells treated with U373vIII derived MVs are then stained for 30 minutes at 4° C. with for example a monoclonal antibody against EGFRvIII (Zymed). After washing, samples are incubated with Alexa Fluor 488 goat anti-mouse secondary antibody (Molecular Probes, Eugene, Oreg.) for 30 minutes at 4° C., washed with phosphate buffered saline (PBS) and analyzed. In the case of treatment with MVs derived from U373vIII-GFP cells fresh cell suspensions are directly analyzed for GFP fluorescence. The data can be acquired using FACScalibur flow cytometer (BD Biosciences, Mountain View, Calif.).
[0110] All in vivo experiments are performed in 6- to 8-week-old severe combined immunodeficiency (SCID) mice (Charles River, Saint-Coustant, QC, Canada). Briefly, 1 to 10×10.sup.6 of U373vIII or U373, cells are injected subcutaneously in 0.2 ml PBS. Blood is collected from mice by cardiac puncture, into heparin sodium solution. Platelet-free plasma was prepared by centrifugation at 2000 g for 15 minutes, 2000 g for 5 minutes, and 16,000 g for 5 minutes to isolate microvesicles.
Example 2
Microvesicle Transfer Assays
[0111] U373 (acceptor) cells are treated with microvesicles for 24 hours and a single-cell suspension is analyzed by flow cytometry or fluorescent microscopy for expression of EGFRvIII or GFP. To detect signaling events, U373 are starved in 0.5% FBS (DMEM) before addition of microvesicles, which are either intact or preincubated with annexin-V, or CI-1033, at the concentrations as indicated. The expression of microvesicle associated molecules (EGFRvIII, TF), and expression of total and activated MAPK and Akt as well as other changes are assayed by immunoblot (BclxL. p27/Kip1), ELISA (VEGF, R&D Systems), or promoter activity assays (VEGF), as described elsewhere (Lopez-Ocejo et al. 2000, Oncogene, 40:4611-4620). For soft agar colony formation assays single cell suspensions are prepared in 0.3% agarose from equal numbers of cells pretreated with microvesicles or control media. Cultures are established in plates precoated with 0.5% agarose and all colonies containing more than 4 cells are counted.
Example 3
Detection of Circulating EGFRvIII in Patients with Glioblastoma Multiforme
[0112] Microvesicles are collected from human plasma in a similar manner as previously described for plasma of tumor bearing mice (AI-Nedawi et al., 2008, Nature Cell Biology, 10: 619-624). Briefly, archival blood samples are subjected to two consecutive centrifugations at 300 g for 5 minutes, and then at 12000 g for 20 minutes to eliminate cells and debris. Finally, microvesicles are obtained after centrifugation for 2 hours at 100 000 g, washed twice with a large volume of phosphate buffered saline (PBS). The protein lysates are prepared in the lysis buffer containing: 10 mMTris, pH 6.8, 5 mM EDTA, 50 mM NaF, 30 mM sodium pyrophosphate, 2% (wt/vol) SDS, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM Na.sub.3VO.sub.4, for 10 minutes on ice. Unless otherwise indicated the lysates are resolved by SDS-PAGE and subjected to immunoblotting with for example a mouse or a sheep anti-human EGFR polyclonal antibody or appropriate mouse monoclonal antibodies. Immunodetection is accomplished using the appropriate HRP-conjugated secondary antibody and chemiluminescence plus kit (ECL kit; Amersham Pharmacia, Buckinghamshire. United Kingdom), after which the blots are scanned and protein bands quantified using for example the Storm 860 scanner (GE healthcare). Both wild type EGFR and EGFRvIII bands are detected in this manner at the expected sizes and resolved using a standard SDS-PAGE protocol.
[0113] The EGFRvIII oncoprotein was also detected in circulating MVs collected from a cohort of 24 GBM patients from the Toronto Tumor Bank using Western analysis (
[0114] The EGFR signal was detected in MVs collected from blood of human xenograft bearing SCID mice using an ELISA assay (
Example 4
[0115] Detection of Multiple Cancer-Related Molecular Targets in MVs We isolated microvesicles released by human tumour cell lines into culture media and used Western analysis to detect several proteins related to malignancy, such as oncogenes, tumour suppressors, and mediators of cellular signalling, in the cargo of the microvesicles. Glioma cell lines (U373, U373vIII, U87 and U87vIII), lung cancer cells (A549), breast cancer cells (MDA-MB-231), prostate cancer cells (PC-3), and colorectal cancer cells (DLD-1, HCT116 and CaCo2) were used. The putative oncoproteins K-ras, c-Met, beta-catenin and PDGFR and the tumour suppressor gene products PTEN, TP53 and E-cadherin were tested in the microvesicles. Results are shown in
[0116] Western blot analysis was performed on total cell lysates of MV preparations, obtained from the cultured cell lines, as indicated. The antibodies used included the following: Pan Ras: Rabbit monoclonal [Y131], Cat. No. (ab32442), Abcam; K-ras: Mouse monoclonal Ab., Cat. No. AT2650a, Biolynx Inc.; C-Met: Rabbit monoclonal (EP145Y], Cat. No. (ab51067), Abcam; PDGFR-b2: Mouse monoclonal. Cat. No. (ab10847-100), Abcam; Beta-catenin: Mouse monoclonal, Cat. No. (MA 1-301), ABR: E-Cadherin: Mouse monoclonal [MB2], Cat. No. (ab8993), Abcam; PTEN: Rabbit monoclonal, Cat. No. (#9188), Cell Signaling; P53: Rabbit monoclonal, Cat. No. (#2527), Cell Signaling. It is noted that cell lines used in this study represent stocks currently used in the laboratory and obtained from various sources (ATCC, collaborators), and may therefore differ in their molecular status from similarly named cell lines maintained elsewhere.
[0117] These results demonstrate the large scope of molecular targets relevant to cancer that are present and detectable in MVs. For example, phosphorylated MV-associated RTKs in vivo can be detected. The results also demonstrate that a multiplicity of human tumour types can be analysed in this manner.
Example 5
Demonstration of the Sustained Functional Status (Phosphorylation/Activation) of MV-Associated Cancer-Related Proteins
[0118] We detected, in vitro, multiple phospho-RTKs in microvesicles released into culture medium by several types of human tumour cell lines, using a Phospho-Protein Antibody Array containing probes for 42 RTKs (R & D Systems) (
[0119] The cell lines used in this study represent stocks currently used in the laboratory and obtained from various sources (ATCC, collaborators), and may therefore differ in their molecular status from similarly named cell lines maintained elsewhere. In addition, it is noted that the results obtained depend on the specific antibodies used for this detection and the phospho-epitopes they target. The antibodies used in the array are unique and may have non-overlapping reactivity with other phospho-specific antibodies directed at the same RTKs.
[0120] These results demonstrate that the receptor tyrosine kinases (RTKs) contained in microvesicles of cancer cells have sustained phosphorylation and therefore have a sustained activation and functional status.
[0121] We also performed large scale in vitro profiling of phospho-proteins contained or enriched in MVs released from EGFRvIII-driven GBM cells. A KinexJ Antibody Microarray composed of over 650 different phospho site-specific antibodies for profiling cell signaling protein expression and phosphorylation was used (Kinexus, Inc.) The array can detect 248 unique phospho-specific antibody phosphorylation sites, 121 kinase phosphorylation sites, 2 phosphatase phosphorylation sites and 125 phosphorylation sites in regulatory subunits and other proteins. Using the Kinex™ Antibody Microarray, we analyzed the phosphoproteome of MVs produced by human GBM cells. In order to identify EGFRvII-dependent changes in. the phosphoproteome of the MVs, both U373P (also referred to as parental U373) cells (non-transfected, non-tumorigenic cells) and U373vIII cells were used. In each case, the values obtained from MVs were normalized to values obtained from cells. Exemplary results are given in Tables 1 and 2 below, where the phospho-protein (P-Protein) is given in the left column and the Normalized Net Median Signal (NNMS) is given in the right column. The most abundant proteins found in the MVs are shown in Tables 1 and 2. Table 3 gives further examples of phosphoproteins detected in U373-MVs using the Kinex™. Antibody Array; these proteins represent a sampling of the over 200 phosphoproteins detected. Table 3.1 shows that phosphorylation of distinct sites, e.g. Y1068 or Y1110 of EGFR, can be monitored in the MVs.
[0122] The results indicate that there are numerous phosphorylated, and therefore activated, proteins in MVs, many of which are known to have a role and/or predictive value in cancer. In addition, the results presented herein using both the Kinex™ antibody array and the phospho-protein antibody array from R & D systems demonstrate that multiple oncogenic, signalling and biologically active molecular targets can be detected simultaneously in cancer cell derived MVs, both for MVs recovered from cell culture conditioned medium and for MVs recovered from the blood of mice harbouring human tumour xenografts.
TABLE-US-00001 TABLE 1 Kinex ™ Arrays - MV-Associated Phosphoproteins with the Highest Net Normalized Median Signal (U373P-Cells vs U373P-MVs). P-Protein Normalized Net Median Signal BLNK 5079 BAD 8520 CrystalliaB 8499 eIF4E 6455 MKK1 6244 PKB/Akt 5505 PKCg 5316 PLCg1 5025 Progesterone Receptor 5013 VEGFR2(KDR) Y1214 5065 VEGFR2(KDR) Y1214 5252 ZAP70/Syk 7160 ZAP70/Syk 7031
TABLE-US-00002 TABLE 2 Kinex ™ Arrays - MV-Associated Phosphoproteins with the Highest Net Normalized Median Signal (U373vIII- Cells vs. U373vIII-MVs). P-Protein NNMS MEK1 6003 MEK 8459 MARCK 6202 Tau (mtub) 5270 IRS1 5064 IRS1 5947 Ret 5861 Jun 8782 Jun 6743 BAD 12142 BAD 5055 Erk-1 5314 MKK1 9113 HER-2 6389 HER-2 6252 HER-2 5779 HER-2 5399 PKCd 9078 PKCg 14431 PKCg 5999 PKCg 15696 PKCg 17197 PKCg 7949 PKCg 7013 Tau 5264 Tau 5117 Chk2 6002 EGFR 5929 EGER 5958 EGFR 5250 VEGFR2 5117 Zap70/Syk 8242 Zap70/Syk 5796 FAK 5242 BLNK (lin) 11480
TABLE-US-00003 TABLE 3 Examples of phosphoproteins detected in U373-MVs. Antibody Target Phospho Site Full Target Codes Protein Name (Human) Protein Name NNMS PN011 Bad S91 Bcl2-antagonist of cell death protein 8520.4 N024 CREB1 S133 cAMP response element binding protein 4935.8 PK121 EGFR T693 Epidermal growth factor receptor-tyrosine kinase 4807.0 PN030-1 eIF4E S209 Eukaryotic translation initiation factor 4 (mRNA 6554.6 cap binding protein) PK125 ErbB2 (HER2) Y877 ErbB2 (Neu) receptor-tyrosine kinase 4922.7 PK014- Erk1 T202 + Y204; Extracellular regulated protein-serine kinase 1 4199.1 PK015-2 T185/Y187 (p44 MAP kinase) PK019-1 FAK Y577 Focal adhesion protein-tyrosine kinase 3457.4 PN048-1 Jun S73 Jun proto-oncogene encoded AP1 transcription 5879.4 factor PK046-3 MEK1 T291 MAPK/ERK protein-serine kinase 1 (MKK1) 6244.5 PK116 mTOR (FRAP) S2448 Mammalian target of rapamycine (FRAP) 1183.9 PN053 NFkappaB p65 S276 NF-kappa-B p65 nuclear transcription factor 1503.1 PK060-3 P38a MAPK T180 + Y182 Mitogen-activated protein-serine kinase p38 alpha 1171.6 PK063 PDGFRa Y754 Platelet-derived growth factor receptor kinase 2315.3 alpha PK065 PDGFRb Y716 Platelet-derived growth factor receptor kinase 3369.6 beta PK073 PKCa S657 Protein-serine kinase C alpha 3019.3 PK079 PKCd S645 Protein-serine kinase C delta 4061.2 PK082-2 PKCg T514 Protein-serine kinase C gamma 4857.1 NN143 PLCg2 Y753 1-phosphatidylinositol-4,5-bisphosphate 2472.8 phosphodiesterase gamma-2 PN104 Progesterone S294 Progesterone receptor 5013.0 Receptor PP003 PTEN S380 + S382 + Phosphatidylinositol-3,4,5-trisphosphate 3- 1623.7 S385 phosphatase and protein phosphatase and tensin homolog deleted on chromosome 10 PK098 Raf1 S259 Raf1 proto-oncogene-encoded protein-serine 2463.0 kinase PN065 Rb T356 Retinoblastoma-associated protein 1 1331.6 PN074-2 Shc1 Y349 + Y350 SH2 domain-containing transforming protein 1 2088.9 PN077 SOX9 S181 SRY (sex determining region Y)-box 9 3355.7 (campomelic dysplasia, autosomal sex-reversal) PN078 STAT1 S727 Signal transducer and activator of transcription 1 2016.7 PK110 VEGFR2 Y1054 Vascular endothelial growth factor receptor- 3542.7 (KDR) tyrosine kinase 2 (Flk1) Histone H1 phospho CDK1 Histone H1 phosphorylated 663.2 sites P27 Kip1 S10 p27 cyclin-dependent kinase inhibitor 1B 27.9 Histone H2A.X S139 Histone H2A variant X 1558.5 BRCA1 S1423 Breast cancer type 1 susceptibility protein 4698.6 Hsp27 S15 Heat shock 27 kDa protein beta 1 (HspB1) 1428.5 Lck S157 Lymphocyte-specific protein-tyrosine kinase 2049.3 Caveolin 2 S36 Caveolin 2 977.9 p53 S392 Tumour suppressor protein p53 (antigen NY-CO- 1535.4 13) IRS1 S639 Insulin receptor substrate 1 5064.2 Ret S696 Ret receptor-tyrosine kinase 5861.1 Rac1 S71 Ras-related C3 botulinum toxin substrate 1 2791.5 FAK S843 Focal adhesion protein-tyrosine kinase 4081.5 SMC1 S957 Structural maintenance of chromosomes protein 470.1 1A Integrin a4 S988 Integrin alpha 4 (VLA4) 2846.1 p38a MAPK T180 + Y182 Mitogen-activated protein-serine kinase p38 alpha 1817.2 MLK3 T277 + S281 Mixed-lineage protein-serine kinase 3 2096.6 PKBa (Akt1) T308 Protein-serine kinase B alpha 2339.6 RSK 1/3 T359 + S363/ Ribosomal S6 protein-serine kinase 1/3 130.0 T356 + S360 MEK1 T385 MAPK/ERK protein-serine kinase 1 (MKK1) 4256.4 (MAP2K1) Chk2 T68 Checkpoint protein-serine kinase 2 6002.0 IR/IGF1R Y1189/Y1190 Insulin receptor/Insulin-like growth factor 1 3420.7 (INSR) receptor Met Y1230 + Y1234 + Hepatocyte growth factor (HGF) receptor-tyrosine 2459.8 Y1235 kinase CDK1/2 Y15 Cyclin-dependent protein-serine kinase 1/2 4737.5 Lck Y191 Lymphocyte-specific protein-tyrosine kinase 2272.8 Shc1 Y239 SH2 domain-containing transforming protein 1 2557.0 GSK3a Y279/Y216 Glycogen synthase-serine kinase 3 alpha 4249.1 Src Y529 Src proto-oncogene-encoded protein-tyrosine 3564.0 kinase Kit Y703 Kit/Steel factor receptor-tyrosine kinase 1653.3 IR (INSR) Y972 Insulin receptor 2594.7
TABLE-US-00004 TABLE 3.1 Kinex ™ - Examples of Redundant and Non-Redundant Detection of Receptor (EGFR) Phosphorylation Sites in MVs Released by Cancer Cells (U373). Target Phospho Site Full Target Protein Name (Human) Protein Name NNMS EGFR T693 Epidermal growth factor 4807.0 receptor-tyrosine kinase EGFR Y1110 Epidermal growth factor 3966.1 receptor-tyrosine kinase EGFR Y1110 Epidermal growth factor 3806.9 receptor-tyrosine kinase EGFR Y1197 Epidermal growth factor 1971.5 receptor-tyrosine kinase EGFR Y1197 Epidermal growth factor 3521.4 receptor-tyrosine kinase EGFR T678 Epidermal growth factor 4535.8 receptor-tyrosine kinase EGFR T678 Epidermal growth factor 4001.3 receptor-tyrosine kinase EGFR T693 Epidermal growth factor 5929.1 receptor-tyrosine kinase
[0123] These results indicate the spectrum of types of molecules that can be found in MVs in phosphorylated form. While this is an in vitro assay, it is noted that the spectrum of oncogene-related changes in MVs (some of which may be MV-associated but not necessarily oncogenic) is unexpectedly large.
[0124] The results also show that not only phosphorylation-dephosphorylation can be determined. but also the status of distinct phosphorylation sites can be monitored (e.g. Y1068 or Y1110 of the EGFR), which are responsible for different functions of the molecule (interactions with different targets, recycling etc). This information is readily available from analysis of MVs, whereas it would be difficult, and perhaps not possible, to obtain this information using a comparable analysis of tumour samples.
Example 6
Determination of Phosphoprotein Profiles of MVs Circulating in Mice with Human Tumour Xenografts
[0125] Next, the human phosphoprotein profiles of MVs circulating in blood of mice harbouring human tumour xenografts was analyzed in vivo (Table 4;
[0126] The results are given in Table 4 below, and an example of the RTK assay for circulating MVs from mice with an A431 xenograft is shown in
TABLE-US-00005 TABLE 4 Phosphoprotein profiles of MVs circulating in blood of mice harbouring human tumour xenografts. Cell line Tumour type Detectable phosphoproteins U373vIII glioblastoma EGFR, ErbB3, ErbB4, FGFR1, FGFR4, InsulinR, IGF- 1R, Dtk, Mer, MSPR, c-Ret, ROR1, ROR2, Tie-1, Tie- 2, TrkA, TrkB, VEGFR1, VEGFR3, EphA1, EphA7, EphB2, EphB4 U87 glioblastoma EGFR, ErbB2, ErbB4, FGFR1, FGFR2a, InsulinR, IGF- 1R, Dtk, Mer, MSPR, PDGFRb, SCFR, c-Ret, Tie-2, TrkA, TrkB, TrkC, VEGFR1, VEGFR2, VEGFR3, EphA7, EphB1, EphB2, EphB4, EphB6 U87vIII glioblastoma EGFR, ErbB2, ErbB4, FGFR1, FGFR2a, Dtk, Mer, MSPR, c-Ret, ROR1, ROR2, Tie-1, Tie-2, TrkA, TrkB, TrkC, VEGFR3, EphA6, EphA7, EphB1, EphB2 A549 lung cancer EGFR, ErbB2, InsulinR, IGF-1R, Dtk, Mer; MSPR, c- Ret, ROR1, Tie-2, TrkA, EphA4, EphA7, EphB2, A431 squamous cell carcinoma EGFR, ErbB2, ErbB4, FGFR1, FGFR2a, FGFR3 InsulinR, Dtk, Mer, MSPR, c-Ret, ROR1, ROR2, Tie-1, TrkC, EphA1 MDA-MB-231 breast cancer EGFR, ErbB2, InsulinR, IGF-1R, Dtk, Mer, MSPR, c- Ret, ROR1, Tie-1, Tie-2, TrkB, EphA1, EphA7, EphB2 PC-3 prostate cancer EGFR, ErbB2, ErbB4, FGFR1, FGFR3, IGF-1R, Dtk, Mer, MSPR, PDGFRb, ROR1, ROR2, Tie-1, Tie-2, TrkA, TrkB, TrkC, VEGFR1, EphA7, EphB2, EphB4 PANC-1 pancreatic cancer EGFR, ErbB2, ErbB4, FGFR1, FGFR2a, InsulinR, IGF- 1R, Axl, Dtk, Mer; HGFR, MSPR, PDGFRa, SCFR, Flt- 3, M-CSFR, c-Ret, ROR1, ROR2, Tie-1, Tie-2, TrkA, TrkC, VEGFR1, VEGFR2, VEGFR3, MuSK, EphA1, EphA4, EphA6, EphA7, EphB2, EphB4, EphB6 DLD-1 colon cancer EGFR, InsulinR, IGF-1R, Axl, Dtk, MSPR c-Ret, ROR1, ROR2, Tie-1, Tie-2, TrkC, EphA7, EphB2 HCT116 colon cancer EGFR, ErbB2, ErbB4, FGFR1, FGFR4, InsulinR, IGF- 1R, Dtk, Mer, MSPR, PDGFRb, M-CSFR, c-Ret, ROR1, ROR2, Tie-1, Tie-2, TrkA, TrkB, TrkC, VEGFR1, VEGFR2, VEGFR3, MuSK, EphA4, EphA6, EphA7, EphB1, EphB2, EphB4, EphB6 CaCo2 colon cancer EGFR, ErbB2, InsulinR, IGF-1R, Dtk, Mer, MSPR, c- Ret, ROR1, Tie-1, TrkB, TrkC, EphA1, EphA7, EphB2
Example 7
Demonstration that Phosphorylation Status of MV-Associated RTKs is Responsive to Targeted Therapy
[0127] In order to determine whether MVs can be used as biomarkers to monitor response to oncogene-directed therapeutics in vivo, we treated mice bearing EGFR-driven subcutaneous tumours with the EGFR inhibitor CI-1033 (
[0128] Our results demonstrate that the phosphorylation status of MV-associated active RTKs, which are oncogenic, is responsive to systemic targeted therapy directed at these receptors, and that status of these oncogenic targets may be monitored using a blood test based on isolating MVs and analyzing their content. These findings indicate that the circulating MVs can be used as biomarkers to monitor response to oncogene-directed therapeutics in vivo.
[0129] The contents of all documents and references cited herein are hereby incorporated by reference in their entirety.
[0130] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.