BIOMARKER, METHOD FOR SEARCHING DISEASE-RELATED GENE, AND RENAL CANCER MARKER

TECHNICAL FIELD

The present invention relates to a method for identifying a biomarker or disease-related gene from separated/purified extracellular vesicles, and a test method. Besides, the present invention relates to a renal cancer marker obtained by using the search method.

BACKGROUND ART

A biomarker refers to an in vivo substance or image data correlating with a normal process or pathological process of a body, or a pharmacological reaction against treatment, and is used as an objective index of a body condition. Biomarkers include various information such as so-called clinical laboratory values of a biochemical test, a blood test and tumor markers, and diagnostic imaging data of CT and MRI. In particular, a biomarker indicating the presence or the progression of a disease as an index of the disease is indispensable for the present medical care for finding, diagnosing and making prognostic prediction of the disease. Besides, a biomarker is used in development of a new drug for selection and evaluation of a compound to be developed.

In recent years, a large number of biomarkers have been discovered as a result of development of techniques for enabling detection of a tiny amount of protein or nucleic acid and development of genome analysis and proteome analysis. Among these, however, a very small number of markers are actually used in clinical practice. Particularly for diseases such as cancers and neurodegenerative diseases exhibiting little subjective symptoms at an early stage of the diseases, a biomarker useful as a tool for diagnosis or drug discovery is desired. Few biomarkers are, however, currently practically used.

For example, renal cell carcinoma causes few symptoms in many cases, and is found after considerable progression in most cases. Although renal cell carcinoma is recently accidentally found in more cases through ultrasonic echography or CT scan carried out for medical examination or another disease, it is still regarded as cancer difficult to find at an early stage. Patent Literatures 1 to 3 report biomarkers for renal cell carcinoma, but these biomarkers have not been put to practical use yet.

In recent years, liquid biopsy (diagnosis using a body fluid) is attracting attention in the field of clinical diagnosis using a biomarker. In the field of cancer diagnosis employing the liquid biopsy, a body liquid is used as a specimen, without collecting tumor tissue as in the conventional biopsy, for detecting a cancer cell or non-invasively or minimally invasively detecting a disease by measuring a biomarker.

As an analysis object of the liquid biopsy, a circulating tumor cell (CTC) and a DNA derived from a cancer cell (circulating tumor DNA; ctDNA) are well known. A CTC or ctDNA is, however, presumed to be detected in metastasis of a cancer cell, and hence is regarded to be suitable for prognostic prediction of cancer but unusable for early diagnosis. Therefore, as an analysis object to be used for the early diagnosis of cancers and other diseases, extracellular vesicles (hereinafter sometimes referred to as EVs) have started to attract attention.

The extracellular vesicles are vesicles released from almost all cells, and are roughly divided, depending on the size and marker molecule to be presented, into exosomes, microvesicles and apoptotic bodies. In recent years, it has been clarified that the extracellular vesicles play various roles. In particular, it has been clarified that the exosomes and the microvesicles contain nucleic acids such as mRNA and microRNA and proteins, and are involved in communication not only between close cells but also between organs (Non Patent Literatures 1 to 4).

It has been reported that the extracellular vesicles are involved, regarding cancer, in development of the cancer including angiogenesis, immunosuppression and metastasis. Besides, based on the composition of a special integrin contained in the extracellular vesicles, it has been suggested that there is a possibility of the extracellular vesicles preparing for metastasis of cancer cells (Non Patent Literature 5). In this manner, the extracellular vesicles are involved also in the development of a disease, and proteins and nucleic acids contained in the extracellular vesicles are changed depending on the state of a living body. Accordingly, a protein or a nucleic acid to be detected using the extracellular vesicles varies depending on the type or state of the disease, and the extracellular vesicles are known to work as biomarkers.

Results of studies conventionally reported are, however, based on mainly cultured cells, and there is no data of comparison of microvesicles obtained from a disease site and a normal site having the same histological background. Therefore, it has been doubted whether or not they function as a biomarker effective for disease diagnosis or new drug development.

CITATION LIST
Patent Literature

Patent Literature 1: Japanese Patent Laid-Open No. 2016-86678

Patent Literature 2: Japanese Patent Laid-Open No. 2013-140030

Patent Literature 3: National Publication of International Patent Application No. 2015-505965

Non Patent Literature

Non Patent Literature 1: Kahlert, C., & Kalluri, R., J. Mol. Med. (Berl)., 2013, Vol. 91(4), p. 431-437.

Non Patent Literature 2: An, T. et al., J. Extracell. Vesicles, 2015, Vol. 4, 27522.

Non Patent Literature 3: Peinado, H. et al., Nat. Med., 2012, Vol. 18(6), p. 883-891

Non Patent Literature 4: Costa-Sliva, B. et al., Nat. Cell Biol., 2015, Vol. 17(6), p. 816-826.

Non Patent Literature 5: Hoshino, A. et al., Nature, 2015, Vol. 527(7578), p. 329-335.

Non Patent Literature 6: Lasser, C., et al, J. Vis. Exp., 2012, Vol. 59, e3037, doi:10.3791/3037.

Non Patent Literature 7: Geissler, K. et al., Oncoimmunology, 2015, Vol. 4, e985082.

Non Patent Literature 8: Teltsh, O., et al., Oncotarget, 2015, Vol. 32, p. 33191-33205.

Non Patent Literature 9: Guldur, M. E. et al., J. Pak. Med. Assoc., 2014, Vol. 3, p. 300-303.

Non Patent Literature 10: Bussolati, B. et al., FASEB J., 2003, Vol. 17(9), p. 1159-1161.

Non Patent Literature 11: Wiklander, O. P. et al., J. Extracell. Vesicles, 2015, Vol. 4, 26316.

Non Patent Literature 12: Tominaga, N. et al., Adv. Drug Deliv. Rev., 2015, Vol. 95, p. 50-55.

Non Patent Literature 13: Lai, C. P. et al., ACS Nano., 2014, Vol. 8(1), p. 483-494.

Non Patent Literature 14: Gruenwald, V. et al., BMC Cancer, 2010, 10:695.

Non Patent Literature 15: Vroling, L. et al., Angiogenesis, 2009, Vol. 12(1), p. 69-79.

Non Patent Literature 16: del Puerto-Nevado, L. et al., Br. J. Cancer, 2014, Vol. 110, p. 2700-2707.

Non Patent Literature 17: Shankhajit, D. et al., International Journal of Nutrition, Pharmacology, Neurological diseases, 2012, Vol. 2, p. 3-7.

SUMMARY OF INVENTION
Technical Problem

An object of the present invention is to provide a method for searching a novel biomarker from biological components contained in extracellular vesicles usable in liquid biopsy. Another object is to provide a novel search method for disease-related gene. Besides, a novel biomarker for renal cell carcinoma, for which an effective biomarker has not been found, and a test method are provided.

Solution to Problem

The present invention relates to a method for searching a biomarker or disease-related gene derived from diseased tissue, a test method for renal cell carcinoma, a biomarker for renal cell carcinoma, and a method for screening a therapeutic agent for renal cell carcinoma.

(1) A search method, comprising: immersing resected diseased tissue in an immersion liquid; analyzing an exuded component derived from the diseased tissue exuded from the diseased tissue in the immersion liquid; and identifying a biomarker and/or disease-related gene derived from the diseased tissue.

(2) The search method according to (1), comprising: immersing normal tissue obtained from around the resected diseased tissue in an immersion liquid; analyzing an exuded component derived from the normal tissue exuded from the normal tissue in the immersion liquid; identifying a biomarker and/or disease-related gene derived from the normal tissue; and selecting a disease-specific biomarker and/or disease-related gene by comparatively examining the biomarker and/or disease-related gene derived from the diseased tissue and the biomarker and/or disease-related gene derived from the normal tissue.

(3) The search method according to (1) or (2), wherein the exuded component is an extracellular vesicle, a protein, a nucleic acid or a lipid.

(4) The search method according to any one of (1) to (3), wherein the exuded component is an extracellular vesicle, and the biomarker and/or disease-related gene contained in the extracellular vesicle is a protein, a nucleic acid or a lipid.

(5) The search method according to any one of (1) to (4), wherein the disease is cancer, neurodegenerative disease, multiple sclerosis, diabetes, liver disease, autism or cerebral infarction.

(6) A test method for renal cell carcinoma, comprising: separating an extracellular vesicle contained in a body fluid; detecting at least one biomarker out of biomarkers listed in Tables 1, 2 and 4 contained in the extracellular vesicle; and comparing with a prescribed value.

(7) The test method for renal cell carcinoma according to (6), wherein the biomarker is AZU1, CA9, STBD1, COMT or GYG1.

(8) The test method for renal cell carcinoma according to (6) or (7), wherein the body fluid is blood or urine.

(9) Biomarkers for renal cell carcinoma, listed in Tables 1, 2 and 4.

(10) A method for screening a therapeutic agent for renal cell carcinoma using AZU1, CA9, STBD1, COMT or GYG1 as a target, comprising: selecting a candidate compound by using AZU1, CA9, STBD1, COMT or GYG1 as an index.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram illustrating the outline of a method for isolating tissue-exudative extracellular vesicles and for searching a biomarker.

FIG. 2 is a diagram illustrating marker analysis and size distribution of obtained tissue-exudative extracellular vesicles.

FIG. 3 is a diagram illustrating the results of analysis, by mass spectrometry, of expression of tetraspanin family molecules in the tissue-exudative extracellular vesicles.

FIG. 4 illustrates the analysis results of the tissue-exudative extracellular vesicles by gene ontology analysis. FIG. 4A illustrates the numbers of proteins identified in tissue-exudative extracellular vesicles obtained from renal cell carcinoma tissue and normal tissue in the vicinity of the carcinoma tissue. FIG. 4B, FIG. 4C and FIG. 4D are diagrams listing biological properties of proteins classified into three categories of cellular components, biological processes and molecular functions, respectively.

FIG. 5 is a volcano plot illustrating the results of analysis, by corresponding t-test, of protein expression in extracellular vesicles derived from normal tissue and tumor tissue.

FIG. 6 is a diagram illustrating expression of AZU1 in extracellular vesicles derived from normal tissue and tumor tissue. FIG. 6A is a diagram illustrating the expression of AZU1 by using samples obtained from the same patient as a pair. FIG. 6B is a diagram illustrating correlation between the stage of the cancer and the expression of AZU1. FIG. 6C is a diagram in which the expression of AZU1 is analyzed by Western blotting. FIG. 6D is a diagram illustrating an amount of AZU1 in a serum sample.

FIG. 7 illustrates disintegration, depending on AZU1, of a vascular endothelial layer caused by extracellular vesicles derived from a renal cell carcinoma cell line. FIG. 7A is a diagram illustrating the expression and localization of AZU1 in immunoelectron microscopic images. FIG. 7B is a diagram illustrating the expression level of AZU1 and its content in secreted extracellular vesicles in the renal cell carcinoma cell line. FIG. 7C is a diagram illustrating results of TEER analysis using extracellular vesicles secreted from the cell line. FIG. 7D is a Western blot diagram illustrating an amount of AZU1 in the extracellular vesicles secreted from ACHN cell line in which AZU1 is forcedly expressed. FIG. 7E illustrates immunoelectron microscopic images of localization of forcedly expressed AZU1. FIG. 7F is a diagram illustrating results of the TEER analysis using extracellular vesicles secreted from the ACHN cell line in which AZU1 is forcedly expressed.

FIG. 8 illustrates disintegration of a vascular endothelial layer caused by extracellular vesicles derived from a patient. FIGS. 8A and 8B are diagrams illustrating results of the TEER analysis using tissue-exudative extracellular vesicles derived from patient tissue. FIG. 8C is a microscopic image illustrating incorporation into HUVEC cells of tissue-exudative extracellular vesicles derived from tumor tissue and normal tissue. FIG. 8D is a diagram illustrating results of the TEER analysis using tissue-exudative extracellular vesicles derived from a patient.

DESCRIPTION OF EMBODIMENTS

Now, a method for isolating extracellular vesicles from tissue and for analyzing a biomarker, and a method for searching disease-related gene will be described in detail by exemplarily describing search for a renal cell carcinoma marker, but it is noted that a search method for a biomarker and disease-related gene of the present invention is applicable to not only renal cell carcinoma but also any disease. In particular, for cancer to be treated by surgical resection of diseased tissue, a useful biomarker and disease-related gene can be searched by the method of the present invention. Besides, as the diseased tissue, not only tissue surgically obtained but also tissue obtained by autopsy can be used, and the present invention is also applicable to various diseases for which effective biomarkers have not been found yet, such as neurodegenerative diseases including amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease, multiple sclerosis, prostate cancer, pancreatic cancer, diabetes, liver disease, developmental disorder such as autism, and cerebral infarction.

In the case of, for example, cancer to be treated by surgical resection, a so-called non-cancerous part (normal tissue) around a cancerous part is also resected. Therefore, tissues of both a disease site and a normal site can be obtained from the same patient, and thus, samples having the same genetic background can be obtained. Accordingly, when extracellular vesicles obtained from these samples are analyzed, proteins and nucleic acids expressed irrespectively of the disease of interest in the individual can be excluded. As a result, a biomarker specific to the disease can be searched for. Besides, in the case where completely normal tissue is unavailable as in neurodegenerative disease, tissues can be obtained from a group of patients different in severity and the degree of progression to be subjected to comparative quantitative analysis, and thus, disease-related gene and a biomarker can be specified. Furthermore, a biomarker exuded from tissue and information of the biomarker obtained from a body fluid such as a serum can be used in combination.

Alternatively, in addition to the tissue obtained from a patient, tissue can be obtained from a model animal for searching a biomarker. Also for a disease such as neurodegenerative disease and diabetes for which no tissue is resected from a patient for treatment, a biomarker can be searched by using a model animal.

Besides, disease-specific biomarkers thus searched are exuded from diseased tissue, and those contained in extracellular vesicles secreted into a body fluid such as urine or blood can be selected among these to be used as a disease marker for use in liquid biopsy.

Furthermore, since a large number of biological components such as proteins are contained in extracellular vesicles, a large number of disease-specific biomarkers can be found. Among the thus obtained biomarkers, a plurality of biomarkers are selected to be measured in combination, and thus, not only diagnostic sensitivity but also specificity can be increased. When a plurality of markers are used, a disease that could not be found at an early stage can be detected.

Besides, through analysis of proteins and nucleic acids expressed specifically to a disease, a biological component that relates to the disease and can be a target of a therapeutic agent for the disease can be found. For example, when the function of a protein highly expressed in a diseased tissue is analyzed, and if it has a function essential for the onset or development of the disease, a pharmaceutical can be developed by using it as a target of a therapeutic agent.

In addition, when tissue derived from a patient who has become resistant to a molecular target drug is used, extracellular vesicles released from molecular target drug-resistant cells can be captured. A way for braking the resistance can be thus found based on an extracellular vesicle regarded as a replica of a cell.

Besides, a biomarker thus searched can be used in screening of candidate compounds in studies for developing new drugs. In high throughput screening, the screening is often carried out using a cell line. As a cell line to be used for the screening, cell lines having expression tendency similar to that of a plurality disease markers having been searched by the method of the present invention are selected in advance, and thus, candidate compounds can be accurately narrowed down. Furthermore, the obtained biomarker can be used, in addition to the cell line, for examining an effect of a candidate compound in an animal model or at a stage of clinical trial.

Now, renal cell carcinoma will be exemplarily described for describing a method for searching a novel biomarker and a test method using the same in detail. It is noted that analysis using human tissue is approved by the ethics committee of each research institution before the analysis. Besides, renal cell carcinoma tissue and normal tissue around the tumor tissue are used for the analysis after informed consent is obtained from a patient.

[Example 1] Method for Isolating Te-EVs

FIG. 1 illustrates the outline of a method for isolating tissue-exudative extracellular vesicles (tissue-exudative EVs, hereinafter sometimes referred to as the Te-EVs) and analyzing a biomarker. As tissue surgically resected, diseased tissue and normal tissue are cut out and immediately immersed in an immersion liquid. In the case of renal cell carcinoma, a serum-free DMEM is used as the immersion liquid, and the immersion liquid can be appropriately selected depending on tissue from which the Te-EVs are extracted. Since extracellular vesicles are contained in a serum, a serum-free medium is preferably used as the immersion liquid, but a medium containing a serum may be used as long as extracellular vesicles are removed therefrom in advance.

A piece of tissue collected from a disease site or a normal site is allowed to stand still in the immersion liquid at 4° C. for about 1 hour to cause extracellular vesicles to be secreted. The time for the standing in the immersion liquid can be adjusted in accordance with the amount of the obtained piece of tissue. When the piece of tissue is allowed to stand still in the immersion liquid for a long period of time, a larger number of extracellular vesicles can be obtained. Besides, a temperature condition for immersing the tissue can be any temperature within a range from 0° C. to 37° C. Since a higher temperature increases a secretion rate and increases the amount of extracellular vesicles and the like to be obtained, the time and the temperature for the immersion may be appropriately determined. Alternatively, the immersion can be performed by, instead of still standing, gently stirring the immersion liquid by shaking, inverting or rotating. After causing extracellular vesicles to be sufficiently secreted, the resultant is centrifuged at 2,000 g for 30 minutes to remove the piece of tissue and cells through precipitation. Next, the resultant is centrifuged at 16,000 g for 30 minutes to remove cell debris through precipitation. Furthermore, a resultant supernatant is centrifuged at 100,000 g for 90 minutes to collect the Te-EVs secreted from the diseased tissue or the normal tissue through precipitation. The thus obtained Te-EVs are suspended in PBS, and washed by centrifugation at 100,000 g for 90 minutes. The thus isolated Te-EVs are extracellular vesicles derived from the diseased tissue and the normal tissue obtained from the same patient, and hence have the same genetic background, and therefore, when these are comparatively analyzed, a disease-specific biomarker can be selected.

The isolated Te-EVs are decomposed, by trypsin digestion, into peptides analyzable by mass spectrometry. The resultant peptides are identified and quantitatively determined by LC/MS (liquid chromatography/mass spectrometry) analysis. Besides, a protein specific to each of the Te-EVs is analyzed by statistical analysis.

[Example 2] Purification of Extracellular Vesicles from Renal Tissue

Te-EVs were collected to be analyzed for renal cell carcinoma patient from whom both tumor tissue and normal tissue (non-cancerous part) were obtained among 20 renal cell carcinoma patients. Histological diagnosis of renal cell carcinoma was carried out by hematoxylin-eosin staining. The disease stages of the patients classified based on AJCC TNM 6th edition were stages T1a to T3c.

The degree of purification of the thus obtained extracellular vesicles was analyzed by the Western blotting method, the immunoelectron microscopy, nanoparticle tracking analysis (FIG. 2) and the mass spectrometry (FIG. 3). The Western blotting method was carried out by an ordinary method using 100 ng of each Te-EVs protein obtained as above. Exosome markers of an anti-CD63 monoclonal antibody (8A12), an anti-CD81 monoclonal antibody (12C4) and an anti-CD9 monoclonal antibody (12A12) (all manufactured by Cosmo Bio Co., Ltd.) were used as primary antibodies, an HRP-labeled goat anti-mouse IgG antibody (manufactured by Santa Cruz Biotechnology, Inc.) was used as a secondary antibody, and detection was performed with ECL (ECL Prime western blotting detection reagent, manufactured by GE Healthcare).

An upper panel of FIG. 2 illustrates the results of the analysis by the Western blotting method of the Te-EVs obtained from three patients. N corresponds to a non-cancerous region (normal tissue), and T corresponds to Te-EVs derived from a cancerous region. In any one of the samples, no matter whether it was obtained from a cancerous region or a non-cancerous region, expression of the exosome markers of the CD63, CD81 and CD9 was found.

The immunoelectron microscopy was performed basically in accordance with a method of Lasser et al., (Non Patent Literature 6) by using the anti-CD9 monoclonal antibody (12A12) and a 20 nm gold colloid-labeled anti-mouse antibody (manufactured by Abcam Plc.), respectively, as a primary antibody and a secondary antibody. Specifically, 1 μg of a Te-EVs sample was allowed to stand still for 1 hour on a formvar support film having carbon deposited thereon, and then was fixed with 2% paraformaldehyde to be reacted with the primary antibody. Thereafter, the resultant was reacted with the secondary antibody, and was observed with an electron microscope H-7650 (manufactured by Hitachi High-Technologies Corporation). A gold colloid bonded to the CD9 was observed as illustrated with an arrow on the Te-EVs derived from either of the normal tissue and the tumor tissue (middle panel of FIG. 2).

It was found through the observation under an electron microscope that particle sizes of the Te-EVs derived from the tumor tissue largely varied. Therefore, the particle sizes of the Te-EVs derived from each tissue were measured by the nanoparticle tracking analysis. It is known that renal cell carcinoma tissue contains not only cancer cells but also non-cancerous cells (immune cells, endothelial cells and mast cells) (Non Patent Literatures 7 to 10). Therefore, it is presumed that EVs derived from normal cells are also secreted in addition to the EVs derived from cancer cells. Accordingly, in addition to the Te-EVs, extracellular vesicles secreted from cell lines established from renal cell carcinoma, that is, 786-O, ACHN and Caki-1 cell lines, were similarly isolated, purified and analyzed for a size distribution.

The 786-O, ACHN and Caki-1 cell lines were all obtained from American Type Culture Collection (ATCC), and cultured in an RPMI medium (manufactured by Wako Pure Chemical Industries Ltd.) supplemented with 10% fetal bovine serum, 100 U/mL penicillin G and 0.1 μg/mL streptomycin. For isolating EVs from the cell line, 5.0×10⁵cells were seeded in a 10 cm culture dish and cultured for 48 hours, and EVs secreted into the culture fluid were collected, by the centrifugation similarly to the Te-EVs, to be used for the analysis.

Te-EVs obtained from 21 normal tissues, Te-EVs obtained from 28 tumor tissues and EVs isolated from the above-described three renal cell carcinoma cell lines were used for analyzing the particle sizes. The nanoparticle tracking analysis was performed under the same conditions by using NanoSight LM10 (manufactured by Malvern Panalytical Ltd.) in which NTA 2.0 analysis software was installed (lower panel in FIG. 2). The value of an average particle size of the EVs obtained from the renal cell carcinoma cell lines fall in a range of the particle size distribution of the Te-EVs derived from the tumor tissues, which suggested that the Te-EVs derived from the tumor tissue contain EVs secreted from cancer cells. Besides, it was clarified that the average particle size is significantly different between the Te-EVs derived from the normal tissue and those derived from the tumor tissue (p<0.001). Since the particle size distribution of the Te-EVs was different between the diseased tissue and the normal tissue obtained from the renal cell carcinoma patient, there is a possibility that the particle size distribution of the Te-EVs is different between those derived from the diseased tissue and those derived from the normal tissue.

Next, Te-EVs derived from tumor tissues obtained from 20 patients and Te-EVs to be paired derived from normal tissues were used for identifying a tetraspanin molecule, which is known as an exosome marker, by the mass spectrometry, and its expression level was analyzed by the LC/MS (FIG. 3).

The isolated Te-EVs were reduced with 20 mM dithiothreitol at 100° C. for 10 minutes, followed by alkylation with 50 mM iodoacetamide at an ambient temperature for 45 minutes. Thereafter, the resultant was digested with 5 μl immobilized trypsin (manufactured by Thermo Fisher Scientific K.K.) by rotating/swinging at 1000 rpm at 37° C. for 6 hours. The thus obtained peptide was extracted with ethyl acetate, then desalted using Oasis HLB μ-elution plate (manufactured by Waters Corporation), and subjected to the mass spectrometry. In the mass spectrometry, an LTQ-Orbitrap-Velos mass spectrometer directly connected to UltriMate 3000 RSLC nano-flow HPLC system (both manufactured by Thermo Fisher Scientific K.K.) was used. Protein was identified and quantitatively determined through analysis using MaxQuant software.

In order to examine properties of the obtained Te-EVs, the expression level of the tetraspanin molecule was compared. Fifteen tetraspanin molecules were detected, and it was revealed that the Te-EVs derived from either tissue had a high degree of purification. Besides, it was clarified that the expression of most molecules of the tetraspanin family was reduced in the Te-EVs derived from the tumor tissue as compared with the Te-EVs derived from the normal tissue.

[Example 3] Analysis of Te-EVs of Tumor Tissue and Normal Tissue in Renal Cell Carcinoma

Te-EVs derived from tumor tissue obtained from 20 patients and Te-EVs to be paired derived from normal tissue around a cancerous region were used for performing the LC/MS analysis in the same manner as described above (FIG. 4). Thus, 3871 proteins in total were identified, and among these, 160 were Te-EVs derived from the normal tissue, 253 were expression specific to Te-EVs derived from the tumor tissue, and 3458 were expressed in both of these tissues (FIG. 4A).

These genes were classified, in accordance with the DAVID gene ontology analysis, into categories of cellular components (CC; FIG. 4B), biological processes (BP; FIG. 4C) and molecular functions (MF; FIG. 4D), and FIGS. 4B to 4D each illustrate the top 6 protein groups specific to the Te-EVs derived from the whole tissue, the normal tissue or the tumor tissue, respectively.

According to the gene ontology enrichment analysis, in the Te-EVs derived from the tumor tissue, membrane protein was characteristically present in the proteins classified as the cellular components (FIG. 4B), metabolism-related protein was characteristically present in those classified as the biological processes (FIG. 4C), and expression of nucleotide binding protein was characteristically present in those classified as the molecular functions (FIG. 4D). That there is a difference between proteins contained in the Te-EVs derived from the tumor tissue and the those contained in the Te-EVs derived from the normal tissue means that proteins contained in the extracellular vesicles are also regulated in accordance with various cancer-related events, and indicates that these proteins are useful as cancer markers.

Among the 3871 proteins thus identified, proteins specifically expressed in the Te-EVs derived from the renal cell carcinoma tissue were analyzed. The Te-EVs derived from the tumor tissue and the normal tissue obtained from the renal tissue of the same patient were analyzed as a pair by a paired t-test. FIG. 5 illustrates proteins found to be significantly different in the expression between the Te-EVs derived from the cancerous region and those derived from the non-cancerous region (p<0.05, fold change ≥2.0, both sides of dotted lines in FIG. 5). Besides, those with large fold change are illustrated with arrows. The contents of carbonic anhydrase 9 (CA9), starch-binding domain-containing protein 1 (STBD1), azurocidin (AZUL), catechol O-methyltransferase (COMT) and glycogenin-1 (GYG1) were remarkably larger in the Te-EVs derived from the tumor tissue than in the Te-EVs derived from the normal tissue.

In the Te-EVs derived from the renal cell carcinoma tissue, as compared with the proteins contained in the Te-EVs derived from the normal tissue, the expression of 106 proteins shown in Table 1 (Tables 1-1 and 1-2) or 291 proteins shown in Table 2 (Tables 2-1 to 2-5) was found to be significantly larger or smaller.

TABLE 1-1

AC number
Protein Description
Gene name

P46976
Glycogenin-1
GYG1

P20160
Azurocidin
AZU1

P09104
Gamma-enolase
ENO2

Q16790
Carbonic anhydrase 9
CA9

Q96HE7
ERO1-like protein alpha
ERO1L

A2PYH4
Probable ATP-dependent DNA helicase HFM1
HFM1

Q5VT79
Annexin A8-like protein 2
ANXA8L2

P21964
Catechol O-methyltransferase
COMT

O95210
Starch-binding domain-containing protein 1
STBD1

P20701
Integrin alpha-L
ITGAL

Q8N386
Leucine-rich repeat-containing protein 25
LRRC25

O75505
Putative double homeobox protein 2
DUX2

Q9NWQ8
Phosphoprotein associated with glycosphingolipid-enriched
PAG1

microdomains 1

P11215
Integrin alpha-M
ITGAM

P32455
Interferon-induced guanylate-binding protein 1
GBP1

P13726
Tissue factor
F3

P19971
Thymidine phosphorylase
TYMP

Q8IZ83
Aldehyde dehydrogenase family 16 member A1
ALDH16A1

P46821
Microtubule-associated protein 1B
MAP1B

Q02928
Cytochrome P450 4A11
CYP4A11

Q8IZJ1
Netrin receptor UNC5B
UNC5B

Q6GTX8
Leukocyte-associated immunoglobulin-like receptor 1
LAR1

P04179
Superoxide dismutase [Mn], mitochondrial
SOD2

P05107
Integrin beta-2
ITGB2

Q6NYC8
Phostensin
PPP1R18

Q14956
Transmembrane glycoprotein NMB
GPNMB

Q96FQ6
Protein S100-A16
S100A16

P32942
Intercellular adhesion molecule 3
ICAM3

Q0JRZ9
FCH domain only protein 2
FCHO2

P30453
HLA class I histocompatibility antigen, A-34 alpha chain
HLA-A

Q8IYT3
Coiled-coil domain-containing protein 170
CCDC170

Q06210
Glutamine-fructose-6-phosphate aminotransferase [isomerizing] 1
GFPT1

Q96A46
Mitoferrin-2
SLC25A28

P13807
Glycogen [starch] synthase, muscle
GYS1

Q9NZR1
Tropomodulin-2
TMOD2

O43752
Syntaxin-6
STX6

Q6UWP8
Suprabasin
SBSN

P30273
High affinity immunoglobulin epsilon receptor subunit gamma
FCER1G

P10321
HLA class I histocompatibility antigen Cw-7 alpha chain
HLA-C

Q5T681
Uncharacterized protein C10orf62
C10orf62

P02794
Ferritin heavy chain
FTH1

P13611
Versican core protein
VCAN

Q8N6N2
Tetratricopeptide repeat protein 9B
TTC9B

P01892
HLA class I histocompatibility antigen, A-2 alpha chain
HLA-A

P01612
Ig kappa chain V-I region Mey
—

P10316
HLA class I histocompatibility antigen, A-69 alpha chain
HLA-A

Q92614
Unconventional myosin-XVIIIa
MYO18A

P49454
Centromere protein F
CENPF

Q92572
AP-3 complex subunit sigma-1
AP3S1

Q13643
Four and a half LIM domains protein 3
FHL3

Q5SNT6
WASH complex subunit FAM21B
FAM21B

P33908
Mannosyl-oligosaccharide 1,2-alpha-mannosidase IA
MAN1A1

P28799
Granulins
GRN

TABLE 1-2

AC number
Protein Description
Gene name

Q01628
Interferon-induced transmembrane protein 3
IFITM3

P20929
Nebulin
NEB

Q9H2L5
Ras association domaim-containing protein 4
RASSF4

Q8TD55
Pleckstrin homology domain-containing family O member 2
PLEKHO2

Q53T59
HCLS1-binding protein 3
HS1BP3

P49207
60S ribosomal protein L34
RPL34

P31146
Coronin-1A
CORO1A

P51397
Death-associated protein 1
DAP

P39060
Collagen alpha-1(XVIII) chain
COL18A1

P02786
Transferrin receptor protein 1
TFRC

Q6ZUB1
Spermatogenesis-associated protein 31E1
SPATA31E1

P28065
Proteasome subunit beta type-9
PSMB9

P02792
Ferrtin light chain
FTL

P34810
Macrosialin
CD68

P16188
HLA class I histocompatibility antigen, A-30 alpha chain
HLA-A

Q9P282
Prostaglandin F2 receptor negative regulator
PTGFRN

Q00151
PDZ and LIM domain protein 1
PDLIM1

P09972
Fructose-bisphosphate aldolase C
ALDOC

Q9Y4A5
Transformation/transcription domain-associated protein
TRRAP

P18084
Integrin beta-5
ITGB5

P30508
HLA class I histocompatibility antigen, Cw-12 alpha chain
HLA-C

P47914
60S ribosomal protein L29
RPL29

P17693
HLA class I histocompatibility antigen, alpha chain G
HLA-G

P16989
Y-box-binding protein 3
YBX3

P11169
Solute carrier family 2, facilitated glucose transporter member 3
SLC2A3

P16949
Stathmin
STMN1

P30685
HLA class I histocompatibility antigen, B-35 alpha chain
HLA-B

Q02388
Collagen aplha-1(VII) chain
COL7A1

P02747
Complement C1q subcomponent subunit C
C1QC

Q9ULU4
Protein kinase C-binding protein 1
ZMYND8

P78324
Tyrosine-protein phosphatase non-receptor type substrate 1
SIRPA

P04430
Ig kappa chain V-I region BAN
—

Q7Z5R6
Amyloid beta A4 precursor protein-binding family B member
APBB1IP

1-interacting protein

P01714
Ig lambda chain V-III region SH
—

P06748
Nucleophosmin
NPM1

Q99571
P2X purinoceptor 4
P2RX4

P08575
Receptor-type tyrosine-protein phosphatase C
PTPRC

Q99426
Tubulin-folding co factor B
TBCB

Q9NYZ2
Mitoferrin-1
SLC25A37

P05534
HLA class I histocompatibility antigen, A-24 alpha chain
HLA-A

Q6ZRP7
Sulfhydryl oxidase 2
QSOX2

Q16555
Dihydropyrimidinase-related protein 2
DPYSL2

O43583
Density-regulated protein
DENR

Q96MI9
Cytosolic carboxypeptidase 4
AGBL1

P62280
40S ribosomal protein S11
RPS11

P27695
DNA-(apurinic or apyrimidinic site) lyase
APEX1

Q07812
Apoptosis regulator BAX
BAX

O15155
BET1 homolog
BET1

P10632
Cytochrome P4502C8
CYP2C8

P08670
Vimentin
VIM

P30622
CAP-Gly domain-containing linker protein 1
CLIP1

P07451
Carbonic anhydrase 3
CA3

P01703
Ig lambda chain V-I region NEWM
—

TABLE 2-1

AC number
Protein Description
Gene name

Q8WWT9
Solute carrier family 13 member 3
SLC13A3

P31639
Sodium/glucose cotransporter 2
SLC5A2

O75264
Transmembrane protein C19orf77
C19orf77

P07148
Fatty acid-binding protein, liver
FABP1

Q9UHI7
Solute carrier family member 1
SLC23A1

P16444
Dipeptidase 1
DPEP1

P05062
Fructose-bisphosphate aldolase B
ALDOB

Q9NZA1
Chloride intracellular channel protein 5
CLIC5

P36269
Gamma-glutamyltransferase 5
GGT5

Q03154
Aminoacylase-1
ACY1

Q92499
ATP-dependent RNA helicase DDX1
DDX1

Q695T7
Sodium-dependent neutral amino acid transporter B(0)AT1
SLC6A19

Q13113
PDZK1-interacting protein 1
PDZK1IP1

Q00592
Podocalyxin
PODXL

P09467
Fructose-1,6-bisphosphatase 1
FBP1

P21695
Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic
GPD1

Q5T2W1
Na(+)/H(+) exchange regulatory co factor NHE-RF3
PDZK1

Q15599
Na(+)/H(+) exchange regulatory co factor NHE-RF3
SLC9A3R2

Q9BYF1
Angiotensin-converting enzyme 2
ACE2

Q8WW52
Protein FAM151A
FAM151A

P51580
Thicourine S-methyltransferase
TPMT

O96013
Serine/threonine-protein kinase PAK 4
PAK4

Q93088
Betaine-homocysteine S-methyltransferase 1
BHMT

P07911
Uromodulin
UMOD

P29972
Aquaporin-1
AQP1

Q07337
Neutral and basic amino add transport protein rBAT
SLC3A1

P08473
Neprilysin
MME

P15144
Aminopeptidase N
ANPEP

P35558
Phosphoenolpyruvate carboxykinase, cytosolic [GTP]
PCK1

O43175
D-3-phosphoglycerate dehydrogenase
PHGDH

P08729
Keratin, type II cytoskeletal 7
KRT7

O14745
Na(+)/H(+) exchange regulatory co factor NHE-RF1
SLC8A3R1

P09758
Tumor-associated calcium signal transducer 2
TACSTD2

P00742
Coagulation factor X
F10

Q9UGT4
Sushi domain-containing protein 2
SUSD2

Q1EHB4
Sodium-coupled monocarboxylate transporter 2
SLC5A12

Q75348
V-type proton ATPase subunit G 1
ATP6VIG1

Q16864
V-type proton ATPase subunit F
ATP6VIF

Q14894
Thiomorpholine-carboxylate dehydrogenase
CRYM

Q8Y2J2
Band 4,1-like protein 3
EPB41L3

Q4V9L6
Transmembrane protein 119
TMEM119

P11465
Pregnancy-specific beta-1-glycoprotein 2
PSG2

Q9H0W9
Ester hydrolase C11orf54
C11orf54

P12821
Angiotensin-converting enzyme
ACE

P13640
Metallothionein-1G
MT1G

P21266
Glutathione S-transferase Mu 3
GSTM3

P52758
Ribonuclease UK114
HRSP12

P16083
Ribosyldihydronicotinamide dehydrogenese [quinone]
NQO2

Q6ZQN7
Solute carrier organic anion transporter family member 4C1
SLCO4C1

P36543
V-type proton ATPase subunit E 1
ATP6VIE1

O75954
Tetraspanin
TSPAN9

O43451
Maltase-glucoamylase, intestinal
MGAM

P11137
Microtubule-associated protein 2
MAP2

P15941
Mucin-1
MUC1

Q9NQ84
G-protein coupled receptor family C group 5 member C
GPRC5C

P07305
Histone H1.0
H1F0

P53990
IST1 homolog
IST1

P05937
Calbindin
CALB1

TABLE 2-2

AC number
Protein Description
Gene name

P50135
Histamine N-methyltransferase
HNMT

O75131
Copine-3
CPNE3

O60262
Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-7
GNG7

Q9Y252
Lambda-crystallin homolog
CRYL1

Q16822
Phosphoenolpyruvate carboxykinase [GTP], mitochondrial
PCK2

P62330
ADP-ribosylation factor 6
ARF6

Q16625
Occludin
OCLN

P22748
Carbonic anhydrase 4
CA4

P18859
ATP synthase-coupling factor 6, mitochondrial
ATP5J

O43895
Xaa-Pro aminopeptidase 2
XPNPEP2

P27487
Dipeptidyl peptidase 4
DPP4

Q9U112
V-type proton ATPase subunit H
ATP6VIH

P29034
Protein S100-A2
S100A2

Q01740
Dimethylaniline monooxygenase [N-oxide-forming] 1
FMO1

P07195
L-lactate dehydrogenase B chain
LDHB

P38606
V-type proton ATPase catalytic subunit A
ATP6VIA

Q8N357
Solute carrier family 35 member F6
SLC35F6

Q14019
Coactosin-like protein
COTL1

Q98X6
TBC1 domain family member 10A
TBC1D10A

P09669
Cytochrome c oxidase subuni 6C
COX6C

Q9BUT1
3-hydroxybutyrate dehydrogenase type 2
BDH2

P21281
V-type proton ATPase subunit B, brain isoform
ATP6VIB2

O75094
Signal recognition particle subunit SRP72
SRP72

Q00796
Sorbitol dehydrogenase
SORD

Q2LD37
Uncharacterized protein KIAA1109
KIAA1109

Q96C23
Aldose 1-epimerase
GALM

Q9Y696
Chloride intracellular channel protein 4
CUC4

Q12929
Epidermal growth factor receptor kinase substrate 8
EPS8

P30086
Phosphatidylethanolamine-binding protein 1
PEBP1

O60749
Sorting nexin-2
SNX2

Q96YE9
Cadherin-related family member 2
CDHR2

B2RUZ4
Small integral membrane protein 1
SMM1

P05413
Fatty acid-binding protein heart
FABP3

P17813
Endogin
ENG

P00966
Argininosuccinate synthase
ASS1

Q96FL8
Multidrug and toxin extrusion protein 1
SLC47A1

Q92820
Gamma-glutamyl hydrolase
GGH

O43181
NADH dehydrogenase [ubiquinonel] iron-sulfur protein 4, mitochondrial
NDUFS4

Q13228
Selenium-binding protein 1
SELENBP1

Q9HBJ8
Collectrin
TMEM27

P13073
Cytochrome c oxidase subunit 4 isoform 1, mitochondrial
COX4I1

P09210
Glutathione S-transferase A2
GSTA2

P35241
Radixin
ROX

P22732
Solute carrier family 2, facilitated glucose transporter member 5
SLC2A5

Q9Y5X3
Sorting nexin-5
SNX5

P98082
Disabled homolog 2
DAB2

Q07075
Glutamyl aminopeptidase
ENPEP

P21291
Cysteine and glycine-rich protein 1
CSRP1

P29992
Guanine nucleotide-binding protein subunit alpha-11
GNA11

P82980
Retinol-binding protein 5
RBP5

Q9UBI6
Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-12
GNG12

Q13277
Syntaxin-3
STX3

O43490
Prominin-1
PROM1

O95747
Serine/threonine-protein kinase OSR1
OXSR1

Q9HD42
Charged multivesicular body protein 1a
CHMP1A

Q8NGM8
Olfactory receptor 6M1
OR6M1

P05026
Sodium/potassium-transporting ATPase subunit beta-1
ATP1B1

Q9NV59
Pyridoxine-5-phosphate oxidase
PNPO

P21912
Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial
SDHB

TABLE 2-3

AC number
Protein Description
Gene name

P16930
Fumarylacetoacetase
FAH

P08195
4F2 cell-surface antigen heavy chain
SLC3A2

P14550
Alcohol dehydrogenase [NADP(+)]
AKR1A1

Q9H0E2
Toll-interacting protein
TOLLIP

P56385
ATP synthase subunit e, mitochondrial
ATP5I

Q9Y5K8
V-type proton ATPase subunit D
ATP6V1D

Q93099
Homogentisate 1,2-dioxygenase
HGD

Q9Y6R1
Electrogenic sodium bicarbonate cotransporter 1
SLC4A4

Q16853
Membrane primary amine oxidase
AOC3

P54710
Sodium/potassium-transporting ATPase subunit gamma
FXYD2

P34896
Serine hydroxymethyltransferase, cytosolic
SHMT1

Q9NVD7
Alpha-parvin
PARVA

Q7Z3B1
Neuronal growth regulator 1
NEGR1

P12277
Creatine kinase B-type
CKB

O95292
Vesicle-associated membrane protein-associated protein B/C
VAPB

P63000
Ras-related C3 botulinum toxin substrate 1
RAC1

O15244
Solute carrier family 22 member 2
SLC22A2

Q92736
Ryanodine receptor 2
RYR2

Q13427
Peptidyl-prolyl cis-trans isomerase G
PPIG

O96019
Actin-like protein 6A
ACTL6A

P50148
Guanine nucleotide-binding protein G(q) subunit alpha
GNAQ

P35611
Alpha-adducin
ADD1

Q99653
Calcineurin B homologous protein 1
CHP1

P00167
Cytochrome b5
CYB5A

Q8NFU3
Thiosulfate sulfurtransferase/rhodanese-like domain-containing protein 1
TSTD1

P19440
Gamma-glutamyltranspeptidase 1
GGT1

Q96IX5
Up-regulated during skeletal muscle growth protein 5
USMG5

P09455
Retinol-binding protein 1
RBP1

P15311
Ezrin
EZR

O94760
N(G),N(G)-dimethylarginine dimethylaminohydrolase 1
DDAH1

O00499
Myc box-dependent-interacting protein 1
BIN1

P48059
LIM and senescent cell antigen-like-containing domain protein 1
LIMS1

Q16775
Hydroxyacylglutathione hydrolase, mitochondrial
HAGH

P05023
Sodium/potassium-transporting ATPase subunit alpha-1
ATP1A1

Q8WU39
Marginal zone B- and B1-cell-specific protein
MZB1

P80723
Brain acid soluble protein 1
BASP1

P54920
Alpha-soluble NSF attachment protein
NAPA

O75309
Cadherin-16
CDH16

P62873
Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1
GNB1

P98164
Low-density lipoprotein receptor-related protein 2
LRP2

Q9Y2Q5
Ragulator complex protein LAMTOR2
LAMTOR2

P20073
Annexin A7
ANXA7

Q96IU4
Alpha/beta hydrolase domain-containing protein 14B
ABHD14B

Q9BZV1
UBX domain-containing protein 6
UBXN6

O75936
Gamma-butyrobetaine dioxygenase
BBOX1

P50053
Ketohexokinase
KHK

P00918
Carbonic anhydrase 2
CA2

P11279
Lysosome-associated membrane glycoprotein 1
LAMP1

O75874
Isocitrate dehydrogenase [NADP] cytoplasmic
IDH1

P00325
Alcohol dehydrogenase 1B
ADH1B

Q14699
Raftlin
RFTN1

Q8N201
Integrator complex subunit 1
INTS1

Q9NZZ3
Charged multivesicular body protein 5
CHMP5

Q8WUM4
Programmed cell death 6-interacting protein
PDCD6IP

P09211
Glutathione S-transferase P
GSTP1

Q10567
AP-1 complex subunit beta-1
AP1B1

Q96KP4
Cytosolic non-specific dipeptidase
CNDP2

Q9H1C7
Cysteine-rich and transmembrane domain-containing protein 1
CYSTM1

P60953
Cell division control protein 42 homolog
CDC42

TABLE 2-4

AC number
Protein Description
Gene name

P24539
ATP synthase subunit b, mitochondrial
ATP5F1

P62070
Ras-related protein R-Ras2
RRAS2

Q9UHE5
Probable N-acetyltransferase 8
NAT8

Q8NFJ5
Retinoic acid-induced protein 3
GPRC5A

Q13596
Sorting nexin-1
SNX1

Q8N3Y1
F-box/WD repeat-containing protein 8
FBXW8

Q9NZ45
CDGSH iron-sulfur domain-containing protein 1
CISD1

Q9H4A4
Arninopeptidase B
RNPEP

Q96DG6
Carboxymethylenebutenolidase homolog
CMBL

Q9ULE6
Paladin
PALD1

Q6UX53
Methyltransferase-like protein 7B
METTL7B

P17174
Aspartate aminotransferase, cytoplasmic
GOT1

P55795
Heterogeneous nuclear ribonucleoprotein H2
HNRNPH2

Q9NQR4
Omega-amidase NIT2
NIT2

P51149
Ras-related protein Rab-7a
RAB7A

P11908
Ribose-phosphate pyrophosphokinase 2
PRPS2

P10599
Thioredoxin
TXN

P05783
Keratin, type I cytoskeletal 18
KRT18

O94903
Proline synthase co-transcribed bacterial homolog protein
PROSC

Q9UJ68
Mitochondrial peptide methionine sulfoxide reductase
MSRA

P15374
Ubiquitin carboxyl-terminal hydrolase isozyme L3
UCHL3

P32119
Peroxiredoxin-2
PRDX2

Q9NVA2
Septin-11
SEPT11

Q6QHC5
Sphingolipid delta(4)-desaturase/C4-hydroxylase DES2
DEGS2

P11233
Ras-related protein Ral-A
RALA

Q96CX2
BTB/POZ domain-containing protein KCTD12
KCTD12

O95154
Aflatoxin B1 aldehyde reductase member 3
AKR7A3

Q13183
Solute carrier family 13 member 2
SLC13A2

P84077
ADP-ribosylation factor 1
ARF1

P21810
Biglycan
BGN

P21796
Voltage-dependent anion-selective channel protein 1
VDAC1

Q16270
Insulin-like growth factor-binding protein 7
IGFBP7

P62834
Ras-related protein Rap-1A
RAP1A

P13473
Lysosome-associated membrane glycoprotein 2
LAMP2

Q00839
Heterogeneous nuclear ribonucleoprotein U
HNRNPU

Q13418
Integrin-linked protein kinase
ILK

P51148
Ras-related protein Rab-5C
RAB5C

P50895
Basal cell adhesion molecule
BCAM

P62820
Ras-related protein Rab-1A
RAB1A

Q96F10
Diamine acetyltransferase 2
SAT2

P21283
V-type proton ATPase subunit C 1
ATP6V1C1

Q9HCU5
Prolactin regulatory element-binding protein
PREB

P68104
Elongation factor 1-alpha 1
EEF1A1

Q9NQV5
PR domain-containing protein 11
PRDM11

Q9UEU0
Vesicle transport through interaction with t-SNAREs homolog 1B
VTI1B

Q9H2A2
Aldehyde dehydrogenase family 8 member A1
ALDH8A1

Q01650
Large neutral amino acids transporter small subunit 1
SLC7A5

P11142
Heat shock cognate 71 kDa protein
HSPA8

Q9NRA2
Sialin
SLC17A5

O75165
DnaJ homolog subfamily C member 13
DNAJC13

P61224
Ras-related protein Rap-1b
RAP1B

P17927
Complement receptor type 1
CR1

Q96A57
Transmembrane protein 230
TMEM230

O94886
Transmembrane protein 63A
TMEM63A

P60981
Destrin
DSTN

P30153
Serine/threonine-protein phosphatase 2A 65 kDa regulatory
PPP2R1A

subunit A alpha isoform

Q06830
Peroxiredoxin-1
PRDX1

Q96BW5
Phosphotriesterase-related protein
PTER

Q6P4A8
Phospholipase B-like 1
PLBD1

TABLE 2-5

AC number
Protein Description
Gene name

P04216
Thy-1 membrane glycoprotein
THY1

P56199
Integrin alpha-1
ITGA1

O95865
N(G),N(G)-dimethylarginine dimethylaminohydrolase 2
DDAH2

Q6IAA8
Ragulator complex protein LAMTOR1
LAMTOR1

P40925
Malate dehydrogenase, cytoplasmic
MDH1

O00560
Syntenin-1
SDCBP

P54707
Potassium-transporting ATPase alpha chain 2
ATP12A

Q02952
A-kinase anchor protein 12
AKAP12

P08183
Multidrug resistance protein 1
ABCB1

Q9Y4F1
FERM, RhoGEF and pleckstrin domain-containing protein 1
FARP1

P02549
Spectrin alpha chain, erythrocytic 1
SPTA1

Q5TZA2
Rootletin
CROCC

P01116
GTPase KRas
KRAS

Q15907
Ras-related protein Rab-11B
RAB11B

Q9H2P9
Diphthine synthase
DPH5

O75223
Gamma-glutamylcyclotransferase
GGCT

Q9H223
EH domain-containing protein 4
EHD4

P30041
Peroxiredoxin-6
PRDX6

P30046
D-dopachrome decarboxylase
DDT

P22352
Glutathione peroxidase 3
GPX3

Q13200
26S proteasome non-ATPase regulatory subunit 2
PSMD2

P62879
Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2
GNB2

P00441
Superoxide dismutase [Cu—Zn]
SOD1

P98172
Ephrin-B1
EFNB1

Q13526
Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1
PIN1

P15090
Fatty acid-binding protein, adipocyte
FABP4

P00491
Purine nucleoside phosphorylase
PNP

P18206
Vinculin
VCL

P14384
Carboxypeptidase M
CPM

Q96KX1
Uncharacterized protein C4orf36
C4orf36

Q93050
V-type proton ATPase 116 kDa subunit a isoform 1
ATP6V0A1

Q93052
Lipoma-preferred partner
LPP

Q13576
Ras GTPase-activating-like protein IQGAP2
IQGAP2

Q8IWA5
Choline transporter-like protein 2
SLC44A2

Q15847
Adipogenesis regulatory factor
ADIRF

Q9Y5Z4
Heme-binding protein 2
HEBP2

Q96CW1
AP-2 complex subunit mu
AP2M1

Q9BX97
Plasmalemma vesicle-associated protein
PLVAP

P26038
Moesin
MSN

P11277
Spectrin beta chain, erythrocytic
SPTB

Q92692
Poliovirus receptor-related protein 2
PVRL2

Q15286
Ras-related protein Rab-35
RAB35

Q01995
Transgelin
TAGLN

P15586
N-acetylglucosamine-6-sulfatase
GNS

Q9UL25
Ras-related protein Rab-21
RAB21

P05106
Integrin beta-3
ITGB3

P13987
CD59 glycoprotein
CD59

P06703
Protein S100-A6
S100A6

O43488
Aflatoxin B1 aldehyde reductase member 2
AKR7A2

Q13423
NAD(P) transhydrogenase, mitochondrial
NNT

Q9UK41
Vacuolar protein sorting-associated protein 28 homolog
VPS28

Q06495
Sodium-dependent phosphate transport protein 2A
SLC34A1

Q9Y266
Nuclear migration protein nudC
NUDC

Q7LBR1
Charged multivesicular body protein 1b
CHMP1B

O43707
Alpha-actinin-4
ACTN4

Q9UBV8
Peflin
PEF1

Among the 106 proteins found to be increased in the expression, the proteins shown in Table 3 including azurocidin (hereinafter referred to as AZUL) are characteristic with a large difference in the expression level from the proteins contained in the Te-EVs derived from the normal tissue, and hence can be more preferably used as the markers for renal cell carcinoma.

TABLE 3

Gene

Protein Description
name
AC number

Glycogenin-1
GYG1
P46976

Azurocidin
AZU1
P20160

Carbonic anhydrase 9
CA9
Q16790

Catechol O-methyltransferase
COMT
P21964

Starch-binding domain-containing protein 1
STBD1
O95210

Phosphoprotein associated with
PAG1
Q9NWQ8

glycosphingolipid-enriched microdomains 1

Tissue factor
F3
P13726

Thymidine phosphorylase
TYMP
P19971

Leukocyte-associated immunoglobulin-like
LAIR1
Q6GTX8

receptor 1

Transmembrane glycoprotein NMB
GPNMB
Q14956

Glutamine--fructose-6-phosphate
GFPT1
Q06210

aminotransferase [isomerizing] 1

Glycogen [starch] synthase, muscle
GYS1
P13807

High affinity immunoglobulin epsilon receptor
FCER1G
P30273

subunit gamma

Versican core protein
VCAN
P13611

Granulins
GRN
P28799

Prostaglandin F2 receptor negative regulator
PTGFRN
Q9P2B2

Solute carrier family 2, facilitated glucose
SLC2A3
P11169

transporter member 3

Tyrosine-protein phosphatase non-receptor type
SIRPA
P78324

substrate 1

Nucleophosmin
NPM1
P06748

Receptor-type tyrosine-protein phosphatase C
PTPRC
P08575

Density-regulated protein
DENR
O43583

DNA-(apurinic or apyrimidinic site) lyase
APEX1
P27695

Cytochrome P450 2C8
CYP2C8
P10632

Vimentin
VIM
P08670

CAP-Gly domain-containing linker protein 1
CLIP1
P30622

Carbonic anhydrase 3
CA3
P07451

In particular, carbonic anhydrase 9, catechol O-methyltransferase, phosphoprotein associated with glycosphingolipid-enriched microdomains 1, leukocyte-associated immunoglobulin-like receptor 1, transmembrane glycoprotein NMB, high affinity immunoglobulin epsilon receptor subunit gamma, solute carrier family 2, facilitated glucose transporter member 3, tyrosine-protein phosphatase non-receptor type substrate 1, receptor-type tyrosine-protein phosphatase C, vimentin and carbonic anhydrase 3 are regarded as particularly useful markers because these have been reported to be highly expressed in renal cancer. Besides, phosphoprotein associated with glycosphingolipid-enriched microdomains 1 and tyrosine-protein phosphatase non-receptor type substrate 1 are regarded as promising candidates for a target for drug discovery because these make contribution to inhibition of T cell activity and immune evasion by cancer cells when highly expressed. One of these markers may be singly used, or when a plurality of markers are used in combination, the specificity and the sensitivity can be increased. Further, an existing biomarker may be used in combination for diagnosis.

Although the analysis results of the proteins contained in the Te-EVs are herein described, any biological component contained in the Te-EVs may be analyzed to be used as a biomarker. Examples of such a biological component include a nucleic acid and a lipid.

[Example 4] Analysis of AZU1

The results of detailed analysis of AZU1 farthest from the origin of the volcano plot (p=2.85×10⁻³, fold-change: 31.59, shown with an arrow AZU1 in FIG. 5) will now be described, and it goes without saying that the other proteins described above can be similarly used as the markers.

FIG. 6A illustrates the concentrations of AZU1 in the Te-EVs derived from the normal tissue and the tumor tissue of 20 cases of renal cell carcinoma patients. In all the 20 renal cell carcinoma patients used for the analysis by the mass spectrometry, the expression of AZU1 increased in the Te-EVs derived from the tumor tissue. FIG. 6B illustrates the concentrations of AZU1 depending on the progression of the cancer, and as renal cell carcinoma progressed, the amount of AZU1 contained in the Te-EVs increased. In a cancerous region of renal cell carcinoma at stage T3 (T3a and T3b), the content of AZU1 was significantly higher than in a non-cancerous region. Besides, as illustrated in an enlarged view in FIG. 6B, the content of AZU1 was different even in early stages of cancer (T1a to T2a) as compared with that in the Te-EVs derived from the normal tissue.

Besides, the increase of the expression of AZU1 in accordance with the progression of cancer was checked by the Western blotting (FIG. 6C). Five hundred ng per lane of EVs protein was separated by electrophoresis and then transferred onto a film, and the detection was performed in the same manner as in Example 2 by using an anti-AZU1 monoclonal antibody (manufactured by Abcam Plc.) as a primary antibody. As a result of the analysis of Te-EVs obtained from four patients at stages of T1a to T3b, it was confirmed, in all samples, that the expression of AZU1 remarkably increased in Te-EVs obtained from a cancerous region (T) as compared with Te-EVs obtained from a non-cancerous region (N).

It has been reported that extracellular vesicles derived from cancer are detected also in a serum (Non Patent Literatures 3 to 5 and 11 to 13). Therefore, the amount of AZU1 in EVs contained in a serum sample was measured by quantitative mass spectrometry (FIG. 6D).

The EVs contained in the serum sample was purified by using an EVSecond column (manufactured by GL Sciences Inc.). AZU1 was not at all detected in EVs contained in serums of 10 cases of healthy persons, but AZU1 was detected in 10 cases out of 19 cases of renal cell carcinoma patients. Although the AZU1 content did not increase in accordance with the progression of cancer as in the Te-EVs derived from the renal cell carcinoma tissue, it was detected at a high ratio in EVs contained in serums of cancer patients at early stages of T1a to T2b. This result reveals that a renal cell carcinoma test for detecting AZU1 using a serum is effective.

That AZU1 was detected in the extracellular vesicles present in the serum of a renal cell carcinoma patient indicates that a protein different in the content in Te-EVs can be detected in the serum. Accordingly, it indicates that a biomarker found by this method can function as a disease marker with which a test can be performed by using a serum.

[Example 5] Analysis of Effect of AZU1 in Renal Cell Carcinoma

Since the expression of AZU1 was detected specifically to the renal cell carcinoma tissue, the biological effect of AZU1 was examined. It is known that angiogenesis is generated largely to construct microenvironment of tumor tissue in renal cell carcinoma (Non Patent Literatures 14 to 17). Therefore, the effect of AZU1 on the form of vascular endothelial cells was examined.

First, localization of intrinsic AZU1 was examined under an immunoelectron microscope. The localization of AZU1 was examined in Te-EVs obtained from normal tissue and tumor tissue of the same patient by using an anti-AZU1 antibody obtained by immunizing a rabbit (manufactured by Abcam Plc.) and an anti-CD9 monoclonal antibody as primary antibodies, and secondary antibodies labeled with colloidal gold particles having different sizes (FIG. 7A).

An antibody labeled with 40 nm colloidal gold particles was used as an anti-rabbit antibody, and an antibody labelled with 20 nm colloidal gold particles was used as an anti-mouse antibody. Accordingly, a large particle corresponds to the expression of AZU1 and a small particle corresponds to the expression of CD9 in FIG. 7A. Although the expression of CD9 was found on the surface of the Te-EVs obtained from either of the normal tissue and the tumor tissue, the expression of AZU1 was found on the surface of the Te-EVs derived from the tumor tissue alone.

The AZU1 expression in a renal cell carcinoma cell line was checked by the Western blotting (FIG. 7B). The expression of AZU1 in EVs isolated from 786-O, ACHN, Caki-1 and Caki-2 (obtained from ATCC) cell lines and in a cell lysate (whole cell lysate) was examined. The expression of AZU1 was found in any of these cells, and although AZU1 was detected in the EVs isolated from culture fluids (cultured media EVs, hereinafter sometimes referred to as CM-EVs) of the three cell lines of 786-O, Caki-1 and Caki-2, AZU1 was minimally detected in CM-EVs derived from the ACHN cell line.

Next, the permeability of the vascular endothelial cells of the CM-EVs obtained from these cell lines was analyzed based on transendothelial electrical resistance (TEER) (FIG. 7C). The TEER was measured by using HUVEC cells (obtained from Gibco). After seeding 4.0×10⁴HUVEC cells in a 24-well culture insert (manufactured by Thermo Fisher Scientific K.K.) with a pore size of 0.4 μm and culturing the cells for 4 days, CM-EVs obtained from each cell line was added thereto in a concentration of 10 μg/ml, and the TEER was measured with Millicell® ERS-2 voltammeter (manufactured by Millipore) to calculate the TEER of each sample in accordance with the following expression:

(Electric resistance (Ω) of sample well−Electric resistance (Ω) of empty well)×culture area (cm²)=TEER (Ωcm²) [Expression 1]

The results are illustrated in FIG. 7C. As a result of measuring the TEER over time, the TEER of the HUVEC cell sheet was reduced 24 hours after the addition of the CM-EVs. In particular, the TEER was found to be remarkably reduced by addition of CM-EVs obtained from the Caki-1 and 786-O cells in which a larger amount of AZU1 was presented on EVs.

Since it was presumed that the EVs having a larger amount of AZU1 presented thereon had an effect of increasing permeability, detailed analysis was performed by using a system in which AZU1 was forcedly expressed. AZU1-FLAG (manufactured by Addgene) was introduced into and forcedly expressed in ACHN cells in which AZU1 was minimally detected in CM-EVs. As illustrated in FIG. 7D, in the ACHN cell line in which AZU1-FLAG was forcedly expressed, it was found that EVs also contained a large amount of AZU1-FLAG.

Besides, it was confirmed under an immunoelectron microscope that the AZU1-FLAG was presented also on the EVs. The detection of the FLAG was performed by using an anti-FLAG monoclonal antibody (manufactured by Sigma-Aldrich) as a primary antibody and using a colloidal gold labeled anti-mouse antibody (FIG. 7E). Although the FLAG was not detected in the CM-EVs obtained from cells to which a vector alone was introduced, the FLAG was detected on the CM-EVs obtained from cells into which the AZU1-FLAG was introduced. It was confirmed based on these results that the forcedly expressed AZU1-FLAG was also presented on the EVs in the same manner as the intrinsic AZU1.

The thus obtained expression system was used to examine the permeability by using HUVEC (FIG. 7F). It was clarified that CM-EVs obtained from cells in which AZU1 is forcedly expressed remarkably reduces the TEER as compared with CM-EVs obtained from cells into which a vector alone is introduced. Accordingly, it is suggested that AZU1 has an effect of disintegrating the form of vascular endothelial cells.

Next, examination was made to check whether or not a similar effect can be exhibited by Te-EVs obtained from renal cell carcinoma patients. The permeability of cells was measured by using Te-EVs derived from normal tissue and tumor tissue obtained from six patients at various stages. FIG. 8A illustrates change, over time, of the TEER in each patient after addition of Te-EVs derived from the normal tissue and the tumor tissue, and FIG. 8B is a diagram in which the respective measurement values are plotted together. When the Te-EVs derived from the tumor tissue were added, the TEER was found to be remarkably reduced from 12 hours after the addition. Besides, when the Te-EVs obtained from a patient with cancer in an advanced stage were added, the TEER was more remarkably reduced.

Examinations were made to check whether or not the difference in the effect on the TEER of the Te-EVs derived from the normal tissue and the tumor tissue was caused by incorporation into cells of the Te-EVs derived from these tissues. The Te-EVs derived from the normal tissue and the tumor tissue were respectively labeled with PKH-67 and PKH-26 (manufactured by Sigma Aldrich) and then mixed, the resultant mixture was added to a culture fluid of HUVEC cells, and the resultant was observed under a microscope 12 hours later. FIG. 8C illustrates the results obtained by using the Te-EVs obtained from two patients. In either sample, Te-EVs derived from the normal tissue looking bright around a nucleus (stained green under a fluorescence microscope) and Te-EVs derived from the tumor tissue recognized as a dark stained image (stained red under a fluorescence microscope; two portions are shown with arrows in each image) were both detected to the same extent. Accordingly, it was confirmed that the incorporation efficiency of Te-EVs did not vary depending on the tissue from which the Te-EVs were derived. In other words, it was revealed that the reduction of the TEER caused by the Te-EVs derived from the tumor tissue was not caused by a difference in the incorporation efficiency of Te-EVs. Therefore, the reduction is probably caused by proteins and the like including AZU1 presented on the Te-EVs.

In blood of a patient, Te-EVs derived from tumor tissue and normal tissue are circulating in a mixed state. It was examined whether or not the reduction of the TEER was induced in such a mixed state. FIG. 8D illustrates the TEER measured over time after adding, to a culture fluid of HUVEC cells, a mixture of Te-EVs derived from normal tissue and tumor tissue obtained from three patients. The reduction of the TEER was observed in the Te-EVs obtained from any one of the patients even if the mixture of the Te-EVs derived from tumor tissue and normal tissue was used. This result seems to reflect a phenomenon actually occurring in a body of a patient, and hematogenous metastasis seems to be induced by AZU1.

Based on the above-described results, it is presumed that AZU1 is a disease-related gene closely involved in the onset and development of renal cell carcinoma. Accordingly, a therapeutic agent can be created by using AZU1 as a target. In this manner, when the function of a biological component such as a protein or a nucleic acid expressed specifically to a disease is analyzed, a target of a novel therapeutic agent can be found.

[Example 6] Analysis Using Serum of Patient

If a protein characteristic to a renal cell carcinoma patient can be detected with EVs contained in a serum, it can be used as a useful marker for early detection of renal cell carcinoma. Therefore, proteins different between a renal cell carcinoma patient and a healthy person were searched for in EVs contained in a serum. Table 4 shows proteins that can be detected in EVs contained in a serum of a renal cell carcinoma patient but cannot be detected in those of a healthy person, excluding those detected as a result of the Te-EVs analysis shown in Tables 1 and 2.

TABLE 4

AC number
Protein Description
Gene name

P11678
Eosinophil peroxidase
EPX

Q8NI35
InaD-like protein
INADL

P68104
Elongation factor 1-alpha 1
EEF1A1

Q63ZY3
KN motif and ankyrin repeat domain-
KANK2

containing protein 2

Q16836
Hydroxyacyl-coenzyme A dehydrogenase,
HADH

mitochondrial

P07437
Tubulin beta chain
TUBB

P55058
Phospholipid transfer protein
PLTP

Q9Y3R5
Protein dopey-2
DOPEY2

P05543
Thyroxine-binding globulin
SERPINA7

P06732
Creatine kinase M-type
CKM

P31946
14-3-3 protein beta/alpha
YWHAB

O75533
Splicing factor 3B subunit 1
SF3B1

P07900
Heat shock protein HSP 90-alpha
HSP90AA1

Q15404
Ras suppressor protein 1
RSU1

Q9UII5
Zinc finger protein 107
ZNF107

P09874
Poly [ADP-ribose] polymerase 1
PARP1

P10619
Lysosomal protective protein
CTSA

P14618
Pyruvate kinase PKM
PKM

P14625
Endoplasmin
HSP90B1

P23284
Peptidyl-prolyl cis-trans isomerase B
PPIB

P60033
CD81 antigen
CD81

P62249
40S ribosomal protein S16
RPS16

Q86XI8
Uncharacterized protein C19orf68
C19orf68

These proteins are characteristic to a renal cell carcinoma patient, but are not detected in all renal cell carcinoma patients. When a plurality of these markers are combined, however, a renal cell carcinoma patient can be detected at an early stage by using a blood sample. In addition to findings obtained from Te-EVs of renal cell carcinoma patients, these proteins specifically detected in the serums of the renal cell carcinoma patients are usable as novel markers for renal cell carcinoma, for which an effective biomarker has not been found.

INDUSTRIAL APPLICABILITY

According to a search method for a biomarker of the present invention, a tissue-specific disease marker contained in extracellular vesicles can be obtained. Since extracellular vesicles are secreted into a body fluid, they are very useful as non-invasive or minimally invasive disease markers. When this method is employed, a biomarker can be obtained for a disease for which an effective biomarker has not been found.

Besides, a renal cell carcinoma marker described herein can be used, in a renal cell carcinoma detecting test, as a novel marker for renal cell carcinoma for which there has been no effective biomarker. Furthermore, since AZU1 has an effect of disintegrating the form of vascular endothelial cells, it is suggested to be significant for metastasis of cancer cells. Therefore, a molecular target drug using AZU1 as a target is expected to have an anticancer metastasis effect.

BIOMARKER, METHOD FOR SEARCHING DISEASE-RELATED GENE, AND RENAL CANCER MARKER

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