Method for diagnosing or assisting in diagnosing bone tumor prognosis

Abstract

To provide a prognosis diagnosis method or a prognosis diagnosis assisting method for a bone tumor.SOLUTION: It has been found that the number of osteoclasts and a specific miRNA expression level of a biological sample of a bone tumor patient are correlated with prognosis, and the present invention has been completed.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for prognostic diagnosis or an auxiliary method for prognostic diagnosis of bone tumors. [Background technology]

[0002] (Bone Tumor) Bone tumors are abnormal cell growths in the bone that may be cancerous (malignant) or non-cancerous (benign). They can originate in the bone (primary cancer) or can originate in another organ and spread to the bone (metastatic cancer). Osteosarcoma is the most common primary malignant bone tumor that occurs primarily in teenagers, but the cause is unknown. The incidence rate in Japan is said to be 1 per 1 million people (130 to 260 per year). In the past, when amputation was the only treatment option, the 2-year survival rate for osteosarcoma was 15 to 20%, and it was a disease with a very poor prognosis (see Patent Document 1). Metastatic bone tumors are cancers that have metastasized to the bone (bone metastasis), and any cancer can cause bone metastasis. The most common types include lung cancer, breast cancer, prostate cancer, and kidney cancer (see the Kanazawa University Hospital Orthopedic Surgery website (Non-Patent Document 1)).

[0003] The following methods have been reported for testing and prognostic diagnosis of bone tumors. Patent Document 2 discloses "a method for diagnosing the prognosis of a patient suffering from cancer, comprising the following step: measuring the expression level of GP88 in a biological sample obtained from the patient." Patent Document 3 describes "Arg of the Gs alpha gene" 201 A method for identifying a group of diseases caused by Gs alpha gene mutations and differentiating them from other tumor diseases by detecting codon mutations with high sensitivity, comprising the steps of: detecting the Arg codon of genomic DNA in a sample; 201The method disclosed herein includes the steps of: performing a first amplification reaction to amplify a region containing a codon; performing a second amplification reaction using a mismatch primer to introduce a cleavage site for the restriction enzyme EagI, with the product obtained by the first amplification reaction as a template; and determining that a mutation has occurred if the product obtained by the second amplification reaction is not cleaved by the restriction enzyme EagI, wherein a PNA (peptide nucleic acid) probe is added in the first and second amplification reactions to suppress amplification of normal alleles. The above patent documents do not disclose or suggest the prognostic diagnosis method or the prognostic diagnosis auxiliary method of the present invention. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Re-tabled publication 2018 / 074451 [Patent Document 2] JP 2015-158498 A [Patent Document 3] JP 2008-092899 A [Non-patent literature]

[0005] [Non-Patent Document 1] http: / / ortho.w3.kanazawa-u.ac.jp / patnt / pages / brn_tmr.html Summary of the Invention [Problem to be solved by the invention]

[0006] The objective of the present invention is to provide a method for prognostic diagnosis or an auxiliary method for prognostic diagnosis of bone tumors. [Means for solving the problem]

[0007] The present inventors discovered that the number of osteoclasts and the expression level of a specific miRNA in a biological sample from a bone tumor patient correlate with prognosis, and completed the present invention.

[0008] That is, the present invention is as follows. 1. A method for diagnosing or assisting in the prognosis of a bone tumor patient, which comprises measuring the number of osteoclasts in a biological sample collected from the bone tumor patient. 2. The method according to item 1 above, characterized in that the measurement involves comparing the number of osteoclasts in the biological sample with the number of osteoclasts in a biological sample obtained from a healthy subject or a cutoff value, and determining or assisting in the determination of poor prognosis if the number is lower. 3. The cutoff value is 0.50 mm 2 ~1.50 mm 2 per 1000μm 2 3. The method according to item 2 above, wherein the number of osteoclasts is 4 or more. 4. The method according to any one of the preceding items 1 to 3, wherein the prognosis is any one of the following: (1) Metastasis (2) Relapse (3) Metastasis-free survival rate (4) Local recurrence-free survival rate (5) Progression-free survival rate (6) Overall survival rate 5. A prognostic kit for bone tumor patients, comprising an antibody that stains osteoclasts. 6. A method for diagnosing or assisting in the prognosis of a bone tumor patient, comprising measuring the expression level of any one or more of the following miRNAs in a biological sample collected from the bone tumor patient: (1)hsa-miR-146a-5p (2) hsa-miR-1260 (3) hsa-miR-487b-3p (4)hsa-miR-6720-3p (5)hsa-miR-1260b (6)hsa-miR-4758-3p (7)hsa-miR-4690-3p (8)hsa-miR-4286 (9)hsa-miR-6765-3p (10)hsa-miR-1261 (11)hsa-miR-7975 (12)hsa-miR-4664-5p (13)hsa-miR-3907 (14)hsa-miR-7977 (15)hsa-miR-1273c (16)hsa-miR-375-5p 7. The method according to item 6 above, characterized in that the measurement involves comparing the expression level of miRNA in the biological sample with the expression level of miRNA in a biological sample obtained from a healthy subject or a cutoff value, and determining or assisting in the determination of poor prognosis if the expression level is higher. 8. The method according to the preceding item 6 or 7, wherein the prognosis is any one of the following: (1) Metastasis (2) Relapse (3) Metastasis-free survival rate (4) Local recurrence-free survival rate (5) Progression-free survival rate (6) Overall survival rate Effect of the Invention

[0009] The present invention provides a method for diagnosing or assisting in the prognosis of bone tumors. [Brief description of the drawings]

[0010] [Figure 1]SEV (Small Extracellular Vesicle)-Inhibited Osteosarcoma (OS) Decreased Malignancy in an Orthotopic Implantation Mouse Model. Mouse OS LM8-WT (Wild Type), TSG101-KO (Knock Out)1 or TSG101-KO2 cells were implanted into the distal femur of C3H / He mice. When the tumors grew to 2,000 mm3, femurs and lungs were harvested from the mice. The femurs were subjected to immunohistochemical staining for Ki67 (A), CD31 (C) or VEGF (E). The number of Ki67-positive cells per total cell count (B), the number of CD31-positive blood vessels per mm2 (D), and the area of ​​VEGF-positive regions per mm2 (F) were measured. (G) Lungs were stained with H&E. The number (H) or area (I) of metastatic lesions was calculated. Magnification of images is 40x for A and E, 20x for C, and 10x for G. Scale bars indicate 50 μm for A and E, 100 μm for C, and 300 μm for G. Values ​​in B, D, F, H, and I are expressed as mean ± standard deviation. * P < 0.05, ** P < 0.01 vs. WT (Student's t test). [Diagram 2]Inhibition of osteoclast formation by OS-SEV. (A) LM8-WT, TSG101-KO1 or TSG101-KO2 cells were implanted into the distal femur of C3H / He mice. When the tumors grew to 2,000 mm3, immunohistochemical staining of femur for cathepsin K was performed. Images were taken at 20x magnification. B, BM, M and T in the images indicate bone, bone marrow, metaphysis and tumor, respectively. Scale bar indicates 100 μm. (B) The area of ​​cathepsin K-positive cells within the field was calculated. Values ​​are shown as mean ± standard deviation. *P <0.05, **P <0.01, ns (not significant) vs. sham or WT (Student's t test with Bonferroni correction). (C) OPCs (1 × 105 cells) were cultured in medium containing each dose of PKH-labeled LM8-SEV for 24 h and analyzed by flow cytometer. (D) OPCs were cultured in medium containing various doses of LM8-SEV for 2 days, and the cell viability of OPCs was measured by WST-8 assay. (E) OPCs were cultured in medium with or without various SEV and with or without RANKL for 5 days. Images of TRAP staining were taken at 4x magnification. Scale bar indicates 500 μm. (F, G) The area of ​​TRAP-positive cells shown in E was measured. The number of small OCs with a TRAP-positive area of ​​1,000-10,000 μm2 or large OCs with a TRAP-positive area of ​​10,000 μm2 or more per field was measured. (H) The relative expression of Trap and Gapdh genes in OPCs cultured for 5 days with or without RANKL or SEV was detected by real-time PCR. The expression level of Trap gene was normalized to Gapdh expression and expressed as a relative value to the RANKL(-) and SEV(-) groups. Values ​​in (D) and (FH) are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01 versus RANKL(+) and SEV(-) groups (Student's t test with Bonferroni correction). nd means not detected. [Diagram 3]Suppression of genes involved in osteoclastogenesis and the NF-κB pathway by LM8-SEV. (A) OPCs were stimulated with or without RANKL or LM8-SEV for 48 h. Expression of Nfatc1, c-fos, Atp6v0d2, Dcstamp, Ocstamp, and Gapdh genes was detected by real-time PCR. The expression level of each gene was normalized to Gapdh expression and shown as a relative value to the RANKL(-) and SEV(-) groups. Values ​​are shown as mean ± standard deviation. *P < 0.01 vs. RANKL(+), SEV(-) groups (Student's t test). (B) After 48 h of stimulation with or without RANKL or LM8-SEV, surface DC-STAMP was stained with anti-DC-STAMP antibody (1A2; Merck) and analyzed by flow cytometer. (C) OPCs were precultured for 12 h in SEV-free medium or medium containing each SEV and stimulated with RANKL for the indicated times. Pphospho-NF-κB, phospho-IκBα, and GAPDH were detected by Western blot. Band intensities were normalized to GAPDH. Bar graphs show relative values ​​to the maximum sample. [Figure 4]Suppression of osteoclast formation dependent on TRAF6 downregulation by miRNA (miR-146a-5p). (A) CD9-, CD63-, or CD81-positive SEVs secreted from 143B or HOS cells were detected by SEV-ELISA. Values ​​are shown as mean ± standard deviation. * P < 0.01 vs. 143B cells (Student's t-test). (B) OPCs were cultured for 5 days in media with or without SEVs or with or without RANKL. The relative expression of Trap, Nfatc1, Ocstamp, and Gapdh genes was detected by real-time PCR. Trap, Nfatc1, and Ocstamp expressions were normalized to Gapdh expression and shown as relative values ​​of RANKL(-) and SEV(-) groups. Values ​​are shown as mean ± standard deviation. *P < 0.01, ns (not significant) vs. RANKL(+), SEV(-) group or RANKL(+), 143B-SEV group (Student's t test with Bonferroni correction). (C) Small RNA was isolated from 143B-SEV or HOS-SEV and used for real-time PCR to detect has miR-146A-5P and U6. hsa miR-146a-5p expression was normalized to U6 expression and expressed as relative values ​​to HOS-SEV. Values ​​are expressed as mean ± SD. *P < 0.01 vs. HOS-SEV (Student's t test). (D) OPCs were cultured in medium containing M-CSF with or without each SEV for 2 days. TRAF6 and GAPDH proteins were detected by Western blot. Band intensity was normalized to GAPDH. Relative values ​​to SEV(-) are shown in bar graphs. (E, F) OPCs were transfected with miR-control (miR-Ctrl) or hsa miR-146a-5p mimic and further cultured in medium containing M-CSF for 2 days. The relative expression of Traf6 and Gapdh genes was detected by real-time PCR. Traf6 expression was normalized to Gapdh expression and expressed as a relative value to miR-Ctrl (E). TRAF6 and GAPDH proteins were detected by Western blot. Band intensity was normalized to GAPDH. The relative value to miR-Ctrl is shown in a bar graph (F).(G) OPCs were transfected with miR-Ctrl or has miR-146a-5p mimic and cultured in medium containing RANKL for 5 days. Images of TRAP staining were taken at 4x magnification. Scale bar indicates 500 μm. (H) The area of ​​TRAP-positive cells shown in (G) was measured. The number of large OCs >10,000 μm2 per field was calculated. Values ​​are expressed as mean ± standard deviation. *P < 0.01 vs. miR-Ctrl (Student's t test). [Diagram 5] The relationship between decreased OC number and poor prognosis in OS patients. (A) Biopsy specimens were collected from chemotherapy-naive OS patients at the time of initial diagnosis and subjected to IHC staining for cathepsin K. Images were taken at 20x magnification. Scale bar indicates 100 μm. OC(+) group indicates “≥5 OC / field” and OC(-) group indicates “<5 OC / field”. Patients with distant metastasis at the time of initial diagnosis are shown as black areas. (B-E) Metastasis-free survival (B), local recurrence-free survival (C), progression-free survival (D) and overall survival (E) were followed up in the OC(+) group or OC(-) group. The proportions versus time were plotted as Kaplan-Meier curves. P values ​​were calculated between the OC(+) group and the OC(-) group (log-rank test). [Figure 6] In patients with high-grade OS, there are many highly inhibitory SEVs that inhibit osteoclastogenesis and promote angiogenesis and metastasis, whereas in patients with low-grade OS, there are few less inhibitory SEVs that do little to inhibit osteoclastogenesis, resulting in a lower incidence of metastasis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] (Subject of the present invention) The present invention is directed to the following: (1) A method for prognostic diagnosis or an auxiliary method for prognostic diagnosis of bone tumors, comprising a step of measuring the number of osteoclasts of a specific size in a biological sample. (2) A method for prognostic diagnosis or an auxiliary method for prognostic diagnosis of bone tumors, comprising a step of measuring the number of osteoclasts containing a specific number or more of nuclei in a biological sample. (3) A method for prognostic diagnosis or an auxiliary method for prognostic diagnosis of bone tumors, comprising a step of measuring the amount of Trap gene in a biological sample. (4) A method for diagnosing or assisting in the prognosis of bone tumors, comprising a step of measuring the expression levels of 16 specific types of miRNA in a biological sample. (5) A method for screening for a compound that suppresses bone tumor growth, angiogenesis, distant metastasis and / or metastatic lesions, comprising detecting a substance that reduces the number and / or inhibits the activity of OS-SEV. (6) A method for screening for a compound that promotes osteoclast formation in bone tumors, comprising detecting a substance that reduces the number and / or inhibits the activity of OS-SEV. (7) A method for screening for a compound that suppresses bone tumor growth, angiogenesis, distant metastasis, and / or metastatic lesions, comprising detecting a substance that increases the number and / or promotes the activity of Traf6. (8) A method for screening for a compound that promotes osteoclast formation in bone tumors, comprising detecting a substance that increases the number and / or promotes the activity of Traf6. (9) A method for screening for a compound that promotes bone tumor growth, angiogenesis, distant metastasis, and / or metastatic lesions, comprising detecting a substance that reduces the number and / or inhibits the activity of Traf6.

[0012] In this specification, the term "gene" is intended to include not only RNA and double-stranded DNA, but also each of the single-stranded DNAs constituting them, such as the positive strand (or sense strand) or complementary strand (or antisense strand), and is not particularly limited by its length. In this specification, unless otherwise specified, the term "microRNA (miRNA)" refers to a 15-25 base RNA that is transcribed as an RNA precursor with a hairpin-like structure, cleaved by a dsRNA cleaving enzyme having RNaseIII cleavage activity, incorporated into a protein complex called RISC, and is involved in the translational inhibition of mRNA. The term "miRNA" refers not only to "miRNA" represented by a specific base sequence (or sequence number), but also to "miRNA" that contains a precursor of the "miRNA" (pre-miRNA, pri-miRNA) and has a biological function equivalent to that of the miRNA encoded by these, such as a homolog (i.e., a homolog), a mutant such as a gene polymorphism, and a derivative. Examples of "miRNA" that encodes such a precursor, homolog, mutant, or derivative include "miRNA" that can be identified by miRBase (http: / / www.mirbase.org / ) and has a base sequence that hybridizes with a complementary sequence of a specific base sequence under stringent conditions.

[0013] (Bone Tumor) The bone tumor of the present invention includes malignant bone tumors, including osteosarcoma and metastatic bone tumors, and benign bone tumors, but is not limited to these, as other tumors also exist.

[0014] (Prognostic diagnosis method (auxiliary method)) "Prognosis" in the present invention means metastasis rate, recurrence rate, metastasis-free survival rate, local recurrence-free survival rate, progression-free survival rate and / or overall survival rate at about 24 months, about 36 months, about 60 months, about 96 months, about 120 months, about 180 months, about 240 months, or about 360 months after the initial bone tumor diagnosis or after surgical operation (curative resection). In other words, the prognostic diagnosis method (auxiliary method) of the present invention is a method for diagnosing (a method for assisting diagnosis) the probability of metastasis, recurrence, metastasis-free survival, local recurrence-free survival, progression-free survival and / or overall survival at the time of initial bone tumor diagnosis or within a specified period after surgical operation (curative resection). In the present invention, "poor prognosis" refers to a significantly worse prognosis in terms of metastasis, recurrence, metastasis-free survival, local recurrence-free survival, progression-free survival and / or overall survival at the time of initial bone tumor diagnosis or within a specified period after surgical operation (curative resection) compared with bone tumor patients exhibiting the cutoff values ​​shown below (see FIG. 5).

[0015] (Biological samples) The "biological sample" of the present invention is not particularly limited as long as it is a biological sample containing osteoclasts, the Trap gene, and / or 16 specific types of miRNA. Such biological samples generally include tissues (including tumor tissues) collected from living organisms (bone tumor patients and individuals suspected of having bone tumors), as well as blood, urine, tears, serum, feces, ejaculated semen, sputum, saliva, cerebrospinal fluid, and the like. Examples of tissues collected from living organisms include tissue resections of tissues (tumors) obtained by surgery or biopsy, and tissue biopsy materials obtained by endoscopic examination before surgery. In particular, the biological sample containing the osteoclast count and the Trap gene is preferably a bone tumor tissue, and the biological sample containing the 16 specific miRNAs is preferably a body fluid such as blood, urine, or tears.

[0016] (Prognostic diagnosis method including a step of measuring the number of osteoclasts of a specific size in a biological sample) The "diagnostic method comprising a step of measuring the number of osteoclasts of a specific size in a biological sample" of the present invention means comparing the number of osteoclasts in a biological sample (particularly, bone tumor lesion tissue) with the number of osteoclasts in a biological sample obtained from a healthy subject or a cutoff value, and determining or assisting in the determination of poor prognosis if the number is lower. More specifically, a biological sample (particularly, a bone tumor lesion tissue) of 0.50 mm 2 ~1.50 mm 2 per 1000μm 2 The number of osteoclasts was 0.50 mm 2 ~1.50 mm 2 per 1000μm 2 When the osteoclast count is lower than the above or the below cutoff value, a poor prognosis is determined or a determination is assisted. The cutoff value used in the step of measuring the number of osteoclasts of a specific size was 0.50 mm based on the results of Example 6. 2 ~1.50 mm 2 per 1000μm 2 For example, when the number of osteoclasts is 4 or less, 3 or less, 2 or less, 1 or less, or 0, the prognosis is judged to be poor. On the other hand, when the number of osteoclasts is 5 or more, 6 or more, 7 or more, 8 or less, 9 or more, or 10 or more, the prognosis is judged to be good.

[0017] (Prognostic diagnosis method including a step of measuring the number of osteoclasts containing a specific number or more of nuclei in a biological sample) The "diagnostic method comprising a step of measuring the number of osteoclasts containing a specific number or more of nuclei in a biological sample" of the present invention means comparing the number of osteoclasts containing a specific number or more of nuclei in a biological sample (particularly, bone tumor lesion tissue) with the number of nuclei of osteoclasts containing a specific number or more of nuclei in a biological sample obtained from a healthy subject or a cutoff value, and determining or assisting in the determination of poor prognosis if the number is lower.

[0018] (Osteoclast staining) In the step of measuring the number of osteoclasts in the present invention, it is preferable to stain osteoclasts (particularly, immunostain) by a method known per se, if necessary, in order to facilitate the measurement of the number of osteoclasts. Examples of the staining method include, but are not limited to, the use of a known antibody that stains osteoclasts (e.g., an antibody against cathepsin K, an antibody against TRAP), etc.

[0019] (Prognostic diagnosis method including a step of measuring the amount of Trap gene in a biological sample) The "prognosis diagnosis method comprising a step of measuring the amount of the Trap gene in a biological sample" of the present invention determines or assists in the determination of poor prognosis when the amount of the Trap gene in a biological sample (particularly bone tumor lesion tissue) is lower than the amount of the Trap gene in a biological sample obtained from a healthy subject or the following cutoff value. When setting the cutoff value based on the results of Example 3, if healthy individuals are set to 100, bone tumor patients with a good prognosis can be set to 75-100, and bone tumor patients with a poor prognosis (including osteosarcoma) can be set to 0-50. As an alternative method for setting the cut-off value, the cut-off value is calculated from the average expression level of the Trap gene in biological samples from individuals with no history of bone tumors (healthy subjects) or the average expression level of the Trap gene in biological samples from bone tumor patients with good and poor prognosis. In general, a patient can be judged to have a poor prognosis if the cut-off value is 90% or more, preferably 80% or more, more preferably 70% or more, even more preferably 60% or more, and most preferably 50% or more of the standard deviation of the cut-off value for healthy subjects. Another method for setting the cut-off value is to create a receiver operating characteristic (ROC) curve using commercially available statistical analysis software based on the values ​​of biological samples from subjects with no history of bone tumors or from bone tumor patients with good and poor prognosis, and determine the optimal sensitivity and specificity. For example, it is possible to set a cut-off value that prioritizes higher sensitivity for purposes such as primary screening, and higher specificity for detailed examination.

[0020] (Prognostic diagnosis method including a step of measuring the expression levels of 16 specific miRNAs in a biological sample) The "prognosis diagnosis method comprising a step of measuring the expression levels of 16 specific miRNAs in a biological sample" of the present invention refers to a method for determining or assisting in the determination of a poor prognosis when the expression levels of 16 specific miRNAs in a biological sample (particularly bodily fluids such as blood, urine, tears, etc.) are higher than the expression levels of 16 specific miRNAs in a biological sample obtained from a healthy subject or the following cutoff values. The 16 specific miRNAs (see Table 1) are hsa-miR-146a-5p (SEQ ID NO: 1), hsa-miR-1260 (SEQ ID NO: 2), hsa-miR-487b-3p (SEQ ID NO: 3), hsa-miR-6720-3p (SEQ ID NO: 4), hsa-miR-1260b (SEQ ID NO: 5), hsa-miR-4758-3p (SEQ ID NO: 6), hsa-miR-4690-3p (SEQ ID NO: 7), hsa-miR-4286 (SEQ ID NO: 8), hsa-miR-6765-3p (SEQ ID NO: 9), hsa-miR-1261 (SEQ ID NO: 10), hsa-miR-7975 (SEQ ID NO: 11), hsa-miR-4664-5p (SEQ ID NO: 12), hsa-miR-3907 (SEQ ID NO: 13), hsa-miR-7977 (SEQ ID NO: 14), hsa-miR-1273c (SEQ ID NO: 15) and hsa-miR-375-5p (SEQ ID NO: 16). When setting the cutoff value based on the results of Example 5, if healthy individuals are set to 100, bone tumor patients with a good prognosis can be set to 100-150, and bone tumor patients with a poor prognosis (including malignant bone tumors such as osteosarcoma) can be set to 150 or more. As an alternative method for setting the cutoff value, the cutoff value is calculated from the average expression levels of 16 specific miRNAs in biological samples from individuals (healthy subjects) with no history of bone tumors, or the average expression levels of 16 specific miRNAs in biological samples from bone tumor patients with good and poor prognosis. In general, a patient can be judged to have a poor prognosis if the cutoff value is 90% or more, preferably 80% or more, more preferably 70% or more, even more preferably 60% or more, and most preferably 50% or more of the standard deviation of a predetermined cutoff value for healthy subjects. Another method for setting the cut-off value is to create a receiver operating characteristic (ROC) curve using commercially available statistical analysis software based on the values ​​of biological samples from subjects with no history of bone tumors or from bone tumor patients with good and poor prognosis, and determine the optimal sensitivity and specificity. For example, it is possible to set a cut-off value that prioritizes higher sensitivity for purposes such as primary screening, and higher specificity for detailed examination.

[0021] (Method for measuring miRNA expression level) The expression level of miRNA can be measured by any method known to those skilled in the art. For example, the expression level of each miRNA can be measured using a primer or probe that specifically hybridizes to each miRNA. Such primers or probes can be appropriately designed by those skilled in the art with reference to information in databases, etc. The Northern blot method is a classic method using such primers or probes. Other methods that have been reported include miRNA microarray (Liu et al., 2004; Lim et al., 2005), improved invader method (Allawi et al., 2004), and bead-based flow cytometer method (Luetal, 2005). Real-time PCR is also a quantitative method (Chen et al., 2005).

[0022] (Screening Method) The screening method of the present invention is directed to: (1) A method for screening for a compound that suppresses bone tumor growth, angiogenesis, distant metastasis and / or metastatic lesions, comprising detecting a substance that reduces the number and / or inhibits the activity of OS-SEV. (2) A method for screening for a compound that promotes osteoclast formation in bone tumors, comprising detecting a substance that reduces the number and / or inhibits the activity of OS-SEV. (3) A method for screening for a compound that suppresses bone tumor growth, angiogenesis, distant metastasis and / or metastatic lesions, comprising detecting a substance that increases the number and / or promotes the activity of Traf6. (4) A method for screening for a compound that promotes osteoclast formation in bone tumors, comprising detecting a substance that increases the number and / or promotes the activity of Traf6. (5) A method for screening for a compound that promotes bone tumor growth, angiogenesis, distant metastasis and / or metastatic lesions, comprising detecting a substance that reduces the number and / or inhibits the activity of Traf6.

[0023] "Detecting a substance that reduces the number and / or inhibits the activity of OS-SEV" may include, for example, any one or more of the following steps. (a-1) measuring the amount of reduction in the number of OS-SEVs and / or the amount of inhibition of the activity of OS-SEVs in a sample containing OS-SEVs in the presence of a test substance; and (a-2) A step of selecting a test substance having the effect of reducing the number and / or inhibiting the activity of OS-SEV based on the results of (a-1) above.

[0024] "Detecting a substance that increases the number and / or promotes the activity of Traf6" may include, for example, any one or more of the following steps. (a-1) measuring the amount of increase in the number and / or the amount of promotion of Traf6 activity in a sample containing Traf6 (gene, protein) in the presence of a test substance; and (a-2) A step of selecting a test substance having an effect of increasing the number and / or promoting the activity of Traf6 based on the results of (a-1) above.

[0025] "Detecting a substance that reduces the number and / or inhibits the activity of Traf6" may include, for example, any one or more of the following steps. (a-1) measuring the amount of reduction in the number and / or the amount of inhibition of the activity of Traf6 in a sample containing Traf6 (gene, protein) in the presence of a test substance; and (a-2) A step of selecting a test substance having an effect of decreasing the number and / or inhibiting the activity of Traf6 based on the results of (a-1) above.

[0026] (Test substance) Any substance can be used as a test substance that is a candidate inhibitor (therapeutic agent) used in the above screening. The type of test substance is not particularly limited, and may be an individual low molecular weight synthetic compound (e.g., siRNA), a compound present in a natural product extract, or a synthetic peptide. The test substance may be a chemical library, a genome editing library, a phage display library, or a combinatorial library. The construction of chemical libraries, phage display libraries, and combinatorial libraries is known to those skilled in the art, and commercially available chemical libraries can also be used.

[0027] (Agents that inhibit bone tumor growth, angiogenesis, distant metastasis and / or metastatic lesions) The agent of the present invention that suppresses bone tumor growth, angiogenesis, distant metastasis and / or metastatic lesions includes a substance that reduces the number and / or inhibits the activity of OS-SEV obtained by the above screening. Furthermore, the agent of the present invention that suppresses bone tumor growth, angiogenesis, distant metastasis and / or metastatic lesions includes a substance that increases the number and / or promotes the activity of Traf6 obtained by the above screening.

[0028] (Promoting osteoclast formation in bone tumors) The agent for promoting osteoclast formation in bone tumors of the present invention includes a substance that reduces the number and / or inhibits the activity of OS-SEV obtained by the above screening. The agent for promoting osteoclast formation in bone tumors of the present invention includes the substance obtained by the above screening that increases the number and / or promotes the activity of Traf6.

[0029] (Administration method, dosage form) The route of administration of the agent of the present invention is not particularly limited, but preferred routes of administration include intravenous, oral, transdermal, and transmucosal (oral, rectal, vaginal, etc.). Examples of preparations for oral administration include tablets, capsules, granules, powders, syrups (dry syrups), oral jellies, etc. Examples of preparations for transdermal or transmucosal administration include patches, ointments, etc. Tablets, capsules, granules, powders, etc. can be made into enteric preparations. For example, tablets, granules, and powders are provided with an enteric coating. As the enteric coating agent, a poorly soluble enteric coating agent in the stomach can be used. In addition to the active ingredient, the inhibitor of the present invention can contain a pharmacologically acceptable carrier depending on the administration form. Examples of the pharmacologically acceptable carrier include excipients, disintegrants or disintegration assistants, binders, lubricants, coating agents, dyes, diluents, bases, solubilizers or solubilization assistants, isotonicity agents, pH regulators, stabilizers, propellants, and adhesives.

[0030] The dosage and frequency of administration of the inhibitor of the present invention may vary as appropriate depending on the conditions of the subject, its age, body weight, sex, purpose (prevention or treatment, etc.), severity of symptoms, dosage form, administration route, etc. When administered to humans, the dosage of the active ingredient in a specific protein is, for example, about 0.0001 mg / kg body weight to 10 mg / kg body weight per day. The frequency of administration may be once or multiple times per day, or once every few days. For example, it may be 1 to 3 times, 1 to 2 times, or once per day. The inhibitor of the present invention can be made into medicines, quasi-drugs, medical devices, sanitary products, foods, beverages, and supplements.

[0031] (Prognostic diagnostic kit for bone tumor patients.) The prognostic diagnostic kit for bone tumor patients of the present invention comprises at least one of the following: (1) Antibody that stains osteoclasts (2) Antibody against cathepsin K (3) Antibody against TRAP EXAMPLES

[0032] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. This example was approved by the Kanazawa University Laboratory Animal Research Facility and the Kanazawa University Ethics Committee. EXAMPLES

[0033] Materials and Methods The following materials and methods were used to carry out Examples 2-7.

[0034] (plasmid) pX330Puroplasmid was constructed by introducing a puromycin resistance gene cassette into the NotI site of pX330-U6-Chimeric BB-CBh-hSpCas9plasmid (Addgene, Watertown, MA,USA). To knock out the mouse Tsg101 gene, pX330Puro-TSG101 was constructed targeting the CCTACTAGTTCAATGACTA sequence (see Colombo M, Moita C, van Niel G, Kowal J, Vigneron J, Benaroch P, et al. Analysis of ESCRT Functions in Exosome Biogenesis, Composition and Secretion Highlights the Heterogeneity of Extracellular Vesicles. JCell Sci (2013) 126:5553-65. doi: 10.1242 / jcs.128868).

[0035] (cell) The highly invasive murine osmotic cell line LM8 was obtained from RIKEN BRC (Ibaraki, Japan). NIH / 3T3 cells, HOS cells (non-aggressive, wild-type k-Ras, human OS), and 143B cells (highly invasive; k-Ras activated, human OS) were obtained from ATCC (Manassas, VA, USA). These cells were cultured in Advanced DMEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 2% heat-inactivated FBS. LM8-WT cells or LM8 TSG101-KO cells were generated by the CRISPR-CAS9 system. pX330Puro or pX330Puro-TSG101 plasmid was transfected into LM8 cells by ViaFect (Promega, Madison, WI, USA). LM8TSG101-KO cells were monocloned to generate two monoclonal LM8TSG101-KO cell lines (K01 and K02).

[0036] (Orthotopic transplant mouse OS model) LM8-WT or LM8 TSG101-100 KO cells (1 x 10 5 The tumors were transplanted into the distal femur of 8-week-old male C3H / He mice (JapanSLC, Tokyo, Japan) using Matrigel. 3 Lungs and femurs were harvested at 4 weeks (sham group) or 4 weeks after transplantation. These tissues were embedded in paraffin and stained with H&E. For immunohistochemistry (IHC), deparaffinized sections were incubated with antibodies against cathepsin K (Abcam, Cambridge, United Kingdom), Ki67 (Abcam), CD31 (Abcam), or vascular endothelial growth factor (VEGF; Santa Cruz Biotechnology, Santa Cruz, CA), followed by HRP-conjugated secondary antibody and DAB-chromogen staining. The number of Ki67-positive cells or the area of ​​VEGF-positive cells were measured using a microscope BZ-X710 and a BZ-X analyzer (KEYENCE, Osaka, Japan).

[0037] (Separation of SEV) To isolate SEVs from LM8, NIH / 3T3, 143B or HOS cells, cells were cultured in Advanced DMEM containing 2% SEV-free FBS (see Curr Protoc Cell Biol (2017) 77:3.45.1-3.45.18.). Conditioned media were harvested at approximately 80% confluency and centrifuged sequentially at 300 × g for 5 min, 2,000 × g for 20 min and 10,000 × g for 30 min. After ultracentrifugation at 100,000 × g for 2 h using an S50ST rotor (Hitachi, Tokyo, Japan), SEVs were collected from the pellet, washed with PBS(-) by repeated ultracentrifugation and finally reconstituted in PBS(-). Size distribution and quantity of SEVs were analyzed using the NanoSIGHT TMThe PKH-labeled LM8-SEVs were determined by nanoparticle tracking analysis using LM10 (Malvern Panalytical, Malvern, United Kingdom). Characterization of isolated SEVs was performed according to the MISEV2018 guidelines (see: Journal of Extracellular Vesicles (2018) 7:1535750.doi:10.1080 / 20013078.2018.1535750). To prepare PKH-labeled LM8-SEVs, isolated SEV pellets were stained with 1 μL of PKH26 (Merck, Darmstadt, Germany) for 3 min, after which the staining reaction was stopped by adding 1% BSA / PBS(-). Excess staining reagent was removed by ultracentrifugation for 2 h, and the pellet of PKH-labeled SEVs was reconstituted in PBS(-).

[0038] (Osteoclast formation) Bone marrow was harvested from the femur and tibia of 8-week-old male C3H / He mice (Japan SLC), and the bone marrow was cultured at 5 × 10 in MEMα (FUJIFILM Wako, Osaka, Japan) supplemented with 10% FBS and 100 ng / mL macrophage colony-stimulating factor (M-CSF; BioLegend, San Diego, CA, USA). 6 The cells were cultured for 3 days at a density of 10 cells / 100 mm dish. To induce osteoclast precursor cells (OPCs), cells were cultured at a density of 5 × 10 5 For osteoclast formation, OPCs were cultured at 1 × 10 cells / 100-mm dish in MEMα containing 10% FBS, 20 ng / mL M-CSF, and 50 ng / mL receptor activator of NF-kappaB ligand (RANKL; Biolegend). 5 To determine the effect of SEV, 1 × 10 cells were cultured at the onset of osteoclastogenesis. 10SEV particles / mL or the indicated doses were added to the medium. To examine the effects of miRNAs, 60 pmol of miRNA mimic transfection control withDy547 (miR-Ctrl) or hsa-miR-135 146a-5p mimic (Horizo ​​Discovery, Cambridgeshire, United Kingdom) were transfected into OPCs using Lipofectamine RNAiMAX (Thermo Fisher Scientific) at the onset of osteoclast formation. Cell proliferation was assessed using WST-8 assay reagent (Nacalai Tesque, Kyoto, Japan). TRAP staining was performed 5 days after osteoclast formation using the Acid Phosphatase Leukocyte Kit (Merck) and evaluated using a microscope BZ-X710 and BZ-X analyzer.

[0039] (Real-time PCR) To detect mRNA, total RNA extraction was performed using the High Pure RNA Isolation Kit (Roche, Basel, Switzerland). RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan), and the cDNA was reacted with Fast SYBR Green Master Mix (Thermo Fisher Scientific) on the LightCycler96 system (Roche). The mRNA expression of each gene was normalized to Gapdh. To detect miRNA, total miRNA extraction was performed using the FastGene RNA Basic Kit and FastGenemiRNA Enhancer (NIPPON Genetics, Tokyo, Japan). miRNA was reverse transcribed to cDNA and detected using Mir-X miRNA First-Strand Synthesis (TakaraBio, Shiga, Japan) on the LightCycler96 system. The expression of hsa-miR-146a-5p was normalized to the expression of U6.

[0040] (Western blot analysis) For NF-κB pathway analysis, OPCs were cultured at 1 × 10 5 Cells were seeded at 1 x 10 / 12-well plate in 10% FBS, 20 ng / mL M-CSF, and 1 x 10 10 The cells were precultured for 12 hours in MEMα containing 1000 particles / mL SEVs. The medium was replaced with MEMα containing 10% FBS, 20 ng / mL M-CSF, and 50 ng / mL RANKL. After RANKL stimulation, the cells were washed with PBS(-) and lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors. For analysis of TRAF6 expression, 1 × 10 5 1 x 10 OPC cells 10 The cells were treated with MEMα containing 10% FBS and 20 ng / mL L-M-CSF containing 60 pmol of SEV or 60 pmol of miRNA mimic. After 48 h of incubation, the cells were washed with PBS(-) and lysed in RIPA buffer containing protease inhibitors. The lysates were used for Western blot analysis. Proteins were detected using SuperSignal West PicoChemiluminescent Substrate (Thermo Fisher Scientific) and ImageQuantLAS4000 Mini (GE Healthcare, Chicago, IL, USA). Band intensity was determined by ImageJ software (NIH, Bethesda, MD, USA). The intensity of each band was normalized to GAPDH.

[0041] (SEV-ELISA) Cells were cultured at 5 × 10 in Advanced DMEM containing 2% SEV-depleted FBS. 4Cells were seeded in 24-well plates. After 24 hours of culture, the culture supernatants were collected and centrifuged sequentially at 300 × g for 5 min, 2,000 × g for 20 min, and 10,000 × g for 30 min. The obtained supernatants were subjected to ELISA according to the same method as described in the literature "Curr Protoc Cell Biol (2017) 77:3.45.1-3.45.18." SEVs from human cell lines were detected using antibodies against human CD9, CD63, or CD81 and HRP-conjugated anti-mouse IgG (JacksonImmunoResearch, West Grove, PA, USA).

[0042] (Microarray analysis of miRNAs in SEVs) SEVs were isolated from the conditioned medium of HOS or 143B cells using the MagCapture Exosome Isolation Kit (FUJIFILM Wako). miRNAs contained in SEVs were detected using Human miRNA Oligo chips (Toray Industries Inc., Tokyo, Japan). The value of each miRNA was normalized by correcting the median signal intensity to 25 to obtain a global normalized value. The ratio of each miRNA between 143B-SEVs and HOS-SEVs was calculated by dividing the global normalized value of 143B-SEVs by the global normalized value of HOS-SEVs.

[0043] (Clinical samples) Biopsy specimens from all chemotherapy-naive patients with primary osteosarcoma were collected from the pathology database between 1999 and 2018. Patients with low-grade osteosarcoma, dedifferentiated osteosarcoma, multicentric osteosarcoma, and secondary osteosarcoma were excluded. Patients who underwent biopsy or any treatment at other hospitals and patients with less than 3 months of follow-up after diagnosis were also excluded. Distant metastases were determined in all patients at the time of initial diagnosis by multimodal radiological examination, including computed tomography, bone scintigraphy, and PET. The presence of OC was determined by IHC in biopsy specimens surrounding osteoid produced by the tumor using an antibody against cathepsin K (Santa Cruz Biotechnology). OC was defined as a tumor with an area of ​​1,000 μm2 or less. 2 OC was defined as more than 1000 cathepsin K-positive cells. Based on the analysis of three IHC images, patients were classified into OC(+) group (≥5 OC / field in at least two images) and OC(-) group (<5 OC / field in at least two images).

[0044] (ethical approval) This retrospective study of patient samples was approved by the Ethics Committee of Kanazawa University Hospital (clinical trial numbers 2310 and 3249) in accordance with the guidelines of the revised 2013 Declaration of Helsinki.

[0045] (statistical analysis) To analyze the oncological outcomes of patients, Kaplan-Meir curves were created and the log-rank test was used to compare the differences between groups. Patient metastasis-free survival, local recurrence-free survival, progression-free survival, and overall survival were defined as the time from definitive diagnosis to distant metastasis or death, from definitive diagnosis to recurrence or death, from definitive diagnosis to distant metastasis, recurrence, disease progression, or death, and from definitive diagnosis to death, respectively. Chi-square test was used for statistical analysis of categorical data. For numerical data, Student's t-test or Mann-Whitney U test (two-tailed) was used as appropriate based on the Shapiro-Wilk test of distribution. EXAMPLES

[0046] We confirmed that OS cells with impaired SEV secretion have low malignant potential in an orthotopic transplantation model. Details are as follows.

[0047] To analyze the effect of SEV in OS, we generated mouse OSLM8 cells with impaired SEV secretion by knocking out the Tsg101 gene. Two independent clones of LM8 TSG101-KO cells (KO1 and KO2) showed approximately 40% reduction in SEV, but cell proliferation in vitro was not affected. To determine the function of OS-SEV in OS progression, LM8 TSG101-KO cells were implanted into the distal femur of mice. Unlike the in vitro results, the proliferation of LM8 TSG101-KO cells was slower than that of LM8WT cells in vivo. To eliminate potential bias, all examinations were performed when the tumors had grown to the same size. Ki67 staining of the primary tumor showed a 75% decrease in expression in LM8 TSG101-KO cells (Figure 1A, B). Considering that there was no difference in cell proliferation in vitro, LM8-SEV may create a favorable microenvironment for tumor growth by influencing surrounding cells. Furthermore, angiogenesis was dramatically reduced by LM8-SEV inhibition, as shown by a 75% reduction in CD31-positive blood vessels (Fig. 1C,D) and a ∼75% reduction in VEGF-positive areas (Fig. 1E,F) in mice bearing TSG101-KO cells, contributing to slowed tumor growth. Regarding the assessment of the effect of OS-SEV on distant metastasis based on lung specimens, when the primary foci of each tumor grew to the same size, mice bearing TSG101-KO cells showed a 15-fold reduction in the number of metastatic foci and a 6-fold reduction in the area of ​​metastatic lesions compared to mice bearing WT cells (Figures 1G, I).

[0048] The above results indicated that OS-SEV altered the tumor microenvironment to promote tumor growth, angiogenesis, and distant metastasis. Therefore, substances that reduce the number and / or inhibit the activity of OS-SEV can suppress bone tumor growth, angiogenesis, distant metastasis, and metastatic lesions. EXAMPLES

[0049] We confirmed that OS-SEV inhibits osteoclast formation. Details are as follows.

[0050] OC is known to be one of the important cells that constitute the tumor microenvironment of OS. Immunohistochemical staining for cathepsin K showed that OC in mice bearing LM8-WT cells was dramatically reduced compared to sham mice, whereas impaired secretion of OS-SEV by TSG101-KO suppressed OC reduction (Fig. 2A, B). In vitro analysis of PKH-labeled SEV uptake into OPCs confirmed that LM8-SEV was dose-dependently taken up by OPCs (Fig. 2C). WST assay showed that LM8-SEV did not show cytotoxicity to OPCs at any dose (Fig. 2D). LM8-SEV treatment suppressed the formation of large OCs (>10,000 μm) containing more than 10 nuclei. 2 ) and small OCs (1,000–10,000 μm) containing approximately 3–9 nuclei. 2 ) increased the number of sarcoplasmic reticulum (Figure 2E-G). Furthermore, LM8-SEV suppressed Trap mRNA expression by approximately 70% compared to mature OCs (Figure 2H). These suppressive effects were not observed in the control NIH / 3T3-SEV-treated group, suggesting that the suppressive effects were due to molecules specifically contained in LM8-SEV.

[0051] The above results indicate that OS-SEV potently inhibits osteoclast formation by increasing the number of small immature OCs and decreasing the number of mature OCs. Therefore, substances that reduce the number and / or inhibit the activity of OS-SEV can promote osteoclast formation in bone tumors, and further suppress bone tumor growth, angiogenesis, distant metastasis, and metastatic lesions. EXAMPLES

[0052] We confirmed that OS-SEVs inhibit osteoclast formation by suppressing the NF-κB pathway.

[0053] To determine the mechanism by which OS-SEV suppressed osteoclastogenesis, we analyzed two important transcription factors (c-fos and Nfatc1) and three important molecules (Dcstamp, Atp6v0d2, and Ocstamp) involved in bone cell formation. Transfer of LM8-SEV into OPCs did not affect the expression of c-fos mRNA, but strongly suppressed the expression of Nfatc1 mRNA (Figure 3A). Real-time PCR analysis showed a decrease in the mRNA expression levels of these markers (Figure 3A). Flow cytometer analysis showed that RANKL stimulation induced the disappearance of DC-STAMP from the cell surface, whereas LM8-SEV suppressed this (Figure 3B). These results indicated that OS-SEV suppressed the expression of key genes involved in osteoclastogenesis and disrupted the ER transduction of DC-STAMP. At 1 to 6 h after RANKL stimulation, phosphorylation of IκBα or NF-κB in OPCs treated with LM8-SEV was lower than that in untreated or NIH / 3T3-SEV-treated OPCs ( Fig. 3C ). These results suggest that OS-SEV inhibits osteoclast formation by affecting proteins upstream of IκBα in the NF-κB pathway. EXAMPLES

[0054] We confirmed that the miRNA contained in OS-SEV inhibits osteoclast formation. The details are as follows.

[0055] We evaluated whether the number of secreted SEVs or the effect of SEVs correlated with tumor grade in human OS. Comparing two human OS cell lines known to differ in malignancy, the highly malignant 143B cells secreted approximately 5-fold more SEVs than the less malignant HOS cells (Fig. 4A), and 143B-SEVs showed a stronger inhibitory effect on osteoclast formation than HOS-SEVs (Fig. 4B). These results were consistent with the results of studies in mice implanted with TSG101-KO cells. To clarify the mechanism by which 143B-SEV suppresses osteoclastogenesis, we comprehensively analyzed the miRNAs contained in HOS-SEV and 143B-SEV using microarray analysis. As a result, we identified 16 miRNAs that were more than 5-fold more abundant in 143B-SEV than in HOS-SEV (Table 1: Microarray analysis of miRNAs contained in 143B-SEV or HOS-SEV). Hsa-miR146a-5p is known to target the TRAF6 gene and is known to signal RANK activation to the NF-kB pathway. Real-time PCR analysis revealed that hsa-miR146a-5p was 22.8-fold more abundant in 143B-SEVs than in HOS-SEVs (Figure 4C). Treatment of 143B-SEVs reduced the expression of TRAF6 protein compared to that of HOS-SEVs in OPCs (Figure 4D). We next evaluated the effect of hsa-miR146a-5p on osteoclast formation. Transfection of hsa-miR146a-5p mimic reduced Traf6 mRNA expression by 37% compared with that of miR-Ctrl, leading to a decrease in TRAF6 protein (Figure 4E, F). Furthermore, TRAP staining analysis also showed that transfection of miR-146a-5p mimic dramatically inhibited osteoclast formation (Figure 4G, H).

[0056] [Table 1]

[0057] The above results suggest that the suppression of TRAF6 expression by miR-146a-5p may be one of the mechanisms by which SEVs reduce mature OC in aggressive OC. Taking into account the results of Example 4 above, substances that increase the number and / or promote the activity of Traf6, a protein upstream of IκBα in the NF-κB pathway, can promote osteoclast formation in bone tumors, and can also suppress the growth, angiogenesis, distant metastasis, and metastatic lesions of bone tumors. EXAMPLES

[0058] We confirmed whether a decrease in the number of mature OCs could predict poor prognosis in OS patients. Details are as follows.

[0059] We hypothesized that high-grade OS would secrete a large number of SEVs and reduce the number of mature OCs in patients. Therefore, to analyze the correlation between the malignancy of OS and the number of OCs, we investigated the number of OCs in patient samples (biological samples) that did not receive chemotherapy, and followed up the clinical outcomes based on 61 OS patients. Based on IHC for cathepsin K, we classified 1) patients with less than 5 mature OCs per field into OC(-) patients (n = 33), and 2) patients with 5 or more mature OCs per field into OC(+) patients (n = 28) (Figure 5A). All patient characteristics were not significantly different between the two groups (Table 2: Patient characteristics). According to the results of multimodal radiological examination, 24% of OC(-) patients and 11% of OC(+) patients had distant metastasis, and there was no significant difference in the metastasis rate between the two groups at the time of initial diagnosis (Figure 5A). Follow-up studies confirmed that OC(+) patients had significantly increased metastasis-free survival, local recurrence-free survival, progression-free survival, and overall survival rates compared with OC(-) patients (Figure 5B-E). Similarly, metastasis and recurrence occurred at significantly higher rates in OC(-) patients compared with OC(+) patients (73% vs. 29%, and 36% vs. 14%, respectively). Mortality from the disease was higher in OC(-) patients (30%) compared with OC(+) patients (4%) (Table 2). These results indicated that the number of mature OCs surrounding OS lesions correlated with OS malignancy. Furthermore, its accuracy for predicting metastasis was evaluated (Table 3: Cross-classification of patients with OS by IHC for cathepsin K and metastasis). The evaluation results confirmed that the measurement of the reduction in mature OC based on cathepsin K IHC is an indicator of metastasis associated with OS, with a sensitivity of 75% and a specificity of 69% (Table 4: Diagnostic accuracy measurements). Furthermore, the diagnostic accuracy of this measurement is 72%, so it can be used to diagnose patients at high risk of metastasis.

[0060] [Table 2]

[0061] [Table 3]

[0062] [Table 4]

[0063] (Conclusion) In conclusion, the above results and the results of Examples 1-6 show that highly inhibitory (active) SEV secreted from highly malignant OS suppresses osteoclast formation and promotes distant metastasis (FIG. 6). In addition, the number of mature OCs around OS lesions can be used to prognose the metastasis, recurrence, metastasis-free survival, local recurrence-free survival, progression-free survival and overall survival of cancer patients. The 16 miRNAs, including hsa-miR146a-5p, listed in Table 1 can be used to prognose patients' metastasis, recurrence, metastasis-free survival, local recurrence-free survival, progression-free survival and overall survival, as well as the number of mature OCs around the OS lesions. Trap can be used to prognose patients' metastasis, recurrence, metastasis-free survival, local recurrence-free survival, progression-free survival and overall survival, as well as the number of mature OCs surrounding OS lesions.

[0064] (General remarks) The results of this example confirmed the following. (1) Analyzing the phenotype of SEV-deleted OS cells, we demonstrated that OPCs are active receivers of OS-SEV in the microenvironment, leading to a decrease in the number of mature OCs and an increase in undifferentiated OPCs. (2) We identified OS-SEV as an OS-derived factor that suppresses osteoclast formation. (3) 143B cells secrete more OS-SEV than HOS cells, and 143B-SEV has a stronger ability to suppress osteoclast formation than HOS-SEV. (4) miR-146a-5p and other miRs contained in OS-SEV inhibit osteoclast formation by suppressing the expression of TRAF6 mRNA. (5) miR-146a-5p affects not only tumor cells and vascular endothelial cells, but also OPCs, which make up the tumor microenvironment. (6) A decrease in OC numbers correlates with accelerated metastasis and poor prognosis in OS patients. (7) The number of OCs in biopsy specimens collected at the time of initial diagnosis can accurately predict future metastasis and overall survival. (8) Because OCs in the tumor microenvironment are the first cells affected by OS-EVs, the reduction of OCs may occur earlier than angiogenesis and metastasis, and thus could be used for early diagnosis to predict metastasis and overall survival in OS patients. [Industrial Applicability]

[0065] A method for prognostic diagnosis or an auxiliary method for prognostic diagnosis of bone tumors is provided.

Claims

1. A method for measuring the number of osteoclasts in a biological sample collected from a bone tumor patient and providing the result of the measurement as an index for prognosis of the bone tumor patient, comprising: The method, wherein the prognosis in the index for prognosis diagnosis is any one of the following: (1) Metastasis (2) Recurrence (3) Metastasis-free survival rate (4) Local recurrence-free survival rate (5) Progression-free survival rate (6) Overall survival rate (7) Survival rate

2. The method according to claim 1, characterized in that the measurement compares the number of osteoclasts in the biological sample with the number of osteoclasts in a biological sample obtained from a healthy subject or a cutoff value obtained from a healthy subject, and if the number is lower, the result is provided as an indicator for determining poor prognosis.

3. The cutoff value obtained from the healthy subject was 0.50 mm 2 ~1.50 mm 2 per 1000μm 2 The method according to claim 2, characterized in that the number of osteoclasts is 4 or more.

4. A prognostic kit for bone tumor patients, comprising an antibody for staining osteoclasts; Here, the prognosis in the prognosis diagnosis of the kit is any one of the following: (1) Metastasis (2) Recurrence (3) Metastasis-free survival rate (4) Local recurrence-free survival rate (5) Progression-free survival rate (6) Overall survival rate (7) Survival rate

5. A method for measuring the expression level of any one or more of the following miRNAs in a biological sample collected from a bone tumor patient, and providing the result of the measurement as an index for prognosis diagnosis of the bone tumor patient: (1) hsa-miR-146a-5p (2) hsa-miR-1260 (3) hsa-miR-487b-3p (4) hsa-miR-6720-3p (5) hsa-miR-1260b (6) hsa-miR-4758-3p (7) hsa-miR-4690-3p (8) hsa-miR-4286 (9) hsa-miR-6765-3p (10) hsa-miR-1261 (11) hsa-miR-7975 (12) hsa-miR-4664-5p (13) hsa-miR-3907 (14) hsa-miR-7977 (15) hsa-miR-1273c (16) hsa-miR-375-5p

6. The method according to claim 5, characterized in that the measurement compares the expression level of miRNA in the biological sample with the expression level of miRNA in a biological sample obtained from a healthy subject or a cut-off value obtained from a healthy subject, and if the result is higher, provides the result as an indicator for determining poor prognosis.

7. The method according to claim 5 or 6, wherein the prognosis in the indicator for prognosis diagnosis is any one of the following: (1) Metastasis (2) Recurrence (3) Metastasis-free survival rate (4) Local recurrence-free survival rate (5) Progression-free survival rate (6) Overall survival rate (7) Survival rate

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