COMPOSITION FOR DIAGNOSING BONE METASTASIS OF CANCER AND METHOD FOR DIAGNOSING BONE METASTASIS OF CANCER USING SAME

Abstract

The present invention relates to a composition for diagnosing bone metastasis of cancer, a method for providing information needed for diagnosis of bone metastasis of cancer using same, a method for providing information needed for monitoring responses to treatment of bone metastasis of cancer using same, and a method for screening a therapeutic agent for bone metastasis of cancer using same. The composition for diagnosing bone metastasis of cancer of the present invention has the effect of effectively diagnosing bone metastasis of cancer at an early stage.

Claims

1. A composition for diagnosis of cancer bone metastasis, comprising: a CD45 detecting agent, a HLA-DR detecting agent, and a CD11 b detecting agent; and (a) a CD14 detecting agent and a CCR2 detecting agent, (b) a CD14 detecting agent, (c) a CD15 detecting agent and a CD33 detecting agent, or (d) a CD14 detecting agent, a CCR2 detecting agent, a CD15 detecting agent, and a CD33 detecting agent.

2. The composition of claim 1, wherein the detecting agents are each independently selected from the group consisting of an antibody, an aptamer, DNA, RNA, a protein, and a polypeptide.

3. The composition of claim 1, wherein the detecting agents are each independently labeled with at least one marker selected from the group consisting of a ligand, a bead, a radionuclide, an enzyme, a substrate, a cofactor, an inhibitor, a fluorescer, a fluorescent protein, a chemiluminescent substance, a magnetic particle, a hapten, and a dye.

4. The composition of claim 1, wherein the detecting agents contained in the composition each have a concentration of 5 μl/2×10.sup.5 cells or more.

5. The composition of claim 1, wherein the cancer is breast cancer, prostate cancer, liver cancer, lung cancer, bladder cancer, stomach cancer, uterine cancer, colorectal cancer, colon cancer, blood cancer, ovarian cancer, pancreatic cancer, spleen cancer, testis cancer, thymic carcinoma, brain cancer, esophageal cancer, kidney cancer, cholangiocarcinoma, ovarian cancer, thyroid cancer, or skin cancer.

6. A method for providing information necessary for diagnosis of cancer bone metastasis, the method comprising: a labeling step of contacting the composition for diagnosis of cancer bone metastasis of claim 1 with a sample; an acquiring step of acquiring a labeled mononuclear cell; and an analyzing step of analyzing the labeled mononuclear cell.

7. The method of claim 6, wherein the sample contains a mononuclear cell isolated from peripheral blood.

8. The method of claim 6, wherein the sample comprises 2×10.sup.5 or more mononuclear cells.

9. The method of claim 6, wherein the sample is obtained through: a first centrifugation step for centrifuging a blood sample taken from a patient; a mononuclear cell layer acquiring step; a mononuclear cell layer washing step; and a second centrifugation step.

10. The method of claim 6, wherein the analyzing step comprises the following steps of: obtaining only single cells; sorting the cells to give a CD45-positive cell population; reclassifying the cells to give a HLA-DR-negative cell population; reclassifying the cells to give a CD11b-positive cell population; and reclassifying the cells to give a CD14-positive and CCR2-positive cell population.

11. A method for providing information for monitoring a response to therapy for cancer bone metastasis, the method comprising: a labeling step of contacting a composition for diagnosis of cancer bone metastasis with a sample; an acquiring step of acquiring a labeled mononuclear cell; and an analyzing step of analyzing the labeled mononuclear cell.

12. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0196] FIG. 1 shows in vivo bioluminescence imaging (hereinafter referred to as “BLI”) results obtained in the procedure of constructing bone micrometastasis mice according to an embodiment of the present disclosure.

[0197] FIG. 2 shows images of normal mouse bone tissues in the procedure of constructing bone micrometastasis mice according to an embodiment of the present disclosure.

[0198] FIG. 3 shows images of bone tissues in the bone micrometastasis model during the construction of bone micrometastasis mice according to an embodiment of the present disclosure.

[0199] FIG. 4 is a plot of overall distribution patterns of mononuclear myeloid-derived suppressor cells in the blood of bone micrometastasis mouse models according to an embodiment of the present disclosure.

[0200] FIG. 5 is a plot of distribution patterns of mononuclear myeloid-derived suppressor cells in the blood of bone micrometastasis mouse models according to the present disclosure.

[0201] FIG. 6 is a plot of distribution patterns of CCR2+ mononuclear myeloid-derived suppressor cells in the blood of bone micrometastasis mouse models according to the present disclosure.

[0202] FIG. 7a is a flow cytometry profile of cancer patients according to an embodiment of the present disclosure.

[0203] FIG. 7b is a flow cytometry profile of cancer patients according to an embodiment of the present disclosure.

[0204] FIG. 7c is a flow cytometry profile of cancer patients according to an embodiment of the present disclosure.

[0205] FIG. 8 is a plot of clinical study results from cancer patient blood samples according to an embodiment of the present disclosure.

DETAILED DESCRIPTION

[0206] Hereinafter, the present disclosure will be described in more detail through examples. The following examples are for illustrative purposes only and it will be apparent to those of ordinary skill in the related art that the scope of this disclosure is not limited by the examples.

Example 1: Construction of Bone Micrometastasis Model

[0207] In order to secure ideal bone metastasis animal models overcoming limits of preexisting bone metastasis mouse models, bone metastasis mouse models were constructed. All experiments relevant to the construction of the mouse models were approved by the Institutional Animal Care and Use Committee in the Korea University College of Medicine and conducted under strict appliance. For constructing the animal models, Balb/c lineage mice were used. The mouse mammary gland cell line 4T1-tdTomato; Luc was subcultured to prepare a sufficient amount of cells to be injected into the mice.

[0208] The cancer cells were seeded into one femur or tibia of each mouse by injection through the iliac artery. In brief, after mice were generally anesthetized with 2-3% vaporized isoflurane in conjunction with oxygen, an incision was made into the medial femoral skin to expose the femoral artery. Blood vessels were dissected using an anatomy microscope. The cells were introduced into the blood vessels with the aid of a syringe. After hemostasis and suturing, the mice were laid on heating pad and allowed to sufficiently recover from the surgery. Every week after injection of the cancer cells, the treated femoral and tibial regions were monitored for the formation or growth of metastatic bone tumors by in-vivo bioluminescence imaging according to bioluminescence intensity (ph/s). The results are depicted in FIG. 1.

[0209] As can be seen in FIG. 1, the construction of bone micrometastasis mouse models was primarily finished.

[0210] Then, in order to examine whether substantial bone metastasis proceeded, the mice were euthanized and the femur and tibia were separated and histologically analyzed for bone metastasis. Bone metastasis regions were roughly determined in the separated bone tissues by X-ray imaging. The tissues were fixed for 3 days in a 4% paraformaldehyde solution and the muscles attached to the bones were peeled off, followed by decalcification for about two weeks in a 0.5 M EDTA solution. The decalcification was carried out by slowly shaking at 4° C., with change of 0.5 M EDTA with a fresh one every three days. Subsequently, the tissues were embedded into paraffin blocks, sectioned into slices, and mounted onto slides, followed by histological staining with hematoxylin and eosin (H&E). Bioluminescence imaging, X-ray imaging, and histological staining analysis results according to tumor growth are depicted in FIGS. 2 and 3.

[0211] As is understood from FIGS. 2 and 3, bone fracture and tumorigenesis were identified in the leg bones of the cancer bone micrometastasis models as measured by X ray and histological staining analysis whereas no tumorigenesis was detected in the bone tissues of normal mice. Therefore, successful construction of cancer bone micrometastasis models was confirmed, as in FIG. 1.

Example 2: Analysis for Myeloid-Derived Suppressor Cell According to Bone Micrometastasis Growth

[0212] For use in analyzing myeloid-derived suppressor cells according to bone metastasis growth by weeks after cancer cell transplantation, blood samples were taken from the bone micrometastasis mouse models constructed in Example 1. A total of 20, 10, and 10 mouse models were prepared for week 1, 2, and 3 after cancer cell transplantation, respectively.

[0213] Before blood sampling, the syringe to be used for blood sampling was coated with a heparin solution to prevent blood coagulation. After general anesthesia of the mice, about 1 ml of whole blood was taken from each of the mice via a heart puncture. Immediately after blood sampling, red blood cells were removed by adding the blood to an RBS lysis buffer to lyse red blood cells. The blood was allowed to stand at room temperature for 15 minutes in the RBC lysis buffer. After centrifugation at 441×g for 5 minutes, the supernatant was removed. These procedures were repeated until complete removal of red blood cells. The hemocytes thus clearly separated were stored in a FBS-supplemented buffer in a refrigerator.

[0214] For flow cytometry, 10.sup.6 hemocytes were transferred to each flow cytometer-specific tube, washed, and incubated for 5 minutes with 200 μl of a buffer containing CD16/CD32 antibody to conduct an Fc blocking process. Then, anti-viability dye 780 (1 μl/10.sup.6 cells), anti-CD45 (0.25 μl/10.sup.6 cells), anti-CD11b (0.25 μl/10.sup.6 cells), anti-Ly-6G (0.5 μl/10.sup.6 cells), anti-Ly-6C (0.5 μl/10.sup.6 cells), and anti-CCR2 (1 μl/10.sup.6 cells) antibodies were added to the tube and incubated at room temperature for 30 minutes for immunostaining.

[0215] Information on antibodies used:

[0216] Viability dye 780 APC-Cy7 (Biogems, #6910-00),

[0217] Anti-CD45 PE-Cy7 (Biolegned, #103114),

[0218] Anti-CD11 b FITC (BD, #553310),

[0219] Anti-Ly-6G Alexa 647 (BD, #12610),

[0220] Anti-Ly-6C Alexa 700 (BD, #562137),

[0221] Anti-C-C chemokine Receptor 2 (CCR2) APC (Biolegend, #150604).

[0222] After completion of the staining, the cells in each tube were washed with about 4 ml of a buffer and then centrifuged at 441×g for 5 minutes to obtain antibody-labeled mononuclear cells. The cells were analyzed by flow cytometry (FACS Canto ± and FACS DIVA software from Beckton-Dickinson).

[0223] As for analysis, only single cells were obtained first and sorted into cell populations in the order of the antibodies enumerated above. Briefly, among sorted single cell populations, selection was made of viable cells which were then classified in terms of CD45+. Subsequently, reclassification was made of the CD45+ cell population according to CD11b+, Ly-6G-, and Ly-6C+ in that order. Finally, the Ly-6G- and Ly-6C+ cell population was sorted again in terms of Ly-6C+CCR2+ and counted as % values. The results are depicted in FIGS. 4 to 6 and summarized in Tables 1 to 3.

TABLE-US-00001 TABLE 1 CD11b (% CD45) Week 1 Week 2 Week 3 78.19 84.05 94.65 78.05 82.03 94.32 77.36 80.88 94.08

TABLE-US-00002 TABLE 2 Ly-6G−/Ly-6C+ (% CD45) Week 1 Week 2 Week 3 0.44 0.52 0.51 0.37 0.49 0.49 0.35 0.44 0.48

TABLE-US-00003 TABLE 3 Ly-6C+CCR2+ on CD45 (%) Week 1 Week 2 Week 3 0.43 0.45 0.44 0.35 0.45 0.41 0.33 0.39 0.4

[0224] As is understood from the data of FIGS. 4 to 6 and Tables 1 to 3, the overall distribution pattern of the myeloid-derived suppressor cells gradually increased with weekly time after cell transplantation. Counts of the mononuclear myeloid-derived suppressor cells and the CCR2+ mononuclear myeloid-derived suppressor cells significantly increased weeks 2 and 3, compared to week 1, but with no significant difference between weeks 2 and 3. This result implies that counts of the mononuclear myeloid-derived suppressor cells already peaked by week 2.

Example 3. Attainment of Peripheral Blood Mononuclear Cell from Cancer Patient Blood Sample and Establishment of Flow Cytometry

[0225] To establish an analysis method for peripheral blood mononuclear cells in human patients, liquid biopsy was conducted to obtained blood samples from cancer patients and to isolate peripheral blood mononuclear cells. For patient blood samples, specimens were attained according to regulations under the approval of the Institutional Review Board (IRB) in the Korea University Anam Hospital Clinical Trial Center.

[0226] Briefly, 8 cc of a blood sample was taken from each patient to be diagnosed and transferred to a specific tube coated with heparin for preventing blood coagulation. Then, density-gradient centrifugation using Ficoll was conducted at 4° C. and 635×g for 20 minutes to split layers of plasma, peripheral blood mononuclear cells (hereinafter referred to as “PBMC”), Ficoll, and red blood cells (RBC) in that order. Among the split layers, only the second layer of PBMC was carefully isolated and transferred to a new tube. The PBMC was washed with PBS and then centrifuged at 783×g for 5 minutes to obtain washed PBMC only.

[0227] The mononuclear cells were subjected to antibody staining in order to establish flow cytometry for the mononuclear cells. For use in flow cytometry, the peripheral blood mononuclear cells were placed at a density of 2×10.sup.5 cell/tube in a FACS-specific tube.

[0228] Then, the cells were stained by incubation with anti-CD45 (0.5 μl/2×10.sup.5 cells), anti-CD14 (0.5 μl/2×10.sup.5 cells), anti-CD15 (5 μl/2×10.sup.5 cells), anti-HLA-DR (0.5 μl/2×10.sup.5 cells), anti-CD33 (1.25 μl/2×10.sup.5 cells), anti-CD11b (2.5 μl/2×10.sup.5 cells), anti-CCR2 (1 μl/2×10.sup.5 cells) antibodies at room temperature for 20 minutes.

[0229] Information on antibodies used:

[0230] Anti-CD45 PerPC-Cy5.5 (BD #564105),

[0231] Anti-HLA-DR Alexa700 (BD #560743),

[0232] Anti-CD11 b APC (BD #55019),

[0233] Anti-CD14 APC-Cy7 (BD #557831),

[0234] Anti-CD15 FITC (BD #555401),

[0235] Anti-CD33 PE-Cy7 (BD #333946),

[0236] Anti-CCR2 PE (Biolegend #357206).

[0237] Thereafter, the cells in each tube were washed with about 4 ml of a buffer and then centrifuged at 441×g for 5 minutes to obtain antibody-labeled mononuclear cells. The cells were analyzed by flow cytometry (FACS Canto ± or Fortessa X-20, and FACS DIVA software from Beckton-Dickinson) as in Example 2. For analysis, only single cells were gated first and sorted into cell populations. From corresponding cell populations, selection was made of a CD45+ cell population which was then reclassified on the basis of HLA-DR- and CD11+ only. Subsequently, the HLA-DR- and CD11+ populations were classified on the basis of CD14+ and CCR2+. The resulting cell populations were counted and CCR2+ mononuclear myeloid-derived suppressor cells were analyzed. The analysis results obtained in the cancer patient specimens are depicted in FIG. 7 and summarized in Table 4.

TABLE-US-00004 TABLE 4 Gating No. of cells Percentage Total 267,758 — Single cells 108,911 — CD45 50,581 46.4% (Single) CD11b, HLA-DR- 17,355 .sup. 34.3% (CD45)) CD14, CCR2 7,447 14.7% (CD45).sup. 

[0238] As can be seen in FIG. 7 and Table 4, a total of 267,758 cells were analyzed. Among them, 108,911 single cells were selected. Then, the single cells were sorted on the basis of CD45+(46.4% relative to the single cells), CD11b+HLA-DR-(34.3% relative to CD45+ cells), and CD14+CCR2+(14.7% relative to CD45+ cells) in that order. For the analytical criteria, a total of 696 breast cancer and prostate cancer patients were analyzed to construct cohorts.

Example 4. Clinical Study of Mononuclear Myeloid-Derived Suppressor Cells

[0239] On the basis of the flow cytometry established in Example 3, mononuclear myeloid-derived suppressor cells present in blood of cancer patients were analyzed, and evaluated for clinically diagnostic value. Counts (%) of individual populations including total counts of myeloid-derived suppressor cells (CD45+CD11b+ cells) and CCR2+ mononuclear myeloid-derived suppressor cells (CD14+CCR2+ cells) were compared.

[0240] Furthermore, the corresponding cancer patients were examined for metastasis and divided into groups: metastasized to bone, metastasized to other organs, and non-metastasis. Cells in each patient group were counted. Between bone metastasis and other metastasis patients and non-metastasis patients, CCR2+ mononuclear myeloid-derived suppressor cells were analyzed to significantly differ in count, demonstrating the clinical diagnostic value thereof. The results are depicted in FIG. 8 and summarized in Table 5.

TABLE-US-00005 TABLE 5 CCR2+ CD14+ M-MDSC Bone metastasis Non-metastasis patient patient 6.63% 4.51% 6.18% 4.04% 6.14% 3.26%

[0241] As can be seen in FIG. 8 and Table 5, CCR2+ mononuclear myeloid-derived suppressor cells were detected at explicitly higher % in the metastasis group, compared to the non-metastasis group and thus evaluated to have a clinically diagnostic value for bone metastasis lesions.