Cancer treatment method using analysis of circulating tumor cells in peripheral blood

Abstract

To provide a method for treating cancer that has high safety and improved therapeutic effect on cancer, and can prevent the metastasis and relapse of cancer, and a monitoring method that determines the therapeutic effect of PPAR-γ agonist in a cancer patient.SOLUTION: A method for treating cancer includes a first analysis step for analyzing circulating tumor cells (CTCs) in a mammal subject, a step for administering an effective amount of PPAR-γ agonist to a subject in need of treatment on the basis of the results of the analysis, and a second analysis step for analyzing circulating tumor cells (CTCs) in the subject who has undergone the administration of the PPAR-γ agonist.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to A cancer treatment method that is highly safe, enhances the therapeutic effect of cancer, and prevents metastasis and recurrence of cancer; and The present invention relates to a method of combination therapy with a PPAR-γ agonist for cancer patients determined to have peripheral blood circulating tumor cells. [Background technology]

[0002] It is known that in advanced cancers and metastatic cancers, cancer cells enter the bloodstream and circulate within the body. Cancer cells that invade blood vessels during this metastasis process are called CTCs (Circulating Tumor Cells), and CTC testing is a method for directly detecting cancer cells circulating in the bloodstream (Patent Documents 1 to 5).

[0003] CTC testing, which is an indicator of cancer metastasis and treatment effectiveness, is also called molecular pathology testing (liquid biopsy) because it can detect minute cancers that cannot be seen with imaging tests. It is known that if the CTC test is positive, it is highly likely that recurrence and metastasis can be predicted one to four years in advance.

[0004] In addition, cancer cells originally have the characteristics of epithelial cells, but in highly malignant cancers, they lose the morphology of epithelial cells and their cell adhesion function with surrounding cells, and are known to undergo a phenomenon called epithelial-mesenchymal transition (EMT), in which the cells lose the ability to migrate and invade other tissues. This phenomenon is thought to be deeply involved in the most important issues in cancer treatment, namely cancer metastasis and recurrence. Therefore, the detection of even one CTC is a sign of suspicion of cancer onset and the possibility of cancer metastasis (Non-Patent Documents 1 to 3).

[0005] It is known that when cancer undergoes EMT, it acquires cancer stem cell-like characteristics, and even the daughter cells of normal cancers are known to change to cancer stem cell-like properties when they undergo EMT. In addition, the treatment with general anticancer drugs is based on the goal of shrinking solid tumors, and targets only differentiated cancer cells that do not function as cancer stem cells, which make up the majority of tumors. It has also been pointed out that some cancer stem cells have acquired drug resistance. Therefore, even if most cancer cells are removed by anticancer drug treatment, if a small number of cancer stem cells survive, recurrence will occur.

[0006] In other words, if cancer stem cells can be removed, it will be possible to prevent cancer metastasis and recurrence (Non-Patent Documents 4-5). From the above, it can be easily inferred that even if mesenchymal cells that have undergone EMT and acquired cancer stem cell-like characteristics are detected by CTC testing, it will be difficult to treat them with general anticancer drugs.

[0007] On the other hand, iodine exerts antitumor effects on various tissues (breast, prostate, thyroid, melanoma, pancreatic cancer, neuroblastoma cell lines, etc.), and recent research suggests that these antitumor effects involve direct or indirect mechanisms (Non-Patent Documents 6 to 9).

[0008] Regarding the direct effects of iodine, it has been reported that the oxidative / antioxidative properties of iodine may disrupt mitochondrial membrane potential and induce mitochondrial-mediated apoptosis (Non-Patent Documents 8 and 11). The indirect pathway involves the production of iodolipid intermediates, one of which is 6-iodolactone (6IL). The presence of 6IL has been reported in normal and tumor mammary glands of rats that were continuously administered iodine in the diet (Non-Patent Document 12).

[0009] It has also been reported that 6IL strongly stimulates peroxisome proliferator-activated receptor gamma (PPARγ) and inhibits PPAR alpha (PPARα) expression in MCF-7 cells, a breast cancer cell line (Non-Patent Document 13).

[0010] While PPARα is associated with the promotion of carcinogenesis (Non-Patent Documents 14 and 15), PPARγ (Non-Patent Documents 16 and 17) has been reported to be an antitumor agent because it inhibits proliferation, induces apoptosis, and promotes differentiation (Non-Patent Documents 18 and 19). In addition, opposing effects of PPARα and PPARγ have been reported in normal and tumor breast tissues and breast cell lines (Non-Patent Documents 20 and 21), and PPARγ has been reported to be involved in arresting cancer cell division, inhibiting EMT (epithelial-mesenchymal transition), inducing apoptosis, etc. (Non-Patent Document 22).

[0011] Regarding the use of iodine in cancer treatment, there have been reports on the use of aqueous solutions of sodium iodide and potassium iodide, but in neither case was there sufficient evidence to prove the mechanism of action of iodine or its therapeutic effect, and the conditions, timing, duration, and dosage of iodine were unclear (Patent Documents 6 and 7). [Prior art documents] [Patent documents]

[0012] [Patent Document 1] JP 2013-17429 A [Patent Document 2] Special Publication No. 2010-530234 [Patent Document 3] Patent No. 5141919 [Patent Document 4] JP 2013-42689 A [Patent Document 5] JP 2011-163830 A [Patent Document 6] JP 2004-315470 A [Patent Document 7] JP 2008-143808 A [Non-patent literature]

[0013] [Non-Patent Document 1] Priya Gogoi:PLOS ONE, January 2016,Volume 11

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[0014] In recent years, various methods have been developed for detecting CTCs in the analysis of circulating tumor cells in peripheral blood. Among them, the FDA-approved Cell Search system is a method for detecting CTCs that uses EpCAM antibodies as a surface marker for cancer cells. However, since tumor cells that express little or no EpCAM or cytokeratin (CK) have been reported, this method, which uses affinity depending on the amount of antigen expression, has limitations on the cancer cells that can be captured.

[0015] On the other hand, the microfluidic chip method is a testing method developed to compensate for the shortcomings of CellSearch. Compared to the CellSearch method's 61% CTC capture rate, the microfluidic chip method has a 94% capture rate, demonstrating its extremely high sensitivity (Non-Patent Documents 23, 24).

[0016] In addition, Abnova's CytoQuest (trademark) CR is a technology that uses a microfluidic chip that can capture and isolate viable CTCs, and can release CTCs captured with antibodies by changing the temperature, making it possible to capture CTCs with high sensitivity, similar to the microfluidic device method (Non-Patent Document 25).

[0017] Although CTC testing devices based on various other principles have been developed, the CTC analysis technology required in the present invention is capable of detailed analysis of the number, properties, and morphology of captured cancer cells. For example, the captured cancer cells are simultaneously stained with DAPI (cell nucleus staining), Cytokeratin (expressed in epithelial cells, epithelial-derived cells, and epithelial-derived malignant tumor cells), CD45 (expressed in all human leukocytes), and Vimentin (expressed in mesenchymal cells that have undergone EMT) to detect (1) CTCs with epithelial traits, (2) CTCs with both epithelial and mesenchymal traits, and (3) CTCs with mesenchymal traits. Alternatively, the properties of cancer stem cells are analyzed in detail by detecting CD44 (Non-Patent Document 26). By analyzing the properties of these cancer cells, it is possible to maximize the therapeutic effect of iodine, a PPAR-γ agonist. [Means for solving the problem]

[0018] The present invention provides a method for treating cancer that is highly safe, enhances the therapeutic effect of cancer, and can prevent metastasis and recurrence of cancer. More specifically, the present invention provides: 1. A method for predicting therapeutic efficacy by analyzing peripheral blood circulating cells of a mammalian subject, comprising: detecting peripheral blood circulating tumor cells in a blood sample obtained from the subject; If peripheral blood circulating tumor cells are detected, classifying the detected peripheral blood circulating tumor cells; To assess the amount of classified circulating tumor cells in peripheral blood and Including, Circulating tumor cells in peripheral blood are Cytokeratin-positive and vimentin-positive Type.2 subtype, and Cytokeratin negative, Vimentin positive, Type 2 are circulating tumor cells in the peripheral blood, The reduction in the Type.2 subtype and Type.2 after treatment indicates that the treatment was effective. Methods (excluding medical procedures for humans) are provided.

[0019] The mammalian subject is preferably a human, a cat, or a dog.

[0020] The analysis of the peripheral blood circulating tumor cells preferably includes quantifying the number of peripheral blood circulating tumor cells, and more preferably includes analyzing the morphology of the peripheral blood circulating tumor cells. The analysis of the peripheral blood circulating tumor cells preferably includes quantifying the number of microenvironments (niches) surrounding the peripheral blood circulating tumor cells, the microenvironments (niches) including one or more selected from the group consisting of platelets, macrophages, lymphocytes, and stromal cells.

[0021] In the analysis of peripheral blood circulating tumor cells, detection of one or more peripheral blood circulating tumor cells per mL of blood can be used as an indicator that a cancer patient has a tumor.

[0022] The present invention is preferably used when a cancer patient has one or more types of tumor selected from the group consisting of epithelial tumors, mesenchymal tumors, tumors having both epithelial and mesenchymal properties, and metastatic tumors.

[0023] The present invention is preferably used when the cancer is one or more types selected from the group consisting of breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer and skin cancer. Effect of the Invention

[0024] The present invention has the remarkable effect of providing a cancer treatment method that is highly safe, enhances the therapeutic effect of cancer, and prevents cancer metastasis and recurrence, as well as a monitoring method for assessing the therapeutic effect of a PPAR-γ agonist in cancer patients. [Brief description of the drawings]

[0025] [Figure 1] Photographs of example images 1 to 5 of tumor-associated macrophages. [Diagram 2] Photographs of example images 1 to 5 of dead cells. [Diagram 3] Photographs of example images 1 to 5 of intact cells of Type.1. [Figure 4] Photographs showing example images 1 to 5 of Type.1 aggregated cells (cluster CTCs). [Diagram 5] Photographs of example images 1 to 5 of Type.1 aggregated cells (CTM). [Figure 6] These are example photographs of images 1 to 5 of intact cells (intact CTCs) of the Type.2 subtype (Metastable Cells). [Figure 7] These are example photographs of images 1 to 5 of amoeboid cells of the Type.2 subtype (Metastable Cell). [Figure 8] These are example photographs of images 1 to 5 of aggregated cells (cluster CTCs) of the Type.2 subtype (Metastable Cells). [Figure 9] These are example photographs of images 1 to 5 of aggregated cells (CTM) of the Type.2 subtype (Metastable Cells). [Figure 10] These are example photographs of images 1 to 5 of Type.2 mesenchymal cells. [Figure 11] Photographs showing example images 1 to 5 of amoeboid cells of Type.2 CTCs. [Figure 12] Photographs showing example images 1 to 5 of Type.2 aggregated cells (cluster CTCs). [Figure 13] Photographs of example images 1 to 5 of Type.2 aggregated cells (CTM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, an embodiment of the present invention will be described with reference to the accompanying drawings. However, the illustrated examples are shown for illustrative purposes only, and it goes without saying that various modifications are possible without departing from the technical concept of the present invention.

[0027] The cancer treatment method of the present invention includes a first analysis step of analyzing peripheral blood circulating tumor cells (CTCs) of a mammalian subject, a step of administering an effective amount of a PPAR-γ agonist to the subject who is deemed to require treatment based on the results of the analysis, and a second analysis step of analyzing peripheral blood circulating tumor cells (CTCs) of the subject after administration of the PPAR-γ agonist.

[0028] The method for analyzing peripheral blood circulating tumor cells used in the present invention can be a wide variety of known CTC analysis methods and is not particularly limited. However, the microfluidic device method (Non-Patent Document 23) and microfluidic chip (Non-Patent Document 25) used in the analysis of peripheral blood circulating tumor cells can capture cancer cells circulating in the peripheral blood, and by staining the cancer cells, it is possible to measure CTCs that have leaked from the primary tumor as Type 1 CTCs and CTCs that may have undergone EMT as Type 2 CTCs.

[0029] It is known that EMT is a process in which epithelial cells lose their cell polarity and adhesive function with surrounding cells, and gain migration and invasive capabilities, thereby transforming into mesenchymal-like cells (Non-Patent Documents 1 to 3). As cancer cells become more malignant, they undergo a metastable cell transformation, which is a subtype of Type 2 (Metastable Cell), and then transform into a mesenchymal cell, which is a Type 2 CTC.

[0030] According to recent research, as the malignancy of the same CTC increases, the cell morphology changes to amoeboid cells, and it has been reported that cluster CTCs, which are formed by the aggregation of cancer cells, are related to metastasis and prognosis, unlike single cancer cells (Non-Patent Document 28). Among cluster CTCs, circulating tumor microemboli (CTM), which form aggregates containing blood cells and platelets, have been suggested to exhibit different phenotypes and molecular characteristics compared to single CTCs, and have been reported to have higher metastatic potential and resistance to apoptosis (Non-Patent Documents 29, 31). As for tumor-associated macrophages (TAM), although they are not cancer cells, they have been reported to form a cancer microenvironment (niche) together with fibroblasts and vascular endothelial cells as macrophages that support cancer cells and have the effect of promoting the proliferation of cancer cells (Non-Patent Document 30).

[0031] In the present invention, based on the results of CTC analysis, an effective amount of a PPAR-γ agonist is administered to a subject who is deemed to require treatment for a specified period of time, and then the subject's CTCs are analyzed again to determine the therapeutic effect of the PPAR-γ agonist in cancer patients.

[0032] The PPAR-γ agonist is preferably one having an inhibitory effect on PPAR-α, and is particularly preferably an iodine-containing compound, with sodium iodide being more preferable from the standpoint of safety and effectiveness.

[0033] Regarding the safety of iodine in iodine therapy, (1) 30 healthy adult males with normal thyroid function were randomly orally administered iodine (as iodide) at doses of 500, 1,500, or 4,500 μg / day for 14 days. As a result, no effect was observed on serum thyroid hormone levels or serum TSH levels at a total iodine intake of 800 μg / day (0.011 mg / kg body weight / day), but at 1,800 μg / day (0.026 mg / kg body weight / day) and 4,800 μg / day (0.069 mg / kg body weight / day), there was a small but significant transient decrease in serum total T4 and free T4 levels compared to before administration ( (10%) and an increase in serum TSH levels (48%) were noted; (2) a study in which 11 healthy adults with normal thyroid function were given iodine in the form of seaweed at a dose of 25 mg / day for 14 days, showing a significant increase in the average serum TSH levels, but these levels were within the normal range; and (3) a long-term study in which 4 healthy adults were given iodine orally at a dose of approximately 1,000 mg / day for 11 consecutive weeks, showing a small but statistically significant decrease in the average serum T4 levels and an increase in serum TSH levels, but these two changes returned to normal within one week after administration was discontinued (Non-Patent Document 32).

[0034] The inventors of the present application have found that in the treatment method of the present invention, by prescribing an effective amount of iodine taking into consideration the safety of the above-mentioned iodine, iodine inhibits not only metastable cells, which are CTCs of the Type.2 subtype (Metastable Cell), and mesenchymal cells, which are CTCs of Type.2, but also cluster CTCs, cluster CTMs, and TAMs.

[0035] In addition, apoptotic CTCs are dead cancer cells that have been damaged and no longer function, and are often detected in patients with a history of anti-cancer drug treatment or when treatment is ongoing. Since iodine has also been reported to have the effect of inducing apoptosis in cancer cells, they are analyzed as one way of assessing the effectiveness of iodine treatment.

[0036] Based on the above, in the combination treatment of PPAR-γ agonists such as iodine-containing compounds of the present invention and CTC analysis, the therapeutic effect of the PPAR-γ agonist is assessed by analysis of Type.1 CTCs, metastable cells which are a subtype of Type.2 (metastable cells), mesenchymal cells which are Type.2 CTCs, as well as amoeboid cells, CTMs, TAMs, and apoptotic CTCs. If a therapeutic effect is observed, further treatment guidelines such as the dosage, frequency, and duration of administration of the PPAR-γ agonist will be determined.

[0037] Based on the test results using the microfluidic device method, an analysis table (Table 1) of peripheral blood circulating tumor cells to be used in combination therapy with the PPAR-γ agonist used in the present invention and its assessment criteria are shown below.

[0038] [Table 1]

[0039] <Explanation of captured cancer cell images> Cancer cells captured using the microfluidic device method are simultaneously stained with different colors: DAPI (cell nucleus stain: blue), fluorescent antibody for CD45 (expressed in all human white blood cells) (red), fluorescent antibody for Cytokeratin (expressed in epithelial cells, epithelial-derived cells, and epithelial-derived malignant tumor cells) (green), and fluorescent antibody for Vimentin (expressed in mesenchymal cells that have undergone EMT) (yellow). The meanings of the staining in each image are as follows: Image 1: Cell nuclei stained with DAPI (blue). Image 2: Human leukocytes stained with CD45 (red). Image 3: Cytokeratin staining of epithelial cells and epithelial-derived cells (green). Image 4: Mesenchymal cells stained with Vimentin (yellow). Image 5: Observation using a phase contrast microscope.

[0040] (1) Criteria for determining tumor-associated macrophages. 1) Large in shape and contains a nucleus (DAPI: blue). 2) They express CD45 and Vimentin weakly or not at all. 3) As a result of phagocytosis of cancer cells, cytokeratin (green), a characteristic of epithelial cells, is present.

[0041] Figure 1 shows example images of tumor-associated macrophages. Figure 1 (a) to (e) are photographs showing the results of images 1 to 5, respectively. As shown in Figure 1, they are large in shape (circled in image 5), their nuclei are located within the cytoplasm (circled in image 1), and they weakly express CD45 (circled in image 2) and Vimentin (circled in image 4). In addition, as a result of phagocytosing cancer cells, they contain cytokeratin (circled in image 3), which is a characteristic of epithelial cells.

[0042] (2) Criteria for determining dead cells (apoptotic CTCs). 1) It has no nucleus. 2) Cytokeratin (green) is present. 3) The shape is irregular (such as cellular condensation).

[0043] Figure 2 shows example images of dead cells (apoptotic CTCs). Figure 2 (a) to (e) are photographs showing the results of images 1 to 5, respectively. As shown in Figure 2, the nucleus is not present in the cytoplasm (circled in image 1).

[0044] (3) Criteria for determining Type.1 intact cells (intact CTCs). 1) Spherical or oval in shape and contains a nucleus (DAPI: blue). 2) No expression of CD45 (red). 3) Cytokeratin (green) is expressed. 4) The shape is intact and taut.

[0045] Figure 3 shows an example image of Type.1 intact cells (intactCTCs). Figure 3 (a) to (e) are photographs showing the results of images 1 to 5, respectively. As shown in Figure 3, the nucleus is located within the cytoplasm (circled in image 1), and cytokeratin (circled in image 3), which is a characteristic of epithelial cells, is present. In addition, there is no expression of CD45 (image 2) and Vimentin (image 4), and the cancer cells are not shrunken (red circle in image 5).

[0046] (4) Criteria for determining Type.1 aggregated cells (cluster CTCs). 1) There are numerous nuclei (DAPI: blue). 2) No expression of CD45 (red). 3) Cytokeratin (green) is expressed. 4) Form clusters.

[0047] Figure 4 shows an example image of Type.1 aggregated cells (cluster CTCs). Figure 4 (a) to (e) are photographs showing the results of images 1 to 5, respectively. As shown in Figure 4, there are many nuclei (circled in image 1), and cytokeratin, a characteristic of epithelial cells, is expressed (circled in image 3). In addition, there is no expression of CD45 (image 2) and Vimentin (image 4), and the cancer cells have formed clusters (circled in images 1, 3, and 5).

[0048] (5) Criteria for determining Type 1 clumped cells (CTM). 1) There are numerous nuclei (DAPI: blue). 2) Weakly expressing CD45 (red). 3) Cytokeratin (green) is expressed. 4) No expression of Vimentin (yellow). 5) Formation of clusters containing blood cells, platelets, etc.

[0049] Figure 5 shows an example image of Type.1 aggregated cells (CTM). Figure 5 (a) to (e) are photographs showing the results of images 1 to 5, respectively. As shown in Figure 5, there are many nuclei (circled in image 1), and cytokeratin (circled in image 3), which is a characteristic of epithelial cells, is present. In addition, because the aggregates (CTM) are formed containing blood cells and platelets, CD45 (circled in image 2) is weakly expressed, there is no expression of Vimentin (image 4), and the cancer cells form clusters (circled in images 1, 2, 3, and 5).

[0050] (6) Criteria for determining intact cells (intact CTCs) of the Type.2 subtype (Metastable Cells). 1) Spherical or oval in shape and contains a nucleus (DAPI: blue). 2) No expression of CD45 (red). 3) Cytokeratin (green) and Vimentin (yellow) are expressed. 4) The shape is intact and taut.

[0051] Figure 6 shows an example image of an intact cell (intactCTC) of the Type.2 subtype (Metastable Cell). Figure 6 (a) to (e) are photographs showing the results of images 1 to 5, respectively. As shown in Figure 6, the nucleus is located within the cytoplasm (circled in image 1), there is no expression of CD45 (image 2), there is expression of Cytokeratin (circled in image 3), which is a characteristic of epithelial cells, and Vimentin (circled in image 4), which is a characteristic of mesenchymal cells, and the cancer cells are not shrunken (circled in image 5).

[0052] Criteria for determining amoeboid cells of the Type.2 subtype (Metastable Cell). 1) Spherical or oval in shape and contains a nucleus (DAPI: blue). 2) No expression of CD45 (red). 3) Cytokeratin (green) and Vimentin (yellow) are expressed. 4) A cell that is shaped like an amoeba.

[0053] Figure 7 shows an example image of an amoeboid cell of the Type.2 subtype (Metastable Cell). Figure 7 (a) to (e) are photographs showing the results of images 1 to 5, respectively. As shown in Figure 7, the nucleus is located within the cytoplasm (circled in image 1), there is no expression of CD45 (image 2), there is expression of Cytokeratin (circled in image 3), which is a characteristic of epithelial cells, and Vimentin (circled in image 4), which is a characteristic of mesenchymal cells, and the cell has an amoeboid morphology (circled in images 3 and 4).

[0054] (8) Criteria for determining Type.2 subtype (Metastable Cell) aggregated cells (cluster CTCs). 1) There are numerous nuclei (DAPI: blue). 2) No expression of CD45 (red). 3) Cytokeratin (green) and Vimentin (yellow) are expressed. 4) Form clusters.

[0055] Figure 8 shows an example image of aggregated cells (cluster CTCs) of the Type.2 subtype (Metastable Cells). Figure 8 (a) to (e) are photographs showing the results of images 1 to 5, respectively. As shown in Figure 8, there are numerous nuclei (circled in image 1), there is no expression of CD45 (image 2), there is expression of Cytokeratin (circled in image 3), which is a characteristic of epithelial cells, and Vimentin (circled in image 4), which is a characteristic of mesenchymal cells, and the cancer cells have formed clusters (circled in images 1, 3, 4, and 5).

[0056] (9) Criteria for determining Type.2 subtype (Metastable Cell) aggregated cells (CTM). 1) There are numerous nuclei (DAPI: blue). 2) Weakly expressing CD45 (red). 3) Cytokeratin (green) and Vimentin (yellow) are expressed. 4) Formation of clusters containing blood cells, platelets, etc.

[0057] Figure 9 shows an example image of aggregated cells (CTM) of the Type.2 subtype (Metastable Cell). Figure 9 (a) to (e) are photographs showing the results of images 1 to 5, respectively. As shown in Figure 9, there are many nuclei (circled in image 1), and there is expression of Cytokeratin (circled in image 3), which is a characteristic of epithelial cells, and Vimentin (circled in image 4), which is a characteristic of mesenchymal cells. In addition, CD45 (circled in image 2) is weakly expressed as the aggregates (CTM) are formed in a form that includes blood cells and platelets, and the cancer cells form clusters (circled in images 1, 3, 4, and 5).

[0058] (10) Criteria for determining Type.2 mesenchymal cells. 1) Spherical or oval in shape and contains a nucleus (DAPI: blue). 2) No expression of CD45 (red). 3) No expression of cytokeratin (green). 4) Vimentin (yellow) is expressed.

[0059] Figure 10 shows an example image of a mesenchymal cell of Type.2 CTC. (a) to (e) in Figure 10 are photographs showing the results of images 1 to 5, respectively. As shown in Figure 10, the nucleus is located within the cytoplasm (circled in image 1), there is no expression of CD45 (image 2) and Cytokeratin (image 3), there is expression of Vimentin (circled in image 4), which is a characteristic of mesenchymal cells, and the cancer cell is not shrunken (circled in image 5).

[0060] (11) Criteria for determining amoeboids in Type.2 CTCs. 1) Nuclei are present (DAPI: blue). 2) No expression of CD45 (red) and cytokeratin (green). 3) Vimentin (yellow) is expressed. 4) A cell that is shaped like an amoeba.

[0061] Figure 11 shows an example image of an amoeboid cell of Type.2 CTC. (a) to (e) in Figure 11 are photographs showing the results of images 1 to 5, respectively. As shown in Figure 11, the nucleus is located within the cytoplasm (circled in image 1), there is no expression of CD45 (image 2) and Cytokeratin (image 3), there is expression of Vimentin (circled in image 4), which is characteristic of mesenchymal cells, and the cell has an amoeboid morphology (red circles in images 4 and 5).

[0062] (12) Criteria for determining Type 2 aggregated cells (clustered CTCs). 1) There are numerous nuclei (DAPI: blue). 2) No expression of CD45 (red) and cytokeratin (green). 3) Vimentin (yellow) is expressed. 4) Form clusters.

[0063] Figure 12 shows an example image of Type.2 aggregated cells (cluster CTCs). Figure 12 (a) to (e) are photographs showing the results of images 1 to 5, respectively. As shown in Figure 12, there are many nuclei (circled in image 1), there is no expression of CD45 (image 2) and Cytokeratin (image 3), there is expression of Vimentin (circled in image 4), which is a characteristic of epithelial cells and mesenchymal cells, and the cancer cells have formed clusters (red circles in images 1, 4, and 5).

[0064] (13) Criteria for determining type 2 clumped cells (CTM). 1) There are numerous nuclei (DAPI: blue). 2) Weakly expressing CD45 (red). 3) No expression of cytokeratin (green). 4) Vimentin (yellow) is expressed. 5) Formation of clusters containing blood cells, platelets, etc.

[0065] Figure 13 shows an example image of Type.2 aggregated cells (CTM). Figure 13 (a) to (e) are photographs showing the results of images 1 to 5, respectively. As shown in Figure 13, there are many nuclei (circled in image 1), no cytokeratin (circled in image 3), and expression of Vimentin (circled in image 4), which is characteristic of mesenchymal cells. In addition, CD45 (circled in image 2) is weakly expressed as the aggregate (CTM) is formed containing blood cells and platelets, and the cancer cells form clusters (red circles in images 1, 4, and 5).

[0066] In the treatment method of the present invention, the criteria for cancer to be administered with a PPAR-γ agonist are, except for cases where a doctor prescribes a PPAR-γ agonist at the request of a patient who has tested positive in a CTC test for the purpose of a health check, stage 3 and stage 4 cancer that has become difficult to treat with standard treatments consisting of surgery, radiation therapy, and chemotherapy such as anticancer drugs, which are known as the three major cancer treatments, and where an analysis of circulating tumor cells in the peripheral blood detects metastable cells, which are CTCs of the Type.2 subtype (Metastable Cells) that may have undergone EMT, and Type.2 CTCs that have undergone EMT, and where the patient's consent has been obtained.

[0067] Analysis of circulating tumor cells in the peripheral blood is performed after obtaining a consent form from the cancer patient explaining the purpose and method of the test, and stating that the treatment plan will be determined based on the test results. For example, 10 mL of blood is collected using a dedicated blood collection tube, and the circulating tumor cells in the peripheral blood are analyzed using a microfluidic device method or the like.

[0068] In the present invention, when iodine therapy is performed using an iodine-containing compound as a PPAR-γ agonist, the prescription must, in principle, be made using stable iodine water produced by a doctor at a hospital himself / herself after the doctor has confirmed that the patient's thyroid function is normal.

[0069] The effective amount of iodine therapy used in the present invention means a safe amount of iodine that is sufficient to bring about a therapeutic response to cancer when used in the manner described herein without excessive harmful side effects. The upper limit of the safe amount of iodine therapy is prescribed with reference to a report on the safety of daily oral administration of about 1,000 mg / day for 11 weeks (Non-Patent Document 32), but the doctor may adjust the dosage and administration at any time depending on the malignancy such as the progression state of the cancer.

[0070] The basic dosage of sodium iodide used in stable iodine water was determined with reference to the following non-patent literature. First, regarding the absorption rate of iodine, in most cases, iodine from ingested sodium iodide is absorbed 100% from the digestive tract, reduced to iodide in the intestine, and absorbed almost completely in the small intestine. In an exposure test involving seven adults with normal thyroid function, 100% of the iodine absorbed orally from a single dose of tracer was 131 Of the radioactive iodine in the feces, less than 1% was detected. From this, it has been reported that the absorption rate of the ingested radioactive iodine is close to 100%. In another exposure test involving 20 adults with normal thyroid function, potassium iodide was ingested every day for 13 weeks, and the daily urinary excretion of iodine was about 80-90% of the estimated daily intake. This also suggests that the absorption rate of iodine is close to 100% (Non-Patent Document 33).

[0071] Regarding the distribution of iodine in the body, in an exposure test in which radiolabeled iodine was orally ingested in humans at a tracer concentration as sodium iodide, approximately 20-30% of the iodine was distributed to the thyroid gland, and approximately 30-60% was excreted in the urine within approximately 10 hours. 131 Essentially the same results were obtained when tracer concentrations of I were orally administered (Non-Patent Document 34).

[0072] Regarding inhalation of iodine from exhaled breath, it has been reported that iodine is easily absorbed from the lungs and digestive tract, but in a test in which volunteers were made to inhale radioactive iodine, the inhaled iodine was almost entirely eliminated from the respiratory tract, with a half-life of about 10 minutes (Non-Patent Document 35), and most of the inhaled iodine was transferred to the digestive tract by mucociliary clearance. The fact that inhaled iodine is absorbed relatively quickly has also been supported by tests using mice, rats, dogs, and sheep (Non-Patent Document 36).

[0073] Concerning the antitumor effects of iodine, a large amount of data from in vitro experiments (Non-Patent Documents 37), in vivo experiments (Non-Patent Documents 6 and 38-41), clinical trials (Non-Patent Documents 42-45), and epidemiological reports (Non-Patent Documents 46-48) supports the view that iodine has the effect of inhibiting tumor progression and improving inflammatory / proliferative lesions. These beneficial effects have been reported at relatively high iodine concentrations (milligrams / day).

[0074] Considering the above reports, for example, the 8,400 ppm stable iodine water preferably used in the present invention is composed of an aqueous solution of 1 w / v% sodium iodide, 0.08 w / v% potassium dihydrogen citrate, and 0.1 w / v% sodium metasilicate nonahydrate, and the pH at this time is 8.3 to 8.7, which is weakly alkaline. The 10,000 ppm stable iodine water is composed of an aqueous solution of 1.2 w / v% sodium iodide, 0.08 w / v% potassium dihydrogen citrate, and 0.1 w / v% sodium metasilicate nonahydrate, and the pH at this time is 8.3 to 8.7, which is weakly alkaline. The potassium dihydrogen citrate and sodium metasilicate nonahydrate prescribed here are safe additives prescribed as pH buffers. In addition, this stable iodine water is ultrafiltered, and then substituted with nitrogen in consideration of the stability of iodine. It is then heated at 85°C and stored at room temperature until use.

[0075] The dosage of the stable iodine water used in the present invention will be determined by a doctor based on the analysis results of peripheral blood circulating tumor cells, and the dosage and administration should be up to 1,000 mg / day. For example, in the case of 8,400 ppm stable iodine water, 30 mL three times a day (756 mg of iodine / day), and in the case of 10,000 ppm stable iodine water, 30 mL three times a day (900 mg of iodine / day), are suitable. However, the doctor may change the dosage and administration or discontinue administration at any time based on the analysis results of peripheral blood circulating tumor cells during the course of treatment.

[0076] The injectable stable iodine water used in the present invention is determined by a physician based on the analysis results of peripheral blood circulating tumor cells. For example, 10 mL of 10 w / v % sodium iodide that has been sterilized by filtration through a 0.02 μm membrane filter is preferably dissolved in 100 mL of injectable physiological saline for infusion, and 110 mL of this solution is preferably infused once a day over 30 minutes. However, the physician may change the number of infusions or stop the infusion at any time based on the analysis results of peripheral blood circulating tumor cells during the course of treatment.

[0077] The stable iodine water for inhalation used in the present invention is determined by a physician based on the analysis results of peripheral blood circulating tumor cells. For example, an aqueous solution of sodium iodide prepared at a concentration of 1 w / v% with physiological saline is sterilized by filtration using a 0.02 μm membrane filter, and 30 mL of the solution is transferred to a dedicated nebulizer and inhaled several times a day. However, the physician may change the dosage or discontinue inhalation at any time based on the analysis results of peripheral blood circulating tumor cells during the course of treatment.

[0078] As used herein, cancer refers to any type of cancer or neoplasm or malignant tumor found in a mammal, including, but not limited to, leukemia, lymphoma, melanoma, carcinoma, and sarcoma. Examples of cancer are cancer of the brain, breast, pancreas, cervix, colon, head and neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus, and medulloblastoma. Other cancers include, for example, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical carcinoma, and prostate cancer. EXAMPLES

[0079] The present invention will be described in more detail below with reference to examples. However, it goes without saying that these examples are given for illustrative purposes and should not be construed as limiting.

[0080] All of the treatment cases shown below are the results of private medical treatment conducted under the direction of the doctor with the patient's consent, and are disclosed with the patient's consent. In the following examples, 10 mL of blood was collected using a special blood collection tube, and the analysis of peripheral blood circulating tumor cells was performed using a microfluidic device method. In addition, iodine treatment was performed using the above-mentioned stable iodine water [8,400 ppm stable iodine water: 1 w / v% sodium iodide, 0.08 w / v% potassium dihydrogen citrate, 0.1 w / v% sodium metasilicate nonahydrate aqueous solution (pH 8.3-8.7), or 10,000 ppm stable iodine water: 1.2 w / v% sodium iodide, 0.08 w / v% potassium dihydrogen citrate, 0.1 w / v% sodium metasilicate nonahydrate aqueous solution (pH 8.3-8.7)] as a PPAR-γ agonist.

[0081] Example 1 Table 2 shows information about Patient A and the course of iodine treatment, and Table 3 shows the analysis results of circulating tumor cells in the peripheral blood.

[0082] [Table 2]

[0083] [Table 3]

[0084] As shown in Table 3, the analysis of peripheral blood circulating tumor cells on June 19, 2018 detected one Type.1 intact cell (intact CTC), one Type.1 clumped cell (cluster CTC), one Type.1 clumped cell (CTM), and one metastable cell amoeboid. Therefore, the patient was started on 8,400 ppm stable iodine water at 20 mL three times a day.

[0085] The analysis of peripheral blood circulating tumor cells on December 3, 2018 showed that three damaged and dead cells (apoptotic CTCs) were detected, but no CTCs were detected. Therefore, from January 7, 2019, the dosage of 8,400 ppm stable iodine water was reduced to 10 mL once (before breakfast), and from January 21, 2019, the dosage was discontinued.

[0086] In the case of Example 1, it is considered that the activation of PPAR-γ and the suppression of PPAR-α by iodine caused the disappearance of the metastable cell amoeboids, Type.1 aggregated cells (cluster CTC), and Type.1 aggregated cells (CTM).

[0087] Example 2 Table 4 shows information about Patient B and the course of iodine treatment, and Table 5 shows the analysis results of circulating tumor cells in the peripheral blood.

[0088] [Table 4]

[0089] [Table 5]

[0090] As shown in Table 5, the analysis results of peripheral blood circulating tumor cells on December 17, 2018 showed that 9 intact cells of the Type.2 subtype (Metastable Cell) and 1 Type.2 mesenchymal cell were detected, so the patient was started on 8,400 ppm stable iodine water at 20 mL x 3 times per day.

[0091] The analysis results of peripheral blood circulating tumor cells on January 21, 2019 showed that no Type.2 mesenchymal cells were detected, but two intact cells with low viability of Type.1 were detected, and five intact cells of the Type.2 subtype (Metastable Cells) were detected. Therefore, stable iodine water of 8,400 ppm was continuously administered at 20 mL x 3 times per day. In addition, the fact that intact cells of metastable cells, which are thought to be in the middle stage of EMT conversion, did not disappear is presumed to be due to the influence of anticancer drug treatment, as seen in the cases of other examples in this specification, but the detailed mechanism of action is unknown.

[0092] Analysis of peripheral blood circulating tumor cells on March 6, 2019 revealed that three Type.1 intact cells (intact CTCs) were detected, but no Type.2 subtype (metastable cells) or Type.2 mesenchymal cells were detected.

[0093] In the case of Example 2, it is presumed that the activation of PPAR-γ possessed by iodine inhibited EMT transformation, resulting in the disappearance of the Type.2 subtype (Metastable Cells) and Type.2 mesenchymal cells.

[0094] Example 3 Table 6 shows information about Patient C and the course of iodine treatment, and Table 7 shows the analysis results of circulating tumor cells in the peripheral blood.

[0095] [Table 6]

[0096] [Table 7]

[0097] As shown in Table 7, the analysis of peripheral blood circulating tumor cells on April 9, 2018 detected 39 intact cells (CTCs) of Type 2 metastable cells, 35 amoeboid cells (amoeboids) of Type 2 metastable cells, 3 clustered cells (CTCs) of Type 2 metastable cells, 4 aggregated cells (CTMs) of Type 2 metastable cells, 12 mesenchymal cells (mesenchymals) of Type 2, and 7 amoeboid cells (amoeboids) of Type 2. Therefore, from April 9, 2018, the patient started taking 15 mL of 8,400 ppm stable iodine water twice a day (after breakfast and dinner).

[0098] The results of the analysis of peripheral blood circulating tumor cells on May 15, 2018 showed that one Type.1 intact cell (intact CTC), one Type.2 metastable cell intact cell (intact CTC), three Type.2 metastable cell amoeboid cells, and two Type.2 metastable cell aggregated cells (cluster CTCs). However, since Type.2 mesenchymal cells and Type.2 amoeboid cells were not detected, the patient was administered a reduced dose of 8,400 ppm stable iodine water at 20 mL three times a day.

[0099] On November 19, 2018, the analysis of peripheral blood circulating tumor cells showed that one damaged and dead cell (apoptotic CTC) was detected, but no CTCs were detected, so the patient was administered a reduced dose of 8,400 ppm stable iodine water to 10 mL three times a day.

[0100] In the case of Example 3, it is speculated that the activation of PPAR-γ possessed by iodine inhibited EMT transformation, resulting in the disappearance of Type.2 mesenchymal cells and Type.2 subtype (Metastable Cells). However, it is speculated that the reason why all peripheral blood circulating tumor cells disappeared may be that the direct effect of iodine, its oxidative / antioxidative properties, disrupted the mitochondrial membrane potential, causing mitochondrial-mediated apoptosis (Non-Patent Documents 8, 11).

[0101] Example 4 Table 8 shows information about Patient D and the course of iodine treatment, and Table 9 shows the analysis results of circulating tumor cells in the peripheral blood.

[0102] [Table 8]

[0103] [Table 9]

[0104] Analysis of peripheral blood circulating tumor cells on June 18, 2018 showed that three tumor-associated macrophages (TAM), two Type 1 intact cells (intact CTC), one Type 1 aggregated cell (CTM), one Type 2 metastable cell intact cell, two Type 2 metastable cell amoeboid cells, one Type 2 metastable cell aggregated cell (cluster CTC), and three Type 2 metastable cell aggregated cells (CTM), so the patient was started on 8,400 ppm stable iodine water at 30 mL three times a day.

[0105] Analysis of peripheral blood circulating tumor cells on September 26, 2018 showed that one Type.1 intact cell (intact CTC), one Type.2 subtype (metastable cell) intact cell (intact CTC), and three Type.2 subtype (metastable cell) aggregated cells (CTM). However, as the Type.1 tumor-associated macrophage aggregated cells (CTM) disappeared, the patient was administered a reduced dose of 8,400 ppm stable iodine water to 10 mL three times a day.

[0106] In the case of Example 4, the activation of PPAR-γ by iodine inhibited EMT conversion and the amoeboid cells of the Type.2 subtype (metastable cells) disappeared, but clusters of cells (clusters, CTM) of the Type.2 subtype (metastable cells) were still detected, so the patient was administered a reduced amount of stable iodine water. On the other hand, the disappearance of the microenvironment (niche) containing tumor-associated macrophages (TAM) and blood cells, platelets, etc. may be due to the disruption of mitochondrial membrane potential caused by the direct effect of iodine, i.e., its oxidative / antioxidative properties, which may lead to mitochondrial-mediated apoptosis (Non-Patent Documents 8, 11), or the involvement of various anti-tumor effects of iodine (Non-Patent Documents 6-9). In addition, in this example, intact metastable cells, which are thought to be in the middle stage of EMT conversion, did not disappear. This is presumably due to the influence of anticancer drug treatment, as seen in the cases in other examples of this specification, but the detailed mechanism of action is unknown.

[0107] Example 5 Table 10 shows information about Patient E and the course of iodine treatment, and Table 11 shows the analysis results of circulating tumor cells in the peripheral blood.

[0108] [Table 10]

[0109] [Table 11]

[0110] As shown in Table 11, the analysis results of peripheral blood circulating tumor cells on June 8, 2018 showed that 2 Type.1 intact cells (intact CTCs), 12 Type.2 subtype (metastable cell) intact cells (intact CTCs), 11 Type.2 subtype (metastable cell) amoeboid cells, and 51 Type.2 subtype (metastable cell) aggregated cells (cluster CTCs) were detected. Therefore, administration of 8,400 ppm stable iodine water was started at 30 mL x 3 times per day.

[0111] Analysis of peripheral blood circulating tumor cells on June 18, 2018 showed that three intact cells (intact CTCs) of Type.2 subtype (metastable cells), two amoeboid cells of Type.2 subtype (metastable cells), and one clustered cell (cluster CTC) of Type.2 subtype (metastable cells) were detected, so the patient was administered a reduced dose of 8,400 ppm stable iodine water to 20 mL three times a day.

[0112] In the case of Example 5, it is presumed that the activation of PPAR-γ by iodine clearly inhibited EMT transformation, resulting in a decrease in the number of intact cells, amoeboid cells, and cell clusters (CTM) of the Type.2 subtype (Metastable Cell).

[0113] Example 6 Table 12 shows information about Patient F and the course of iodine treatment, and Table 13 shows the analysis results of circulating tumor cells in the peripheral blood.

[0114] [Table 12]

[0115] [Table 13]

[0116] As shown in Table 13, the analysis results of peripheral blood circulating tumor cells on November 19, 2018 showed that 4 Type.1 intact cells (intact CTCs), 1 Type.1 aggregated cell (CTM), 8 Type.2 subtype (metastable cell) intact cells (intact CTCs), 1 Type.2 subtype (metastable cell) aggregated cell (cluster CTC), and 1 aggregated cell (CTM) were detected. Therefore, administration of 8,400 ppm stable iodine water was started at 30 mL x 3 times per day.

[0117] The analysis of peripheral blood circulating tumor cells on January 7, 2019 showed that no Type.1 intact cells (intact CTCs) or Type.1 aggregated cells (CTMs) were detected, but two Type.2 mesenchymal cells were detected. Therefore, the patient continued to take 8,400 ppm stable iodine water at 30 mL three times a day.

[0118] Analysis of peripheral blood circulating tumor cells on February 14, 2019 showed that three Type.1 intact cells with high intact CTCs, four Type.2 metastable cell intact cells, one Type.2 metastable cell aggregate (CTM), and one Type.2 mesenchymal cell were detected, so the patient continued to take 8,400 ppm stable iodine water at 30 mL three times a day.

[0119] Analysis of peripheral blood circulating tumor cells on March 8, 2019 showed that one Type.1 intact cell (intact CTC) and three Type.2 mesenchymal cells were detected, so the patient continued to take 8,400 ppm stable iodine water at 30 mL three times a day.

[0120] In this example, the fact that intact metastable cells, which are thought to be in the middle stage of EMT conversion, and EMT-converted Type.2 mesenchymal cells did not disappear is presumed to be due to the influence of anticancer drug treatment, as seen in the cases in other examples herein, but the detailed mechanism of action is unknown.

[0121] Example 7 Table 14 shows information about Patient G and the course of iodine treatment, and Table 15 shows the analysis results of circulating tumor cells in the peripheral blood.

[0122] [Table 14]

[0123] [Table 15]

[0124] As shown in Table 15, the analysis results of peripheral blood circulating tumor cells on December 4, 2018 showed that 5 Type.1 intact cells (intact CTCs), 13 Type.2 subtype (metastable cell) intact cells (intact CTCs), 26 Type.2 subtype (metastable cell) amoeboid cells, and 15 Type.2 subtype (metastable cell) aggregated cells (cluster CTCs) were detected. Therefore, the patient was started on 8,400 ppm stable iodine water at 30 mL x 3 times per day.

[0125] In addition, because there were many intact cells of Type.2 subtype (metastable cells), amoeboid cells of Type.2 subtype (metastable cells), and cell clusters (CTM) of Type.2 subtype (metastable cells), intravenous drip (100 mL / saline + 10 mL of injectable iodine) was administered 13 times, and the concentration of stable iodine water was increased from 8,400 ppm to 10,000 ppm, with the patient taking 30 mL three times a day.

[0126] The analysis of peripheral blood circulating tumor cells on January 7, 2019 showed that no intact cells (intact CTCs) of Type.2 subtype (metastable cells), amoeboid cells (amoeboids) of Type.2 subtype (metastable cells), or clustered cells (cluster CTCs) of Type.2 subtype (metastable cells) were detected, but one clustered cell (cluster CTC) of Type.1 and four clustered cells (CTMs) of Type.1 were detected. Therefore, the concentration of stable iodine water was kept at 10,000 ppm, and the frequency was increased to 30 mL x 5 times per day.

[0127] In the case of Example 7, it is presumed that the activation of PPAR-γ possessed by iodine clearly inhibited EMT transformation, resulting in the disappearance of intact cells of Type.2 subtype (metastable cells), amoeboid cells of Type.2 subtype (metastable cells), and cell clusters (CTM) of Type.2 subtype (metastable cells).

[0128] Example 8 Table 16 shows information about Patient H and the course of iodine treatment, and Table 17 shows the analysis results of circulating tumor cells in the peripheral blood.

[0129] [Table 16]

[0130] [Table 17]

[0131] As shown in Table 17, the analysis results of peripheral blood circulating tumor cells on September 27, 2018 showed that three Type.1 intact cells (intact CTCs), nine Type.2 subtype (metastable cell) intact cells (intact CTCs), and two Type.2 mesenchymal cells were detected. Therefore, the patient was started on 8,400 ppm stable iodine water at 30 mL x 3 times a day.

[0132] The analysis of peripheral blood circulating tumor cells on November 19, 2018 showed that no Type.2 mesenchymal cells were detected, but three intact cells (metastable cells) of the Type.2 subtype were detected, so the patient continued to take 8,400 ppm stable iodine water at 30 mL three times a day.

[0133] On December 17, 2018, the results of an analysis of peripheral blood circulating tumor cells showed that the number of intact cells (intact CTCs) of the Type.2 subtype (Metastable Cell) had decreased to one, so the patient was administered a reduced dose of 8,400 ppm stable iodine water at 20 mL three times a day.

[0134] The results of the analysis of peripheral blood circulating tumor cells on January 21, 2019 showed that type 2 mesenchymal cells, which had disappeared until then, were detected, and 9 intact cells (CTCs) of the type 2 subtype (metastable cells), which had been decreased, were detected. Therefore, the concentration of stable iodine water was increased to 10,000 ppm, and 30 mL was administered three times a day.

[0135] In the case of Example 8, since anticancer drug treatment was continued in parallel during iodine treatment, it is difficult to accurately evaluate the effect of iodine treatment. However, in light of other Examples of the present disclosure, it is speculated that the effect of the anticancer drug on the revival of cancer stem cell-like mesenchymal cells through EMT conversion is due to the effect of the anticancer drug.

[0136] Example 9 Table 18 shows information about Patient I and the course of iodine treatment, and Table 19 shows the analysis results of circulating tumor cells in the peripheral blood.

[0137] [Table 18]

[0138] [Table 19]

[0139] As shown in Table 19, the analysis results of peripheral blood circulating tumor cells on November 6, 2018 showed that one intact cell (intact CTC) of Type.1, one aggregated cell (cluster CTC) of Type.1, four amoeboid cells of the Type.2 subtype (metastable cell), and two mesenchymal cells of Type.2 were detected. Therefore, the patient was started on 8,400 ppm stable iodine water at 20 mL x 3 times per day.

[0140] Analysis of peripheral blood circulating tumor cells on December 18, 2018 showed that 12 Type 1 intact cells (intact CTCs) were detected, but Type 1 aggregated cells (cluster CTCs), Type 2 metastable cell amoeboid cells, and Type 2 mesenchymal cells were not detected, so the dosage was reduced to 20 mL x 3 times per day.

[0141] In the case of Example 8, it is presumed that the activation of PPAR-γ by iodine inhibited EMT transformation, resulting in the disappearance of amoeboid cells, a subtype of Type.2 (metastable cells), and mesenchymal cells, Type.2.

[0142] Example 10 Table 20 shows information about Patient J and the course of iodine treatment, and Table 21 shows the analysis results of circulating tumor cells in the peripheral blood.

[0143] [Table 20]

[0144] [Table 21]

[0145] As shown in Table 21, the analysis results of peripheral blood circulating tumor cells on September 4, 2018 showed that one Type.1 intact cell (intact CTC) and four Type.2 subtype (metastable cell) intact cells (intact CTCs) were detected, so the patient was started on 8,400 ppm stable iodine water at 30 mL x 3 times per day.

[0146] Analysis of peripheral blood circulating tumor cells on October 16, 2018 showed that one Type.1 intact cell (intact CTC), 34 Type.2 metastable cell intact cells (intact CTC), and two Type.2 mesenchymal cells were detected, so the patient was administered an increased amount of 8,400 ppm stable iodine water to 40 mL three times a day.

[0147] Analysis of peripheral blood circulating tumor cells on December 4, 2018 showed that Type.2 mesenchymal cells had disappeared and intact cells (intact CTCs) of the Type.2 subtype (metastable cells) had decreased, so the patient was administered a reduced dose of 8,400 ppm stable iodine water at 30 mL three times a day.

[0148] In the case of Example 10, since anticancer drug treatment was continued in parallel during iodine treatment, it is difficult to accurately evaluate the iodine treatment. However, in light of other Examples in this specification, it is easily inferred that the analysis results of the peripheral blood circulating tumor cells on October 16, 2018 showed that cancer stem cell-like mesenchymal cells were revived through EMT conversion under the influence of the anticancer drug.

[0149] Example 11 Table 22 shows information about Patient K and the progress of iodine treatment, and Table 23 shows the analysis results of circulating tumor cells in the peripheral blood.

[0150] [Table 22]

[0151] [Table 23]

[0152] As shown in Table 23, the analysis results of peripheral blood circulating tumor cells on November 19, 2018 showed that 5 Type.1 intact cells (intact CTCs), 37 Type.2 subtype (metastable cell) intact cells (intact CTCs), 68 Type.2 subtype (metastable cell) aggregated cells (cluster CTCs), 2 Type.2 subtype (metastable cell) aggregated cells (CTMs), 4 Type.2 mesenchymal cells (mesenchymal), and 1 Type.2 aggregated cell (cluster CTC) were detected. Therefore, from November 27, 2018, 8,400 ppm stable iodine water was administered 30 mL x 5 times / day and infusion (100 mL / saline + 10 mL injectable iodine water) was performed a total of 7 times.

[0153] Analysis of peripheral blood circulating tumor cells on December 28, 2018 showed that 10 Type.1 intact cells (intact CTCs) were detected, but Type.2 subtype (metastable cell) intact cells (intact CTCs), Type.2 subtype (metastable cell) aggregated cells (cluster CTCs), Type.2 subtype (metastable cell) aggregated cells (CTMs), Type.2 mesenchymal cells, and Type.2 cell clusters (CTMs) had disappeared, so the patient was administered a reduced dose of 8,400 ppm stable iodine water to 30 mL three times a day.

[0154] Analysis of peripheral blood circulating tumor cells on January 8, 2019 revealed that eight intact cells (intact CTCs) of Type.1, four intact cells (intact CTCs) of the Type.2 subtype (metastable cells), and two aggregated cells (cluster CTCs) of the Type.2 subtype (metastable cells) were detected.

[0155] In the case of Example 11, intact cells (intact CTCs) of the subtype of Type.2 (Metastable Cells) and aggregated cells (cluster CTCs) of the subtype of Type.2 (Metastable Cells), which had disappeared due to infusion of stable iodine water (100 mL / saline + 10 mL of injectable iodine), were re-detected. The cause is thought to be a reduction in the amount of stable iodine water, but the patient is continuing to receive 8,400 ppm stable iodine water at 30 mL x 3 times per day and is being observed.

[0156] Example 12 Table 24 shows information about Patient L and the course of iodine treatment, and Table 25 shows the analysis results of circulating tumor cells in the peripheral blood.

[0157] [Table 24]

[0158] [Table 25]

[0159] As shown in Table 25, the analysis results of peripheral blood circulating tumor cells on November 20, 2018 showed that one Type.1 intact cell (intact CTC), 12 Type.2 metastable cell intact cells (intact CTC), and seven Type.2 mesenchymal cells were detected. Therefore, 8,400 ppm stable iodine water was administered 30 mL x 3 times per day, and infusion (100 mL / saline + 10 mL injectable iodine water) was performed a total of 7 times.

[0160] Analysis of peripheral blood circulating tumor cells on December 18, 2018 showed that eight Type.1 intact cells (intact CTCs) were detected, but no Type.2 subtype (metastable cell) intact cells (intact CTCs) or Type.2 mesenchymal cells were detected. Therefore, the patient was administered a reduced dose of 8,400 ppm stable iodine water to 20 mL three times a day.

[0161] In the case of Example 12, it is presumed that EMT conversion was inhibited by the activation of PPAR-γ possessed by iodine, resulting in the disappearance of Type.2 mesenchymal cells and Type.2 subtype (metastable cells).

[0162] Example 13 Table 26 shows information about Patient M and the progress of iodine treatment, and Table 27 shows the analysis results of circulating tumor cells in the peripheral blood.

[0163] [Table 26]

[0164] [Table 27]

[0165] As shown in Table 27, the analysis results of peripheral blood circulating tumor cells on April 17, 2018 showed that 2 Type.1 intact cells (intact CTCs), 9 Type.2 subtype (metastable cell) intact cells (intact CTCs), 1 Type.2 subtype (metastable cell) amoeboid cell, 4 Type.2 subtype (metastable cell) aggregated cells (CTM), and 1 Type.2 mesenchymal cell were detected. Therefore, administration of 8,400 ppm stable iodine water was started at 30 mL x 3 times per day.

[0166] The analysis of peripheral blood circulating tumor cells on June 6, 2018 showed that two Type.1 intact cells (intact CTCs) were detected, but no Type.2 subtype (metastable cell) intact cells (intact CTCs), Type.2 subtype (metastable cell) amoeboid cells, Type.2 subtype (metastable cell) cell clusters (CTM), or Type.2 mesenchymal cells were detected. Chest X-ray results also returned to normal, so from June 18, 2018, the patient was administered a reduced dose of 8,400 ppm stable iodine water at 20 mL three times a day.

[0167] Analysis of peripheral blood circulating tumor cells on July 31, 2018 showed that one intact cell (intact CTC) of Type.1 and one intact cell (intact CTC) of the Type.2 subtype (metastable cell) were detected, so the patient was administered 20nL three times a day.

Claims

1. A method for predicting the efficacy of a tumor treatment by analyzing peripheral blood circulating cells of a mammalian subject, comprising: detecting peripheral blood circulating tumor cells in a blood sample obtained from the subject; If peripheral blood circulating tumor cells are detected, classifying the detected peripheral blood circulating tumor cells; To assess the amount of classified circulating tumor cells in peripheral blood and Including, Circulating tumor cells in peripheral blood are Cytokeratin-positive and vimentin-positive Type. 2 subtype, and Cytokeratin negative, Vimentin positive Type. 2 are circulating tumor cells in the peripheral blood, A reduction in the Type. 2 subtype and Type. 2 following treatment indicates that the treatment was effective. Methods (excluding medical procedures on humans).

2. A method for determining a drug dosage by analyzing peripheral blood circulating cells of a mammalian subject, comprising: detecting peripheral blood circulating tumor cells in a blood sample obtained from the subject; If peripheral blood circulating tumor cells are detected, classifying the detected peripheral blood circulating tumor cells; To assess the amount of classified circulating tumor cells in peripheral blood and Including, Circulating tumor cells in peripheral blood are Cytokeratin-positive and vimentin-positive Type. 2 subtype, and Cytokeratin negative, Vimentin positive Type. 2 are circulating tumor cells in the peripheral blood, A decrease in Type. 2 subtype and Type. 2 with treatment indicates that the dosage may be decreased, and an increase in Type. 2 subtype and Type. 2 with treatment indicates that the dosage may be increased. Methods (excluding medical procedures on humans).

3. The method of claim 1 or 2, wherein the classification further comprises classifying the peripheral blood circulating tumor cells based on whether they have an amoeboid morphology, whether the cancer cells have aggregated into clusters, or whether they form circulating tumor microemboli that form aggregates containing blood cells or platelets.

4. The method according to any one of claims 1 to 3, further comprising evaluating the amount of cytokeratin-positive, vimentin-negative Type. 1 peripheral blood circulating tumor cells.

5. The method according to claim 4, wherein the treatment increases Type. 1 circulating tumor cells in peripheral blood.

6. A method according to any one of claims 1 to 5, wherein the classification of peripheral blood circulating tumor cells includes classification of peripheral blood circulating tumor cells that have multiple nuclei, are CD45 positive, are Cytokeratin negative and Vimentin positive, and form clusters containing blood cells or platelets.

7. A method according to any one of claims 1 to 5, wherein the classification of peripheral blood circulating tumor cells includes classification of peripheral blood circulating tumor cells that have multiple nuclei, are CD45 negative, are Cytokeratin negative and Vimentin positive, and form clusters.

8. The method according to any one of claims 1 to 5, wherein the classification of peripheral blood circulating tumor cells includes classification of peripheral blood circulating tumor cells that have a nucleus, are CD45 negative, are Cytokeratin negative and Vimentin positive, and have an amoeba-like shape.

9. A method according to any one of claims 1 to 5, wherein the classification of peripheral blood circulating tumor cells includes classification of peripheral blood circulating tumor cells having a spherical or elliptical nucleus, being CD45 negative, and being Cytokeratin negative and Vimentin positive.

10. The classification of the peripheral blood circulating tumor cells is that they have multiple nuclei, are CD45 positive, are cytokeratin positive and vimentin positive, and form clusters containing blood cells or platelets. The method according to any one of claims 1 to 5, comprising sorting of peripheral blood circulating tumor cells.

11. The classification of the peripheral blood circulating tumor cells is that they have multiple nuclei, are CD45 negative, are cytokeratin positive and vimentin positive, and form clusters. The method according to any one of claims 1 to 5, comprising sorting of peripheral blood circulating tumor cells.

12. The classification of the peripheral blood circulating tumor cells is that they have nuclei, are CD45 negative, are cytokeratin negative and vimentin positive, and have an amoeba-like shape. The method according to any one of claims 1 to 5, comprising sorting of peripheral blood circulating tumor cells.

13. A method according to any one of claims 1 to 5, wherein the classification of peripheral blood circulating tumor cells includes classification of peripheral blood circulating tumor cells having a spherical or elliptical nucleus, being CD45 negative, and being Cytokeratin positive and Vimentin positive.

14. The classification of the peripheral blood circulating tumor cells is that they have multiple nuclei, are CD45 positive, are cytokeratin positive and vimentin negative, and form clusters containing blood cells or platelets. The method according to any one of claims 1 to 5, comprising sorting of peripheral blood circulating tumor cells.

15. The classification of the peripheral blood circulating tumor cells is that they have multiple nuclei, are CD45 negative, are cytokeratin positive and vimentin negative, and form clusters. The method according to any one of claims 1 to 5, comprising sorting of peripheral blood circulating tumor cells.

16. The classification of the peripheral blood circulating tumor cells is that they have a spherical or elliptical nucleus, are CD45 negative, are cytokeratin positive and vimentin negative, and form clusters containing blood cells or platelets. The method according to any one of claims 1 to 5, comprising sorting of peripheral blood circulating tumor cells.

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