Production method of cancer model animal

Abstract

To provide a cancer model animal and a production method of the cancer model animal.SOLUTION: Provided is a production method of a cancer model animal including: a step A of collecting a circulating tumor cell (CTC) from a subject; a step B of culturing the CTC to prepare spheroid; and a step C of transplanting the spheroid to an animal other than a human. A step P of measuring the number of nucleotide mutation on genomic DNA by using a part of the CTC is performed after the step A and before the step C.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cancer model animal, and in particular to a method for producing a cancer model animal using patient-derived circulating cancer cells. [Background technology]

[0002] Cancer cell kinetic analysis and drug efficacy evaluation using general cancer cell lines do not reflect individual patient differences. Therefore, there is a need to perform patient-specific cancer tissue kinetic analysis and drug efficacy evaluation. Previously, analysis of cancer cell dynamics and evaluation of the effectiveness of treatment methods using patient-derived xenograft (PDX) models, in which cancer tissue collected from patients was transplanted into mice, have been reported (Non-Patent Documents 1 and 2). However, collecting cancer tissue directly from patients is highly invasive and poses challenges such as the burden on patients and the risk of cancer tissue metastasis.

[0003] Patent Document 1 reports the creation of a model in which circulating cancer cells (CTCs), which are cancer cells released from a patient's blood, are expanded and cultured into spheroids, and then transplanted into mice to transplant CTC-derived tissue. This type of model is less invasive because cancer tissue is not directly collected from the patient, but the success rate of creation is extremely low. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2017079632 [Non-patent literature]

[0005] [Non-Patent Document 1] Patient-derived tumor xenografts as models for oncology drug development. Nature Reviews Clinical Oncology,Vol.9,2012,pp338-350 [Non-Patent Document 2] The future of patient-derived xenografts in prostate cancer research. NatureReviews Urology volume,Vol.20,2023,p.p.371-384 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] An object of the present invention is to provide a cancer model animal and a method for producing the same. In particular, an object of the present invention is to provide a cancer model animal using patient-derived circulating cancer cells (CTCs) and a method for producing the same. [Means for Solving the Problems]

[0007] As a result of intensive studies to solve the above problems, the present inventors have found that when the number of nucleotide mutations on the genomic DNA of CTCs derived from a certain subject is small, spheroids or organoids can be cultured from the CTCs derived from the subject and transplanted into an animal to produce a cancer model animal, or the production efficiency of the cancer model animal is improved, and the present invention has been completed. The present invention includes the following aspects.

[0008] [1] A method for producing a cancer model animal, comprising: step A of collecting circulating cancer cells (CTCs) from a subject; step B of culturing the CTCs to produce spheroids; step C of transplanting the spheroids into an animal other than a human, and characterized in that a step P of measuring the number of nucleotide mutations on genomic DNA is performed using a part of the CTCs after step A and before step C. [2] The method according to [1], wherein the cancer model animal and the animal other than a human are mice.[3] The method of [1] or [2], wherein the subject is a human cancer patient. [4] The method according to any one of [1] to [3], wherein the number of nucleotide mutations on the genomic DNA is the number of nucleotide mutations on the genomic DNA of the CTCs, based on the genomic DNA of normal cells collected from the subject. [5] The method of [4], wherein the normal cells are white blood cells. [6] The method according to any one of [1] to [5], wherein in step P, the number of nucleotide mutations present at any locus among the nucleotide mutations on the genomic DNA is measured. [7] The method according to any one of [1] to [6], wherein in step P, the number of nucleotide mutations present at any locus among the nucleotide mutations on the genomic DNA and causing one or more amino acid mutations in the amino acid sequence encoded by the locus where the mutation is present is measured. [8] The method according to any one of [1] to [7], wherein in step P, the number of nucleotide mutations present at any locus among the nucleotide mutations on the genomic DNA and causing one or more amino acid mutations that change the function of the protein having the amino acid sequence encoded by the locus where the mutation is present is measured. [9] The method according to any one of [6] to [8], wherein the locus is one or more loci selected from the following (i); (i) CDK6, FOXP1, FANCA, SF3B1, ARID2, ZMYM3, ATR, FANCD2, BRCA2, PIK3CB, MDM2, AKT2, AKT1, AKT3, PALB2, CTNNB1, FANCC, MET, USP7, FANCG, CCND1, FANCD2OS, NBN, BRIP1, MRE11, RYBP, and PIK3CA.

[10] In the step P, instead of measuring the number of nucleotide mutations on genomic DNA, any method according to any one of [1] to [5], which measures the number of loci at which one or more nucleotide mutations are present.

[11] In the step P, instead of measuring the number of nucleotide mutations on genomic DNA, any method according to any one of [1] to [5] and

[10] , which measures the number of loci at which the nucleotide mutations present at the loci are nucleotide mutations that cause amino acid mutations of one or more residues in the amino acid sequence encoded by the locus where the nucleotide mutation is present.

[12] In the step P, instead of measuring the number of nucleotide mutations on genomic DNA, any method according to any one of [1] to [5] and

[10] to

[11] , which measures the number of loci at which the nucleotide mutations present at the loci are nucleotide mutations that cause amino acid mutations of one or more residues in the amino acid sequence encoded by the locus where the nucleotide mutation is present and change the function of the protein having the amino acid sequence.

[13] Any method according to any one of

[10] to

[12] , wherein the locus is one or more loci selected from the following (i); (i) CDK6, FOXP1, FANCA, SF3B1, ARID2, ZMYM3, ATR, FANCD2, BRCA2, PIK3CB, MDM2, AKT2, AKT1, AKT3, PALB2, CTNNB1, FANCC, MET, USP7, FANCG, CCND1, FANCD2OS, NBN, BRIP1, MRE11, RYBP, and PIK3CA.

Brief Description of Drawings

[0009]

Figure 1

Figure 2

Figure 3

Embodiments for Carrying Out the Invention

[0010] <The method of the present invention> One aspect of the method of the present invention is step A of collecting circulating tumor cells (CTCs) from a subject, step B of culturing the CTCs collected from the subject to produce spheroids, step C of transplanting the spheroids into non-human animals, and A method for producing a cancer model animal, characterized in that a step P of measuring the number of nucleotide mutations on genomic DNA is carried out using a part of the CTCs after step A and before step C.

[0011] The "animal" in "cancer model animal" is not particularly limited as long as it is a non-human mammal. Non-human mammals may specifically be, for example, mice, rats, cows, pigs, sheep, goats, chickens, and preferably mice.

[0012] The "cancer" in "cancer model animal" may be any cancer. Specifically, the cancer may be, for example, prostate cancer, pancreatic cancer, liver cancer, breast cancer, gastric cancer, or colorectal cancer, but is not limited thereto.

[0013] In step A, CTCs are collected from the subject. Step A may be a step of preparing CTCs collected from the subject.

[0014] The "subject" may be a human or a non-human mammal and is not particularly limited, but is preferably a human. When the subject is a human and suffers from a disease or the like, the subject may be referred to as a patient. In the present invention, the subject is preferably a subject suffering from any cancer. When the subject is a human and suffers from any cancer, the subject may be referred to as a cancer patient.

[0015] "Non-human mammals" may specifically be, for example, mice, rats, cows, pigs, sheep, goats, chickens, and are preferably mice.

[0016] "Circulating tumor cells (CTC)" in the blood refer to cancer cells found in the blood. CTCs are typically cancer cells that have detached from a cancer tissue into the blood. Any known method may be used to collect CTCs from a subject. Specifically, the collection of CTCs from a subject may be performed, for example, by collecting blood from the subject, concentrating CTCs by performing hemolysis of red blood cells and removal of white blood cells using a white blood cell surface marker antibody on the collected blood, and collecting C TCs using a cancer marker antibody. The concentration of CTCs may more specifically be performed, for example, by the method described in Patent No. 6364946. Step A may include a step of collecting CTCs from the blood of a subject. Step A may include a step of concentrating CTCs collected from the blood of a subject. Step A may include a step of collecting and concentrating CTCs from the blood of a subject.

[0017] "Cancer" in "circulating tumor cells (CTC)" is consistent with "cancer" in "cancer model animal". The details are as previously described.

[0018] The present invention is characterized by performing step P of measuring the number of nucleotide mutations on genomic DNA using a part of the CTCs collected from a subject after step A and before step C. A part of the CTCs collected from a subject may mean a part of the number of cells among a plurality of CTCs collected from the subject.

[0019] Step P may be performed after step A and before step C, and the order with respect to step B is not limited. For example, step P may be performed before step B, after step B, or concurrently with step B in terms of the time axis, but is not particularly limited.

[0020] "Genomic DNA" refers to the DNA in a cell that encodes all of the genetic information of an organism. Genomic DNA can be recovered from cells. The method for recovering genomic DNA from cells may use any known technique and is not particularly limited. Specifically, genomic DNA may be recovered, for example, by disrupting the cell membrane of the cells and removing contaminants. Such techniques include, but are not limited to, the organic solvent method, the spin column method, the magnetic bead method, and the like.

[0021] "Nucleotide variation on genomic DNA" refers to a variation found by comparing the base sequence of the genomic DNA of a certain cell with a reference base sequence. The reference base sequence is not particularly limited as long as it is a base sequence to be used as a comparison target for determining the presence or absence of a variation. Specifically, the reference base sequence may be, for example, the base sequence of genomic DNA on a database or the base sequence of genomic DNA of normal cells. The normal cells may be cells collected from the same type of subject as the subject, preferably somatic cells collected from the subject, and more preferably leukocytes collected from the subject.

[0022] "Nucleotide mutations on genomic DNA" may be substitutions, insertions, or deletions of one or more nucleotides. Nucleotide mutations on genomic DNA may be mutations contained in the entire genomic DNA, i.e., the whole genomic DNA, or may be mutations contained in a specific region of genomic DNA. The specific region of genomic DNA may specifically be, for example, a region containing a region having a cancer-related gene. The specific region of genomic DNA may be, for example, a region containing one or more, five or more, ten or more, twenty or more, thirty or more, fifty or more, seventy or more, eighty or more, one hundred or more, five hundred or more, or one thousand or more regions having cancer-related genes. Note that the upper limit of the specific region of genomic DNA may be the entire genomic DNA, may be a region covering all genes contained in the genomic DNA, or may be a region covering a region having a cancer-related gene. The cancer-related gene may be any cancer-related gene and is not particularly limited, but is preferably a cancer-related gene of cancer derived from CTC (for example, when CTC is a cell derived from prostate cancer, prostate cancer). The specific region of genomic DNA may specifically be, for example, a region containing one or more genes selected from CDK6, FOXP1, FANCA, SF3B1, ARID2, ZMYM3, ATR, FANCD2, BRCA2, PIK3CB, MDM2, AKT2, AKT1, AKT3, PALB2, CTNNB1, FANCC, MET, USP7, FANCG, CCND1, FANCD2OS, NBN , BRIP1, MRE11, RYBP, and a region containing one or more genes selected from PIK3CA. Also, when one or more arbitrary nucleotide mutations exist in a specific region on the genome, the entire range per unit of the specific region may be regarded as one unit of nucleotide mutation.

[0023] The "cancer-related gene" is, for example, a gene having a relevance to cancer, such as causing the occurrence, maintenance, and / or progression of cancer through loss of function or constitutive activation due to mutations or the like. The cancer-related gene may be a gene whose relevance to cancer is known, or may be a gene newly suggested to have a relevance to cancer.

[0024] Nucleotide mutations on genomic DNA may be present at any locus. Examples of loci include the loci of the genes exemplified above, such as cancer-related genes. In one aspect of the present invention, in step P, the number of nucleotide mutations present at any locus among the nucleotide mutations on genomic DNA may be measured. In another aspect of the present invention, in step P, instead of the number of nucleotide mutations on genomic DNA, the number of loci at which one or more arbitrary nucleotide mutations are present may be measured. In this case, the region per locus at which one or more arbitrary nucleotide mutations are present may be regarded as one unit of nucleotide mutation as a whole.

[0025] The nucleotide mutation on genomic DNA may be a mutation that causes one or more amino acid mutations in the amino acid sequence encoded by the locus where the mutation is present when it is present at any locus. Further, the nucleotide mutation on genomic DNA may be a nucleotide mutation present at any locus in the above-specified region, or a nucleotide mutation present at any locus that causes one or more amino acid mutations in the amino acid sequence encoded by the locus where the nucleotide mutation is present. "Causing one or more amino acid mutations in the amino acid sequence encoded by the locus where the nucleotide mutation is present" means that when a mutation is present in the DNA of a certain locus, the amino acid sequence transcribed and translated from that locus contains one or more amino acid substitutions, insertions, and / or deletions as compared with the case where there is no mutation at that locus. Such mutations typically include, for example, a change in the codon due to a substitution of the base sequence that specifies another amino acid or a stop codon, or a frameshift of the codon due to a deletion or insertion of the base sequence, but are not limited thereto. Also, such a mutation may be called a missense mutation.

[0026] The nucleotide variation on genomic DNA may further be a nucleotide variation at any locus in the above-specified region, or a nucleotide variation at any locus that is involved in regulating the expression level of the encoded protein. Examples of the region of the locus involved in regulating the expression level of the protein include the 5'-untranslated region of the locus. The region of the locus involved in regulating the expression level of the protein may include, for example, a transcription factor binding motif, a transcription initiation motif, and the like.

[0027] The nucleotide variation on genomic DNA may further be a variation such that the amino acid variation changes the function of the protein having the amino acid sequence in which the amino acid variation exists. Specifically, when the nucleotide variation on genomic DNA exists at any locus, it may be a variation that causes one or more amino acid variations that change the function of the protein having the amino acid sequence encoded by the locus where the variation exists in the amino acid sequence. Such an amino acid variation may be referred to as a LoF (Loss of Function) variation. At this time, "changing the function of the protein" means that the protein having the amino acid sequence in which the amino acid variation exists changes its function as compared with the case where such an amino acid variation does not exist. Note that, "changing the function" may include cases where the activity is lost, or not only the activity decreases but also the activity is constitutively activated, the threshold for activation decreases, etc., that is, cases where the regulatory function is lost or the regulatory function changes.

[0028] The measurement of the number of nucleotide mutations on the genomic DNA of CTCs is not particularly limited as long as the number of nucleotide mutations on the genomic DNA of CTCs can be obtained. For example, it may be carried out by analyzing the base sequence of the genomic DNA of CTCs, identifying mutations by comparing the differences with a reference base sequence, and counting the number of mutations. Note that the number of nucleotide mutations on genomic DNA, even if the mutations on genomic DNA are substitutions, insertions, or deletions of two or more bases, continuous mutations are counted as one mutation. The analysis of the base sequence of genomic DNA is not particularly limited as long as mutations can be identified and may be carried out by any known method. Specifically, the analysis of the base sequence of genomic DNA may be carried out, for example, by amplifying the recovered genomic DNA, preparing a library, and sequencing it.

[0029] The present invention includes step B of culturing CTCs collected from a subject to produce spheroids.

[0030] A "spheroid" is a mass of cells having a three-dimensional structure. In the present invention, the spheroid includes not only a three-dimensional cell mass composed of a simple aggregate of cells, but also an organoid having a complex structure similar to that of a tissue and / or organ, or a cell mass similar thereto.

[0031] The method for culturing CTCs in step B is not particularly limited as long as spheroids can be produced. Specifically, the method for culturing CTCs may be, for example, seeding CTCs on a non-adherent or low-adherent surface plate and culturing until spheroids are formed. The culturing period of CTCs is not particularly limited as long as spheroids are formed. Specifically, the culturing period of CTCs may be, for example, 1 day or more, 3 days or more, 7 days or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 6 weeks or more, or 2 months or more, and may be 6 months or less, 4 months or less, 3 months or less, or 2 months or less, and may be any non-contradictory combination thereof.

[0032] The medium for culturing CTCs is not particularly limited as long as it can produce spheroids. The medium for culturing CTCs may be, for example, a serum-free medium. Specifically, as the medium for culturing CTCs, for example, DMEM / F-12 medium or Advanced DMEM / F-12 medium may be used as the basal medium. When the medium for culturing CTCs is a serum-free medium, although not limited thereto, it may contain any serum substitute. The serum substitute is not particularly limited and may be a commercially available one. Specifically, for example, it may be B-27 supplement. The medium for culturing CTCs may contain other known additives. Specifically, the medium for culturing CTCs may contain, for example, a buffer such as HEPES, an amino acid such as L-glutamine, a vitamin such as nicotinamide, a physiologically active substance such as EGF, FGF (such as FGF2 and FGF10), Noggin, R-Spondin, dihydrotestosterone (DHT), and / or prostaglandin (PGF: such as PGF2), a compound such as A83-01 and / or Y-27632, or any combination thereof. min, and may contain a physiologically active substance such as EGF, FGF (such as FGF2 and FGF10), Noggin, R-Spondin, dihydrotestosterone (DHT), and / or prostaglandin (PGF: such as PGF2), a compound such as A83-01 and / or Y-27632, or any combination thereof.

[0033] The CTCs used for culturing in step B are the CTCs recovered from the same subject as the subject from which the CTCs whose nucleotide mutations on genomic DNA are measured in step P are recovered. The CTCs used for culturing in step B are preferably the remaining part of the CTCs recovered from the subject, with a part of the CTCs recovered from the subject being subjected to step P, that is, the CTCs of the same lot as the CTCs whose mutation number on genomic DNA was measured in step P.

[0034] The present invention further includes step C of transplanting the spheroids produced in step B into an animal.

[0035] "Animal" is consistent with "animal" in "cancer model animal". The details are as described above.

[0036] Any method can be used to transplant spheroids into animals. Specifically, for example, transplantation of spheroids into animals may be performed by subcutaneous injection of the spheroids into immunodeficient mice.

[0037] The present invention is based on the finding that when the number of mutations on the genomic DNA of CTCs collected from a subject is below a certain level, CTCs collected from the subject can be cultured to produce spheroids, and a cancer model animal can be produced or the production efficiency can be improved by transplanting the spheroids into animals other than humans.

[0038] Therefore, in the present invention, when the number of mutations on the genomic DNA of CTCs measured in step P is below a certain level, CTCs may be cultured to produce spheroids, and the spheroids may be transplanted into animals other than humans, that is, steps B and C may be performed. By measuring the number of nucleotide mutations on the genomic DNA of CTCs derived from a certain subject in step P, it is possible to determine whether it is possible to produce a model animal transplanted with CTC-derived cells, cell masses or tissues with a significantly low production success rate, or whether the production efficiency can be improved, when using CTCs collected from the subject.

[0039] The number of nucleotide mutations on the genomic DNA of CTCs measured in step P may be below a certain level. Specifically, when the number of nucleotide mutations on the genomic DNA of CTCs measured in step P is below a certain level, for example, the number of nucleotide mutations on the genomic DNA may be 120 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 3 or less, 2 or less, 1 or less, or 0.

[0040] In addition, when the nucleotide mutation on the genomic DNA is a mutation present at any locus and causes one or more amino acid mutations in the amino acid sequence encoded by the locus where the mutation is present, the number of nucleotide mutations on the genomic DNA of CTC measured in step P may specifically be, for example, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 3 or less, 2 or less, 1 or less, or 0. Further, when the nucleotide mutation on the genomic DNA is a mutation present at any locus and causes one or more amino acid mutations that change the function of the protein having the amino acid sequence in the amino acid sequence encoded by the locus where the mutation is present, the number of nucleotide mutations on the genomic DNA of CTC measured in step P may specifically be, for example, 20 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 3 or less, 2 or less, 1 or less, or 0.

[0041] The locus in the present invention may be one or more loci selected from the following (i); (i) CDK6, FOXP1, FANCA, SF3B1, ARID2, ZMYM3, ATR, FANCD2, BRCA2, PIK3CB, MDM2, AKT2, AKT1, AKT3, PALB2, CTNNB1, FANCC, MET, USP7, FANCG, CCND1, FANCD2OS, NBN, BRIP1, MRE11, RYBP, and PIK3 CA.

[0042] <2>The cancer model animal of the present invention The cancer model animal produced by the method of the present invention is expected to have a cancer tissue derived from the CTC of the subject. Further, by the method of the present invention, it is expected to enable the production of a cancer model animal using the CTC of the subject or to increase the production efficiency.

[0043] The spheroids and / or cancer model animals produced in the present invention can be used for analyzing the behavior of cancer and evaluating the efficacy of drugs in vitro or in vivo. Therefore, the method of the present invention may further include a step of performing analysis of the behavior of cancer, evaluation of the therapeutic efficacy of cancer, and / or evaluation of the efficacy of cancer therapeutic drugs using the produced cancer model animals. In addition, the cancer model animals produced by the method of the present invention may be used for analysis of the behavior of cancer, evaluation of the therapeutic efficacy of cancer, and / or evaluation of the efficacy of cancer therapeutic drugs.

[0044] Tumor tissue may be removed from the cancer model animals produced in the present invention, and a part of it may be transplanted into animals of another newly prepared individual (for example, mice) to passage the cancer tissue derived from the target CTC. Maintaining the cancer tissue derived from the target CTC in the cancer model animal by passage may be referred to as the establishment of a CTC transplantation model animal. For example, by the method of the present invention, a CTC transplantation model animal, particularly a CTC transplantation mouse, may be established.

Examples

[0045] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following embodiments. After collecting CTC, genomic DNA analysis was performed, and an example with a large number of nucleotide mutations in the genomic DNA was used as a comparative example, and an example with a small number was used as an example for explanation. The collection of CTC, the production of spheroids, the production of cancer model mice, and the examples of genomic DNA were all performed by the common methods shown below.

[0046] Collection of CTC CTC was collected by the following method. (1) 180 μL of physiological saline containing 0.5 mg / mL tirofiban and 1 mg / mL ethylenediaminetetraacetic acid dipotassium (EDTA-2K) was added to 3 mL of blood collected from a prostate cancer patient who had given informed consent to prepare a blood sample. (2) 200 μL of magnetic particles modified with an antibody against CD15, a surface antigen of leukocytes (Dynabeads CD15, manufactured by ThermoFisher) and 200 μL of magnetic particles modified with an antibody against CD45, a surface antigen of leukocytes (Dynabeads CD45, manufactured by ThermoFisher) were added to 800 μL of PBS containing 2% (w / v) BSA, and magnetic particle blocking was performed by rotating and stirring at room temperature for 10 minutes. After the blocking was completed, the magnetic particle solution was placed near a magnet for 3 minutes, and after removing the solution, 100 μL of PBS containing 2% (w / v) BSA was added to resuspend the magnetic particles. (3) An aqueous solution containing 0.9% (w / v) ammonium chloride and 0.1% (w / v) potassium hydrogen carbonate was added to the blood sample prepared in (1) until the total volume reached 90 mL, and then left standing at room temperature for 5 minutes to lyse the red blood cells (hemolysis). After the hemolysis treatment, centrifugation was performed at 900×g for 5 minutes at 25 °C. (4) After removing 88 mL of the supernatant after centrifugation, the pellet containing CTCs and contaminating cells (such as leukocytes and platelets) was resuspended in the remaining supernatant by pipetting. (5) PBS containing 2% (w / v) BSA was added to the cell suspension in (4) until the total volume reached 30 mL, and then centrifuged at 600×g for 5 minutes at 25 °C. 29 mL of the obtained supernatant was removed, and the pellet was resuspended in the remaining supernatant by pipetting. (6) After mixing the magnetic particle solution prepared in (2) with the total amount of the cell suspension obtained in (5), leukocytes were bound to the magnetic particles by rotating and stirring at room temperature for 5 minutes. The magnetic particles to which leukocytes were bound were brought close to a magnet for 1 minute, and the supernatant containing CTCs was collected. (7) PBS was added to the cell suspension collected in (6) until the total volume reached 30 mL, and then centrifuged at 600×g for 5 minutes at 25 °C. The obtained supernatant was removed to obtain CTCs.

[0047] Preparation of spheroids Using the CTCs obtained in the above "Collection of CTCs", spheroids were prepared by the following method. (1) Add 1.5 mL of Matrigel to CTC, mix by pipetting, and drop approximately 40 μL per well into a 24-well plate. Incubate the Matrigel containing CTC at 37 °C for 45 minutes in a 5% CO2 environment to solidify it into a dome shape. (2) Add 500 μL of Advanced DMEM / F-12 medium (used as the culture solution) containing 10 mM HEPES, 2 mM Glutamax, 2% (w / v) B-27, 10 mM nicotinamide, 5 ng / mL EGF, 500 nM A83-01, 100 ng / mL Noggin, 500 ng / mL R-Spondin, 1 nM DHT, 5 ng / mL FGF2, 10 ng / mL FGF10, 1 μM PGE2, and 10 μM Y-27632 to the CTC Matrigel obtained in (1), and culture at 37 °C in a 5% CO2 environment. Replace the culture solution once every 2 to 3 days and passage once every 1 to 4 weeks to obtain CTC spheroids.

[0048] Preparation of cancer model mice Collect the CTC spheroids prepared in the above "Preparation of spheroids", and centrifuge at 2500 rpm for 5 minutes at room temperature. Remove the supernatant, and transplant the pellet containing CTC spheroids (about 1×10 7 cells) into the subcutaneous tissue of 2 4-week-old male immunodeficient mice (NSG mice) to prepare cancer model mice. Approximately 6 months after transplantation, when a palpable tumor formation of about 1 cm was confirmed, the mice were euthanized and tumor tissue pieces were excised.

[0049] Analysis of genomic DNA The genomic DNA analysis of CTC was performed on a per-cell basis. First, CTC single cells were collected by the following method. (1-1) Polyethylene glycol with a molecular weight of 5000 (mPEG-NHS) where one end is a methoxy group and the other end is an N-hydroxysuccinimide ester group, and bovine serum albumin (BSA) (300 mg, 0.3 mmol) were dissolved in sodium bicarbonate buffer (0.1 M, 15 mL), and the solution was stirred at around 25 °C for 3 hours to prepare BSA conjugated with polyethylene glycol (PEG-BSA). When preparing, the molar ratio of mPEG-NHS to BSA (mPEG-NHS / BSA) was set to 2. After preparation, solution replacement with pure water was carried out for 3 days using a dialysis membrane with a molecular weight cut-off of 10,000. (1-2) 0.75 mL of a stabilizer was added to 5 mL of blood collected from a prostate cancer patient and stored at 4 °C for 5 days. The stabilizer was prepared by dissolving 3.8 g of imidazolidinyl urea, 384 mg of polyethylene glycol (PEG) with a molecular weight of 600, 50 mg of EDTA-2K, and 32 mg of tirofiban in PBS to make 50 mL as a solution. (1-3) 100 μL of magnetic particles modified with an antibody against CD15, a surface antigen of leukocytes (Dynabeads CD15, manufactured by ThermoFisher) and 100 μL of magnetic particles modified with an antibody against CD45, a surface antigen of leukocytes (Dynabeads CD45, manufactured by ThermoFisher) were added to 800 μL of PBS containing 2% (w / v) BSA, and magnetic particle blocking was performed by rotating and stirring at room temperature for 10 minutes. After the blocking was completed, the magnetic particle solution was placed near a magnet for 3 minutes, the solution was removed, and then 100 μL of PBS containing 2% (w / v) BSA was added to resuspend the magnetic particles. (1-4) 3 mL of the blood sample prepared in (1-2) was used, and an aqueous solution containing 0.9% (w / v) ammonium chloride and 0.1% (w / v) potassium bicarbonate was added until the total volume reached 90 mL, and then left standing at room temperature for 5 minutes to lyse the red blood cells (hemolysis). After the hemolysis treatment, centrifugation was performed at 900×g for 5 minutes at 25 °C. (1-5) After removing 88 mL of the supernatant after centrifugation, the pellet containing CTCs and contaminating cells (such as leukocytes and platelets) was suspended in the residual supernatant by pipetting. (1-6)(1-5)'s cell suspension was added with PEG-BSA prepared in (1-1) (0.1% (w / v) as BSA) and PBS containing 2% (w / v) BSA until the total volume reached 30 mL, and then centrifuged at 600×g for 5 minutes at 25°C. 29 mL of the obtained supernatant was removed, and the pellet was resuspended in the remaining supernatant by pipetting. After mixing the magnetic particle solution prepared in (1-7)(1-3) with the total amount of the cell suspension obtained in (1-6), the magnetic particles were allowed to bind to white blood cells by rotating and stirring at room temperature for 5 minutes. The magnetic particles bound with white blood cells were brought close to a magnet for 1 minute, and the supernatant containing CTC was collected. (1-8) To the cell suspension collected in (1-7), an aqueous solution containing PEG-BSA (0.1% (w / v) as BSA) and 280 mM xylitol (used as a substitution solvent) was added until the total volume reached 30 mL, and then centrifuged at 600×g for 5 minutes at 25°C. This operation is to reduce the salt concentration in the solution and concentrate the target CTC. After removing 25 mL of the supernatant after centrifugation in (1-9), the substitution solvent was added until the total volume reached 30 mL, and then centrifuged at 600×g for 5 minutes at 25°C.

[0050] (1-10) The same operation as in (1-9) was performed again. After removing the supernatant after centrifugation in (1-11) and making the remaining volume 650 μL, the pellet containing CTC was resuspended by pipetting to obtain a cell suspension. After holding the suspension in the cell holding device 100 shown in FIGS. 1 and 2 by the method shown below, the target cells were detected. The cell holding device 100 is provided with a holding portion 170 having a diameter of φ30 μm and a depth of 40 μm. After introducing the cell suspension from the introduction portion 131 in (1-12), an alternating voltage (voltage 20 Vpp, frequency 1 MHz, rectangular wave) was applied from the AC power supply 160 to each of the electrodes 141 and 142, and the cells were held in the holding portion 170 by dielectrophoretic force. (1-13) An aqueous solution of 280 mM xylitol containing 0.01% (w / v) poly-L-lysine was introduced from the introduction section 131 while applying the alternating voltage. After standing for 3 minutes, the application of the alternating voltage was stopped, and the aqueous solution was aspirated and removed from the discharge section 132. (1-14) A PBS solution containing 1% formaldehyde was introduced from the introduction section 131, and the cells were fixed by standing for 10 minutes. Then, the reagent was aspirated and removed from the discharge section 132. Thereafter, a PBS solution containing 0.05% (w / v) Tween 20 (trade name) (hereinafter referred to as PBS-T) was introduced from the introduction section 131 to wash the remaining reagent. (1-15) An aqueous solution containing 95% (v / v) ethanol was introduced from the introduction section 131, and the cells were permeabilized by standing for 10 minutes. Then, the reagent was aspirated and removed from the discharge section 132. Thereafter, PBS-T was introduced from the introduction section 131 to wash the remaining reagent. (1-16) A PBS solution containing 10% (v / v) Goat Serum and 3% (w / v) BSA was introduced from the introduction section 131, and the cells were blocked by standing for 10 minutes. Then, the reagent was aspirated and removed from the discharge section 132. (1-17) A cell labeling reagent obtained by mixing an anti-cytokeratin (CK) mouse antibody (manufactured by Miltenyi Biotec), 10% (v / v) Goat Serum, and 3% (w / v) BSA was introduced from the introduction section 131. After labeling CTCs by standing for 30 minutes, the reagent was aspirated and removed from the discharge section 132. Thereafter, PBS-T was introduced from the introduction section 131 to wash the remaining reagent. (1-18) From the introduction section 131, Alexa Fluor 488-labeled anti-mouse IgG1 antibody (manufactured by Thermo Fisher Scientific), PE (phycoerythrin)-labeled anti-CD45 antibody (manufactured by Miltenyi Biotec), DAPI (4’,6-DiAmidino-2-PhenylIndole) (manufactured by Dojindo Laboratories), 1 A cell staining reagent mixed with 0% (v / v) Goat Serum and 3% (w / v) BSA was introduced, and the cells were stained by allowing them to stand for 20 minutes. Then, the cell staining reagent was aspirated and removed from the discharge port 132. Thereafter, the remaining reagent was washed by introducing PBS-T from the introduction part 131. (1-19) To observe all the cells held in the holding part 170, bright-field images and fluorescence images of all the holding parts were taken using a fluorescence microscope (IX71 manufactured by Olympus Corporation) equipped with a computer-controlled electric stage and a CMOS camera (ORCA-Flash4.0 manufactured by Hamamatsu Photonics K.K.). (1-20) The images taken in (1-19) were analyzed using the analysis software LabVIEW (manufactured by National Instruments Corporation). Cells stained with DAPI (having cell nuclei), stained with Alexa Fluor 488 (expressing CK), and not stained with PE (not expressing CD45) were defined as CK-positive CTCs. Cells stained with DAPI, not stained with Alexa Fluor 488, not stained with PE, and having a cell diameter of 15 μm or more were defined as CK-negative large-diameter CTCs. Cell clusters in which multiple cells were aggregated in a cluster shape, stained with DAPI, not stained with Alexa Fluor 488, and not stained with PE were defined as CK-negative cluster CTCs. Table 1 shows the detected numbers of CK-positive CTCs, CK-negative large-diameter CTCs, and CK-negative cluster CTCs in the patients of Comparative Example 1 and Example 1 described below. The threshold value shown in Table 1 is a value obtained by adding the standard deviation of the number of false-positive cells multiplied by 3 to the average value of the number of false-positive cells detected from the blood of 10 healthy individuals. When the number of CTCs exceeding the threshold value is detected from a patient blood sample, it can be said that the patient is likely to have cancer. In particular, CK-negative CTCs are likely to be cancer cells that have lost epithelial properties and acquired mesenchymal properties. Therefore, it was confirmed that they are cells derived from cancers with higher metastatic potential and malignancy.

[0051]

Table 1

[0052] (1-21) As shown in Fig. 3, using the micro manipulator 600 which is a suction means, one by one, some of the CK-positive CTCs, CK-negative large-diameter CTCs, and CK-negative cluster CTCs held in the holding part 170 of the particle holding device 100 from which the upper electrode substrate 142 has been removed were suctioned, and discharged into a 0.2 mL microtube, thereby collecting CTCs one by one.

[0053] Next, for the CTCs collected one by one, genomic DNA was analyzed for each cell by the following method. (2-1) Using the Ampli1 WGA kit (manufactured by Silicon Biosystems), whole genome amplification was performed for each of the CTCs collected in (1-21), and respective whole genome amplification products were obtained. (2-3) Using 200 μL of the blood sample prepared in (1-2), genomic DNA was extracted from white blood cells using NucleoSpin Tissue (manufactured by Takara Bio Inc.). (2-4) For the obtained whole genome amplification products of CTCs and the genomic DNA of white blood cells, library preparation was performed using the Prostate Tumor Custom Panel and the Ion AmpliSeq Library Kit Plus (both manufactured by Thermo Fisher Scientific). The Prostate Tumor Custom Panel was designed to target the exons and UTR regions of 88 genes related to prostate cancer. (2-5) For the prepared library, after template preparation using the Ion Chef system (manufactured by Thermo Fisher Scientific), gene mutations were analyzed using the Ion GeneStudio S5 system (manufactured by Thermo Fisher Scientific). (2-6) Using the data analysis software VarSeq (manufactured by Golden Helix), by subtracting the gene mutations detected from white blood cells (germline mutations) from the gene mutations detected from each single-cell CTC, single-cell CTC-specific gene mutations were detected. (2-6) Using the data analysis software VarSeq (manufactured by Golden Helix), by subtracting the gene mutations detected from white blood cells (germline mutations) from the gene mutations detected from each single-cell CTC, single-cell CTC-specific gene mutations were detected.

[0054] Collection of CTCs from patients, their analysis, and generation of cancer model mice <Comparative Example 1> Genomic DNA analysis was performed on one prostate cancer patient as described above. This patient was used as the patient in Comparative Example 1. A total of 7 CTCs were collected from the patient in Comparative Example 1, and genomic DNA was analyzed for each of these CTCs, one cell at a time (N = 7). The detected numbers of CK-positive CTCs, CK-negative large-diameter CTCs, and CK-negative clustered CTCs in the patient of Comparative Example 1 are as described in Table 1. The genomic DNA analysis results of CTCs were compared with those of white blood cells, and the number of genomic DNA mutations in each of the 7 CTCs was measured. Table 2 shows the types and numbers of CTC types and genomic DNA mutations.

[0055]

Table 2

[0056] A large number of mutations were found in the genomic DNA of the CTCs collected from the patient in Comparative Example 1. Subsequently, for the CTCs of this patient, an attempt was made to generate a cancer model animal using spheroids derived from the CTCs according to the above "spheroid generation" and "cancer model animal generation". However, at least palpable tumor formation was never confirmed, and no successful cases of such cancer model animals were obtained.

[0057] <Example 1> Genomic DNA analysis was performed on one prostate cancer patient different from Comparative Example 1 as described above. This patient was used as the patient in Example 1. A total of 11 CTCs were collected from the patient in Example 1, and genomic DNA was analyzed for each of these CTCs, one cell at a time. The detected numbers of CK-positive CTCs, CK-negative large-diameter CTCs, and CK-negative clustered CTCs in the patient of Example 1 are as described in Table 1. The genomic DNA analysis results of CTCs were compared with those of leukocytes, and the number of genomic DNA mutations in each of the 11 CTCs was measured. Table 3 shows the types and numbers of CTCs and genomic DNA mutations.

[0058]

Table 3

[0059] It was confirmed that the patient in Example 1 had few mutations in the genomic DNA of CTCs. Specifically, the number of mutations (missense mutations in the table) that caused amino acid mutations of one or more residues in the amino acid sequence encoded by the gene locus where the mutation existed was 0 to 8, and 0 to 5 except for one case. The number of mutations (LoF mutations in the table) that caused amino acid mutations that changed the protein function was within 0 to 3. Also, the total number of mutations not limited to such types of mutations was also within 10.

[0060] Next, for the CTCs of the patient in Example 1, according to the above "spheroid preparation" and "cancer model mouse preparation", an attempt was made to prepare spheroids and cancer model mice using the spheroids. As a result, palpable tumor formation was observed, and a successful case of a cancer model mouse using the spheroids derived from the CTCs of the patient in Example 1 was obtained.

[0061] For the cancer model mouse using the spheroids derived from the CTCs of the patient in Example 1, when palpable tumor formation of about 1 cm was confirmed about 6 months after transplantation of the spheroids, the mouse was euthanized and tumor tissue pieces were excised. A part (about 1 cm in diameter) of the excised tumor tissue piece was transplanted again into the subcutaneous tissue of NSG mice. About 1 month after transplantation, palpable tumor enlargement of 1 cm or more was observed. Therefore, it was confirmed that the tumor tissue piece was passaged in mice.

[0062] Furthermore, another part of the excised tumor tissue pieces (two tissues with a diameter of about 3 mm) was added to the collagenase solution and shaken at 37°C for 2 hours. After sufficiently decomposing the tissue, it was suspended in Matrigel. The Matrigel containing the decomposed tissue (spheroid) was dropped into a 24-well plate at about 40 μL per well and incubated at 37°C for 45 minutes in a 5% CO2 environment until it solidified into a dome shape. Then, 500 μL of the culture medium was added, and in a 5% CO2 environment, cultured at 37°C. Six days after seeding, it was confirmed that the spheroids in the Matrigel had increased in size and formed organoids (with a diameter of about 100 μm). In addition, when PSA (prostate-specific antigen) in the culture supernatant was measured by the ELISA method, 154 ng / mL was detected. Therefore, it was confirmed that the tumor tissue pieces were derived from the prostate.

[0063] Mutation analysis of CTCs in the patient of Comparative Example 1 and the patient of Example 1 From the analysis results of genomic DNA of CTCs in the patient of Comparative Example 1, the genes with mutations, the total number of mutation detections, and the number of mutation detections per CTC are shown in Table 4 below.

[0064]

Table 4

[0065] Also, from the analysis results of genomic DNA of CTCs in the patient of Example 1, the genes with mutations, the total number of mutation detections, and the number of mutation detections per CTC are shown in Table 5 below. Note that for all the genes with mutations in the CTCs of the patient of Example 1, mutations were also found in the CTCs of the patient of Comparative Example 1. For reference, the total number of mutation detections and the number of mutation detections per CTC in the CTCs of the patient of Comparative Example 1 are also shown in Table 5.

Table 5

[0066] ​As described above, for all the genes with mutations in the CTCs of the patient in Example 1, mutations were also observed in the CTCs of the patient in Comparative Example 1. On the other hand, there were also a large number of genes with mutations only in the CTCs of the patient in Comparative Example 1. For example, genes with mutations only in the CTCs of the patient in Comparative Example 1 and with a mutation number / analysis number of 1.00 or more include CDK6, FOXP1, FANCA, SF3B1, ARID2, ZMYM3, ATR, FANCD2, BRCA2, PIK3CB, MDM2, AKT2, AKT1, AKT3, PALB2, CTNNB1, FANCC, MET, USP7, FANCG, CCND1, FANCD2OS, NBN, BRIP1, MRE11, RYBP, and PIK3CA. In addition, there were also genes that were mutated in the CTCs of the patient in Example 1 and showed a high mutation number / analysis number in the CTCs of the patient in Comparative Example 1. Such genes include, for example, APC, MED12, ARID1A, KMT2D, AR, and KMT2C. It was confirmed that these genes were involved in the mechanism by which successful examples of cancer model animals were obtained only in spheroids derived from CTCs obtained from the patient in Example 1 in which CTCs with a small number of mutations were confirmed.

[0067] Results The generation of CTC-transplanted cancer model mice in the patient of Example 1 was successful, and furthermore, since the tumor tissue of the cancer model mice could be passaged in the mice, the establishment of CTC-transplanted mice was successful. On the other hand, the generation of CTC-transplanted cancer model mice in the patient of Comparative Example 1 failed. Both patients had bone metastases and were in a poor pathological condition. Also, as shown in Table 1, since a large number of CK-negative cluster CTC detection numbers significantly exceeding the threshold were confirmed from each of the two patients, it is suggested that there may be many CTCs with mesenchymal properties with high metastatic and malignant degrees in the blood of both patients. Therefore, it is considered that most of the CTCs transplanted into the mice in both patients were CK-negative cluster CTCs. That is, the establishment of CTC-transplanted mice was not affected by the pathological condition of the cancer patients from which they were derived or the properties of the CTCs.

[0068] To verify the factors determining the success of establishing CTC-transplanted mice, gene expression analysis of single-cell CTCs from both patients was performed. As a result, the CTCs obtained from the patient in Example 1 had a small number of detected mutations (Table 4). On the other hand, a large number of mutations were detected in the CTCs obtained from the patient in Comparative Example 1 regardless of the type of mutation (Table 3). As shown in Table 6, all the gene mutations in the CTCs obtained from the patient in Example 1 were also detected in the patient in Comparative Example 1, and the number of mutations detected per CTC of common genes other than TP53 and MSH2 was higher in the patient in Comparative Example 1 than in the patient in Example 1. Both TP53 and MSH2 are genes related to maintaining genomic stability, but their relationship with engraftment into CTC-transplanted mice is unclear. Therefore, it was confirmed that the success or failure of establishing CTC-transplanted mice is affected by the number of gene mutations in CTCs, and that a small number of the aforementioned gene mutations is a requirement for establishing CTC-transplanted mice. Generally, since CTCs circulate in a resting state in the patient's blood, it is presumed that they are difficult to proliferate or do not proliferate even when taken out of the body. That is, it was found that CTCs with a lower gene mutation frequency are more likely to turn into proliferation than CTCs with a higher gene mutation frequency.

Explanation of Signs

[0069] 100: Cell holding device 110: Insulating film 120: Light-shielding film 111·121: Through-hole 130: Spacer 131: Introduction part 132: Discharge part 141·142: Electrode 150: Conductive wire 160: AC power supply 170: Holding part 200: Detection part 300: Cell 400: Dielectrophoretic force 500: Light 600: Suction means (micromanipulator)

Claims

1. A method for producing a cancer model animal, comprising: Step A of collecting circulating tumor cells (CTCs) from a subject; Step B of culturing the CTCs to produce spheroids; Step C of transplanting the spheroids into an animal other than a human, wherein a step P of measuring the number of nucleotide mutations on genomic DNA is performed using a part of the CTCs after step A and before step C.

2. The method according to claim 1, wherein the cancer model animal and the animal other than the human are mice.

3. The method according to claim 1 or 2, wherein the subject is a human cancer patient.

4. The method according to claim 1 or 2, wherein the number of nucleotide mutations on genomic DNA is the number of nucleotide mutations on the genomic DNA of the CTCs based on the genomic DNA of normal cells collected from the subject.

5. The method according to claim 4, wherein the normal cells are white blood cells.

6. The method according to claim 1 or 2, wherein in step P, the number of nucleotide mutations present at any locus among the nucleotide mutations on genomic DNA is measured.

7. The method according to claim 1 or 2, wherein in step P, the number of nucleotide mutations present at any locus among the nucleotide mutations on genomic DNA and causing one or more amino acid mutations in the amino acid sequence encoded by the locus where the mutation is present is measured.

8. The method according to claim 1 or 2, wherein in step P, the number of nucleotide mutations present at any locus among the nucleotide mutations on genomic DNA and causing one or more amino acid mutations that change the function of the protein having the amino acid sequence encoded by the locus where the mutation is present is measured.

9. The method according to claim 6, wherein the locus is one or more loci selected from the following (i); (i) CDK6, FOXP1, FANCA, SF3B1, ARID2, ZMYM3, ATR, FANCD2, BRCA2, PIK3CB, MDM2, AKT2, AKT1, AKT3, PALB2, CTNNB1, FANCC, MET, USP7, FANCG, CCND1, FANCD2OS, NBN, BRIP1, MRE11, RYBP, and PIK3CA.

10. The method according to claim 1 or 2, wherein in the step P, instead of the number of nucleotide mutations on the genomic DNA, the number of loci at which one or more arbitrary nucleotide mutations are present is measured.

11. The method according to claim 10, wherein in the step P, instead of the number of nucleotide mutations on the genomic DNA, the nucleotide mutations present at the locus are nucleotide mutations that cause amino acid mutations of one or more residues in the amino acid sequence encoded by the locus at which the nucleotide mutation is present, and the number of the loci is measured.

12. The method according to claim 10, wherein in the step P, instead of the number of nucleotide mutations on the genomic DNA, the nucleotide mutations present at the locus are nucleotide mutations that cause amino acid mutations of one or more residues in the amino acid sequence encoded by the locus at which the nucleotide mutation is present, and the amino acid mutations change the function of the protein having the amino acid sequence, and the number of the loci is measured.

13. The method according to claim 10, wherein the locus is one or more loci selected from the following (i); (i) CDK6, FOXP1, FANCA, SF3B1, ARID2, ZMYM3, ATR, FANCD2, BRCA2, PIK3CB, MDM2, AKT2, AKT1, AKT3, PALB2, CTNNB1, FANCC, MET, USP7, FANCG, CCND1, FANCD2OS, NBN, BRIP1, MRE11, RYBP, and PIK3CA.

Images