JP2021018154A - Tumor cell cluster detection method, tumor cell cluster collection method, reagent used in the methods, and carrier immobilization molecule - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting a tumor cell cluster, which can detect a tumor cell cluster which is usually contained in a small amount in a sample separately from a contaminating cell. SOLUTION: A gene encoding a polypeptide containing at least one of 576 amino acid sequences including (i) STEAP1 (GenBank No. NP_03658.1) in a sample, (ii) the above-mentioned (i). A gene encoding a polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence, (iii) In the amino acid sequence of (i) above, one or more amino acids are substituted, deleted, inserted and /. Alternatively, a method for detecting tumor cell clusters contained in a sample by detecting the expression of any gene of a gene encoding a polypeptide containing at least an added amino acid sequence. [Selection diagram] None

Description

The present invention relates to a method for detecting and recovering tumor cell clusters contained in a sample, a reagent for detecting tumor cell clusters used therein, a reagent for capturing, and a carrier-immobilized molecule. More specifically, the present invention uses a protein expressed by the tumor cell cluster or a gene thereof to detect and recover the tumor cell cluster contained in the sample separately from the contaminating cells contained in the sample. Regarding the method.

When a tumor cell leaves the primary lesion, it penetrates into a blood vessel or lymph vessel, circulates in blood or lymph, and finally invades other organs or tissues to form a metastatic lesion. Here, tumor cells that circulate in the blood are also called CTCs (Circulating Tumor cells), and many clinical trials and studies have been conducted. For example, the number of CTCs contained in blood collected from a patient can be measured (Japanese Patent Laid-Open No. 2008-533487 (Patent Document 1), etc.), and protein expression, gene mutation or translocation in the CTC can be examined. It is known that this provides information on early detection of metastatic malignancies, prediction of the patient's condition and prediction of recurrence after tumor detection and treatment.

It is known that among tumor cells, a plurality of tumor cells called tumor cell clusters exist in a state of forming agglomerates, and these circulate in blood or lymphatic vessels. For example, it has been reported that the CTC cluster, which is a cluster of CTCs circulating in the blood, has a higher metastatic ability than the CTCs circulating in the blood with one cell (Japanese Patent Application Laid-Open No. 2016-536303 (Patent Document 2), etc.). ), Clinical analysis is of high importance. However, since the number of CTC clusters contained in blood is very small and the blood also contains a wide variety of other cells such as erythrocytes and white blood cells, when detecting / analyzing CTC clusters, , A technique for distinguishing and recovering tumor cells constituting a small number of CTC clusters from a large amount of cells is required.

Conventionally, tumor cell clusters are detected by using a protein (tumor marker) expressed in tumor cells in the same manner as when detecting one tumor cell, but more specific tumor cell clusters are detected. And the development of methods for recovery is required.

Japanese Patent Publication No. 2008-533487 Special Table 2016-536303

An object of the present invention is a method for detecting and / or recovering a tumor cell cluster, which can detect and / or recover a tumor cell cluster which is usually contained in a small amount in a sample separately from a contaminating cell, and a method thereof. To provide reagents and carrier-immobilized molecules used in the above.

In order to solve the above problems, the present inventors have developed a next-generation sequencer of a circulating tumor cell cluster (CTC cluster), which is an example of a tumor cell cluster, and a leukocyte, which is an example of contaminating cells contained in a blood sample. As a result of comparative expression analysis using, a polypeptide specifically expressed in the tumor cells constituting the tumor cell cluster was found. Furthermore, it was found that the tumor cell cluster contained in the sample can be detected and / or recovered separately from other contaminating cells contained in the sample by using such a polypeptide as a marker, and the present invention is completed. I arrived.

That is, the present invention is as follows.

[1]
A method for detecting a tumor cell cluster, which comprises a step of detecting the expression of at least one gene selected from the group consisting of the following (i) to (iii) in a sample.
(I) A gene encoding a polypeptide containing at least the amino acid sequence set forth in any of SEQ ID NOs: 1 to 576.
(Ii) A gene encoding a polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1 to 576.
(Iii) A gene encoding a polypeptide containing at least one or more amino acids substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 1 to 576.

[2]
The detection of the expression of the gene is the detection of the polypeptide encoded by the gene, and the detection of the polypeptide is the detection using an antibody or an aptamer that specifically recognizes the polypeptide. , [1]. The method for detecting a tumor cell cluster.

[3]
The method for detecting a tumor cell cluster according to [1] or [2], wherein the sample is a blood sample.

[4]
A step of detecting or capturing at least one polypeptide selected from the group consisting of the following (iv) to (vi) in a sample to detect or capture a tumor cell cluster, and the detected or captured tumor. The process of collecting cell clusters and
A method for recovering a tumor cell cluster, which comprises.
(Iv) A polypeptide comprising at least the amino acid sequence set forth in any of SEQ ID NOs: 1-153.
(V) A polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1 to 153.
(Vi) A polypeptide comprising at least an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted and / or added in the amino acid sequence set forth in any of SEQ ID NOs: 1 to 153.

[5]
The method for recovering a tumor cell cluster according to [4], wherein the detection or capture of the polypeptide is detection or capture using an antibody or aptamer that specifically recognizes the polypeptide.

[6]
The method for recovering a tumor cell cluster according to [4] or [5], wherein the sample is a blood sample.

[7]
A reagent for detecting tumor cell clusters, which comprises a molecule that binds to an expression product of at least one gene selected from the group consisting of the following (i) to (iii) as an active ingredient.
(I) A gene encoding a polypeptide containing at least the amino acid sequence set forth in any of SEQ ID NOs: 1 to 576.
(Ii) A gene encoding a polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1 to 576.
(Iii) A gene encoding a polypeptide containing at least one or more amino acids substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 1 to 576.

[8]
The reagent according to [7], wherein the molecule that binds to the expression product of the gene is an antibody or aptamer that specifically recognizes the polypeptide encoded by the gene.

[9]
A reagent for detecting or capturing tumor cell clusters, which comprises a molecule that binds to at least one or more polypeptides selected from the group consisting of the following (iv) to (vi) as an active ingredient.
(Iv) A polypeptide comprising at least the amino acid sequence set forth in any of SEQ ID NOs: 1-153.
(V) A polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1 to 153.
(Vi) A polypeptide comprising at least an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted and / or added in the amino acid sequence set forth in any of SEQ ID NOs: 1 to 153.

[10]
The reagent according to [9], wherein the molecule that binds to the polypeptide is an antibody or aptamer that specifically recognizes the polypeptide.

[11]
Carrier immobilization for capture of tumor cell clusters, characterized in that a molecule that binds to at least one polypeptide selected from the group consisting of (iv) to (vi) below is supported on the carrier. molecule.
(Iv) A polypeptide comprising at least the amino acid sequence set forth in any of SEQ ID NOs: 1-153.
(V) A polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1 to 153.
(Vi) A polypeptide comprising at least an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted and / or added in the amino acid sequence set forth in any of SEQ ID NOs: 1 to 153.

[12]
The carrier-immobilized molecule according to [11], wherein the molecule that binds to the polypeptide is an antibody or aptamer that specifically recognizes the polypeptide.

According to the present invention, a method for detecting and / or recovering a tumor cell cluster, which is capable of detecting and / or recovering a tumor cell cluster which is usually contained in a small amount in a sample separately from a contaminating cell, and a method thereof. It becomes possible to provide the reagent and the carrier-immobilized molecule used in the above.

(A) Bright-field microscopy images of CTC clusters detected from blood samples derived from prostate cancer patients, and (b) Hoechst 33342, (c) FITC-labeled anti-CD45 antibody (CD45-FITC antibody), and ( d) It is a fluorescence microscope image which shows the result of staining with PE-labeled tumor cell detection antibody (anti-TM4SF1 antibody, anti-TNFRSF12A antibody and anti-EpCAM antibody) respectively. It is a graph which shows the expression value of STEAP1 gene in leukocyte and CTC cluster. In FIG. 2, WBC1 and WBC2 show the expression values in each leukocyte, and Cruster1 to Cruster16 show the expression values in each CTC cluster.

Hereinafter, the present invention will be described in detail according to its preferred embodiment.

<Tumor cell cluster detection method and tumor cell cluster recovery method>
The method for detecting a tumor cell cluster of the present invention is a method for detecting at least one gene (hereinafter, in some cases, a “tumor cell cluster-specific gene”” selected from the group consisting of the following (i) to (iii) in a sample. It is a method including a step (detection step) of detecting the expression of (referred to as).
(I) A gene encoding a polypeptide containing at least the amino acid sequence set forth in any of SEQ ID NOs: 1 to 576.
(Ii) A gene encoding a polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1 to 576.
(Iii) A gene encoding a polypeptide containing at least one or more amino acids substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 1 to 576.

In addition, the method for recovering a tumor cell cluster of the present invention is a method for collecting at least one or more polypeptides selected from the group consisting of the following (iv) to (vi) in a sample (hereinafter, in some cases, "tumor cell cluster specific". A step of detecting or capturing a tumor cell cluster (referred to as "transmembrane polypeptide") to detect or capture a tumor cell cluster, and a step of recovering the detected or captured tumor cell cluster.
It is a method including.
(Iv) A polypeptide comprising at least the amino acid sequence set forth in any of SEQ ID NOs: 1-153.
(V) A polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1 to 153.
(Vi) A polypeptide comprising at least an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted and / or added in the amino acid sequence set forth in any of SEQ ID NOs: 1 to 153.

According to the present invention, the tumor cell cluster contained in the sample can be detected by using the detection of the expression of the tumor cell cluster-specific gene as an index. Further, according to the present invention, the tumor cell cluster contained in the sample is detected or captured using the detection of the tumor cell cluster-specific transmembrane polypeptide among the expression products of the tumor cell cluster-specific gene as an index. , Can be recovered.

In the present invention, the "tumor cell" is not particularly limited, and examples thereof include prostate cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, bone cancer, head and neck cancer, liver cancer, and biliary tract. Tumor cells such as esophageal cancer, gastric cancer, kidney cancer, bladder cancer, ovarian cancer, and skin cancer can be mentioned.

In the present invention, the "circulating tumor cell" refers to a tumor cell that has penetrated into a blood vessel or a lymphatic vessel from a primary tumor (primary tumor) or a metastatic tumor (metastatic lesion), and among these, a circulating tumor that circulates in the blood. The cells are referred to as "CTC (Circulating Tumor cell)". CTCs exist, for example, as tumor cells that circulate in the peripheral bloodstream of the cancer patients described above.

In the present invention, the "tumor cell cluster" refers to a cluster (aggregate) composed of tumor cells, preferably composed of two or more tumor cells. The tumor cell cluster according to the present invention is preferably a cluster composed of the circulating tumor cells (circulating tumor cell cluster), and is not particularly limited, and examples thereof include a CTC cluster which is a CTC cluster. In the present invention, the term "tumor cell cluster" simply refers to the tumor cell cluster itself forming the agglomerate, the tumor cells constituting the tumor cell cluster, and the tumor cell cluster. Tumor cells are also included.

In the present invention, the "sample" is not particularly limited as long as it is a sample in which the tumor cell cluster can exist, but for example, a target (for example, a human and a non-human animal (mouse, rat, dog, cat, bovine)). , Horses, pigs, birds, etc., preferably humans). Examples thereof include biological tissues (cells, tissues, organs, body fluids, whole blood, ascites, etc.), which are not limited to cancer patients. A healthy person may be used. As the sample, a diluted solution obtained by diluting the tissue, whole blood, ascites, etc. with an appropriate buffer; components derived from whole blood such as serum, plasma, umbilical cord blood, and component blood; It may be a suspension in which a piece of tissue containing blood such as liver, lung, spleen, kidney, tumor, and lymph node is suspended in an appropriate buffer, and further, these are separated and collected by centrifugation or the like. The fraction containing tumor cells may be obtained. Among these, the sample according to the present invention includes whole blood, whole blood diluent, and components derived from whole blood (serum, plasma, umbilical cord blood, etc.). It is preferable that the blood sample is such as a component blood collection, etc.), a suspension of tissue containing blood, and a fraction containing tumor cells obtained from these.

In the present invention, the "contaminated cells" contained in the sample refer to cells other than the tumor cells constituting the tumor cell cluster, and also include 1-cell tumor cells (tumor cells not constituting the tumor cell cluster). When the sample is the blood sample, examples thereof include red blood cells, white blood cells, and platelets. Among these, the method for detecting and recovering tumor cell clusters of the present invention is, for example, a method excellent in distinguishing from leukocytes contained in a blood sample.

In the present invention, the "transmembrane polypeptide" refers to a polypeptide containing at least one or more transmembrane regions. In the present invention, among the tumor cell cluster markers, by targeting the transmembrane polypeptide, a tumor cell cluster expressing the transmembrane polypeptide on the cell membrane can be detected, and the transmembrane polypeptide can be detected. It can be captured and recovered via a type polypeptide.

In the present invention, if the gene whose expression is detected (hereinafter, referred to as “target gene” in some cases) is of human origin, it is typically the amino acid according to (i) SEQ ID NO: 1 to 576. Although it is a gene encoding a polypeptide containing at least a sequence, in the present invention, a homologue of these genes (for example, a counterpart gene in an organism other than human) can be targeted (detection target). In addition, since the DNA sequence of a gene can be mutated in nature (that is, non-artificially) due to its mutation or the like, such a natural mutant can also be targeted in the present invention.

The amino acid sequence of the homolog is 70% or more, more preferably 75% or more, 80% or more, 85% or more, 90 with respect to the entire amino acid sequence shown in (ii) SEQ ID NO: 1 to 576. It may be an amino acid sequence having homology of% or more and 95% or more (for example, 96, 97, 98, 99% or more). The homology of the sequence can be determined using, for example, a BLASTP program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990).

Further, as the amino acid sequence of the mutant, the amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in any of (iii) SEQ ID NOs: 1 to 576. It may be. Here, "several numbers" are 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or two integers. Means. The 1 to several amino acid residues preferably have 1 or more and 10 or less amino acid residues, more preferably 1 or more and 5 or less, still more preferably 1 or more and 3 or less, and particularly preferably 2 or less. Is.

In the present invention, the tumor cell cluster-specific gene is more specific to the tumor cell cluster than the contaminating cells. As used herein, "highly specific" means that tumor cell clusters are more highly expressed at transcriptional or translational levels than at least one contaminating cell. Preferably, the tumor cell cluster has a tumor cell cluster-specific gene expression 1.5-fold or higher, more preferably 2-fold or higher, still more preferably 3-fold or higher, and particularly preferably 5 times higher than the contaminating cells. It is more than twice as high (for example, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more).

In Tables 1 to 24 below, for each of the amino acid sequences of SEQ ID NOs: 1 to 576, the sequence number of each amino acid sequence, the name of the protein corresponding to the polypeptide consisting of each amino acid sequence, and the GenBank accession of each amino acid sequence. The number (GenBank No.);, the sequence number of the gene encoding the polypeptide consisting of each amino acid sequence, its open reading frame position (ORF position), and the GenBank accession number (GenBank No.) of each gene are shown. Tables 1 to 24 below also show the results of gene expression level analysis obtained in the following examples as an example. In addition, Tables 25 to 73 below show official names (full names) and aliases for the names of the proteins listed in Tables 1 to 24, respectively.

In the present invention, "detecting gene expression" means to include both detection of the presence or absence of gene expression and detection of the degree of expression, and in the present invention, the expression of the target gene is detected. The expression level of a gene can be grasped as an absolute amount or a relative amount. When grasping the relative amount, for example, it can be determined by comparing with the expression level of the gene of the prepared standard sample. The "standard sample" is a sample for which whether or not the target gene is expressed is specified in advance, and for example, erythrocytes, leukocytes, and platelets, more preferably leukocytes, can be used as the standard sample. When the subject is a cancer patient, a tissue not affected by the cancer (normal tissue) can also be used as the standard sample.

In the present invention, "gene expression" means to include both transcription and translation of a gene. Therefore, "detection of gene expression" in the present invention includes detection at the transcription level (mRNA level) and detection at the translation level (protein level) (that is, detection of the polypeptide encoded by the gene). Both are included.

In eukaryotic cells, introns in pre-mRNA are removed during gene transcription, and a reaction (splicing) occurs in which anterior and posterior exons are recombined, but exon recombination may be diverse. , Which produces various mature mRNAs. As a result, various proteins are translated. Various mRNAs and proteins produced by such differences in splicing are called "splicing variants". Therefore, the detection of gene expression in the present invention includes the detection of the splicing variant as long as it is specifically recognized as a molecule that binds to the expression product of the target gene below.

For the detection of gene expression in the present invention, a known method or a method similar thereto can be appropriately used. For detection at the transcription level, for example, a probe corresponding to an appropriate position in the base sequence (polynucleotide) of the transcript (mRNA) of the target gene is designed, and then Northern blotting, dot blot, and RNase protection assay. , DNA microarray analysis method, in situ hybridization method, etc., and after designing a primer set corresponding to an appropriate position in the polynucleotide, for example, PCR method, RT-PCR method, TRC ( Transcription Reversation Concerted) method, NASBA (Nucleic Acid Sequence-Based Amplification) method, TMA (Transcription-Mediated Amplification) method, TMA (Transcription-Mediated Amplification) method, TMA (Transcription-Mediated Amplification) method It may be detected by directly applying it to a sequencer.

In addition, detection at the translation level includes, for example, immune cell staining method, imaging cytometry, flow cytometry, ELISA method, radioimmunoassay, immunoprecipitation method, immunoblotting (Western blotting, etc.), antibody array, in vivo imaging. A method of detecting using an antibody such as (immunological method); a method of detecting using an aptamer instead of the antibody can be mentioned. Further, the immunological method may be automatically performed using an enzyme immunoassay device such as AIA-900 or AIA-CL2400 (both manufactured by Tosoh Corporation) after preparing necessary reagents.

In the present invention, the detection of the expression of the gene is preferably at the translation level (detection of the polypeptide encoded by the target gene) from the viewpoint of convenience, and in particular, the polypeptide is specific. A method of detecting the polypeptide using an antibody that recognizes the polypeptide (hereinafter, sometimes referred to as "anti-polypeptide antibody") or an aptamer that specifically recognizes the polypeptide (hereinafter, simply referred to as "aptamer"). Preferably, a method of detecting the polypeptide using the anti-polypeptide antibody is more preferable.

In the present invention, the "antibody" may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of the antibody. In addition, "antibodies" include all classes and subclasses of immunoglobulins. The "functional fragment" of an antibody means a part (partial fragment) of the antibody that specifically recognizes the polypeptide encoded by the target gene in the present invention. Specifically, Fab, Fab', F (ab') ₂ , variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) ₂ , diabody, multispecific antibody, And these polymers and the like.

If the anti-polypeptide antibody according to the present invention is a polyclonal antibody, it immunizes an immune animal with an antigen (a polypeptide encoded by a target gene, a partial peptide thereof, a cell expressing these, etc.) and its antiserum. Can be purified and obtained from the above by conventional means (salting, centrifugation, dialysis, column chromatography, etc.). In addition, the monoclonal antibody can be produced by a hybridoma method or a recombinant DNA method. Examples of the hybridoma method include a Caller & Milstein method (Kohler & Milstein, Nature, 256: 495 (1975)), and examples of the recombinant DNA method include a hybridoma or a DNA encoding the above-mentioned anti-polypeptide antibody. It is cloned from B cells or the like, incorporated into an appropriate vector, introduced into host cells (mammalian cell line, Escherichia coli, yeast cells, insect cells, plant cells, etc.), and the anti-polypeptide antibody is produced as a recombinant antibody. (For example, PJ Delves, Antibodies Production: Essential Technologies, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD, 2000 OXFORD. Biochem. 192: 767-775 (1990)).

In the present invention, as the molecule that binds to the expression product of the target gene, such as the anti-polypeptide antibody and the aptamer, a molecule to which a labeling substance is bound can be used. By using the molecule to which the labeling substance is bound and detecting the labeling substance, the amount of the molecule bound to the expression product encoded by the target gene can be directly measured. The labeling substance is not particularly limited as long as it can bind to the molecule and can be detected by a chemical or optical method. For example, phycoerythrin (PE) and fluorescein isothiocyanate (FITC) are not particularly limited. ), Fluorescent substances such as rhodamine isothiocyanate (RITC), enzymes such as peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP), alkaline phosphatase, and radioactive substances.

Further, in the present invention, the detection of the expression of the gene is the detection of the polypeptide encoded by the gene, and when the anti-polypeptide antibody is used, the secondary antibody to which the labeling substance is bound is used. An indirect detection method such as a method of using a polymer in which a secondary antibody and the labeling substance are bound can also be used. Here, the "secondary antibody" is an antibody that exhibits specific binding properties to the anti-polypeptide antibody. For example, when the anti-polypeptide antibody is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used as a secondary antibody. Labeled secondary antibodies that can be used are commercially available for antibodies derived from various species such as rabbits, goats, and mice, and appropriate secondary antibodies are available depending on the species from which the anti-polypeptide antibody used is derived. Antibodies can be selected and used. Further, instead of the secondary antibody, it is also possible to use protein G, protein A or the like to which a labeling substance is bound.

In the present invention, "detection of tumor cell clusters" refers to detection of the presence or absence of tumor cell clusters in a sample (qualitative detection) using the expression of the tumor cell cluster-specific gene as an index, and the degree of the amount thereof. It is meant to include both detection (quantitative detection), and also includes detection of whether or not the cells collected from the sample are tumor cells constituting the tumor cell cluster.

The criteria for determining the detection of the tumor cell cluster can be appropriately set according to the purpose, and is not particularly limited. For example, if there is even a small amount of detection signal according to the number of tumor cells and the expression level of the target gene. , It may be determined that tumor cell clusters are present in the sample, and if there is a detection signal above a certain threshold value, the determination may be made. In addition, the amount of tumor cell clusters may be detected according to the intensity of the detection signal. Furthermore, when cell clusters are collected from the sample and then detected, it may be determined that the collected cell clusters are tumor cell clusters if there is even a small amount of the detection signal, and the value is equal to or higher than a certain threshold. If there is a detection signal, the judgment may be made, and the signal contained in the contaminating cells collected from the sample (for example, 1-cell tumor cell, erythrocyte, leukocyte, etc. in the case of the blood sample) has a certain value or more or If there is a signal of 2SD or more and 3SD or more or 4SD or more of the average value of the signal, the judgment may be made.

Of the above Tables 1 to 24, the amino acid sequences of SEQ ID NOs: 1 to 153 shown in Tables 1 to 7 are the amino acid sequences of the tumor cell cluster-specific transmembrane polypeptides identified by the present inventors. In the method for recovering tumor cell clusters of the present invention,
(Iv) A polypeptide comprising at least the amino acid sequence set forth in any of SEQ ID NOs: 1-153.
(V) A polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1 to 153 (that is, a homologue of (iv)), and (vi) SEQ ID NOs: 1 to 1. A polypeptide comprising at least one or more amino acid substitutions, deletions, insertions and / or additions of the amino acid sequence according to any of 153 (ie, variants of (iv)).
A method comprising a step of detecting or capturing a tumor cell cluster by detecting or capturing at least one polypeptide selected from the group consisting of, and a step of recovering the detected or captured tumor cell cluster. Is.

The step (detection step) for detecting the tumor cell cluster is as described above. Examples of the method for recovering the detected tumor cell cluster include a method using a cell sorter and a method for recovering the detected tumor cell cluster using a pipette under a microscope (for example, the micropick system described in this example).

Further, in the step of capturing the tumor cell cluster (capture step), the capture of the tumor cell cluster comprising a carrier and a molecule that binds to the tumor cell cluster-specific transmembrane polypeptide supported on the carrier. Can be used for carrier-immobilized molecules. In this case, the recovery method of the present invention is:
A capture step of bringing the carrier-immobilized molecule into contact with the sample and capturing the tumor cell cluster with the carrier-immobilized molecule.
A recovery step of removing impurities such as contaminant cells that are not bound to the carrier-immobilized molecule and recovering the tumor cell cluster captured by the carrier-immobilized molecule.
It is preferable that the method includes.

The recovery step includes a washing step of removing impurities that are not bound to the carrier-immobilized molecule by injecting and removing a washing solution, and a free tumor cell cluster by releasing the tumor cell cluster from the carrier-immobilized molecule. May further include a liberation step to obtain.

Examples of the carrier of the carrier-immobilized molecule include plates, fibers, membranes, particles, microchannel chips and the like. As the molecule that binds to the tumor cell cluster-specific transmembrane polypeptide in the carrier-immobilized molecule, an antibody that binds to the tumor cell cluster-specific transmembrane polypeptide is preferable. In addition, the molecule that binds to the carrier or tumor cell cluster-specific transmembrane polypeptide may be labeled with the labeling substance. As such a carrier-immobilized molecule, conventionally known molecules can be appropriately used, and commercially available ones can also be appropriately used.

The method for recovering the tumor cell cluster captured by the carrier-immobilized molecule is not particularly limited, and a conventionally known method or a method similar thereto can be appropriately adopted. For example, the carrier is a plate. In some cases, a method of removing the liquid phase (supernatant) from the plate after the capture step, or when the carrier is particles, the particles are recovered by centrifugation or magnetic collection after the capture step to obtain a liquid. A method of removing the phase (supernatant) can be mentioned. Further, the recovery device or the like described in JP-A-2016-142616 may be appropriately used.

The number of tumor cell clusters obtained by the method for recovering tumor cell clusters of the present invention can be measured and can be used for various analyzes.

In addition, the information obtained by carrying out the method for detecting and recovering tumor cell clusters of the present invention can be used, for example, in the case of targeting a cancer patient, for evaluation or understanding of the pathological condition of the patient and evaluation of the therapeutic effect. It can be used for such purposes. For example, if the method of the present invention is carried out in parallel with the treatment of cancer, recurrence prediction and therapeutic effect can be evaluated based on the information obtained as a result. Specifically, by implementing the method for detecting and recovering tumor cell clusters of the present invention after drug administration, changes in the expression of tumor cell cluster-specific genes in patients are investigated, and the therapeutic effect is determined from the increase or decrease or transition. can do. As described above, the method of the present invention may be used for predicting recurrence and monitoring the therapeutic effect.

<Reagents for detection of tumor cell clusters and carrier-immobilized molecules for capture>
The reagent for detecting a tumor cell cluster of the present invention (hereinafter, sometimes referred to as "detection reagent") is a detection reagent containing a molecule that binds to an expression product of the tumor cell cluster-specific gene as an active ingredient. .. By using this, the tumor cell cluster detection method of the present invention can be carried out to detect the tumor cell cluster in the sample.

In the present invention, "an expression product of at least one gene selected from the group consisting of (i) to (iii)" is a transcript (mRNA) or translation of the tumor cell cluster-specific gene (target gene). It refers to a product (protein). Examples of the "molecule that binds to the transcript" of the target gene include primers used in the above-mentioned RT-PCR method and the like, and probes used for Northern blotting and the like. The molecule is a polynucleotide containing a base sequence complementary to the base sequence of the transcript of the target gene. Preferably, it is a polynucleotide containing a continuous base sequence of at least 15 bases or more, more preferably 20 bases or more, which is complementary to the base sequence of the transcript of the target gene. As used herein, "complementary" does not have to be 100% complementary as long as specific hybridization with the transcript occurs. It preferably has 90% or more, more preferably 95% or more (eg, 96% or more, 97% or more, 98% or more, 99% or more) complementarity. In addition, examples of the "molecule that binds to the translation product" of the target gene include the above-mentioned anti-polypeptide antibody and aptamer. In the present invention, the anti-polypeptide antibody and the aptamer are preferable, and the anti-polypeptide antibody is more preferable, as the molecule that binds to the expression product of the target gene.

In addition to the molecule that binds to the expression product of the target gene, the detection reagent of the present invention includes a buffer solution, physiological saline; phycoerythrin (PE), fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC) and the like. Fluorescein; Secondary antibody that recognizes and binds to the molecule that binds to the expression product of the target gene; Peroxidase modified to the secondary antibody, β-D-galactosidase, Microperoxidase, Horseradish peroxidase (HRP), Alkaline phosphatase Other components such as the enzyme and its substrate may be added.

When the expression product of the tumor cell cluster-specific gene is a tumor cell cluster-specific transmembrane polypeptide, the molecule that binds to the polypeptide targets the tumor cell cluster in which the polypeptide is present on the cell surface. At the same time, cells expressing the polypeptide as a membrane protein, that is, tumor cells constituting the tumor cell cluster, can be captured through the binding between the molecule and the polypeptide. Therefore, among the detection reagents of the present invention, it binds to at least one or more polypeptides selected from the group consisting of molecules ((iv) to (vi) above) that bind to the tumor cell cluster-specific transmembrane polypeptide. A reagent for detecting a tumor cell cluster containing a molecule) as an active ingredient can also be used as a reagent for capturing a tumor cell cluster (hereinafter, referred to as “capture reagent”).

Examples of the "molecule that binds to the tumor cell cluster-specific transmembrane polypeptide" include the above-mentioned anti-polypeptide antibody and aptamer, and the above-mentioned anti-polypeptide antibody is preferable.

Similar to the detection reagent, the capture reagent of the present invention includes a buffer, physiological saline; phycoerythrin (PE), in addition to a molecule that binds to a tumor cell-specific transmembrane polypeptide as an active ingredient. Fluorescein isothiocyanate (FITC), Rhodamine isothiocyanate (RITC) and other fluorescent substances; secondary antibody that recognizes and binds to a molecule that binds to the expression product of the target gene; peroxidase modified to a secondary antibody, β-D- Other components such as enzymes such as galactosidase, microperoxidase, horseradish peroxidase (HRP), and alkaline phosphatase and their substrates may be added.

In capturing tumor cell clusters, it is convenient to immobilized a molecule that binds to a tumor cell cluster-specific transmembrane polypeptide. Therefore, the present invention comprises a carrier-immobilized molecule for capturing tumor cell clusters (hereinafter, optionally) comprising a carrier and a molecule that binds to a tumor cell cluster-specific transmembrane polypeptide carried on the carrier. Also provided (referred to as "capture carrier"). The carrier-immobilized molecule is as described above.

The detection reagent, capture reagent, and capture carrier of the present invention can be made into a kit by combining with other standards and instruction manuals. Examples of other preparations include a control reagent, a diluted solution of a sample, a washing solution, and the like. In addition, when combined with a labeled preparation, a molecule necessary for detecting the label (for example, if the label is an enzyme, its substrate, etc.) and the like can be included.

Hereinafter, the present invention will be described in more detail by taking the case where the sample is a blood sample and the tumor cell cluster is a CTC (blood circulating tumor cell) cluster as an example, but the present invention is not limited to this example. Absent.

(Example 1) Gene expression analysis of CTC clusters Gene expression analysis of CTC clusters and leukocytes contained in blood samples derived from prostate cancer patients was performed by the following method.

(1) A blood sample (whole blood) is collected from a prostate cancer patient, 10 times the amount of 1 × BD Pharm Lyse (Becton Dickinson) is added to the collected amount, and the mixture is incubated at room temperature for 10 minutes and then 0.5%. (W / v) D-PBS (-) containing BSA and 2 mM EDTA (Dulbecco's Prostate-Buffered Saline, Mg ²⁺ and Ca ²⁺ free) (hereinafter simply referred to as "buffer") in the blood sample. The cells contained in the cells were washed and suspended in a buffer.

(2) 50 μL (Miltenyi Biotec) of FcR Blocking Reagent was added to 50 μL of the cell suspension of (1) and mixed. Then, 50 μL (PE-anti-human TM4SF1 Antibody, R & D systems) of PE (phycoerythrin) -labeled anti-TM4SF1 antibody solution, 2.5 μL (PE-anti-human TNFRSF12A Antibody) of PE-labeled anti-TNFRSF12A antibody solution, Antibody, Anti-EpCAM antibody solution 10 μL (PE-anti-human EpCAM Antibody, Miltenyi Biotec), FITC (fluorescein isothiocyanate) -labeled anti-CD45 antibody solution 10 μL (CD45-BB515, BD), and 1 mg / mL Hoechst 33 Chemical Laboratory) was added and mixed. After incubating for 10 minutes in the refrigerator, the cells were washed with buffer and suspended in buffer.

(3) To 80 μL of the cell suspension of (2), 20 μL of Anti-PE Microbeads UltraPure (Miltenyi Biotec) was added, and after incubating for 15 minutes in a refrigerator, the cells were washed with a buffer and suspended in a buffer.

(4) The MS column (Miltenyi Biotec) was placed on the MACS Separator (Miltenyi Biotec), washed with a buffer, 500 μL of the cell suspension of (3) was added, and the cells were washed 3 times with 500 μL of buffer.

(5) The washed column was removed from the MACS Separator, 1 mL of buffer was added to the column, and cells were collected by pushing a plunger into the column.

(6) The collected cells were seeded on a slide glass, and CTC clusters were detected under a fluorescence microscope. An example thereof is shown in FIG. In FIG. 1, (a) is a bright-field image of the collected cells, and (b) to (d) are images taken at the same location as (a) using wavelengths suitable for each detection. The PE-labeled tumor cell detection antibody (PE-labeled anti-TM4SF1 antibody, PE-labeled anti-TNFRSF12A antibody, and PE-labeled anti-EpCAM antibody) stains tumor cells, and CD45-FITC antibody (the above-mentioned FITC-labeled antibody). The CD45 antibody) stains leukocytes, and Hoechst 33342 stains the nucleus of cells. Cells in which the PE-labeled tumor cell detection antibody and Hoechst 33342 were confirmed and the CD45-FITC antibody was not stained were detected as tumor cells constituting the CTC cluster.

The detected CTC clusters were collected using a micropick system (Neppagene) (16 populations as clusters). In addition, a plurality of (about 100 cells) leukocytes were also collected using a micropick system (Neppagene).

(7) CTC clusters of all collected (16 populations) and leukocytes (2 samples) were synthesized and amplified by SMART-Seq v4 Ultra Low Output RNA Kit for Sequencing (Clontech).

(8) Using 1 ng of each cDNA obtained in (7), a library was prepared by Nextera XT DNA Library Preparation Kit (Illumina) and Nextera XT v2 Index Kit Set A (Illumina) to prepare a library with Next-sec 500. By performing sequence analysis under the conditions of read length 150 bp and paired end read using illumina), nucleotide sequences of 10 million or more reads per sample were decoded.

(9) The nucleotide sequence (sequence data) decoded in (8) was mapped to the human genome sequence using TopHat2 (Johns Hopkins University) and Bowtie2 (Johns Hopkins University). As the human genome sequence and human gene information, BUILD GRCh38 published by NCBI (National Center for Biological Information) was used. For the mapped nucleotide sequence, the expression value for each gene with FPKM (Fragments Per Kilobase of exon per Million reads mapped) as a unit was determined from the number of reads of each gene decoded by Cufflinks (University of Washington).

(10) Expression comparison was performed for CTC clusters (16 populations) and leukocytes (2 samples).

Tables 1 to 24 above show the average expression value (FPKM value) (CTC cluster) in the CTC cluster (16 populations) of each gene, the average expression value (FPKM value) in the leukocyte (2 samples) (leukocyte), and And their ratio (CTC cluster / leukocyte). All of the genes shown in Tables 1 to 7 are genes having the above ratio (CTC cluster / leukocyte) of 10.00 or more and encoding a transmembrane polypeptide. The genes shown in Tables 8 to 24 are genes having a ratio (CTC cluster / leukocyte) of 10.00 or more and encoding a polypeptide other than the transmembrane polypeptide.

Of the genes, the expression value (FPKM value) for each sample (each population in the CTC cluster) for the gene encoding STEAP1 (SEQ ID NO: 1) is shown in FIG. In FIG. 2, WBC1 and WBC2 show the expression values in each leukocyte, and Cruster1 to Cruster16 show the expression values in the CTC cluster of 16 populations.

As shown in Table 1, the average expression value (FPKM value) of the STEAP1 gene (SEQ ID NO: 577) in the CTC cluster (16 populations) is 600 times or more the average of the FPKM value of leukocytes (2 samples). , STEAP1 gene is expressed specifically and at a high level in the tumor cells constituting the CTC cluster. Moreover, since STEAP1 (SEQ ID NO: 1) is a transmembrane protein, it is suggested that the CTC cluster contained in the sample can be recovered by capturing it.

As described above, according to the present invention, a method for detecting and / or recovering a tumor cell cluster, which can detect and / or recover a tumor cell cluster that is usually contained in a small amount in a sample separately from a contaminating cell. , And reagents and carrier-immobilized molecules used in those methods can be provided.

Claims (12)

A method for detecting a tumor cell cluster, which comprises a step of detecting the expression of at least one gene selected from the group consisting of the following (i) to (iii) in a sample.
(I) A gene encoding a polypeptide containing at least the amino acid sequence set forth in any of SEQ ID NOs: 1 to 576.
(Ii) A gene encoding a polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1 to 576.
(Iii) A gene encoding a polypeptide containing at least one or more amino acids substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 1 to 576. The detection of the expression of the gene is the detection of the polypeptide encoded by the gene, and the detection of the polypeptide is the detection using an antibody or an aptamer that specifically recognizes the polypeptide. The method for detecting a tumor cell cluster according to claim 1. The method for detecting a tumor cell cluster according to claim 1 or 2, wherein the sample is a blood sample. A step of detecting or capturing at least one polypeptide selected from the group consisting of the following (iv) to (vi) in a sample to detect or capture a tumor cell cluster, and the detected or captured tumor. The process of collecting cell clusters and
A method for recovering a tumor cell cluster, which comprises.
(Iv) A polypeptide comprising at least the amino acid sequence set forth in any of SEQ ID NOs: 1-153.
(V) A polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1 to 153.
(Vi) A polypeptide comprising at least one or more amino acid substitutions, deletions, insertions and / or additions in the amino acid sequence set forth in any of SEQ ID NOs: 1-153. The method for recovering a tumor cell cluster according to claim 4, wherein the detection or capture of the polypeptide is detection or capture using an antibody or aptamer that specifically recognizes the polypeptide. The method for recovering a tumor cell cluster according to claim 4 or 5, wherein the sample is a blood sample. A reagent for detecting tumor cell clusters, which comprises a molecule that binds to an expression product of at least one gene selected from the group consisting of the following (i) to (iii) as an active ingredient.
(I) A gene encoding a polypeptide containing at least the amino acid sequence set forth in any of SEQ ID NOs: 1 to 576.
(Ii) A gene encoding a polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1 to 576.
(Iii) A gene encoding a polypeptide containing at least one or more amino acids substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 1 to 576. The reagent according to claim 7, wherein the molecule that binds to the expression product of the gene is an antibody or aptamer that specifically recognizes the polypeptide encoded by the gene. A reagent for detecting or capturing tumor cell clusters, which comprises a molecule that binds to at least one or more polypeptides selected from the group consisting of the following (iv) to (vi) as an active ingredient.
(Iv) A polypeptide comprising at least the amino acid sequence set forth in any of SEQ ID NOs: 1-153.
(V) A polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1 to 153.
(Vi) A polypeptide comprising at least an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted and / or added in the amino acid sequence set forth in any of SEQ ID NOs: 1 to 153. The reagent according to claim 9, wherein the molecule that binds to the polypeptide is an antibody or aptamer that specifically recognizes the polypeptide. Carrier immobilization for capture of tumor cell clusters, characterized in that a molecule that binds to at least one polypeptide selected from the group consisting of (iv) to (vi) below is supported on the carrier. molecule.
(Iv) A polypeptide comprising at least the amino acid sequence set forth in any of SEQ ID NOs: 1-153.
(V) A polypeptide containing at least an amino acid sequence having 70% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1 to 153.
(Vi) A polypeptide comprising at least one or more amino acid substitutions, deletions, insertions and / or additions in the amino acid sequence set forth in any of SEQ ID NOs: 1-153. The carrier-immobilized molecule according to claim 11, wherein the molecule that binds to the polypeptide is an antibody or aptamer that specifically recognizes the polypeptide.