Detection method and detection device

Abstract

Provided are a detection method and a detection device that allow highly accurate detection of exosomes or rare cells. Exosomes derived from first cells are acquired (S10), and the surfaces of the exosomes are modified (S11). Second cells of the same type as the first cells are acquired (S12), and through homotyping, exosomes having modified surfaces are selectively incorporated into the second cells together with labels (S13). A signal correlated with the number of the second cells in which the surface-modified exosomes have been incorporated is acquired from a label signal (S14), and the exosomes or the second cells are detected.

Description

Detection method and detection device

[0001] The present invention relates to a detection method and a detection device.

[0002] Exosomes are extracellular vesicles secreted by cells and are composed of proteins, nucleic acids, lipids, etc. Exosomes have a granular structure with a diameter of 50 to 150 nm and are stably dispersed in body fluids such as blood. Physiologically, exosomes play roles such as removing excess or unnecessary components from cells and regulating intercellular communication.

[0003] Exosomes contain a wealth of information about the cells that secreted them. If it becomes possible to detect and analyze exosomes with high accuracy, it will advance our understanding of cellular processes in biology. In medicine, based on the concept of liquid biopsy, it is expected that exosomes can be extracted from non-invasively collected body fluid samples such as blood and urine, enabling early diagnosis of various diseases, particularly cancer. Therefore, there is a demand for a highly accurate method for detecting exosomes.

[0004] Patent Document 1 discloses a method for detecting extracellular vesicles, which includes a complex formation step of forming a complex by contacting a sample with a peptide supported on a carrier, a reaction step of contacting the complex with a detection probe that specifically binds to the extracellular vesicles, and a detection step of detecting the detection probe that has bound to the complex.

[0005] On the other hand, rare cells such as circulating tumor cells (CTCs), circulating fetal cells, antigen-specific T cells, and hematopoietic stem cells (peripheral blood stem cells) are specific cell groups that exist at very low frequencies in a large cell population. Rare cells exist in extremely small amounts in a cell population; for example, CTCs are said to exist in the blood at a rate of only one in one billion cells.

[0006] Rare cells contain a great deal of biologically and medically important information, and if it becomes possible to reliably detect and analyze rare cells, progress in biology and medicine is expected, and there is a demand for highly accurate methods for detecting rare cells.

[0007] JP 2021-099291 A JP 2013-167582 A International Publication No. 2018 / 047815

[0008] Exosomes are highly heterogeneous in terms of properties such as size, content, cell of origin, and function, making it difficult to detect and analyze individual exosome groups. Previous techniques have been unable to distinguish specific exosomes from others due to issues with sensitivity and specificity, resulting in limited analysis of proteins and nucleic acids contained in total exosome extracts. The partially shared protein and nucleic acid properties among different exosomes make the analysis of individual exosome groups even more challenging using such methods.

[0009] However, in order to detect rare cells with high accuracy, there were challenges, such as the lack of highly specific biomarkers for accurate rare cell identification, the need for huge amounts of sample (blood) to obtain statistically significant rare cell populations, the possibility that rare cells may be lost during sample processing, and the fact that rare cells of a particular type do not all have the same appearance or behavior, and may exhibit heterogeneous characteristics.

[0010] The present invention has been made in consideration of such problems, and aims to provide a detection method and detection device that enable exosomes or rare cells to be detected with high accuracy.

[0011] The detection method of the present invention involves obtaining exosomes derived from a first cell, modifying the surface of the exosomes, obtaining second cells of the same type as the first cell, selectively taking up the surface-modified exosomes together with a label by the second cell, obtaining a signal that correlates with the label taken up by the second cell, and detecting the exosomes or the second cell.

[0012] The detection device of the present invention comprises an exosome acquisition unit that acquires exosomes derived from a first cell, a surface modification unit that modifies the surface of the exosomes, a cell acquisition unit that acquires second cells of the same type as the first cells, a homotyping unit that selectively takes up the surface-modified exosomes together with a label by the second cells, and a detection unit that acquires a signal that correlates with the label taken up by the second cells, and detects the exosomes or the second cells.

[0013] According to the present invention, a detection method and a detection device are provided that enable exosomes or rare cells to be detected with high accuracy.

[0014] 1 is a schematic diagram illustrating a detection method according to the first embodiment. It is a diagram illustrating incubation time relative to the number of exosomes captured per host cell according to the first embodiment. It is a flowchart illustrating a detection method according to the first embodiment. It is a block diagram illustrating the configuration of a detection device according to the first embodiment. It is a diagram illustrating incubation time relative to fluorescence intensity according to Example 1. It is a diagram illustrating LSCM of mixed cancer cells co-cultured with exosomes according to Example 2. It is a diagram illustrating the fluorescence intensity released from labeled exosomes captured per host cell according to Example 2. It is a schematic diagram illustrating a detection method according to the second embodiment. It is a schematic diagram illustrating a bimetal complex used in the detection method according to the second embodiment. It is a schematic diagram illustrating the effect of lanthanoid ions in the detection method according to the second embodiment. It is a diagram illustrating PKH67 intensity relative to the number of host cells according to Example 3. It is a diagram illustrating the number of host cells that captured exosomes relative to the concentration of TNBC-derived exosomes according to Example 3. It is a diagram illustrating 100-specificity relative to sensitivity according to Example 3. It is a diagram illustrating the number of host cells that captured TNBC-derived exosomes per 1 mL of serum sample and the estimated number of TNBC-derived exosomes detected according to Example 4. FIG. 10 is a schematic diagram illustrating a detection method according to a third embodiment. FIG. 11 is a schematic diagram illustrating modification of the exosome surface in the detection method according to the third embodiment. FIG. 12 is a diagram illustrating the incubation time versus fluorescence intensity according to Example 5. FIG. 13 is a diagram illustrating PpIX intensity versus reporter cell count according to Example 6. FIG. 14 is a diagram illustrating the number of CTC-like cells mixed in 1 mL of whole blood versus the number of measured reporter cells according to Example 6. FIG. 15 is a diagram illustrating the number of CTC-like cells mixed in 1 mL of whole blood versus the number of measured reporter cells according to Example 6. FIG. 16 is a diagram illustrating 100-specificity versus sensitivity according to Example 6. FIG. 17 is a diagram illustrating the whole blood volume versus the number of measured reporter cells according to Example 6. FIG. 18 is a diagram illustrating the estimated number of CTCs per mL of whole blood according to Example 7.

[0015] (First Embodiment) (Detection Method for Detecting Exosomes or Cells (Second Cells) of the Same Type as Target Cells (First Cells)) FIG. 1 is a schematic diagram illustrating a detection method according to this embodiment. The detection method according to this embodiment is a method for detecting exosomes or cells (second cells) of the same type as target cells (first cells). Here, cells of the same type as target cells are cells of the same lineage as the target cells. Preferably, they are a group of cells with the same function, and more preferably, cells with the same properties, such as receptor expression and proliferation ability. The left portion 1 of FIG. 1 shows cell 1A secreting exosome 2A. In this embodiment, cell 1A is the target cell. The left portion 1 of FIG. 1 also shows cell 1B secreting exosome 2B and cell 1C secreting exosome 2C. Cell 1B and cell 1C are different types of cells from cell 1A. The right portion 3 of FIG. 1 shows exosome 2A being taken up by cell 3A. In this embodiment, cell 3A is a cell of the same type as cell 1A, which is a target cell. In the presence of exosomes 2A, 2B, and 2C secreted from cell 1A, cell 1B, and cell 1C, respectively, cell 3A selectively takes up exosomes 2A derived from cell 1A, which is of the same type, due to the property of a specific cell selectively taking up exosomes derived from cells of the same type (hereinafter referred to as homotyping property or homotypic targeting property).

[0016] By homotyping, the time it takes for cell 3A to selectively incorporate exosome 2A derived from cell 1A of the same type is called the homotyping target time.

[0017] In this embodiment, the surface of exosome 2A is modified before exosome 2A is taken up by cell 3A. This enhances the homotyping property. Surface-modified exosome 2A' is taken up by cell 3A' of the same type as the cell from which exosome 2A' is derived. Cell 3A' is also referred to as a host cell that takes up exosome 2A'. The enhanced homotyping property as described above enhances the selectivity when cell 3A' takes up a specific type of exosome 2A', thereby shortening the homotyping targeting time.

[0018] In this embodiment, when a specific type of exosome 2A' is taken up by a cell 3A', the cell 3A' also takes up a fluorescent label or the like along with the exosome 2A'. After the cell 3A' has taken up the fluorescent label or the like along with the exosome 2A', the cell 3A' that has taken up the fluorescent label or the like is detected by flow cytometry or the like, thereby detecting the specific type of exosome 2A' taken up by the cell 3A'. Alternatively, the fluorescent label or the like taken up by the cell 3A' is taken up by a reporter cell or the like, and the reporter cell or the like that has taken up the fluorescent label or the like is detected by flow cytometry or the like, thereby detecting the cell 3A' that has taken up the specific type of exosome 2A'.

[0019] As shown in Figure 1, exosomes 2A, 2B, and 2C secreted from cells 1A, 1B, and 1C, respectively, are incubated with host cells 3A of the same type as the cells from which exosome 2A was derived. These cells are then taken up by the host cells. Figure 2 is a schematic diagram showing the number of exosomes captured per host cell versus incubation time. In Figure 2, graphs A, B, and C show the numbers of exosomes 2A, 2B, and 2C taken up by the host cells, respectively. As shown in graphs A, B, and C, as the incubation time is increased, the number of exosomes 2A taken up by the host cells increases compared to exosomes 2B and 2C, demonstrating the selective uptake of exosome 2A by host cells due to homotyping characteristics.

[0020] 2 further shows the number of exosomes captured per host cell versus incubation time when surface-modified exosomes 2A' were incubated with host cells (Graph A'). Comparing Graph A and Graph A', it can be seen that modifying the exosome surface boosts homotyping properties and shortens homotyping target time.

[0021] FIG. 3 is a flowchart illustrating a detection method according to this embodiment. In this embodiment, exosomes derived from target cells (first cells) are obtained (S10), and then the surface of the exosomes is modified (S11). Meanwhile, cells of the same type as the target cells (second cells) are obtained (S12). The step of obtaining cells of the same type as the target cells (S12) may be performed in any order: before the step of obtaining exosomes (S10), between the step of obtaining exosomes (S10) and the step of surface modifying the exosomes (S11), or after the step of surface modifying the exosomes (S11). Next, the surface-modified exosomes are selectively taken up by cells of the same type as the target cells by homotyping, together with a fluorescent label or the like (S13). A signal such as fluorescence is obtained from the fluorescent label or the like by a detection method such as flow cytometry, which correlates with the number of cells of the same type as the target cells that have taken up the surface-modified exosomes (S14), thereby detecting the exosomes or cells of the same type as the target cells. The acquisition of the signal (S14) is not limited to flow cytometry using fluorescent labels, but other detection methods that are capable of detecting cells, such as other detection methods using fluorescent labels or other detection methods using labels other than fluorescent labels, can be applied.

[0022] (Detection Device for Detecting Exosomes or Cells of the Same Type as Target Cells) FIG. 4 is a block diagram showing the configuration of a detection device according to this embodiment. The detection device 4 includes an exosome acquisition unit 5, a surface modification unit 6, a cell acquisition unit 7, a homotyping unit 8, and a detection unit 9. The exosome acquisition unit 5 acquires exosomes derived from target cells (first cells). The surface modification unit 6 modifies the surface of the exosomes. The cell acquisition unit 7 acquires cells of the same type as the target cells (second cells). The homotyping unit 8 introduces the surface-modified exosomes and cells of the same type as the target cells, and selectively incorporates the surface-modified exosomes together with the fluorescent label by cells of the same type as the target cells through homotyping. The detection unit 9 acquires a signal correlating with the fluorescent label, etc., incorporated into cells of the same type as the target cells. As described above, the detection device of this embodiment detects exosomes or cells of the same type as the target cells using the detection method of this embodiment.

[0023] In this embodiment, for example, cells of the same type as the target cells acquired by the cell acquisition unit 7 are cells acquired from a known cell line, and exosomes taken up by cells acquired from the known cell line are detected.

[0024] Alternatively, in this embodiment, for example, the exosomes derived from the target cells obtained by the exosome obtaining unit 5 are exosomes obtained from cells of a known cell line, and cells of the same type as the target cells that have taken up the exosomes obtained from cells of the known cell line are detected.

[0025] In this embodiment, preferably, in the homotyping unit 8, a fluorescent label or the like that has been selectively taken up together with surface-modified exosomes by cells of the same type as the target cells is taken up into reporter cells, and in the detection unit 9, a signal such as fluorescence that correlates with the number of cells of the same type as the target cells that have taken up the surface-modified exosomes is obtained from the fluorescent label or the like taken up by the reporter cells, thereby detecting cells of the same type as the target cells that have taken up exosomes obtained from cells of a known cell line.

[0026] In this embodiment, the surface of the exosomes is preferably modified with a bimetallic complex containing a lanthanoid ion in the surface modification unit 6. The lanthanoid ion contains, for example, at least one of Eu and Tb.

[0027] In this embodiment, the detection unit 9 preferably comprises a flow cytometer, which acquires a fluorescent signal from the fluorescent label that correlates with the number of cells of the same type as the target cells that have taken up the surface-modified exosomes. Flow cytometry is a device that guides suspended cells one by one into a measurement region using a sheath flow and irradiates them with excitation light to measure the fluorescence, etc., of each cell (see, for example, Patent Document 2), and standard flow cytometry commonly used in research institutions, etc., can be used. Alternatively, imaging flow cytometry (see, for example, Patent Document 3) can be used, which acquires images of fluorescence, etc., and acquires information about each cell by cell image processing.

[0028] (Actions and effects of the detection method and detection device for detecting exosomes or cells of the same type as target cells) According to the detection method and detection device of this embodiment, the surface of the exosome 2A is modified before the exosome 2A is taken up into the cell 3A, thereby enhancing the homotyping characteristics. Therefore, a detection method and detection device can be provided that can detect exosomes or cells of the same type as the target cells with high accuracy, and by selecting rare cells as cells of the same type as the target cells, rare cells can be detected with high accuracy and the homotyping target time can be shortened.

[0029] Example 1: In the laboratory, a serum sample containing exosomes (Exo-MDA) derived from triple-negative breast cancer (TNBC), a serum sample containing exosomes (Exo-MCF7) derived from breast cancer other than triple-negative breast cancer, and a serum sample containing exosomes derived from blood cells (Exo-BLD) were obtained. Furthermore, the TNBC-derived exosomes were enriched with lanthanide ions (Eu ３＋ ), TDA (2,2'-thiodiacetic acid), and cobalt ions (Co ２＋ The surface of the exosomes was modified using a bimetallic complex consisting of MDA-Eu and MDA-MB-436. A serum sample containing surface-modified TNBC-derived exosomes (Exo-MDA-Eu) was obtained. A fluorescent label was incorporated into each exosome. Each serum sample was mixed with TNBC cell line (MDA-MB-436) cells (host cells) and incubated. After incubation, the fluorescent signal of each serum sample was captured using a laser scanning confocal microscope (LSCM).

[0030] Figure 5(a) shows the fluorescence intensity versus incubation time for Example 1. Figure 5(b) is an enlarged view of Figure 5(a) around the 3-hour incubation time. Figure 5 shows the fluorescence intensities corresponding to TNBC-derived exosomes (Exo-MDA), BC-derived exosomes (Exo-MCF7), blood cell-derived exosomes (Exo-BLD), and surface-modified TNBC-derived exosomes (Exo-MDA-Eu). Fluorescence intensity correlates with the number of exosomes taken up by host cells. Comparison of TNBC-derived exosomes (Exo-MDA), BC-derived exosomes (Exo-MCF7), and blood cell-derived exosomes (Exo-BLD) revealed that with increasing incubation time, the number of TNBC-derived exosomes (Exo-MDA) taken up by host cells increased compared to BC-derived exosomes (Exo-MCF7) and blood cell-derived exosomes (Exo-BLD), indicating that the homotyping properties of TNBC-derived exosomes (Exo-MDA) are selectively taken up by host cells. Comparison of TNBC-derived exosomes (Exo-MDA) and surface-modified TNBC-derived exosomes (Exo-MDA-Eu) revealed that surface modification of the exosomes increased the fluorescence intensity by 25.6-fold after 3 hours of incubation. Thus, the homotyping properties were enhanced (boosted) and the homotyping targeting time was shortened.

[0031] (Example 2) The capture of different surface-modified exosomes derived from different cells by mixed cells was further verified using LSCM.

[0032] Laboratory-derived MDA-MB-436 cells and non-triple-negative breast cancer cell line (MCF-7) cells were labeled with Cell Tracker Far Red reagent, and the cells were mixed at a 1:1 ratio and co-cultured on glass-bottom plates for 2 hours. These cell groups were then co-cultured with TNBC-derived exosomes (Exo-MDA), BC-derived exosomes (Exo-MCF7), and lanthanide ions (Eu ３＋TNBC-derived exosomes (Exo-MDA-Eu), Eu ３＋ The cells were incubated for 20 hours with a suspension of surface-modified BC-derived exosomes (Exo-MCF7-Eu) or a suspension containing only the fluorescent dye (PKH67) as a negative control. After washing and fixation, the nuclei were stained with DAPI.

[0033] Figure 6 (a) shows TNBC-derived exosomes (Exo-MDA), Eu ３＋ The images were taken with LSCM after co-culture of TNBC-derived exosomes (Exo-MDA-Eu) or PKH67 (control) with MDA-MB-436 and MCF-7 cells. Figure 6(b) shows the results of co-culture of BC-derived exosomes (Exo-MCF7), Eu ３＋ These images were taken with LSCM after co-culture of BC-derived exosomes (Exo-MCF7-Eu) or PKH67 (control) with MDA-MB-436 and MCF-7 cells, whose surfaces had been modified by introducing PKH67. In all experiments except the control, the same experiment was repeated, but with the MDA-MB-436 and MCF-7 cells stained with Cell Tracker interchangeably. The arrows indicate exosomes captured by the cells. The scale bar (top) is 100 μm, and the scale bar (bottom) is 20 μm. It was shown that MDA-MB-436-derived exosomes (Exo-MDA) were selectively captured by cells of the same lineage (MDA-MB-436 cells). This targeting property was due to the lanthanide ion (Eu) ３＋ This trend was maintained even after surface modification by the introduction of exosomes. A similar trend was observed in LSCM images of cells co-cultured with MCF-7-derived exosomes, and Exo-MCF7 and Exo-MCF7-Eu were selectively captured by MCF-7 cells.

[0034] Figure 7(a) shows the statistical results of the intensity of fluorescence emitted from exosomes (Exo-MDA or Exo-MDA-Eu) captured by MDA-MB-436 or MCF7. Figure 7(b) shows the statistical results of the intensity of fluorescence emitted from exosomes (Exo-MCF7 or Exo-MCF7-Eu) captured by MDA-MB-436 or MCF7. Data were analyzed using one-way ANOVA with Tukey's post-hoc test. Both types of exosomes showed significantly higher uptake rates in cells derived from the same cell line in co-cultured cells. ３＋ These results suggest that the exosomes modified with lanthanide ions (Eu) exhibited significantly higher uptake rates than the unmodified exosomes. ３＋ ) enhanced the interaction between exosomes and cells without compromising the selective targeting properties of exosomes.

[0035] (Second embodiment) (Exosome detection method) Exosomes exist in body fluids in a state that contains a large amount of information about the cells from which they are secreted. Because exosomes containing disease characteristics are secreted from abnormal cells, they are expected to become important biomarkers for various diseases, particularly cancer diagnosis, and the detection and analysis of exosomes is expected to lead to diagnosis and treatment. Because molecules specifically present in cancer cells can be used to evaluate the molecular or cellular profile of cancer in real time, they are ideal for long-term monitoring of a patient's disease progression and treatment response.

[0036] Exosomes have traditionally been detected and analyzed using various methods, such as nanoparticle tracking analysis (NTA) or transmission electron microscopy (TEM), but it has been difficult to detect specific types of exosomes with high accuracy.

[0037] The exosome detection method of this embodiment aims to detect exosomes with high accuracy. In this embodiment, a method for selectively capturing and isolating a specific type of exosome is established, and in particular, practical exosome detection is achieved in a short time using analytical equipment commonly used in research institutions, etc., and specific exosomes are extracted with high detection sensitivity and specificity from a sample containing a large amount of heterogeneous exosomes.

[0038] The exosomes to be detected by the exosome detection method of this embodiment are not particularly limited, but may be, for example, exosomes derived from triple-negative breast cancer (TNBC). Triple-negative breast cancer is a subtype of breast cancer (BC) that is most difficult to detect, as it lacks the three most common breast cancer cell surface receptors: estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The exosome detection method of this embodiment enables highly accurate detection of exosomes from the presence of other exosomes.

[0039] FIG. 8 is a schematic diagram illustrating the detection method according to this embodiment. In the detection method of this embodiment, a blood sample 11 is first obtained from a subject 10 shown in FIG. 8(a), as shown in FIG. 8(b). The subject 10 may have, for example, triple-negative breast cancer (TNBC) 10-1, other types of breast cancer (BC) 10-2, breast cancer (BC) metastasis to the lungs 10-3, breast cancer (BC) metastasis to the bones 10-4, and breast cancer (BC) metastasis to the liver 10-5. Hereinafter, a case will be described in which the exosome detection method targets TNBC-derived exosomes and the target cells (first cells) are TNBC cells. To detect TNBC-derived exosomes, cells (second cells) of the same type as the target cells described below are TNBC cells obtained from a known cell line.

[0040] Next, as shown in (c) of Figure 8, the obtained blood sample is subjected to a process such as centrifugation to obtain serum 12. The serum 12 contains an exosome population 13 as shown in (d) of Figure 8. The exosome population 13 contains, for example, TNBC-derived exosome 13-3, BC-derived exosome 13-2, and blood cell-derived exosome 13-1.

[0041] Next, as shown in FIG. 8(e), a bimetallic complex 14 containing a lanthanoid ion 14-1 (described below) is used to introduce the lanthanoid ion 14-1 into sialic acid SA expressed on the surface of various exosomes (TNBC-derived exosome 13-3, BC-derived exosome 13-2, and blood cell-derived exosome 13-1) in the exosome population 13, thereby modifying the surface of the various exosomes. The preferred concentration of the bimetallic complex 14 is 0.05 mg / mL or more and 0.2 mg / mL or less. The lanthanoid ion 14-1 can be, for example, Eu ３＋ Or Tb ３＋ These ions include lanthanoid ions 14-1 and 14-2. By modifying the surface of various exosomes, the homotyping property is enhanced, as described below. Here, when modifying the surface of various exosomes with a bimetallic complex 14, a fluorescent label 15, such as PKH67, is simultaneously introduced into the various exosomes. Hereinafter, the fluorescent label 15 is not shown. Subsequently, various exosomes whose surfaces have been modified by the introduction of lanthanoid ions 14-1 are mixed with TNBC cells 16, which are the same type of cells as the target cells, and incubated for a predetermined period of time. By homotyping, the surface-modified TNBC-derived exosomes 13-3 are selectively taken up into the TNBC cells 16 together with the fluorescent label (not shown). In this embodiment, cells of the same type as the target cells that take up specific exosomes are also referred to as host cells. The TNBC cells 16 are obtained from a known cell line and serve as host cells for taking up the surface-modified TNBC-derived exosomes 13-3. Using host cells, specific exosomes (exosomes derived from surface-modified TNBC) are selectively isolated. The concentration of host cells is preferably 1 x 10 per mL of serum. ４ More than 1×10 pieces ８ There are less than 100 pieces.

[0042] In the above-mentioned homotyping, homotyping is enhanced by introducing lanthanoid ions into exosomes expressing sialic acid on their surface. The composition of exosomes is not limited as long as homotyping is enhanced by modifying the surface of exosomes by introducing lanthanoid ions. In addition, Eu as a lanthanoid ion is also usable. ３＋ and Tb ３＋ However, other lanthanide ions can be used. Furthermore, the surface modification of exosomes may be achieved by introducing a substance other than lanthanide ions, as long as it enhances homotyping.

[0043] 8(f), TNBC cells 16 that have selectively taken up TNBC-derived exosomes 13-3, the surfaces of which have been modified by the introduction of lanthanoid ions 14-1, together with a fluorescent label (not shown), are detected using flow cytometry 17. This allows the detection of TNBC-derived exosomes in the obtained blood sample.

[0044] 9 is a schematic diagram illustrating a bimetal complex 14 used in the detection method according to this embodiment. The bimetal complex 14 is a complex consisting of a lanthanoid ion 14-1, TDA (2,2'-thiodiacetic acid) 14-2, and a cobalt ion 14-3. As shown in FIG. 9(a), in water, a lanthanoid ion 14-1, TDA (2,2'-thiodiacetic acid) 14-2, and a cobalt ion (Co ２＋ ) 14-3 is mixed to obtain a bimetallic complex 14 as shown in FIG. 9(b). The lanthanide ion 14-1 is, for example, Eu ３＋With reference to (c) of Figure 9, the case where sialic acid SA is present together with the bimetallic complex 14 will be described. Sialic acid SA is a monosaccharide that is overexpressed on the membrane of both TNBC-derived exosomes 13-3 and TNBC cells 16. When sialic acid SA is present together with the bimetallic complex 14, the lanthanoid ion 14-1 in the bimetallic complex 14 coordinates with the sialic acid SA, dissociates from the bimetallic complex 14, and moves toward the sialic acid SA. As described above, the lanthanoid ion 14-1 is introduced into the sialic acid SA expressed on the surface of the TNBC-derived exosomes 13-3, thereby modifying the surface of the TNBC-derived exosomes 13-3.

[0045] An example of bimetallic complex 14 is (TDA). ｘ (Co ２＋ ) ｙ EU ３＋ For example, a method for preparing an aqueous solution of TDA (0.60 g) and Co(NO ３ ) ２ 6H ２ 0 (0.89 g) was dissolved in 5.0 mL of water and shaken for 0.5 h, while Eu(NO ３ ) ３ ・6H ２ Dissolve 0.59 g of TDA in 5.0 mL of water, mix these solutions, and shake the mixture for 1 hour. In this way, a solution of 0.15 g / mL of TDA was obtained. ｘ (Co ２＋ ) ｙ EU ３＋ The aqueous solution of the bimetallic complex obtained is stored at 4°C.

[0046] 10 is a schematic diagram illustrating the effect of lanthanoid ions in a detection method according to an embodiment. When TNBC-derived exosomes 13-3, the surface of which has been modified by the introduction of lanthanoid ions 14-1, are mixed with TNBC cells 16, which are the same type of cells as the target cells, the surface-modified TNBC-derived exosomes 13-3 are selectively taken up by the TNBC cells 16 through homotyping. At this time, the lanthanoid ions 14-1 coordinately bond to sialic acid SA expressed on the surface of the TNBC-derived exosomes 13-3 and sialic acid SA expressed on the surface of the TNBC cells 16. By introducing lanthanoid ions 14-1 into the surface of the TNBC-derived exosomes 13-3 and modifying it, the interaction between the TNBC cells 16 and the TNBC-derived exosomes 13-3 is significantly amplified, enhancing the selectivity of the homotyping characteristics and shortening the homotyping target time to approximately one hour.

[0047] (Exosome detection device) In the detection device described in the first embodiment, cells of the same type as the target cells acquired by the cell acquisition unit 7 are cells acquired from a known cell line. In this configuration, exosomes taken up by cells acquired from the known cell line are detected.

[0048] (Actions and Effects of Detection Method and Detection Device for Detecting Exosomes) According to the detection method and detection device of this embodiment, the surface of the TNBC-derived exosomes 13-3 is modified before the TNBC-derived exosomes 13-3 are taken up by the TNBC cells 16, thereby enhancing the homotyping characteristics, and therefore, a detection method and detection device that can detect exosomes with high accuracy can be provided.

[0049] (Example 3) 2 x 10 in 1 mL of serum in the laboratory ５ TNBC-derived exosomes (Exo-MDA) and 4 × 10 ８ A serum sample A containing exosomes (Exo-BLD) derived from blood cells was obtained, and lanthanide ions (Eu ３＋ ), TDA, and cobalt ions (Co ２＋The exosomes were mixed with a bimetallic complex consisting of β-lactam β-lactam (B-lactam β ... In Figure 11(a), the vertical axis represents the number of host cells, and the horizontal axis represents PKH67 intensity (relative value). In the hatched region on the right side of Figure 11(a), it was confirmed that a fluorescent signal correlated with the number of TNBC cells that had taken up exosomes derived from the surface-modified TNBC was obtained, based on the difference from the fluorescent signal of a serum sample (control) that did not contain exosomes.

[0050] For serum sample A, 5 x 10 ７ Serum sample B containing BC-derived exosomes (Exo-MCF7) was obtained, and the exosomes were surface-modified as described above. The surface-modified TNBC-derived exosomes were selectively taken up by TNBC cells by homotyping, and fluorescent signals were obtained by flow cytometry. The obtained fluorescent signal is shown by the solid line in Figure 11(b). The fluorescent signal of the serum sample (Control) containing no exosomes is also shown by the dotted line in Figure 11(b). Similarly, Figure 11(b) confirms that a fluorescent signal was obtained that correlates with the number of TNBC cells that had taken up surface-modified TNBC-derived exosomes.

[0051] For serum sample A and serum sample B, the surface of exosomes was modified in the same manner as described above. The surface-modified TNBC-derived exosomes were selectively incorporated into TNBC cells by homotyping, and fluorescent signals were obtained by imaging flow cytometry. A homemade imaging flow cytometry device, as described in Patent Document 3, etc., was used. The obtained fluorescent signals are shown by solid lines in Figures 11(c) and 11(d), respectively. The fluorescent signals of the exosome-free serum sample (Control) are also shown by dotted lines in Figures 11(c) and 11(d), respectively. Similarly, Figures 11(c) and 11(d) confirm that fluorescent signals correlated with the number of TNBC cells that had incorporated surface-modified TNBC-derived exosomes.

[0052] 4 x 10 in 1 mL of serum ８ Serum samples containing blood cell-derived exosomes (Exo-BLD) and TNBC-derived exosomes (Exo-MDA) at different concentrations were obtained. The exosomes were surface-modified as described above. The surface-modified TNBC-derived exosomes were selectively taken up by TNBC cells by homotyping, and fluorescent signals were obtained by flow cytometry (FC) and imaging flow cytometry (IFC). The number of host cells that captured TNBC-derived exosomes was determined from the obtained fluorescent signals. The results are shown in Figure 12(a). In Figure 12(a), the vertical axis represents the number of host cells that captured TNBC-derived exosomes, and the horizontal axis represents the exosome concentration (number of exosomes per mL of serum). Error bars represent standard deviation (n = 3). Figure 12(b) shows the logarithm of the number of host cells that captured TNBC-derived exosomes and the exosome concentration in Figure 12(a). Host cell counts detected by flow cytometry and imaging flow cytometry showed a strong linear correlation with increasing exosome concentration. The detection limit for TNBC-derived exosomes was 750 cells / mL of serum when fluorescently detected by flow cytometry and 65 cells / mL of serum when fluorescently detected by imaging flow cytometry.

[0053] For a serum sample containing blood cell-derived exosomes (Exo-BLD) and TNBC-derived exosomes (Exo-MDA), the receiver operating characteristic (ROC) curves were obtained by flow cytometry (FC) and imaging flow cytometry (IFC) in the same manner as described above. The results are shown in Figure 13(a).

[0054] Furthermore, for serum samples containing blood cell-derived exosomes (Exo-BLD), TNBC-derived exosomes (Exo-MDA), and BC-derived exosomes (Exo-MCF7), ROC curves were obtained by obtaining fluorescence signals using flow cytometry (FC) and imaging flow cytometry (IFC) in the same manner as described above. The results are shown in Figure 13(b).

[0055] 13(a) and (b), the vertical axis represents sensitivity (%), and the horizontal axis represents 100-specificity (%). Figures 13(a) and 13(b) show that the exosome detection method of this example has very high sensitivity and specificity, based on the AUC (area under the ROC curve) obtained from the ROC curve.

[0056] Example 4: Preclinical evaluation was performed using mouse specimens bearing TNBC. Serum samples containing TNBC-derived exosomes (Exo-MDA) along with excess blood cell-derived exosomes (Exo-BLD) per mL of serum were obtained from TNBC-affected mouse specimens. The exosomes were surface-modified as in Example 3 and mixed with 3,000 TNBC cells (host cells) obtained from a known cell line. The surface-modified TNBC-derived exosomes were selectively incorporated into the TNBC cells by homotyping, and fluorescent signals were obtained by flow cytometry. The number of host cells capturing TNBC-derived exosomes per mL of serum sample was determined from the resulting fluorescent signals. A similar test was performed on serum samples from healthy mice, and the number of host cells capturing TNBC-derived exosomes per mL of serum sample was determined. The results are shown in Figure 14(a). From the obtained host counts, the estimated number of TNBC-derived exosomes detected per mL of serum sample was calculated with reference to the data shown in Figure 12(b), and is shown on the right vertical axis in Figure 14(a). In Figure 14(a), A represents a serum sample obtained from a healthy mouse, and B represents a serum sample obtained from a mouse specimen with TNBC. Five tests were performed for each serum sample. To evaluate the statistical significance of the difference between the two groups of healthy mice and TNBC-affected mice, a two-tailed t-test was used to calculate a p-value. A statistical significance of p<0.001 was confirmed for the two groups of healthy mice and TNBC-affected mice.

[0057] The same test was performed using imaging flow cytometry instead of flow cytometry to obtain fluorescent signals. The number of host cells capturing TNBC-derived exosomes per mL of serum sample and the estimated number of TNBC-derived exosomes detected per mL of serum sample were obtained. The results are shown in Figure 14(b). In Figure 14(b), A represents a serum sample obtained from a healthy mouse, and B represents a serum sample obtained from a mouse specimen with TNBC. Five tests were performed for each serum sample. A statistical significance of p<0.0001 was confirmed between the two groups of healthy mice and TNBC-affected mice.

[0058] The above-described exosome detection method reduced the time required for specific cells to selectively take up exosomes derived from the same cell type by more than 10-fold. This enhanced targeting property enabled the realization of an exosome detection method exhibiting high detection sensitivity and specificity, with an AUC value of 0.93 in the receiver operating characteristic curve. Furthermore, the method demonstrated a significant detection limit of approximately 750 exosomes from a 1 mL sample solution, and was able to distinguish specific exosomes from other exosome populations with an accuracy of p<0.001. Compared to conventional TNBC detection methods such as magnetic resonance imaging (MRI) and immunohistochemistry, the method demonstrated outstanding performance in terms of speed, sensitivity, specificity, and cost-effectiveness.

[0059] (Third embodiment) (Method for detecting rare cells) Rare cells refer to a specific group of cells that exist at a very low frequency in a large cell population. Such cells, which appear very rarely in the blood, are believed to provide a wealth of biologically and medically important information. Due to their importance in many biomedical fields, such as oncology, prenatal diagnosis, and immunology, there is a need for the development and improvement of techniques for reliably detecting rare cells.

[0060] Examples of rare cells include circulating tumor cells (CTCs), circulating fetal cells, antigen-specific T cells, and hematopoietic stem cells (peripheral blood stem cells). Circulating tumor cells (CTCs) are cancer cells circulating in the bloodstream that have detached from primary tumors. They are considered to be an indicator of the potential spread and metastasis of cancer. They are an important target for detection in liquid biopsies, which aim to diagnose cancer and other diseases early using non-invasively collected body fluid samples. Circulating fetal cells are fetal cells that migrate from the fetus into the maternal bloodstream during pregnancy. Their detection and analysis can enable non-invasive assessment of the fetus's health and genetic status. Antigen-specific T cells are cells that are tuned to detect specific pathogens and tumor antigens in the adaptive immune system. They are attracting attention in basic research on immune system activation and specificity, including immune responses to infections and tumors. Hematopoietic stem cells (peripheral blood stem cells) are versatile progenitor cells that can differentiate into any type of blood cell in the bloodstream. They contribute to our understanding of the mechanisms of blood cell production and advances in transplantation and regenerative medicine. Endothelial progenitor cells, which are essential for vascular repair and angiogenesis, serve as indicators of cardiovascular health and the progression of several diseases.

[0061] Detecting rare cells is extremely challenging due to their extremely low abundance in a cell population: for example, circulating tumor cells are thought to be present in the blood at a rate of only one in every billion cells.

[0062] In order to detect rare cells with high accuracy, there were challenges such as the lack of highly specific biomarkers for accurate rare cell identification, the need for huge amounts of sample (blood) to obtain statistically significant rare cell populations, the possibility that rare cells may be lost during sample processing, and the fact that rare cells of a specific type do not all have the same appearance or behavior and may exhibit heterogeneous characteristics.

[0063] Flow cytometry, a powerful tool for analyzing cell populations, is the most common conventional method, but it lacks the sensitivity and specificity required for detecting rare cells. Furthermore, other specialized techniques are not practical due to their high cost and complex setup.

[0064] The purpose of the rare cell detection method of this embodiment is to detect rare cells with high accuracy. In this embodiment, a new rare cell detection method is constructed that detects specific types of rare cells with high sensitivity and single-cell resolution, and in particular, the purpose is to achieve practical rare cell detection in a short time using analytical equipment that is standard and widely used in research institutions, etc., and to extract specific rare cells from samples containing large cell populations with high detection sensitivity and specificity.

[0065] The rare cells to be detected by the rare cell detection method of this embodiment are not particularly limited, but are, for example, CTC cells separated from TNBC. According to the rare cell detection method of this embodiment, it is possible to detect CTC cells that are very rarely present in blood with high accuracy.

[0066] FIG. 15 is a schematic diagram illustrating the detection method according to this embodiment. In the detection method according to this embodiment, a blood sample 21 is first obtained from a subject 20 shown in FIG. 15(a), as shown in FIG. 15(b). The subject 20 may have, for example, TNBC 20-1, metastasis from TNBC 20-2, and CTC cells 20-3 detached from TNBC. Hereinafter, the detection target in the rare cell detection method is TNBC-derived CTC cells, and the exosomes used to detect TNBC-derived CTC cells are exosomes obtained from cells (first cells) of a known TNBC cell line.

[0067] Next, as shown in (c) of Figure 15, the obtained blood sample is subjected to a process such as centrifugation to obtain a centrifuged blood sample 22. The centrifuged blood sample 22 contains, for example, CTC cells 22-1 and PBMCs (peripheral blood mononuclear cells) 22-2. The CTC cells 22-1 are cells of the same type (second cells) as cells of the TNBC cell line (first cells). The detection limit for the CTC cells to be detected is one, and single-cell resolution is possible.

[0068] Next, as shown in (d) of FIG. 15, a signal amplification step is performed. The signal amplification step includes the steps shown in (d-1), (d-2), and (d-3) of FIG. 15, which will be described later. Prior to the signal amplification step, the steps shown in (f-1) and (f-2) of FIG. 15 are performed. First, as shown in (f-1) of FIG. 15, TNBC-derived exosomes 23 are obtained from known TNBC cells. Next, as shown in (f-2) of FIG. 15, a bimetallic complex 24 containing a lanthanoid ion 24-1 is used to introduce the lanthanoid ion 24-1 into sialic acid SA expressed on the surface of the TNBC-derived exosomes 23, thereby modifying the surface of the TNBC-derived exosomes 23 to obtain surface-modified TNBC-derived exosomes 23X. A preferred concentration of the bimetallic complex 24 used to modify the surface of the exosomes 23 is 0.05 mg / mL or more and 0.2 mg / mL or less. The lanthanide ion 24-1 is, for example, Eu ３＋ Or Tb ３＋ etc. Modification of the surface of TNBC-derived exosomes 23 enhances homotyping properties. Here, when modifying the surface of TNBC-derived exosomes 23 with bimetallic complexes 24, a fluorescent label, HAL (hexaminolevulinate) 25, is simultaneously introduced into the TNBC-derived exosomes 23. In this way, HAL 25 is introduced into the surface-modified TNBC-derived exosomes 23X. HAL 25 not incorporated into the exosomes is removed at some stage.

[0069] The surface-modified TNBC-derived exosomes 23X are obtained as described above, and then, as shown in (d-1) of Figure 15, the surface-modified TNBC-derived exosomes 23X introduced with lanthanoid ions 24-1 are mixed with the centrifuged blood sample 22 containing CTC cells 22-1 and PBMCs 22-2. The concentration of the surface-modified TNBC-derived exosomes 23X is preferably 1 x 10 per mL of serum. ５ More than 1×10 pieces ９ There are less than 100 pieces.

[0070] Next, by performing incubation for a predetermined time, the surface-modified TNBC-derived exosomes 23X are selectively taken up into CTC cells 22-1 together with HAL 25 by homotyping. Thereafter, the surface-modified TNBC-derived exosomes 23X that have not been taken up into CTC cells 22-1 are removed.

[0071] Next, as shown in (d-3) of FIG. 15, CTC cells 22-1 and PBMC 22-2 are lysed by ultrasonic treatment to form lysed cells 26, which releases HAL25 incorporated into TNBC-derived exosomes incorporated into CTC cells 22-1. The HAL25 released from the lysed cells is then incorporated into TNBC cells (reporter cells) 27. The reporter cells 27 are present in sufficient amounts relative to the released HAL25. HAL25 is converted to PpIX (Protoporphyrin IX) 28 in the reporter cells 27.

[0072] 15(e), reporter cells 27 that have incorporated PpIX 28 are detected using flow cytometry 29. In this way, CTC cells in the obtained blood sample are detected.

[0073] The HAL 25 taken up by the CTC cells 22-1 together with the surface-modified TNBC-derived exosomes 23X is released by cell lysis 26 and then taken up by a sufficient amount of reporter cells 27, thereby amplifying the signal from the fluorescent label, and a signal correlating with the CTC cells 22-1 that have taken up the TNBC-derived exosomes can be obtained by flow cytometry 29.

[0074] FIG. 16 is a schematic diagram illustrating the modification of exosome surfaces in a detection method according to an embodiment. As shown in FIG. 16(a), exosomes are isolated from a known TNBC cell line 23A to prepare exosomes for detection, yielding TNBC-derived exosomes 23 as shown in FIG. 16(b). The surfaces of the resulting TNBC-derived exosomes 23 are modified with a bimetallic complex. For example, by mixing the TNBC-derived exosomes 23 with a bimetallic complex consisting of lanthanoid ion 24-1, TDA, and cobalt ions, lanthanoid ion 24-1 is introduced into sialic acid SA expressed on the surface of the TNBC-derived exosomes 23, resulting in surface-modified TNBC-derived exosomes, as shown in FIG. 16(c).

[0075] When CTC cells 22-1 are mixed with TNBC-derived exosomes 23X whose surfaces have been modified by the introduction of lanthanoid ion 24-1, the surface-modified TNBC-derived exosomes 23X are selectively taken up by CTC cells 22-1 through homotyping. The surface-modified TNBC-derived exosomes 23X are modified by the introduction of lanthanoid ion 14-1 onto their surfaces, which significantly amplifies the interaction between CTC cells 22-1 and the surface-modified TNBC-derived exosomes 23X, enhancing the selectivity of the homotyping properties and shortening the homotyping target time to approximately 1 hour.

[0076] (Device for detecting rare cells) In the detection device described in the first embodiment, the exosomes acquired by the exosome acquisition unit 5 are exosomes acquired from cells of a known cell line. In this configuration, rare cells that have taken up exosomes acquired from cells of a known cell line are detected.

[0077] (Actions and effects of the detection method and detection device for detecting rare cells) According to the detection method and detection device of this embodiment, TNBC-derived exosomes 23 obtained from a known cell line are converted into surface-modified TNBC-derived exosomes 23X before being taken up by CTC cells 22-1, thereby enhancing the homotyping properties, and it is therefore possible to provide a detection method and detection device that can detect rare cells with high accuracy.

[0078] Example 5: Samples containing TNBC-derived exosomes (Exo-MDA) were obtained in the laboratory and mixed with TNBC cell line cells. Fluorescence signals were obtained by flow cytometry for the samples after different incubation times. Similarly, samples containing BC-derived exosomes (Exo-MCF7) were obtained and tested. The results are shown in Figure 17(a). In Figure 17(a), the vertical axis represents fluorescence intensity, and the horizontal axis represents incubation time. Figure 17(a) indicates that a homotyping targeting time of 10 hours or more was required.

[0079] A sample containing TNBC-derived exosomes (Exo-MDA) was obtained and mixed with a bimetallic complex containing Tb to attach Tb to the surface of the exosomes. ３＋ A modified sample was obtained by introducing Tb (Exo-MDA-Tb). Furthermore, a sample containing TCT (a Tb-containing bimetallic complex) was obtained (Exo-MDA-Tb-TCT). Furthermore, a sample containing BC-derived exosomes (Exo-MCF7) was obtained and mixed with a Tb-containing bimetallic complex to form Tb on the surface of the exosomes. ３＋A modified sample was obtained by introducing Tb (Exo-MFC7-Tb). Each of the above samples was mixed with cells of the TNBC cell line, and the fluorescent signals of the samples after different incubation times were obtained by flow cytometry. The results are shown in Figure 17(b). Figure 17(b) shows that a sample containing TNBC-derived exosomes (Exo-MDA) was obtained and mixed with a Tb-containing bimetallic complex to immobilize Tb on the surface of the exosomes. ３＋ In the sample modified by introducing MDA (Exo-MDA-Tb) and the sample further containing TCT (Exo-MDA-Tb-TCT), cells of the TNBC cell line could be detected, and a homotyping targeting time of about 1 hour was sufficient.

[0080] Example 6: Whole blood samples were obtained by mixing 0 to 20 TNBC cell line cells as CTC-like cells into 1 mL of whole blood extracted from a mouse specimen. Each obtained whole blood sample was centrifuged to extract the layer containing PBMCs (peripheral blood mononuclear cells), obtaining a PBMC sample. This was then mixed with exosomes derived from TNBC cell lines whose surfaces had been previously modified with a Tb-containing bimetallic complex and incorporated with a fluorescent label (HAL). After incubation for a predetermined period of 1 hour, the surface-modified TNBC cell line-derived exosomes were selectively incorporated into CTC-like cells by homotyping. The obtained PBMC samples were subjected to ultrasonic treatment to induce cell lysis, releasing the HAL incorporated into the TNBC-derived exosomes incorporated into the CTC-like cells, and 2 x 10 ６The HAL was incorporated into reporter cells. The HAL was converted to PpIX in the reporter cells, and the resulting reporter cells were analyzed by flow cytometry to obtain a fluorescent signal correlating with the number of reporter cells containing PpIX. The resulting fluorescent signals are shown in Figure 18 (a) to (f). For CTC-like cells in 1 mL of whole blood, Figure 18 shows (a) 0, (b) 1, (c) 3, (d) 5, (e) 10, and (f) 20 cells, with the number of CTC-like cells indicated at the top of each graph. In Figure 18, the vertical axis represents the number of reporter cells, the horizontal axis represents PpIX intensity, and the solid line represents the fluorescent signal from the reporter cells obtained from whole blood containing the CTC-like cells shown at the top of each graph. The dotted line represents the fluorescent signal from a control sample that did not contain CTC-like cells. In the gated area Gated on the right side of each graph, the greater the number of CTC-like cells in the whole blood sample, the more significant the difference between the fluorescent signal and that of a sample (control) that does not contain CTC-like cells, and a signal corresponding to the number of CTC-like cells contained in the whole blood sample can be obtained from the signal in the gated area.

[0081] Figure 19(a) shows the number of CTC-like cells mixed in 1 mL of whole blood relative to the number of reporter cells measured in the gated region obtained from the above fluorescent signal. Figure 19(b) is an enlarged view of a portion of Figure 19(a). The error bars indicate standard deviation (n=3). It was confirmed that the greater the number of CTC-like cells mixed in 1 mL of whole blood, the greater the number of reporter cells measured. The error bar size increases as the number of CTC-like cells increases because the HAL concentration in the reporter cells increases according to the Poisson distribution.

[0082] Figure 20 shows the number of CTC-like cells in 1 mL of whole blood relative to the number of reporter cells measured by acquiring fluorescent signals when different CTC-like cells (MCF-7, HL-60, MDA-MB-436) were mixed in at 1, 5, or 10 cells. The CTC-like cells (MCF-7) were derived from BC, the CTC-like cells (HL-60) were derived from leukemia, and the CTC-like cells (MDA-MB-436) were derived from TNBC. It was confirmed that the greater the number of CTC-like cells mixed in 1 mL of whole blood (MDA-MB-436), the greater the number of reporter cells measured. For CTC-like cells (MCF-7, HL-60), which are not derived from TNBC, even if the number of CTC-like cells mixed into 1 mL of whole blood was increased, no significant increase in the number of measured reporter cells was confirmed, demonstrating that the rare cell detection method of this example has very high specificity.

[0083] ROC curves were obtained when fluorescent signals were acquired from samples containing 1, 3, 5, 10, and 20 CTC-like cells in 1 mL of whole blood. The results are shown in Figure 21. In Figure 21, the vertical axis represents sensitivity (%), and the horizontal axis represents 100 - specificity (%). The AUC obtained from the ROC curve in Figure 21 demonstrated that the rare cell detection method of this example has very high sensitivity and specificity.

[0084] 22 is a graph showing the relationship between the number of reporter cells and the whole blood volume when fluorescent signals were obtained from samples containing 0 (control) and 1 CTC-like cell in the whole blood. The number of reporter cells measured from the sample containing 0 (control) CTC-like cells (i.e., background noise) intersects with the number of reporter cells in a sample containing 1 CTC-like cell at a whole blood volume of approximately 18.9 mL. This result demonstrates the high detection sensitivity of this example, which allows detection of 1 CTC cell from a whole blood sample of approximately 19 mL.

[0085] Example 7: Fluorescent signals were obtained from 1 mL whole blood samples collected on days 2 and 8 after TNBC transplantation into healthy mice in the same manner as in Example 5. The number of reporter cells was detected from the obtained fluorescent signals, and the estimated number of CTC-like cells per mL of whole blood was obtained by referring to the data shown in Figure 19. The results are shown in Figure 23. A similar test was also performed on whole blood samples obtained from healthy mice, and the estimated number of CTC-like cells per mL of whole blood was obtained. In Figure 23, A shows the results from a sample obtained from a healthy mouse, B shows the results from a whole blood sample obtained on day 2 after TNBC transplantation, and C shows the results from a whole blood sample obtained on day 8 after TNBC transplantation. Five tests were performed on each whole blood sample. To evaluate the statistical significance of differences between the three groups of samples from healthy mice, those obtained on day 2 after TNBC transplantation, and those obtained on day 8 after TNBC transplantation, a two-tailed t-test was used to determine p values. The results were p<0.01 for the two groups of healthy mice and those on day 2 after TNBC transplantation, and p<0.001 for the two groups of healthy mice and those on day 8 after TNBC transplantation, confirming statistical significance.

[0086] The above-mentioned method for detecting rare cells was confirmed to be capable of detecting rare cells with an accuracy of p<0.001, and showed outstanding performance in terms of speed, sensitivity, specificity, and cost-effectiveness compared to conventional TNBC detection methods such as MRI and immunohistochemistry.

[0087] It should be noted that the present invention is susceptible to various embodiments and modifications without departing from the broad spirit and scope of the present invention. Furthermore, the above-described embodiments are intended to illustrate the present invention and are not intended to limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not the embodiments. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are deemed to be within the scope of the present invention.

[0088] 1A, 1B, 1C Cells 2A, 2B, 2C Exosomes 2A', 2B', 2C' Surface-modified exosomes 3A, 3A' Cells 4 Detection device 5 Exosome acquisition section 6 Surface modification section 7 Cell acquisition section 8 Homotyping section 9 Detection section 10 Subject to detection 10-1 Triple-negative breast cancer (TNBC) 10-2 Breast cancer (BC) 10-3 Breast cancer (BC) lung metastasis 10-4 Breast cancer (BC) bone metastasis 10-5 Breast cancer (BC) liver metastasis 11 Blood sample 12 Serum 13 Exosome population 13-1 Exosomes derived from blood cells 13-2 Exosomes derived from BC 13-3 Exosomes derived from TNBC 14 Bimetallic complex 14-1 Lanthanide ion 14-2 TDA 14-3 Cobalt ion 15 Fluorescent label 16 TNBC cells (host cells), which are the same type of cells as the target cells 17 Flow cytometry 20 Subject to detection 20-1 TNBC 20-2 Metastasis from TNBC 20-3 CTC cells 21 Blood sample 22 Blood sample after centrifugation 22-1 CTC cells 22-2 PBMC 23 TNBC-derived exosomes 23A Known TNBC cell line for preparing exosomes for detection 23X Surface-modified TNBC-derived exosomes 24 Bimetallic complex 24-1 Lanthanide ion 25 HAL 26 Lysed cells 27 TNBC cells (reporter cells) for uptake of HAL released from lysed cells 28 PpIX 29 Flow cytometry

Claims

1. A detection method comprising: obtaining exosomes derived from a first cell; modifying the surface of the exosomes; obtaining second cells of the same type as the first cells; selectively taking up the surface-modified exosomes together with a label by the second cells; obtaining a signal correlating with the label taken up by the second cells; and detecting the exosomes or the second cells.

2. The detection method according to claim 1, wherein the second cells are cells obtained from a known cell line, and the exosomes taken up by the cells obtained from the known cell line are detected.

3. The detection method according to claim 1, wherein the exosomes derived from the first cells are exosomes obtained from cells of a known cell line, and the second cells that have taken up the exosomes obtained from cells of the known cell line are detected.

4. The detection method described in claim 3, comprising: incorporating into reporter cells the label selectively incorporated by the second cells together with the surface-modified exosomes; obtaining from the label incorporated into the reporter cells a signal that correlates with the number of the second cells that have incorporated the surface-modified exosomes; and detecting the second cells that have incorporated the exosomes obtained from cells of the known cell line.

5. The detection method according to claim 1, wherein the surface of the exosome is modified with a bimetallic complex containing a lanthanide ion.

6. The detection method according to claim 5, wherein the lanthanoid ions contain at least one of Eu and Tb.

7. The detection method according to claim 1, wherein a fluorescent label is used as the label, and a fluorescent signal correlated with the number of the second cells that have taken up the surface-modified exosomes is obtained from the fluorescent label by flow cytometry.

8. A detection device for detecting the exosomes or the second cells, comprising: an exosome acquisition unit that acquires exosomes derived from a first cell; a surface modification unit that modifies the surface of the exosomes; a cell acquisition unit that acquires second cells of the same type as the target cells; a homotyping unit that selectively takes up the surface-modified exosomes together with a label by the second cells; and a detection unit that acquires a signal that correlates with the label taken up by the second cells.

9. The detection device according to claim 8, wherein the cell acquisition unit acquires cells from a known cell line, and detects the exosomes taken up by the cells acquired from the known cell line.

10. The detection device according to claim 8, wherein the exosome acquisition unit acquires exosomes from cells of a known cell line, and detects the second cells that have taken up the exosomes acquired from the cells of the known cell line.

11. The detection device described in claim 10, wherein the homotyping unit incorporates the label selectively incorporated by the second cells together with the surface-modified exosomes into reporter cells; the detection unit obtains a signal from the label incorporated into the reporter cells that correlates with the number of the second cells that have incorporated the surface-modified exosomes; and detects the second cells that have incorporated the exosomes obtained from cells of the known cell line.

12. The detection device according to claim 8, wherein the surface of the exosome is modified in the surface modification unit with a bimetallic complex containing a lanthanoid ion.

13. The detection device according to claim 12, wherein the lanthanoid ions contain at least one of Eu and Tb.

14. The detection device according to claim 8, wherein a fluorescent label is used as the label, and the detection unit acquires a fluorescent signal from the fluorescent label by flow cytometry that correlates with the number of the second cells that have taken up the exosomes whose surfaces have been modified.

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