KR20210026981A - Method for Providing Information Necessary to Diagnose the Risk of Cancer Metastasis Using Biochip - Google Patents

Abstract

Disclosed is a method for providing information necessary to diagnose the risk of metastasis of breast cancer, which comprises the steps of: preparing a first sample containing circulating tumor cells; preparing a second sample by performing immunofluorescence staining specific for estrogen receptor, progesterone receptor, and cytokeratin on the first sample; preparing a third sample by treating the second sample with human epidermal growth factor receptor 2 (Her2) specific magnetic nanoparticles; injecting the third sample into the biochip and collecting circulating tumor cells; and determining the metastasis by checking the location and type of a fluorescence signal.

Description

Method for Providing Information Necessary to Diagnose the Risk of Cancer Metastasis Using Biochip}

The present invention relates to a method of providing information necessary for diagnosing the risk of metastasis of breast cancer using a biochip.

Breast cancer is known clinically and pathologically as a highly complex and diverse cancer. Recently, with the development of various targeted therapeutics that are effective in the treatment of breast cancer, breast cancer is also classified according to the presence or absence of molecular markers targeted for treatment. Currently well-known molecular markers of breast cancer are ER (estrogen receptor), PR (progesterone receptor), and Her2 (human epithelial cell growth factor 2).

The subtypes of breast cancer are classified as follows according to the expression of Her2, ER, and PR. If it is ER+ or PR+, it is classified as Luminal type, and is classified into Luminal A type (Her2-) or Luminal B type (Her2+) depending on whether or not Her2 is positive. ER-/PR-/Her2+ is referred to as'HER2 type'. ER-/PR-/Her2- (triple negative) and CK5/6 and EGFR positive types are referred to as Basal-like types, and Her2, ER, PR, CK5/6, and EGFR negative types are classified as Unclassified. do. Alternatively, if all of Her2, ER, and PR are negative, they are classified as triple positive, and if any of them are positive, they are classified as non-triple negative.

The survival rate of invasive breast cancer is the highest in the Luminal type, and among them, it is known that the metastasis of the Luminal A type is low and the survival rate is high. Among them, it is known that the ER-/PR+ phenotype has a relatively good survival rate, and the ER+/PR+ phenotype has a poor survival rate (J Breast Cancer 2010 March; 13(1): 74-82). Because triple-negative breast cancer does not have all three receptors targeted for anti-cancer drugs, there are no special therapeutic drugs other than general anti-cancer drugs. Clinically, recurrence and distant metastasis are common, and the prognosis is known to be poorer than that of non-triple-negative breast cancer.

Diagnosis of the molecular marker is mainly performed by tissue microarray (TMA). In immunohistochemistry using TMA, the definition of ER-positive and PR-positive is when the nuclear staining in tumor cells exceeds 10%. Her2 overexpression is a case where more than 10% of tumor cells show strong membrane staining (3+), and if intermediate staining (2+) is shown, an additional Her2 gene amplification analysis, FISH (fluorescence in situ hybridization), is performed and the Her2 gene If amplification is observed, it is defined as Her2 positive. It is known that the determination of the expression of molecular markers such as ER, PR, and Her2 in immunohistochemistry is largely different for each laboratory due to technical problems occurring during tissue fixation and receptor staining, and false negatives often appear. Therefore, there is a need for a more rapid and accurate method of diagnosing breast cancer metastasis.

Korean Patent Registration No. 10-1740015 (2017.05.25)

One embodiment provides a method of providing information necessary to determine the risk of metastasis of breast cancer according to the location and type of a fluorescent signal.

The biochip according to an embodiment of the present invention includes a sample tube through which a sample including cells to which magnetic particles are specifically attached, flows, a bar-shaped magnet arranged in parallel with a part of the sample tube, and a sample tube. And a plate on which the magnet is disposed.

The sample tube includes a collection channel portion that is formed to protrude from the surface of the sample tube so that cells are collected, and includes a plurality of protrusions each having a different distance from the magnet.

The collection channel portion may include a plurality of uncelled collection channels disposed parallel to the length direction of the magnet and connected to each other to form one flow path.

The sample tube may further include a filtration channel portion for removing impurities of a sample flowing into the collection channel portion by forming a plurality of pillars protruding in a direction crossing a direction in which the sample flows.

The filtration channel unit may include a plurality of fine filtration channels connected to each other to form one flow path.

The plurality of pillars may have a width of greater than 0 and less than or equal to 100 μm.

The collection channel portion may have a width greater than 0 and less than or equal to 250 μm, and the filtering channel portion may have a width greater than 0 and less than or equal to 800 μm.

The plate may include a first plate disposed above the sample tube and a second plate disposed below the sample tube to face the first plate.

The sample tube may include an inlet formed at one end of the sample tube and through which a sample is introduced, and an outlet formed at the other end of the sample tube and through which a sample passing through the sample tube is discharged to the outside.

The cells may include metastatic cancer cells and non-metastatic cancer cells having different numbers of magnetic particles attached according to the amount of surface protein expression.

The sample tube and plate may be made of a transparent material.

According to another embodiment of the present invention, preparing a first sample containing circulatory tumor cells; Preparing a second sample by performing immunofluorescence staining specific for estrogen receptor, progesterone receptor, and cytokeratin on the first sample; Preparing a third sample by treating the second sample with magnetic nanoparticles specific to Her2 (Human Epidermal Growth Factor Receptor 2); Injecting the third sample into a biochip and collecting circulatory tumor cells; And determining the metastaticity by checking the location and type of the fluorescent signal, wherein the biochip includes a plurality of microcell collection channels and magnets for collecting the circulatory tumor cells, and the plurality of microcell collection channels each include a magnet and It provides a method of providing information necessary for diagnosing the risk of metastasis of breast cancer, which is arranged at the same distance and at different distances.

If the method of providing information necessary for diagnosing metastasis of breast cancer according to an embodiment is used, the risk of metastasis of breast cancer can be determined by analyzing the location and type of the fluorescent signal. You can increase it.

1 shows a biochip according to an embodiment of the present invention.
2 is a plan view showing a sample tube and a magnet according to an embodiment of the present invention.
FIG. 3 is an enlarged view of area A of FIG. 2.
FIG. 4 schematically shows an enlarged view of a state in which cells are separated and collected in area A of FIG. 2.
FIG. 5 shows a state in which green fluorescent cells and red fluorescent cells each simulating metastatic cancer cells and non-metastatic cancer cells in area A of FIG. 2 are separated and collected according to the intensity of magnetic force.
6 is a magnetic nanoparticle (MNPs) that specifically binds Her2 to circulating tumor cells, ER and PR-specific fluorescent staining is treated, and the expression of ER/PR and Her2 is confirmed using a biochip according to an embodiment of the present invention. It shows the principle of doing.
7 is a biochip according to an embodiment of the present invention, showing a biochip having a total of 12 non-cell collection channels (12 CTCs trapping segment) and 57 sub-segments per one non-cell collection channel. The distance between the microcell collection channel and the magnet is 0.7 mm, 1.54 mm, 2.38 mm, 3.22 mm, 4.06 mm, 4.9 mm, 5.74 mm, 6.58 mm, 7.42 mm, 8.26 mm, 9.1 mm and 9.94 mm, respectively, and each microcell collection channel The gap between them is 0.84mm, and the strength of the magnet is 4.75 kG.
8 is a result of ER-specific fluorescence staining using GF (green fluorescentn) for four cell lines (BT-474, SK-BR-3, MCF-7, and MDA-MB-231), RF (red fluorescent). The results of PR-specific fluorescence staining used and the results of overlapping them are shown.
9 shows the results of separating the MCF-7 (ER+/PR+/Her2-) cell line according to the magnetic field and confirming the distribution of fluorescence staining.
10 shows the results of separating the BT-474 (triple positive) cell line according to the magnetic field and confirming the distribution of fluorescence staining.
11 shows the results of confirming the distribution by separating MDA-MB-231 (triple negative) and SK-BR-3 (ER-/PR-/Her2+) cell lines according to a magnetic field and performing nuclear staining (DAPI).
12 shows the results of separating BT-474 (green), SK-BR-3 (purple), MDA-MB-231 (red), and MCF-7 (yellow) cell lines according to differences in fluorescence staining and magnetic force. Is shown.
13 is a graph showing the results of separation and detection of SK-BR-3 (purple) and BT-474 (green), which are Her2-positive cells in FIG. 12.
14 is a graph showing the results of separation and detection of MDA-MB-231 (red) and MCF-7 (yellow), which are Her2 negative cells in FIG. 12.
FIG. 15 is a graph showing the separation and detection results of four cells of BT-474 (green), SK-BR-3 (purple), MDA-MB-231 (red), and MCF-7 (yellow) in FIG. 12 It is represented by

According to an aspect, there is provided a biochip capable of separately collecting and detecting cells to which magnetic particles are attached differently from each other by using a difference in magnetic force according to a distance from a magnet.

1 shows a biochip 100 according to an embodiment of the present invention. As shown in FIG. 1, the biochip 100 according to an embodiment of the present invention includes a sample tube 110, a magnet 120, and a plate 130.

The sample tube 110 forms a path through which a sample is introduced and flowed and then discharged to the outside, and is formed in a shape of an elongated pipe with an empty inside for the flow of the sample.

An inlet 116 is formed at one end of the sample tube 110 so that the sample can be introduced, and at the other end of the sample tube 110, the sample of this embodiment flows along the sample tube 110 and then external An outlet 118 may be formed to be discharged into the furnace.

A sample including cells to which magnetic particles and/or fluorescent particles are specifically attached is introduced into the sample tube 110 according to the present embodiment. Cells to which magnetic particles are attached may be affected by magnetic force within the range of the magnetic field.

At this time, as an example, the cells according to the present embodiment may include non-metastatic cancer cells (a) and/or metastatic cancer cells (b) having different adhesion numbers of magnetic particles or adhesion numbers of fluorescent particles according to the amount of surface protein expression. have.

More specifically, the cell according to this embodiment is a magnetic particle having a size of nanometers combined with an antibody (Anti-Her2) capable of specifically binding to a surface protein (Her2) present in the cell membrane of cancer cells. , A fluorescent particle capable of specifically binding to ER, and a fluorescent particle capable of specifically binding to PR may be specifically attached cancer cells, or cancer cells to which neither of them is attached. have.

Metastasis of breast cancer cells depends on the expression levels of surface proteins such as ER, PR, and Her2, and the number of magnetic particles and fluorescent particles attached to the cell membrane may also vary according to the expression level of the surface protein. Therefore, if cancer cells with different expression levels of ER, PR, and Her2 are placed in the same magnetic field, they can be separated according to the difference in magnetic force acting on each cell because the number of attached magnetic particles is different according to the difference in surface protein expression level. In addition, since the number of attached fluorescent particles is different from each other, the distribution of fluorescence detection may also vary. Therefore, the metastasis of cancer can be determined by separating a sample containing cancer cells according to magnetic force and checking the distribution of a fluorescent reagent. For example, FIG. 7 is a biochip according to an embodiment of the present invention, and has a total of 12 non-cell collection channels (12 CTCs trapping segment) and 57 sub-segments per one non-cell collection channel. When the biochip of FIG. 7 is used to determine metastasis of cancer, 12 microcell collection channels are divided into six close to the magnet and six far from the magnet based on the distance, and cells are mainly collected in six microcell collection channels close to the magnet. If the fluorescence staining intensity of ER and PR is low, it can be judged that the metastasis of circulatory tumor cells is high. It can be judged that the metastasis is low.

The biochip 100 according to the present embodiment may include a magnet 120 that provides magnetic force to collect cells to which magnetic particles are attached. The magnet 120 according to the present embodiment may be formed in a bar shape, and may be disposed in parallel with a part of the sample tube 110 through which the sample flows. The shape of the sample tube 110 and the arrangement of the magnet 120 will be described in more detail later.

Meanwhile, the biochip 100 according to the present embodiment includes a plate 130 on which the sample tube 110 and the magnet 120 are disposed. The plate 130 may prevent damage by protecting the sample tube 110 and the magnet 120 from an external environment.

According to this embodiment, the plate 130 may include a first plate 132 and a second plate 134. The first plate 132 may be disposed above the sample tube 110, and the second plate 134 may be disposed below the sample tube 110 so as to face the first plate 132.

At this time, according to the present embodiment, as shown in FIG. 1, the first plate 132 has a hole in the first plate 132 so that the positions corresponding to the inlet 116 and the outlet 118 are opened so that the sample can be introduced. Can be formed.

Meanwhile, the magnet 120 according to the present embodiment may be disposed separately from the first plate 132 on the second plate 134 as shown in FIG. 1, but is not limited thereto, and the first plate It may be disposed on the upper part of the 132 or between the first plate 132 and the second plate 134 together with the sample tube 110, or may be disposed under the second plate 134.

Hereinafter, according to an embodiment of the present invention, a detailed description will be given of how cells are separated and collected according to the shape of the sample tube 110 and the arrangement of the magnet 120. In this regard, FIG. 2 is a plan view showing the shape of the sample tube 110 and the arrangement of the magnet 120 according to an embodiment of the present invention.

As shown in FIG. 2, the sample tube 110 according to the present embodiment may be divided into a collection channel unit 112 and a filtration channel unit 114 according to functions and arrangement positions.

The collection channel unit 112 is a part of the sample tube 110 according to the present embodiment, and is a portion in which a plurality of protrusions 113 through which cells to which magnetic particles are specifically attached are collected are formed. The plurality of protrusions 113 protrude toward the magnet 120 from the surface of the sample tube 110, and are arranged so that the distance from the magnet 120 is different so that cells affected by magnetic forces of different sizes according to the distance are Each can be captured.

The width W1 of the collection channel portion 112 according to the present embodiment may be greater than 0 and less than 250 μm, and the protrusion 113 formed on the surface of the collection channel portion 112 is, for example, 400 μm in length and 100 μm in width. It may have a square column shape of μm and height of 80 μm. In other words, the protrusion according to the present embodiment is disposed in parallel with the side protruding from the surface of the sample tube and parallel to the surface of the sample tube, is connected to the sample tube by the side, collects cells contained in the sample, and is blocked from the adjacent sample tube. It may be formed in the shape of a square column including a blocking surface to block the flow. However, this is only an example, and the width of the collection channel part 112 and the shape and specific dimensions of the protrusion 113 may be designed by any number of modifications in consideration of the size of the cells to be collected and the characteristics of various samples.

3 to 5 illustrate a collection channel unit 112 according to an embodiment of the present invention. FIG. 3 is an enlarged view of area A of FIG. 2, FIG. 4 is an enlarged schematic view showing a state in which cells are separated and collected in area A of FIG. 2, and FIG. 5 is a non-transferable view in area A of FIG. This is a photograph of green fluorescent cells and red fluorescent cells, which simulate cancer cells (a) and metastatic cancer cells (b), respectively, separated and collected according to the intensity of magnetic force, and observed through a microscope by classifying them using a fluorescent material.

At this time, according to this embodiment, as shown in FIGS. 3 to 5, the collection channel unit 112 includes a plurality of uncelled collection channels 112a arranged parallel to the length direction of the magnet 120, and a plurality of The microcell collection channels 112a of may be connected to each other to form one flow path.

The plurality of microcell collection channels 112a are arranged so that the distances from the magnets 120 are different, respectively. That is, as the distance from the inlet 116 of the sample tube 110 to the magnet 120 gradually approaches, a part of the sample tube 110 is bent in a zigzag shape having a square end, and thus a magnet It is possible to form a plurality of non-cell collection channels (112a) arranged in parallel with the 120.

As described above, a plurality of protrusions 113 protruding toward the magnet 120 may be formed on the surface of each non-cell collecting channel 112a according to the present embodiment. According to the present embodiment, in a sample flowing along the inside of the sample tube 110, cells to which magnetic particles are attached may be collected into the protrusion 113 formed on the surface of the sample tube 110.

At this time, according to the present embodiment, as shown in FIG. 4, since cells to which relatively many magnetic particles are attached can be greatly affected by a relatively small magnetic force, a collection of microcells disposed farther from the magnet 120 Cells to which relatively few magnetic particles are attached to the protrusion 113 formed in the channel 112a are collected in the protrusion 113 of the microcell collection channel 112a disposed at a closer distance from the magnet 120. Is captured.

FIG. 5 shows a photograph of observing through a microscope how green fluorescent cells and red fluorescent cells each simulating non-metastatic cancer cells (a) and metastatic cancer cells (b) are separated and collected according to the strength of the magnetic force as described above. have. As shown in FIG. 5, green fluorescent cell particles photographed by a green fluorescent filter and red fluorescent cell particles photographed by a red fluorescent filter can be distinguished with the naked eye due to different colors. The images captured by the filter are combined.

The green fluorescent cell particles photographed by the green fluorescent filter mimic non-metastatic cancer cells (a), and are spherical cells with a diameter of 5 μm. This has the same intensity of magnetic force that occurs when magnetic nanoparticles having a diameter of 100 nm are all combined with surface proteins expressed on the cell membrane of non-metastatic cancer cells (a).

The red fluorescent cell particles photographed by the red fluorescent filter mimic metastatic cancer cells (b), and are spherical cells having a diameter of 1 μm. This has the same intensity of magnetic force that occurs when magnetic nanoparticles having a diameter of 100 nm are all combined with surface proteins expressed on the cell membrane of metastatic cancer cells (b).

As shown in FIG. 5, the non-metastatic cancer cells (a) photographed by a green fluorescent filter to which a relatively large number of magnetic particles are attached are collected in a protrusion 113 disposed at a greater distance from the magnet 120, Conversely, metastatic cancer cells (b) to which relatively few magnetic particles are attached are collected in the protrusion 113 disposed at a closer distance from the magnet 120.

As described above, the biochip 100 according to an exemplary embodiment of the present invention may separate and collect cells with different numbers of magnetic particles attached to each cell membrane according to the characteristics of cells using magnetic force.

In particular, with respect to a disease called cancer, the cause of cancer is not clear until now, and the treatment method is also not clear. However, the earlier it is detected, the lower the probability of metastasis to other sites and the higher the likelihood of cure, so early diagnosis is an important disease. Therefore, there is a need for a device capable of diagnosing cancer in a simpler way.

At this time, the non-metastatic cancer cells (a) and the metastatic cancer cells (b) have different levels of surface protein expression on the cell membrane, and accordingly, the number of magnetic particles attached to each cell may be different. Therefore, as in the present embodiment, the strength of the magnetic force applied to each cell according to the distance can be determined by a simple method by a different biochip 100, whether it is a non-metastatic cancer cell (a) or a metastatic cancer cell (b). .

5 is an example of this embodiment, the non-metastatic cancer cells (a) are simulated as green fluorescent cells, and metastatic cancer cells (b) are simulated as red fluorescent cells and separated and collected by observing with a microscope, green fluorescence filter and red fluorescence. It is a composite of photos taken using filters.

As shown in FIGS. 4 and 5, according to the biochip 100 of the present embodiment, non-metastatic cancer cells (a) and metastatic cancer cells (b) can be simultaneously separated and collected using one bio chip 100, It is more advantageous for rapid diagnosis. However, this is only an example, and is not limited to non-metastatic cancer cells (a) and metastatic cancer cells (b), and if a sample containing a plurality of cell groups capable of varying the number of magnetic particles specifically attached to the cell membrane It will be possible to separate and collect any number of times by the biochip 100 of the present invention.

Meanwhile, according to an exemplary embodiment of the present invention, the filtration channel portion 114 may be further formed at a position of the sample tube 110 before it enters the inlet 116 and reaches the collection channel portion 112.

As shown in FIG. 2, the filtration channel part 114 is a part of the sample tube 110 for filtering impurities contained in the sample to be introduced into the collection channel part 112, and flows into the collection channel part 112. It is formed in a position where the sample before it can pass.

In the filtration channel unit 114 according to the present embodiment, from the sample flowing into the collection channel unit 112 by protruding from the surface of the sample tube 110 toward the inside of the sample tube 110 in a direction crossing the direction in which the sample flows. A plurality of pillars 115 for removing impurities may be formed.

As an example, the plurality of pillars 115 according to the present exemplary embodiment may be formed to protrude from the second plate 134 toward the inside of the sample tube 110, but is not limited thereto. Therefore, if impurities can be removed from the sample flowing inside the sample tube 110, any direction crossing the flow direction of the sample may be included in the scope of the present invention.

At this time, the width W2 of the filtration channel unit 114 according to the present embodiment may be greater than 0 and less than or equal to 800 μm, and the width of each of the plurality of pillars 115 formed in the filtration channel unit 114 is greater than 0 and 100 μm. It can be below. Accordingly, the plurality of pillars 115 according to the present embodiment may be arranged in a plurality of rows in the filtration channel portion 114 to more effectively filter impurities, and the plurality of pillars 115 are provided in the filtration channel portion 114. Even if the filter channel portion 114 is arranged in a row of columns, the width W2 of the filter channel portion 114 is sufficiently wide so that the cells with magnetic particles attached thereto pass between the pillars 115 and can be smoothly transferred to the collection channel portion 112.

Meanwhile, the filtration channel unit 114 according to the present embodiment may also include a plurality of fine filtration channels 114a that are connected to each other to form one flow path, similar to the collection channel unit 112. As the distance from the inlet 116 of the sample tube 110 to the magnet 120 gradually approaches, the microfiltration channel 114a of the present embodiment has a zigzag end portion of the sample tube 110 having a square shape. It is bent in a (zigzag) shape and arranged in parallel with the magnet 120, so that the path of the filtering channel portion 114 can be further increased.

As shown in FIG. 2, according to the present embodiment, a plurality of pillars 115 are formed for each of the plurality of fine filtration channels 114a, so that impurities can be more effectively removed from the sample passing through the filtration channel unit 114. I can.

In the above, the biochip 100 according to an embodiment of the present invention has been described. According to the present invention, a biochip 100 capable of detecting and separating and collecting cells to which magnetic particles are attached differently from each other at a time by using a difference in the intensity of magnetic force according to the difference in distance from the magnet 120 is provided. can do. Accordingly, it is possible to diagnose a disease more easily and quickly, it is possible to provide a more rapid and accurate treatment for a patient, it is possible to enable early diagnosis of the disease, it is possible to increase the treatment rate.

Another aspect provides a method of providing information necessary for diagnosing metastasis of breast cancer using the biochip.

The method of providing information necessary for diagnosing metastatic breast cancer may include: preparing a first sample containing circulatory tumor cells; Preparing a second sample by performing immunofluorescence staining specific to estrogen receptor (ER), progesterone receptor (PR), and cytokeratin on the first sample; Preparing a third sample by treating the second sample with magnetic nanoparticles specific to Her2 (Human Epidermal Growth Factor Receptor 2); Injecting the third sample into a biochip and separating and collecting circulating tumor cells according to the expression level of Her2; And determining the metastaticity by checking the location and type of the fluorescent signal.

The biochip may include a plurality of microcell collection channels and magnets for collecting the circulatory tumor cells, and each of the plurality of microcell collection channels may be arranged at equal intervals with a different distance from the magnet.

The step of determining the metastaticity is to determine Her2 positive or negative by the position of the fluorescent signal, determine ER/PR positive or negative by the type of the fluorescent signal, and risk score 1 in the case of ER/PR positive and Her2 negative. , ER/PR positive and Her2 positive, risk score 2, ER/PR negative and Her2 positive, risk score 3, and ER/PR negative and Her2 negative, risk score 4. The relationship between the expression pattern and the risk score is shown in Table 1 below. The higher the risk score, the higher the risk of metastasis.

Positive means when a specific reaction occurs in the subject when a chemical or biological test is performed for diagnosis, and negative means a case where it is not. Positive may mean the expression of a biomarker or whether it is expressed above a certain standard, for example, if the expression is positive, if it is not expressed as negative, or overexpression higher than a certain standard is positive, a certain standard Lower expression can be distinguished as negative.

The location of the fluorescent signal is as the distance to the magnet increases as the amount of Her2 expression in circulatory tumor cells decreases, and the distance to the magnet increases as the amount of Her2 expression increases, so that the positive or negative Her2 can be determined using this.

The type of the fluorescent signal may be one or more of an estrogen receptor and a progesterone receptor, or a fluorescent signal of cytokeratin. The ER/PR-positive means that at least one of the estrogen receptor and the progesterone receptor is detected, and when circulatory tumor cells express ER and/or PR, the signal by immunofluorescence staining of ER and PR is detected. Position and intensity can be judged. When circulatory tumor cells do not express ER and PR, the position and intensity of the signal can be determined by detecting an immunofluorescent staining signal of cytokeratin, which can be stained specifically for cancer cells. The detection of the fluorescent signal refers to the presence or absence of a signal emitted from immunofluorescence staining of estrogen receptor, progesterone receptor, or cytokeratin, or to confirm that it has a certain intensity or higher.

Signals emitted from immunofluorescence staining of ER and PR become weaker as the expression levels of ER and PR decrease, and when the expression of ER and PR is very low or not expressed, it may not be detected. If the signal strength of ER and PR is not detected, or if the strength of the ER and/or PR signal is weaker than that of the ER and/or PR positive control, ER and/or PR can be determined as negative. . If it is difficult to determine whether Her2 is positive or negative by ER and PR signals, it can be confirmed by checking the position of the signal emitted from immunofluorescence staining of cytokeratin instead.

The positive Her2 may be determined by the position of the fluorescent signal. The location of the fluorescent signal can be specified by the distance between the microcell collection channel in which the fluorescent signal is detected and the magnet. As the amount of Her2 expression decreases, the position of the fluorescent signal gets closer to the magnet, and as the amount of Her2 expression increases, the position of the fluorescent signal moves away from the magnet. For example, in a plurality of microcell collection channels arranged at equal intervals in parallel with the magnet by varying the distance to the magnet, based on half the maximum distance to the magnet, the group is divided into a group close to the magnet and a group far away, and the location of the fluorescent signal. If is located in the area far from the magnet, Her2 can be positive, and if the signal is located in the area close to the magnet, it can be judged as Her2 negative. Referring to FIG. 9, in the biochip, 12 microcell collection channels are arranged side by side at different distances from the magnet, and as a result of injecting a sample containing MCF-7 (ER+/PR+/Her2-) cells, 6 cells located close to the magnet. Since the signal was mainly detected in the uncelled channel, it can be confirmed that Her2 was negative. As another example, according to FIG. 10, since BT-474 (triple-positive) cells mainly detect signals in 6 uncelled cluster channels far from the magnet, Her2 positive and negative can be determined by checking the positions of the ER and PR fluorescent signals. Can be seen. In addition, referring to FIG. 11, it can be seen that even when ER and PR are not expressed, the location of the nuclear staining signal can be identified and Her2 positive and negative can be determined. 11, nuclear staining was used, but the same result can be obtained by replacing it with cytokeratin staining (TRITC).

The Her2 positivity may have an m value greater than 1.0, 1.1, or 1.12 (mm/kG) in Equation 1 below. The present inventors experimented with various cancer cells with different ER/PR and Her2 expression patterns and analyzed the intensity of the magnet and the detection distance of the fluorescence signal. It was confirmed that the cells were Her2 negative, and the cells collected in the non-cell collection channel of 5.74 mm or more were Her2 positive. Therefore, as a result of dividing the magnetic force based on the average value of 4.9 and 5.74, which is 5.32, a reference value of 1.12 was obtained. (1.12 = (5.32 mm/4.75kG))

[Equation 1]

m = distance between the microcell collection channel in which the fluorescence signal was detected and the magnet (mm)/magnet strength (kG)

The maximum value of m may be 2.0 to 2.2 mm/kG, and the minimum value may be 0 to 0.2 mm/kG.

The distance between the microcell collection channel and the magnet may be measured from a surface of the magnet in the microcell collection channel direction to the center of the microcell collection channel.

The distance between the microcell collection channel and the magnet may be 0.5 to 15 mm, 0.5 to 10 mm, 0.7 to 15 mm, or 0.7 to 9.94 mm based on the strength of the magnet at 4.75 kG, and may be appropriately changed according to the strength of the magnet. You can do it.

The plurality of microcell collection channels may include microcell collection channels having a distance from the magnet of (1.12×magnet strength (kG)) mm or more, and may be a maximum of (2.24×magnet strength (kG) mm).

Since the number of circulatory tumor cells detected in the blood of a breast cancer patient is less than 100 per sample, the number of collected cells can be checked and the metastaticity of each of the collected cells can be determined. When both Her2-positive cells and Her2-negative cells are detected, the distribution of Her2-positive cells ((number of circulatory tumor cells present in the microcell cluster channel close to the magnet/number of total circulatory tumor cells)×100(%)) and Her2 It is also possible to analyze the distribution of negative cells ((number of circulatory tumor cells present in non-magnetary channels far from magnets/number of total circulatory tumor cells) × 100 (%)) and identify both trends and individual characteristics of the cancer cell population. Metastaticity can be evaluated by multiplying the distribution of Her2-positive cells and the distribution of Her2-negative cells and the risk score, and calculating the average value, or based on the detection result with the highest risk score regardless of the distribution.

The determination of positive or negative from the location and type of the fluorescent signal may be performed by comparing the signal of ER/PR+Her2- circulatory tumor cells known in the past. For example, as a control, a sample containing ER/PR+Her2- circulatory tumor cells, which is the weakest metastatic, is injected into a biochip and the position and intensity of the fluorescence signal are measured as a reference point, and an unknown sample is injected into the biochip to obtain a fluorescence signal. It is possible to determine the metastaticity by measuring the location and intensity of the two, and comparing the two data. The control group does not necessarily have to be ER/PR+Her2- circulatory tumor cells, and it is possible to compare them based on the most metastatic triple negative circulatory tumor cells.

The first sample may be spinal fluid, lymph fluid, blood, or separately cultured circulatory tumor cells.

The same spacing may be 0.5 to 1.0mm, or 0.84mm.

The magnetic force of the magnet may be 4.0 to 5.5 kG, 4.5 to 5.0 kG, or 4.75 kG.

The estrogen receptor and progesterone receptor-specific immunofluorescence staining may be treatment with an estrogen receptor-specific primary antibody and a progesterone receptor-specific primary antibody, and treatment with a primary antibody-specific secondary antibody.

Injecting the third sample into the biochip may be injecting the biochip sample at a flow rate of 40 to 60 μl/min, 45 to 55 μl/min, or 50 μl/min.

The fluorescence signal may be detected by photographing an estrogen receptor with a DAPI wavelength, a progesterone receptor with a FITC wavelength, and a cytokeratin with a TRITC wavelength.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Preparation of four types of breast cancer cells

Four types of breast cancer cell lines (BT-474, SK-BR-3, MCF-7, MDA-MB-231) having different expression patterns of Her2, ER, and PR were prepared. The expression patterns of ER, PR, and Her2 of the cell line, and the amount of Her2 expression are shown in Table 2 below.

Cell line Her2, ER, PR expression pattern Her2 expression level BT-474 Her2(+), ER(+), PR(+) Triple Positive 6.9 ×10 ⁵ binding sites/cell (overexpression) SK-BR-3 Her2(+), ER(-), PR(-) 9.8 ×10 ⁵ binding sites/cell (overexpression) MCF-7 Her2(-), ER(+), PR(+) 0.25×10 ⁵ binding sites/cell MDA-MB-231 Her2(-), ER(-), PR(-)Triple Negative 0.14×10 ⁵ binding sites/cell

Example 2: Confirmation of cell line discrimination using a biochip

The experiment was conducted as a single cell discrimination experiment and a mixed cell discrimination experiment.

2-1. Single cell discrimination experiment

Each of the four cells of Example 1 was injected into a biochip, and it was confirmed whether they can be distinguished by the location and intensity of the signal.

(1) Dyeing process

Since SK-BR-3 and MDA-MB-231 did not express ER or PR, nuclear staining was performed with a 1% DAPI (4′,6-diamidino-2-phenylindol) solution (see FIG. 8). 100 μl of 1% DAPI solution per 106 cells/ml was added and reacted at room temperature for 15 minutes, and then washed three times with 1x DPBS (DPBS, Thermo Scientific, Massachusetts).

Since both ER and PR were expressed in BT474 and MCF-7, ER and PR were stained (see Fig. 8). Using 1 mg/ml of estrogen receptor primary antibody (Monoclonal, ab66102, Abcam, Cambridge, UK) and 1 mg/ml of progesterone receptor primary antibody (Polyclonal, ab63605, Abcam), ER and PR on the cell surface and room temperature ( room temperature) for 1 hour. In order to remove the unreacted primary antibody, it was washed 3 times with DPBS. Afterwards, fluorescence staining was performed at room temperature for 1 hour using a secondary antibody (DyLight 350 anti-rabbit, Alexa Fluor 488 anti-mouse; all staining reagents were purchased from Abcam, Cambridge, UK).

(2) Evaluation of signal location and strength

Each of the four stained breast cancer cells was independently injected into the biochip, and the location and intensity of the signal were evaluated. The microscope used for the biochip experiment was Olympus IX73, and all experiments were performed at a flow rate of 50 μl/min under a magnetic force of 4.75 kG.

According to FIG. 9, as a result of injecting and flowing the MCF-7 (ER+/PR+/Her2-) cell line into the biochip, the signals of ER and PR were strongly detected, and the position of the signal was concentrated in seg 7 to seg 12 close to the magnet. Yes, it was confirmed that it was Her2 negative.

According to FIG. 10, as a result of injecting and flowing the BT-474 (triple-positive) cell line into the biochip, the signals of ER and PR were strongly detected, and the position of the signal was concentrated in seg 1 to seg 6 far from the magnet, indicating that Her2 was positive. I could confirm.

According to the top of FIG. 11, as a result of injecting and flowing the MDA-MB-231 (triple-negative) cell line into the biochip, the location of the nuclear staining (DAPI) signal is concentrated in seg 8, seg 11, and seg 12 close to the magnet. It was confirmed that it was Her2 negative.

According to the bottom of FIG. 11, as a result of injecting and flowing the SK-BR-3 (ER-/PR-/Her2+) cell line into the biochip, the location of the nuclear staining (DAPI) signal is concentrated in seg 1 to seg 6 far from the magnet. Yes, it was confirmed that Her2 was positive.

According to Example 2-1, circulatory tumor cells with different expression levels of ER, PR, and Her2 have different locations to be collected depending on the level of Her2 expression, and different wavelengths for each of the labels for ER, PR, and circulatory tumor cells themselves. It was confirmed that the metastaticity of circulatory tumor cells present in an unknown sample can be determined by performing immunofluorescence staining.

2-2. Mixed cell biochip test staining process

When each of the four breast cancer cells of Example 1 was stained, mixed, and injected into a biochip, it was confirmed whether or not it could be separated smoothly.

(1) Dyeing process

SK-BR-3 cell staining

Deep Red staining (CellMask™ Deep Red plasma membrane stain, Thermo Scientific, Massachusetts) was performed. After adding 1 μl CellMask deep red to the cell, it was reacted at room temperature for 10 minutes, and then washed 3 times with 1x DPBS.

BT474 cell staining

Only nuclear staining was performed with 1% DAPI (4′,6-diamidino-2-phenylindol) solution. 100 μl of 1% DAPI solution per 106 cells/ml was added, reacted at room temperature for 15 minutes, and washed 3 times with 1x DPBS (DPBS, Thermo Scientific, Massachusetts).

MCF-7 cell staining

Green staining (CellMask™ Green plasma membrane stain, Thermo Scientific, Massachusetts) was performed. CellMask green 1μL was added to MCF-7 and reacted at room temperature for 10 minutes, and then washed 3 times with 1x DPBS.

MDA-MB-231 cell staining

Orange staining (CellMask™ orange plasma membrane stain, Thermo Scientific, Massachusetts) was performed. After adding 1μl of cellmark orange to the cell, it was reacted at room temperature for 10 minutes, and then washed 3 times with 1x DPBS.

(2) Evaluation of signal location and strength

The microscope used for the biochip experiment was Olympus IX73, and all experiments were conducted at a flow rate of 50 μl/min under a magnetic force of 4.75 kG.

12 and 15, the signals of the SK-BR-3 and BT-474 cell lines that are Her2 positive among the four cells were detected in seg 1 to seg 6, which are far from the magnet, and MCF-7 and MDA that are Her2 negative. The signal of the -MB-231 cell line was detected in seg 6 to seg 12, which is close to the magnet.

13 is a result of confirming the location and intensity of signals of Her2 positive SK-BR-3 (ER(-), PR(-), Her2(+)) and BT-474 (triple positive) cell lines using a program. The purple color is SK-BR-3, and the green color is BT-474. The positions of the signals of both cells are distributed in seg 1 to 6.

14 is a result of confirming the location and intensity of signals of Her2 negative MDA-MB-231 (triple negative) and MCR-7 (ER(+), PR(+), Her2(-)) cell lines using a program . The red color is MDA-MB-231, and the yellow color is MCF-7. The positions of the signals of both cells are mainly distributed in seg 7 to 12.

Through this, it was confirmed that a mixture of various types of circulatory tumor cells having different levels of ER, PR, and Her2 expression could be used as a biochip.

Example 3: Detection using an unknown sample

3-1. ER, PR, Cytokeratin immunofluorescence staining

Immunofluorescence staining was performed by 1) staining to confirm the expression of ER and PR in the cell membrane, and 2) Cytokeratin immunofluorescence staining to confirm the presence of cancer cells.

ER, PR, and Cytokeratin were stained to detect different wavelengths. Specifically, ER was stained to detect DAPI wavelength, PR was FITC wavelength, and Cytokeratin was stained to detect TRITC wavelength.

Materials used in the immunofluorescent staining process are shown in Table 3 below.

common -Sample (3ml) mixed with unknown living cells
-1x DPBS (Gibco, Dulbecco's Phosphate Buffered Saline, 14190-144)
-4% Formaldehyde solution (Sigma Aldrich, F8775-500ML)
-0.25% Triton x-100 (Sigma Aldrich, X100-100ML)
-Blocking buffer (3mg/ml Bovine serum albumin, affymetrix USB, 10857)
-Anti-cytokeratin, PE (BD science, 347204) Primary antibody 1.Estrogen receptor alpha antibody (abcam, ab66102, mouse) 2. Progesterone receptor antibody(abcam, ab63605, rabbit) Secondary antibody 1.Alexa Fluor ^TM 488 goat anti-mouse IgG(H+L) (ThermoFisher scientific, A11001, 2mg/ml) 2. Goat Anti-rabbit IgG (H+L), DyLight ^® 350 conjugated, 1mg (1mg/ml) (ThermoFisher scientific, 62270)

To fix the biological state of the sample containing unknown circulatory tumor cells, centrifuge the cells at 1300 rpm for 3 minutes to remove the supernatant, then add 3 ml of 4% formaldehyde solution to room temperature (25 degrees Celsius). ) To react for 20 minutes.

When the reaction was completed, the remaining formaldehyde solution was washed three times using 3 ml of 1x DPBS solution. Washing was carried out by centrifugation at 1300 rpm for 3 minutes to remove the supernatant, and then add 1x DPBS to mix the solution and cells.

In order to stain cytokeratin, a protein inside the cells, the cells were punctured by reacting with 1 ml of 0.25% Triton x-100 solution for 5 minutes. After the reaction with the Triton x-100 solution was completed, it was washed 3 times with 1x DPBS to prevent damage to the cell membrane.

In order to prevent non-specific binding of the protein, it was reacted for 1 hour at room temperature using 3 ml of blocking buffer, and washed 3 times with 1x DPBS to remove the remaining blocking buffer.

To dispense the antibody, a primary antibody and an anti-cytokeratin pre-mix were prepared in advance. 1 μl of ER antibody, 1 μl of PR antibody, and 20 μl of anti-cytokeratin were dispensed into 1 ml of 1x DPBS and mixed evenly (hereinafter referred to as a primary antibody pre-mix).

The supernatant of the unknown cell sample was removed by centrifugation at 1300 rpm for 3 minutes, and the first antibody pre-mix prepared above was mixed and reacted at room temperature for 1 hour. After completion of the reaction, it was washed 3 times with 1x DPBS.

In order to perform fluorescence secondary staining on the sample to which the primary antibody is bound, a secondary antibody pre-mix was prepared. Alexa FluorTM 488 goat anti-mouse IgG 0.5 μl, Goat Anti-rabbit IgG (H+L), DyLight® 350 conjugated 1 μl were dispensed into 1 ml of 1x DPBS and mixed evenly (hereinafter referred to as secondary antibody pre-mix).

The supernatant of the unknown cell sample was removed by centrifugation at 1300 rpm for 3 minutes, and the cells and the prepared secondary antibody pre-mix were mixed and reacted at room temperature for 1 hour. When the reaction was completed, it was washed 3 times with 1x DPBS, and the final volume was 1 ml.

3-1. Attachment of magnetic nanoparticles targeting HER-2

The Her2-specific magnetic nanoparticles were bound to the cells on which immunofluorescence staining was completed in Example 3-1. The materials used to attach magnetic nanoparticles are as follows.

-1 ml of immunofluorescent staining sample mixed with unknown samples

-Anti-ErbB antibody (HER-2 antibody, abcam, ab16901)

-1x DPBS (Gibco, Dulbecco's Phosphate Buffered Saline, 14190-144)

-Blocking buffer (3mg/ml Bovine serum albumin, affymetrix USB, 10857)

-Ethanol (EMD Millipore Corporation, 1.00983.1011)

-(3-Glycidyloxypropyl)trimethoxysilane (Sigma Aldrich, 440167)

-200nm diameter magnetic nano-particles (0.1mg of magnetic nanoparticles are required per sample of unknown cells)

0.1 mg of magnetic nanoparticles were prepared. The magnetic nanoparticles were immersed in water, and the surfaces of the magnetic nanoparticles were washed three times with ethanol to convert the surface of the magnetic nanoparticles into hydroxy groups (-OH). The magnetic nanoparticles were washed by fixing the particles using a magnet, removing the solvent, and adding a new solvent. During the washing process, the amount of solvent was maintained at 1 ml. After the last third washing, it was allowed to react at room temperature for 10 minutes.

An organosilane coupling agent solution was prepared by dissolving 0.5 μl of (3-Glycidyloxypropyl)trimethoxysilane in 1 ml of ethanol. The organosilane coupling agent is for attaching the Her2 antibody to the magnetic nanoparticles. Ethanol was removed from the magnetic nanoparticle solution after the reaction with ethanol, and the organic silane coupling agent solution prepared above was added, mixed well, and then reacted at room temperature for 30 minutes.

The magnetic nanoparticle solution after the reaction was washed 3 times with ethanol, and washed again 3 times with 1x DPBS to prevent the remaining ethanol component from damaging the cells.

1x DPBS 1 μg of HER-2 antibody was added to the washed magnetic nanoparticles and reacted at room temperature for 1 hour to prepare magnetic nanoparticles with Her2 antibody. After completion of the reaction, the magnetic nanoparticles were washed 3 times with 1x DPBS, and the final volume was prepared to be 100 μl.

Into 1 ml of an unknown cell sample on which immunofluorescence staining was completed, 100 μl of the previously prepared Her2 antibody-attached magnetic nanoparticle solution was mixed, and reacted at room temperature for 1 hour to attach the magnetic nanoparticles to circulating tumor cells. In order to prevent non-specific binding between the cells and the magnetic nanoparticles with Her2 antibody, the mixture was centrifuged at 1300 rpm for 3 minutes to remove the supernatant, and then 1 ml of blocking buffer was added and reacted for 30 minutes. The sample with magnetic nanoparticles attached was centrifuged at 1300 rpm for 3 minutes to remove the supernatant, and the washing process of replacing with 1x DPBS solution was repeated 3 times to complete a cell sample for application to the biochip.

3-2. Biochip test

The cell samples on which the immunofluorescent staining and magnetic nanoparticles were attached were separated from the inside of the biochip according to the magnetic force. The materials used in the biochip test are as follows.

-TYGON® flexible plastic tubing (Saint-Gobain PPL Corp, AAD04119)

-Biochip (see Fig. 7)

-Syringe pump (Kd Scientific, Model number: LEGATO 210)

-Microscope (Olympus IX73)

-Microscope control software (Micro-manager 1.4)

-1mg/ml Bovine serum albumin (affymetrix USB, 10857)

Connect the TYGON tubing to the Inlet and Outlet of the biochip, and flow 1mg/ml Bovine serum albumin solution using a syringe pump at a flow rate of 800μl/min to remove water droplets and gases inside the biochip. gave.

A permanent magnet, neodymium magnet (25mm x 11mm x 7mm, 4.75kG) is installed at the bottom of the biochip, and a syringe filled with cell solution is connected to the syringe pump. It was injected at a flow rate of μl/min.

After completion of the cell injection, cells collected on the biochip were photographed with a bright field, DAPI wavelength, FITC wavelength, and TRITC wavelength at 4 and 10 magnifications. The fluorescence signal was detected by photographing the estrogen receptor at the DAPI wavelength, the progesterone receptor at the FITC wavelength, and the cytokeratin at the TRITC wavelength. The exposure time of the microscope software was set according to Table 4 below.

Bright field DAPI FITC TRITC Exposure time(ms) 10ms 100ms 800ms 800ms

As a result of checking the position of the signal and comparing it with the signal of the MCF-7 (ER/PR+Her2-) cell, most of the circulatory tumor cells contained in the unknown sample are captured in seg 1 to seg 5, which are far from the magnet. And the intensity of the signal for ER and PR was similar to that of MCF-7 cells. Therefore, the circulatory tumor cells included in the unknown sample were triple-positive cells, the risk score was 2, and the metastaticity was judged to be higher than that of MCF-7 cells. The unknown sample was a blood sample taken from a patient diagnosed with stage 2 breast cancer among hospitalized patients at Dongguk University Medical Center, and it was consistent with the medical staff's diagnosis result and determination of metastasis potential.

100: biochip
110: sample tube
112: collection channel portion
112a: uncelled channel
113: protrusion
114: filtration channel portion
114a: fine filtration channel
115: pillar
116: inlet
118: outlet
120: magnet
130: plate
132: first plate
134: second plate
W1: width of the collection channel
W2: the width of the filtration channel portion
a: non-metastatic cancer cells
b: metastatic cancer cells

Claims (10)

Preparing a first sample containing circulatory tumor cells;
Preparing a second sample by performing immunofluorescence staining specific for estrogen receptor, progesterone receptor, and cytokeratin on the first sample;
Preparing a third sample by treating the second sample with magnetic nanoparticles specific to Her2 (Human Epidermal Growth Factor Receptor 2);
Injecting the third sample into a biochip and collecting circulatory tumor cells; And
Including the step of determining the metastaticity by checking the location and type of the fluorescent signal,
The biochip includes a plurality of microcell collection channels and magnets for collecting the circulatory tumor cells,
The plurality of microcell collection channels are each arranged at equal intervals with a different distance from the magnet,
How to provide the necessary information for diagnosing the risk of metastasis of breast cancer. The method of claim 1,
The step of determining the metastaticity,
It is determined that Her2 is positive or negative by the position of the fluorescent signal,
ER/PR positive or negative is determined by the type of the fluorescent signal,
Risk score 1 for ER/PR positive and Her2 negative, risk score 2 for ER/PR positive and Her2 positive, risk score 3 for ER/PR negative and Her2 positive, and risk score 4 for ER/PR negative and Her2 negative Is judged by
How to provide the necessary information for diagnosing the risk of metastasis of breast cancer. The method of claim 2,
The ER/PR positive is one in which at least one of the estrogen receptor and the progesterone receptor is detected,
How to provide the necessary information for diagnosing the risk of metastasis of breast cancer. The method of claim 2,
The Her2 positive is that the m value of the following formula 1 is greater than 1.12,
How to provide the information you need to diagnose your risk of breast cancer metastasis:
[Equation 1]
m = distance between the microcell collection channel in which the fluorescence signal was detected and the magnet (mm)/magnet strength (kG) The method of claim 1,
The plurality of microcell collection channels include microcell collection channels having a distance from the magnet of (1.12×magnet strength (kG)) mm or more,
How to provide the necessary information for diagnosing the risk of metastasis of breast cancer. The method of claim 1,
The same spacing is 0.5 to 1.0 mm,
How to provide the necessary information for diagnosing the risk of metastasis of breast cancer. The method of claim 1,
The magnetic force of the magnet is 4.0 to 5.5 kG,
How to provide the necessary information for diagnosing the risk of metastasis of breast cancer. The method of claim 1,
The first sample is blood,
How to provide the necessary information for diagnosing the risk of metastasis of breast cancer. The method of claim 1,
The immunofluorescence staining is to treat an estrogen receptor-specific primary antibody and a progesterone receptor-specific primary antibody, and to treat a primary antibody-specific secondary antibody,
How to provide the necessary information for diagnosing the risk of metastasis of breast cancer. The method of claim 1,
Injecting the third sample into the biochip,
Injecting a biochip sample at a flow rate of 40 μl/min to 60 μl/min,
How to provide the necessary information for diagnosing the risk of metastasis of breast cancer.

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