Method for detecting influence of medicine on trace cell activity and application of method

Abstract

The invention relates to a method for detecting influence of drugs on trace cell activity and application of the method. The detection method comprises the following steps: treating trace cells by adopting a to-be-detected medicine, and introducing a biological orthogonal group to the trace cells through glycometabolism engineering to obtain treated cells; and analyzing the characteristics of the biological orthogonal groups on the treated cells so as to determine the influence condition of the to-be-detected drug on the activity of the trace cells. According to the detection method, the biological orthogonal groups are introduced into the trace cells through glucose metabolism engineering, so that the influence of the medicine on the activity of the trace cells can be quantitatively detected by analyzing the characteristics of the biological orthogonal groups on the trace cells. According to the trace cell activity quantitative test method based on the biological orthogonal substitution sugar engineering disclosed by the invention, a novel technology for performing real-time chemotherapy drug sensitivity test by using CTC is developed on the basis of the trace cell activity quantitative test method. And through individualized screening of chemotherapeutic drugs, precise cancer treatment is realized.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical technology, in particular to a method for detecting the effect of a drug on trace cell activity and an application thereof. Background Art

[0002] In 1869, Australian doctor Ashworth first discovered and proposed the concept of circulating tumor cells (CTC) in the blood of breast cancer patients. In 1976, Nowell revised the definition of CTC to: tumor cells that originate from primary tumors or metastatic tumors, gain the ability to detach from the basement membrane and invade into blood vessels through the tissue matrix. Currently, CTC refers to a general term for various types of tumor cells present in peripheral blood, and its growth and spread run through all stages of tumor growth.

[0003] At present, some progress has been made in the use of CTCs for chemotherapy drug screening (https: / / doi.org / 10.1016 / j.bcp.2021.114589). However, it generally requires the cultivation of isolated CTCs, a process that often takes weeks or even months and is very expensive. In contrast, cancer progresses rapidly, and patients cannot wait so long for treatment. Therefore, in clinical practice, there is an urgent need for a shorter "real-time" CTC pre-evaluation treatment plan method.

[0004] The biggest feature of CTC is its scarcity, with only a few to dozens of CTCs per milliliter of blood. In order to directly screen drugs through these trace amounts of CTCs, the first scientific problem that needs to be solved is how to perform quantitative cell activity tests on trace cells. This is because existing conventional cell activity test methods, including counting, CCK-8, MTT, calcein-PI, etc., are not good for quantitative activity tests of trace cells. Summary of the invention

[0005] Based on this, the present application provides a method for detecting the effect of drugs on the activity of trace cells, through which the activity of trace cells can be quantitatively tested.

[0006] A method for detecting the effect of a drug on trace cell activity comprises the following steps:

[0007] Treating the trace cells with the drug to be tested, and introducing bioorthogonal groups into the trace cells through sugar metabolism engineering to obtain treated cells;

[0008] The properties of the bioorthogonal groups on the treated cells are analyzed to determine the effect of the drug to be tested on the activity of the trace cells.

[0009] In the above detection method, bioorthogonal groups are introduced into trace cells through sugar metabolism engineering, so that the effect of drugs on the activity of trace cells can be quantitatively detected by analyzing the characteristics (such as quantity, etc.) of the bioorthogonal groups on the trace cells.

[0010] In some embodiments, the step of treating the trace cells with the drug to be tested and introducing the bioorthogonal groups into the trace cells by sugar metabolism engineering comprises:

[0011] The trace cells, the drug to be tested and the sugar molecules modified with the bioorthogonal groups are mixed and placed for a predetermined time to obtain the treated cells.

[0012] In some embodiments, the bioorthogonal group comprises an azide group;

[0013] and / or, the sugar molecules include sugar units and their derivatives;

[0014] Optionally, the sugar unit includes any one of a 2-aminomannose unit, a 2-aminogalactose unit, a 2-aminoglucose unit and a 6-azido-L-fucose unit; further, the 2-aminomannose unit, the 2-aminogalactose unit, the 2-aminoglucose unit and the 6-azido-L-fucose unit respectively have the structures shown in the following formulas, wherein X=(CH2)n: n is any number between 1 and 20:

[0015]

[0016] In some embodiments, the detection method further comprises the following steps: introducing bioorthogonal groups to the trace cells that have not been treated with the drug to be tested by sugar metabolism engineering to obtain control cells;

[0017] The step of analyzing the characteristics of the bioorthogonal groups on the treated cells to determine the effect of the drug to be tested on the activity of the trace cells comprises:

[0018] performing fluorescent staining on the bioorthogonal groups on the treated cells and the control cells;

[0019] Randomly select n treated cells and m control cells, count the cell area of ​​each corresponding cell and the luminescence intensity of the bioorthogonal group of each corresponding cell, and calculate the average luminescence intensity RFI per unit perimeter of each corresponding cell;

[0020] Calculate the average RFI value of n treated cells and m control cells, and calculate the ratio of the two values, tRFI 实验组 / tRFI 对照组, according to the ratio tRFI 实验组 / tRFI 对照组 Determine the effect of the drug to be tested on the activity of the trace cells.

[0021] In some embodiments, the average luminous intensity RFI of each corresponding cell unit perimeter is calculated using Formula 1, and the Formula 1 is as follows:

[0022] RFI=FI / {2π[(S / π)^0.5]}

[0023] Wherein, S is the cell area of ​​each corresponding cell, and FI is the luminescence intensity of the bioorthogonal group of each corresponding cell;

[0024] In some embodiments, according to the ratio tRFI 实验组 / tRFI 对照组 The step of determining the effect of the drug to be tested on the activity of the trace cells comprises:

[0025] If the ratio approaches 1, the drug to be tested has no effect on the activity of the trace cells;

[0026] If the ratio approaches 0, the drug to be tested has an infinite effect on the activity of the trace cells.

[0027] In some embodiments, the bioorthogonal group comprises an azide group;

[0028] And / or, the number of the trace cells is less than 100;

[0029] And / or, the trace cells are trace tumor cells, and the drug to be tested is a chemotherapy drug or an anti-tumor drug.

[0030] A method for detecting sensitivity of chemotherapy drugs comprises the following steps:

[0031] The above detection method is used to detect the effect of chemotherapy drugs of different drug concentrations on the activity of trace tumor cells, and a curve of the effect of drug concentration on the activity of the trace tumor cells is drawn, and then the test drug concentration of the chemotherapy drug is obtained according to the activity effect curve, and the test drug concentration is the standard drug concentration for chemotherapy drug sensitivity testing;

[0032] The blood sample to be tested is treated with the corresponding chemotherapy drug at the test drug concentration, and the blood sample to be tested is subjected to sugar metabolism engineering treatment to obtain a treated blood sample;

[0033] performing circulating tumor cell enrichment treatment on the treated blood;

[0034] The activity of circulating tumor cells enriched in the blood after the treatment is detected to determine the drug sensitivity of the body corresponding to the blood sample to be tested to the chemotherapy drug.

[0035] In some embodiments, the chemotherapy drug sensitivity detection method further comprises the following steps: performing a glucose metabolism engineering treatment on the blood sample to be tested that has not been treated with the chemotherapy drug to obtain a control blood sample; performing a circulating tumor cell enrichment treatment on the control blood sample;

[0036] The step of detecting the activity of circulating tumor cells enriched in the treated blood to determine the drug sensitivity of the body corresponding to the blood sample to be tested to the chemotherapeutic drug comprises:

[0037] Performing immunofluorescence staining and bioorthogonal group fluorescence staining on the circulating tumor cells enriched in the treated blood sample and the control blood sample;

[0038] Randomly select n circulating tumor cells after staining from the two blood samples, count the cell area of ​​each corresponding cell and the luminescence intensity of the bioorthogonal group of each corresponding cell, and calculate the average luminescence intensity RFI per unit perimeter of each corresponding cell; calculate the average RFI of the n circulating tumor cells after staining of the two blood samples and calculate the ratio RCV of the two, that is, obtain the relative activity of the circulating tumor cells enriched in the blood after the treatment;

[0039] The drug sensitivity of the body corresponding to the blood sample to be tested to the chemotherapy drug is determined according to the ratio RCV.

[0040] In some embodiments, the test drug concentration of the chemotherapeutic drug is the ratio tRFI 实验组 / tRFI 对照组 The drug concentration corresponding to 65%;

[0041] Furthermore, the step of determining the drug sensitivity of the body corresponding to the blood sample to be tested to the chemotherapy drug according to the ratio RCV includes:

[0042] If the ratio RCV is higher than 70%, the drug sensitivity to the chemotherapy drug is drug resistance;

[0043] If the ratio RCV is lower than 60%, the drug sensitivity to the chemotherapy drug is sensitive.

[0044] In some embodiments, the chemotherapy drug includes at least one of doxorubicin, paclitaxel and cisplatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The blood cell / CTC detection result diagram of the existing scheme and the scheme of this application;

[0046] Figure 2 A comparison chart of the technical routes of the existing solution and the solution of this application;

[0047] Figure 3 This is a curve diagram showing the effect of different concentrations of DOX, CDDP, and PTX on the activity of MCF-7 cells in Example 1;

[0048] Figure 4 This is the result of the drug sensitivity test of artificial CTC samples (spike-in);

[0049] Figure 5 This is the result of drug sensitivity test of clinical samples. DETAILED DESCRIPTION

[0050] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described in the application, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0051] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used in this application and in the specification of this application are only for the purpose of describing the implementation methods and embodiments and are not intended to limit this application.

[0052] Unless otherwise specified or there is a contradiction, the terms and phrases used in this application have the following meanings:

[0053] The terms "and / or", "or / and", and "and / or" used in this application include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0054] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0055] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0056] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0057] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0058] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0059] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In the present application, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in the present application should be understood to include any and all sub-ranges included therein.

[0060] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0061] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0062] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0063] It is understood that the only real-time pre-evaluation method for cancer treatment plans using CTCs is the "Using 2-NBDG as a probe for cellular glucose metabolic activity detection" program conducted by Polaris Biology (i.e. Shanghai Polaris Biotechnology Co., Ltd., https: / / polarisbiology.com / home), CN202110153914.1 A device for monitoring living circulating tumor cells labeled with 2-NBDG. However, ordinary blood cells also have normal glucose metabolism, so the technical solution has serious background noise and low data credibility. At the same time, the program is to first treat whole blood with a treatment plan, and then use a 2-NBDG probe to detect glucose. When the laboratory is required to test, CTC still needs to retain cell activity, which is demanding on the sample.

[0064] In a first aspect, the present application provides a method for detecting the effect of a drug on trace cell activity, comprising the following steps S110-S120:

[0065] S110, treating the trace cells with a drug to be tested, and introducing bioorthogonal groups into the trace cells through sugar metabolism engineering to obtain treated cells;

[0066] S120, analyzing the characteristics of the bioorthogonal groups on the treated cells to determine the effect of the drug to be tested on the activity of the trace cells.

[0067] Bioorthogonal groups are chemical groups that can react chemically under specific conditions in vivo. They are highly inert to other biomolecules in vivo and react only with specific paired groups. This reaction has a high degree of selectivity and efficiency and does not interfere with normal biological processes. In the above-mentioned detection method of the present application, bioorthogonal groups are introduced into trace cells through sugar metabolism engineering, so that the characteristics (such as quantity, etc.) of the bioorthogonal groups on the trace cells can be analyzed to quantitatively detect the influence of drugs on the activity of trace cells.

[0068] In some embodiments, the step of treating the trace cells with the drug to be tested and introducing bioorthogonal groups into the trace cells through sugar metabolism engineering comprises:

[0069] The trace cells, the drug to be tested and the sugar molecules modified with the bioorthogonal groups are mixed and placed for a predetermined time to obtain the treated cells.

[0070] Wherein, the bioorthogonal group includes an azide group; and / or, the sugar molecule includes a sugar unit and its derivatives.

[0071] The sugar unit includes any one of a 2-aminomannose unit, a 2-aminogalactose unit, a 2-aminoglucose unit and a 6-azido-L-fucose unit. Specifically, the 2-aminomannose unit, the 2-aminogalactose unit, the 2-aminoglucose unit and the 6-azido-L-fucose unit have the following structures, respectively, wherein X=(CH2)n: n is any number between 1 and 20:

[0072]

[0073] Derivatives (i.e., all or part of the acetyl groups are removed and replaced with other protective groups). The above-mentioned deprotection refers to the molecule with exposed hydroxyl groups obtained by removing all / part of the acetyl groups; the above-mentioned other protective groups refer to other acyl groups, including propionyl, butyryl, etc.

[0074] It should be noted that the bioorthogonal group is not limited to an azide group, but may also be other bioorthogonal groups.

[0075] In this application, the metabolic pathways involved include all pathways involved in "metabolic glycoengineering" in a broad sense (Chem. Soc. Rev., 2023, 52, 510-535). This scheme mainly uses the activity of metabolic glycoengineering to determine cell activity. Therefore, all sugar molecules that meet the definition of "metabolic glycoengineering" can be used as substitutes, not limited to the sugar molecules mentioned above.

[0076] In a specific example, the sugar molecule is Ac4ManNAz. Azide-modified mannose (Ac4ManNAz) is an azide-containing metabolic glycoprotein labeling reagent that can selectively modify proteins and can be used for cell labeling, tracking and proteomics analysis. The working concentration of Ac4ManNAz is 100 μM.

[0077] Wherein, the step of placing for a predetermined time includes placing at 2-8°C for 24 hours.

[0078] In some embodiments, the detection method further comprises the following step: introducing bioorthogonal groups into the trace cells that have not been treated with the drug to be tested through sugar metabolism engineering to obtain control cells.

[0079] Control cells are used to determine the effect of the drug on trace tumor cells by relative cell viability.

[0080] Among them, the operation process for obtaining control cells is basically similar to the operation process for obtaining treated cells. The main difference is whether the treated cells are treated with the drug to be tested. Other operation descriptions can be referred to above and will not be repeated here.

[0081] Further, the step of analyzing the characteristics of the bioorthogonal groups on the treated cells to determine the effect of the drug to be tested on the activity of the trace cells includes S121-S123:

[0082] S121. Performing fluorescent staining on the bioorthogonal groups on the treated cells and the control cells.

[0083] The bioorthogonal groups are fluorescently stained to facilitate the analysis of their properties.

[0084] S1222. Randomly select n treated cells and m control cells, count the cell area of ​​each corresponding cell and the luminescence intensity of the bioorthogonal group of each corresponding cell, and calculate the average luminescence intensity RFI per unit perimeter of each corresponding cell.

[0085] Wherein, n and m are both natural numbers greater than 0. n and m may be equal or unequal. For example, n and m are both 50.

[0086] The average luminous intensity RFI of each corresponding cell unit perimeter is calculated using Formula 1, and the Formula 1 is as follows:

[0087] RFI=FI / {2π[(S / π)^0.5]}

[0088] Wherein, S is the cell area of ​​each corresponding cell, and FI is the luminescence intensity of the bioorthogonal group of each corresponding cell.

[0089] S123, calculating the average RFI value of n treated cells and the average RFI value of m control cells, and calculating the ratio tRFI of the two 实验组 / tRFI 对照组 , according to the ratio tRFI 实验组 / tRFI 对照组 Determine the effect of the drug to be tested on the activity of the trace cells.

[0090] According to the ratio tRFI 实验组 / tRFI 对照组 The step of determining the effect of the drug to be tested on the activity of the trace cells comprises:

[0091] If the ratio approaches 1, the drug to be tested has no effect on the activity of the trace cells;

[0092] If the ratio approaches 0, the drug to be tested has an infinite effect on the activity of the trace cells.

[0093] In some embodiments, the bioorthogonal group comprises an azide group;

[0094] And / or, the number of the trace cells is less than 100;

[0095] And / or, the trace cells are trace tumor cells, and the drug to be tested is a chemotherapy drug or an anti-tumor drug.

[0096] This application uses the activity of abnormal sugar metabolism pathways of tumor cells themselves to evaluate cell activity. When tumor cells are highly active, the metabolism of this pathway is vigorous; when tumor cells die, the metabolic pathway is completely inactivated. The advantage of this pathway is that due to abnormal metabolism of tumor cells, some chemical groups (azide, etc.) can be anchored to the cell membrane through this pathway and quantitatively determined by fluorescent staining, thereby providing a method for measuring the activity of trace cells.

[0097] Based on the above-mentioned detection method of the effect of drugs on trace cell activity, the present application further develops a "real-time" chemotherapy drug sensitivity testing technology using CTC.

[0098] The second aspect of the present application provides a method for detecting sensitivity of chemotherapy drugs, comprising the following steps S210-S240:

[0099] S210. Use the detection method of the first aspect to detect the effect of chemotherapy drugs at different drug concentrations on the activity of trace tumor cells, and draw a curve of the effect of drug concentration on the activity of the trace tumor cells, and then obtain the test drug concentration of the chemotherapy drug according to the activity effect curve, wherein the test drug concentration is the standard drug concentration for chemotherapy drug sensitivity testing.

[0100] The detection method of the first aspect is described above in detail and will not be repeated here.

[0101] In some specific examples, the tumor cells are MCF-7 cells, and the corresponding test drug concentrations are as follows: the test drug concentration of DOX (i.e., doxorubicin) is 624 nM, the test drug concentration of CDDP (i.e., cisplatin) is 139 μM, and the test drug concentration of PTX (i.e., paclitaxel) is 20 nM.

[0102] S220, using corresponding chemotherapy drugs at the test drug concentration to treat the blood sample to be tested, and performing sugar metabolism engineering treatment on the blood sample to be tested to obtain a treated blood sample.

[0103] The operation of performing sugar metabolism engineering on the blood sample to be tested is similar to the operation of introducing bioorthogonal groups into the trace cells through sugar metabolism engineering in the detection method of the first aspect. The specific details are as mentioned above and will not be repeated here.

[0104] Among them, the blood sample to be tested is a fresh blood sample.

[0105] In a specific example, step S220 includes: pre-placing 100 μM Ac4ManNAz (i.e., 0.5 μmol per 5 ml of blood) and corresponding drugs in a blood collection tube, mixing the blood after blood collection, and placing the blood at 2-8 degrees Celsius for 24 hours to obtain a processed blood sample.

[0106] S230, performing circulating tumor cell enrichment treatment on the treated blood.

[0107] Methods for enriching and processing circulating tumor cells include but are not limited to the methods described in Patent ZL 2021 1 1494317.1.

[0108] S240, detecting the activity of circulating tumor cells enriched in the blood after the treatment to determine the drug sensitivity of the body corresponding to the blood sample to be tested to the chemotherapy drug.

[0109] In some embodiments, the chemotherapy drug sensitivity detection method further comprises the following steps: performing a sugar metabolism engineering treatment on the blood sample to be tested that has not been treated with the chemotherapy drug to obtain a control blood sample; performing a circulating tumor cell enrichment treatment on the control blood sample;

[0110] The steps of S240 include S241-S243:

[0111] S241, performing immunofluorescence staining and bioorthogonal group fluorescence staining on the circulating tumor cells enriched in the treated blood sample and the control blood sample;

[0112] S242, randomly selecting n circulating tumor cells after staining from the two blood samples, counting the cell area of ​​each corresponding cell and the luminescence intensity of the bioorthogonal group of each corresponding cell, and calculating the average luminescence intensity RFI per unit perimeter of each corresponding cell; calculating the average RFI of the n circulating tumor cells after staining from the two blood samples and calculating the ratio RCV of the two, that is, obtaining the relative activity of the circulating tumor cells enriched in the blood after the treatment;

[0113] S243. Determine the drug sensitivity of the body corresponding to the blood sample to be tested to the chemotherapy drug according to the ratio RCV.

[0114] Wherein, the test drug concentration of the chemotherapy drug is the ratio tRFI 实验组 / tRFI 对照组 The drug concentration corresponding to 65%;

[0115] Furthermore, the step of determining the drug sensitivity of the body corresponding to the blood sample to be tested to the chemotherapy drug according to the ratio RCV includes:

[0116] If the ratio RCV is higher than 70%, the drug sensitivity to the chemotherapy drug is drug resistance;

[0117] If the ratio RCV is lower than 60%, the drug sensitivity to the chemotherapy drug is sensitive.

[0118] In some embodiments, the chemotherapy drug comprises at least one of doxorubicin, paclitaxel, and cisplatin.

[0119] The technical solution of this application mainly develops a quantitative test method for the effect of drugs on the activity of trace tumor cells (the so-called trace in this solution refers to less than 100 cells), and based on this method, further develops a "real-time" chemotherapy drug sensitivity test technology using CTC. It mainly includes:

[0120] 1. Quantitative test method for the effect of drugs on trace tumor cell activity based on bioorthogonal metabolic glycoengineering;

[0121] 2. Based on the above method, use CTC for "real-time" chemotherapy sensitivity testing technology.

[0122] Comparison of blood cell / CTC detection results between the existing scheme and the scheme of this application is shown in Figure 1 , Figure 1 The left picture is a promotional color picture collected from Chenan Biological Company, and the right picture is a fluorescence detection image of this application scheme. Figure 1 It can be seen that the noise of this application is very low (green channel).

[0123] The technical route comparison between the existing solution and the solution of this application is shown in Figure 2 , Figure 2 Among them, a) is the technical route of Chenan Biotechnology Co., Ltd., and b) is the technical route of this application. Figure 2 It can be seen that when performing laboratory fluorescent staining, the samples of the present application scheme do not require CTCs to be in a living state, so the requirements for sample storage / transportation are low, which is more convenient and economical.

[0124] It can be seen that the above scheme of the present application evaluates the cell activity by targeting the activity of abnormal sugar metabolism pathways in tumor cells, and is less affected by normal blood cells. At the same time, the cell activity evaluation process begins when the blood enters the blood collection tube. Even if the CTC in the blood is completely inactivated when it reaches the laboratory, it will not affect the test results, and the sample requirements are relaxed.

[0125] The following are specific embodiments.

[0126] Unless otherwise specified, the drugs and instruments used in the examples are all conventionally selected in the art. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions, such as those described in literature or books or methods recommended by kit manufacturers.

[0127] Example 1

[0128] 1. Quantitative test of the effect of drugs on trace tumor cell activity

[0129] The quantitative test method for the effect of drugs on the activity of trace tumor cells specifically refers to the use of 100μM Ac4ManNAz for metabolic glycoengineering while treating trace cells with chemotherapy drugs, and after 24 hours, quantitatively evaluating the number of azide groups introduced by metabolic glycoengineering on the cell membrane surface based on the final cell activity. The specific operation is as follows:

[0130] (1) The experiment was divided into an experimental group and a control group. The cell treatment process was as follows:

[0131] Cell treatment of the experimental group: Trace cells (i.e., MCF-7 cells, 100 cells), 100μM Ac4ManNAz and different concentrations of corresponding chemotherapeutic drugs were mixed and placed at 2-8 degrees Celsius for 24 hours. The chemotherapeutic drugs were DOX (i.e., doxorubicin), CDDP (i.e., cisplatin), and PTX (i.e., paclitaxel). The different concentrations of DOX were 0nM, 20nM, 100nM, 200nM, 500nM, 1000nM, and 1500nM. The different concentrations of CDDP were 0, 5μM, 10μM, 20μM, 100μM, 200μM, and 300μM. The different concentrations of PTX were 0nM, 1nM, 2nM, 5nM, 10nM, 20nM, and 40nM.

[0132] Cell treatment of the control group: a trace amount of cells (ie, MCF-7 cells, 100 cells) and 100 μM Ac4ManNAz were mixed and placed at 2-8 degrees Celsius for 24 hours.

[0133] (2) The two groups of cells treated above were subjected to specific staining of azide groups. The azide group-specific staining protocol used was as follows: cells were treated with DBCO-biotin (100 μM, 1 hour) without fixation or other treatments, washed three times with PBS, treated with Alexa Fluor 488-labeled streptavidin (50 μM, 15 minutes), washed three times with PBS, and cells were centrifuged at 450 g in each step.

[0134] (3) The azide-specific fluorescence intensity FI and cell area S on each tumor cell membrane were counted by software such as imageJ. The average fluorescence intensity per unit perimeter of each tumor cell was obtained by the formula RFI = FI / {2π[(S / π)^0.5]}. The RFI of 50 randomly selected cells was re-averaged to obtain the tRFI (total RFI) of the drug-treated group and the control group. Finally, the ratio tRFI was calculated. 实验组 / tRFI 对照组 , through tRFI 实验组 / tRFI 对照组 The ratio of the drug to the trace tumor cells is used to obtain the parameter RCV (relative cell viability). If there is no effect at all, IR approaches 1, and if the effect is infinite, IR approaches 0. The RCV obtained at different drug concentrations is plotted, and the drug concentration corresponding to RCV = 65% is selected as the standard parameter for the next step of chemotherapy drug sensitivity test parameters (see Figure 3 ). Figure 3 This is a curve diagram showing the effects of different concentrations of DOX, CDDP, and PTX on the activity of MCF-7 cells in Example 1.

[0135] The drug concentrations of the chemotherapy regimen obtained by screening using MCF-7 cells were: DOX was 624 nM, CDDP was 139 μM, and PTX was 20 nM.

[0136] 2. Treatment plan pretreatment CTC

[0137] By plotting the effect curves of different chemotherapy drug treatments on the activity of trace tumor cells, the drug concentration suitable for chemotherapy drug sensitivity testing can be selected. Figure 3 As shown in the figure, the drug concentrations of the chemotherapy regimen screened using MCF-7 cells are: DOX is 624nM, CDDP is 139μM, and PTX is 20nM

[0138] While using the above steps to screen out the conditions for processing fresh blood samples, metabolic sugar engineering treatment was performed. That is, 100 μM Ac4ManNAz (i.e., 0.5 μmoL per 5 mL of blood) and the corresponding drug (no drug was added to the control group) were pre-placed in the blood collection tube, 5 mL of blood sample was added to the above blood collection tube, mixed, and placed at 2-8 degrees Celsius for 24 hours.

[0139] Among them, the fresh blood samples are from 10 patients (stage 3 breast cancer patients). The 10 patients are numbered P1-P10, of which P1-P7 are random patients, and P8-P10 are patients who have received paclitaxel + cisplatin combination therapy.

[0140] 3. CTC Enrichment

[0141] The blood obtained in the above steps is subjected to CTC enrichment treatment, and the method includes but is not limited to the chitosan coating designed in patent ZL 2021 11494317.1.

[0142] 4. Conduct individualized drug sensitivity testing for patients through CTC activity detection

[0143] The obtained CTCs were subjected to immunofluorescence staining and chemical group fluorescence staining, and the data of the drug treatment group and the control group were statistically analyzed by the method described in "1. Quantitative test of the effect of drugs on trace tumor cell activity", and the RCV of each drug on the sample was calculated. The test results are as follows Figure 4-Figure 5 And Table 1.

[0144] like Figure 4 As shown, in the artificial CTC samples (tumor cell line cells mixed into healthy human blood), it is clearly visible that doxorubicin (DOX)-resistant tumor cell samples are more resistant to doxorubicin treatment.

[0145] like Figure 5 As shown in Table 1 , the population (P8-P10) that received paclitaxel and cisplatin combination therapy clinically showed 100% complete resistance to the two-drug therapy.

[0146] The results showed that: 1) At the cell line level, doxorubicin was 100% resistant to "artificial CTC samples prepared from doxorubicin-resistant strains", while paclitaxel and cisplatin were mostly not resistant. 2) At the clinical level, patients showed a relatively scattered random resistance / sensitivity, but for patients P8-P10 who had received paclitaxel + cisplatin combination therapy, they generally showed 100% resistance to paclitaxel and cisplatin.

[0147] It can be seen that the method is highly reliable. At present, the present application believes that the standard drug concentration (i.e., the above tRFI 实验组 / tRFI 对照组 When the RCV of clinical samples is higher than 70%, they are resistant, and lower than 60%, they are sensitive.

[0148] Table 1 Statistical results of the sensitivity of blood samples from 10 patients from P1 to P10 to different drugs

[0149] Patient number PTX CDDP DOX P1 Drug resistance generally sensitive P2 sensitive sensitive generally P3 Drug resistance Drug resistance generally P4 Drug resistance Drug resistance generally P5 Drug resistance sensitive sensitive P6 generally generally generally P7 generally Drug resistance Drug resistance P8 Drug resistance Drug resistance generally P9 Drug resistance Drug resistance generally P10 Drug resistance Drug resistance generally

[0150] In summary, the present invention is based on the trace cell activity quantitative test method based on bioorthogonal metabolic sugar engineering, and on this basis, a new technology for real-time (less than 2 days) chemotherapy drug sensitivity testing using CTCs has been developed. Through individualized screening of chemotherapy drugs, precise cancer treatment can be achieved.

[0151] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0152] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for detecting the effect of a drug on trace cell activity, characterized in that: The steps include: Treating the trace cells with the drug to be tested, and introducing bioorthogonal groups into the trace cells through sugar metabolism engineering to obtain treated cells; The properties of the bioorthogonal groups on the treated cells are analyzed to determine the effect of the drug to be tested on the activity of the trace cells.

2. The detection method according to claim 1, characterized in that: The step of treating the trace cells with the drug to be tested and introducing the bioorthogonal groups into the trace cells through sugar metabolism engineering comprises: The trace cells, the drug to be tested and the sugar molecules modified with the bioorthogonal groups are mixed and placed for a predetermined time to obtain the treated cells.

3. The detection method according to claim 2, characterized in that: The bioorthogonal group includes an azide group; and / or, the sugar molecules include sugar units and their derivatives; Optionally, the sugar unit includes any one of a 2-aminomannose unit, a 2-aminogalactose unit, a 2-aminoglucose unit and a 6-azido-L-fucose unit; further, the 2-aminomannose unit, the 2-aminogalactose unit, the 2-aminoglucose unit and the 6-azido-L-fucose unit respectively have the structures shown in the following formulas, wherein X=(CH2)n: n is any number between 1 and 20:

4. The detection method according to claim 2, characterized in that: The detection method further comprises the following steps: introducing bioorthogonal groups to the trace cells that have not been treated with the drug to be tested by sugar metabolism engineering to obtain control cells; The step of analyzing the characteristics of the bioorthogonal groups on the treated cells to determine the effect of the drug to be tested on the activity of the trace cells comprises: performing fluorescent staining on the bioorthogonal groups on the treated cells and the control cells; Randomly select n treated cells and m control cells, count the cell area of ​​each corresponding cell and the luminescence intensity of the bioorthogonal group of each corresponding cell, and calculate the average luminescence intensity RFI per unit perimeter of each corresponding cell; Calculate the average RFI value of n treated cells and m control cells, and calculate the ratio of the two values, tRFI 实验组 / tRFI 对照组 , according to the ratio tRFI 实验组 / tRFI 对照组 Determine the effect of the drug to be tested on the activity of the trace cells.

5. The detection method according to claim 4, characterized in that: The average luminous intensity RFI of each corresponding cell unit perimeter is calculated using formula 1, and the formula 1 is as follows: RFI=FI / {2π[(S / π)^0.5]} Wherein, S is the cell area of ​​each corresponding cell, and FI is the luminescence intensity of the bioorthogonal group of each corresponding cell.

6. The detection method according to claim 4, characterized in that: According to the ratio tRFI 实验组 / tRFI 对照组 The step of determining the effect of the drug to be tested on the activity of the trace cells comprises: If the ratio approaches 1, the drug to be tested has no effect on the activity of the trace cells; If the ratio approaches 0, the drug to be tested has an infinite effect on the activity of the trace cells.

7. The detection method according to any one of claims 1 to 6, characterized in that: The bioorthogonal group includes an azide group; And / or, the number of the trace cells is less than 100; And / or, the trace cells are trace tumor cells, and the drug to be tested is a chemotherapy drug or an anti-tumor drug.

8. A method for detecting sensitivity of chemotherapy drugs, characterized in that: The steps include: The detection method according to any one of claims 1 to 7 is used to detect the effect of different drug concentrations of chemotherapy drugs on the activity of trace tumor cells, and a curve of the effect of drug concentration on the activity of the trace tumor cells is drawn, and then the test drug concentration of the chemotherapy drug is obtained according to the activity effect curve, wherein the test drug concentration is a standard drug concentration for chemotherapy drug sensitivity testing; The blood sample to be tested is treated with the corresponding chemotherapy drug at the test drug concentration, and the blood sample to be tested is subjected to sugar metabolism engineering treatment to obtain a treated blood sample; performing circulating tumor cell enrichment treatment on the treated blood; The activity of circulating tumor cells enriched in the blood after the treatment is detected to determine the drug sensitivity of the body corresponding to the blood sample to be tested to the chemotherapy drug.

9. The method for detecting sensitivity of chemotherapy drugs according to claim 8, characterized in that: The method for detecting sensitivity of chemotherapy drugs also includes the following steps: performing a sugar metabolism engineering treatment on the blood sample to be tested that has not been treated with the chemotherapy drug to obtain a control blood sample; performing a circulating tumor cell enrichment treatment on the control blood sample; The step of detecting the activity of circulating tumor cells enriched in the treated blood to determine the drug sensitivity of the body corresponding to the blood sample to be tested to the chemotherapeutic drug comprises: Performing immunofluorescence staining and bioorthogonal group fluorescence staining on the circulating tumor cells enriched in the treated blood sample and the control blood sample; Randomly select n circulating tumor cells after staining from the two blood samples, count the cell area of ​​each corresponding cell and the luminescence intensity of the bioorthogonal group of each corresponding cell, and calculate the average luminescence intensity RFI per unit perimeter of each corresponding cell; calculate the average RFI of the n circulating tumor cells after staining of the two blood samples and calculate the ratio RCV of the two, that is, obtain the relative activity of the circulating tumor cells enriched in the blood after the treatment; The drug sensitivity of the body corresponding to the blood sample to be tested to the chemotherapy drug is determined according to the ratio RCV.

10. The method for detecting sensitivity of chemotherapy drugs according to claim 9, characterized in that: The test drug concentration of the chemotherapy drug is the ratio tRFI 实验组 / tRFI 对照组 The drug concentration corresponding to 65%; Furthermore, the step of determining the drug sensitivity of the body corresponding to the blood sample to be tested to the chemotherapy drug according to the ratio RCV includes: If the ratio RCV is higher than 70%, the drug sensitivity to the chemotherapy drug is drug resistance; If the ratio RCV is lower than 60%, the drug sensitivity to the chemotherapy drug is sensitive.

11. The method for detecting sensitivity of chemotherapeutic drugs according to any one of claims 8 to 10, characterized in that: The chemotherapy drug includes at least one of doxorubicin, paclitaxel and cisplatin.

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