Method for testing effect of drug on activity of trace cells, and use thereof

Abstract

A method for testing the effect of a drug on the activity of trace cells, and a use thereof. The testing method comprises the following steps: using a drug under test to treat trace cells, and introducing a bioorthogonal group onto the trace cells by means of metabolic glycoengineering, so as to obtain treated cells; and analyzing characteristics of the bioorthogonal group on the treated cells, so as to determine the effect of the drug under test on the activity of the trace cells. In the testing method, the bioorthogonal group is introduced onto the trace cells by means of metabolic glycoengineering, so that the effect of the drug on the activity of the trace cells can be quantitatively tested by analyzing the characteristics of the bioorthogonal group on the trace cells. On the basis of the quantitative testing method for the activity of the trace cells based on bioorthogonal metabolic glycoengineering, a novel technique for real-time chemosensitivity testing using CTCs is developed. By means of individualized chemotherapeutic drug screening, accurate cancer treatment is achieved.

Description

Detection methods and applications of drug effects on trace cell viability Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a method for detecting the effect of drugs on trace cell activity and its application. Background Technology

[0002] In 1869, Australian physician Ashworth first discovered and proposed the concept of circulating tumor cells (CTCs) in the blood of breast cancer patients. In 1976, Nowell revised the definition of CTCs to: tumor cells originating from primary or metastatic tumors that have acquired the ability to detach from the basement membrane and invade blood vessels through the tissue matrix. Currently, CTCs refer to a collective term for various types of tumor cells present in peripheral blood, and their growth and spread occur throughout all stages of tumor development.

[0003] Significant progress has been made in using CTCs for chemotherapy drug screening (https: / / doi.org / 10.1016 / j.bcp.2021.114589). However, this method generally requires culturing the isolated CTCs, a process that often takes weeks or even months and is extremely costly. In contrast, cancer progresses rapidly, and patients cannot wait such a long time before treatment. Therefore, there is an urgent clinical need for a faster, "real-time" CTC pre-assessment and treatment approach.

[0004] The most significant characteristic of CTCs is their scarcity; only a few to dozens of CTCs are present per milliliter of blood. To directly screen drugs using these trace amounts of CTCs, the primary scientific challenge is how to quantify cell viability in such minute quantities. This is because existing conventional cell viability assays, including counting, CCK-8 assays, MTT assays, and calcein-PI assays, are not well-suited for quantifying the viability of trace cells.

[0005] Summary of the Invention

[0006] Based on this, this application provides a method for detecting the effect of drugs on trace cell activity, which enables quantitative testing of trace cell activity.

[0007] A method for detecting the effect of a drug on trace cell activity, comprising the following steps:

[0008] Trace cells were treated with the drug to be tested, and bioorthogonal groups were introduced onto the trace cells through glucose metabolism engineering to obtain treated cells;

[0009] The characteristics of the bioorthogonal groups on the treated cells were analyzed to determine the effect of the test drug on the trace cell activity.

[0010] In the above detection method, bioorthogonal groups are introduced onto trace cells through glucose metabolism engineering, so that the effect of drugs on trace cell activity can be quantitatively detected by analyzing the characteristics (e.g., quantity) of the bioorthogonal groups on the trace cells.

[0011] In some embodiments, the step of treating trace cells with the drug to be tested and introducing bioorthogonal groups onto the trace cells through glucose metabolism engineering includes:

[0012] The trace cells, the test drug, and sugar molecules modified with the bioorthogonal group are mixed and left to stand for a predetermined time to obtain the treated cells.

[0013] In some embodiments, the bioorthogonal group includes an azide group;

[0014] And / or, the sugar molecule includes sugar units and their derivatives;

[0015] Optionally, the sugar unit includes any one of 2-aminomannose, 2-aminogalactose, 2-glucosamine, and 6-azido-L-fucose; further, the 2-aminomannose, 2-aminogalactose, 2-glucosamine, and 6-azido-L-fucose units each have the structure shown in the following formula, where X = (CH2)n: n is any number between 1 and 20:

[0016] In some embodiments, the detection method further includes the step of introducing bioorthogonal groups into the trace cells that have not been treated with the test drug through glucose 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 test drug on the trace cell activity includes:

[0018] Fluorescent staining was performed 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 of n treated cells and the average RFI of m control cells, and calculate the ratio tRFI between the two. 实验组 / tRFI 对照组 According to the ratio tRFI 实验组 / tRFI 对照组 To determine the effect of the test drug on the activity of the trace cells.

[0021] In some embodiments, the average luminous intensity (RFI) per corresponding cell unit perimeter is calculated using Formula 1, which is as follows:

[0022] RFI = FI / {2π[(S / π)^0.5]}

[0023] Where 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 steps for determining the effect of the test drug on the trace cell activity include:

[0025] If the ratio approaches 1, then 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 infinitely large effect on the activity of the trace cells.

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

[0028] And / or, the number of cells in 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 antitumor drug.

[0030] A method for detecting chemotherapy drug sensitivity includes the following steps:

[0031] The above detection method was 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 was plotted. Then, the test drug concentration of the chemotherapy drug was obtained based on the activity effect curve. The test drug concentration is the standard drug concentration used for chemotherapy drug sensitivity testing.

[0032] The blood sample to be tested was processed with the corresponding chemotherapy drug at the test drug concentration, and the blood sample to be tested was subjected to glucose metabolism engineering to obtain the processed blood sample.

[0033] The treated blood was then subjected to a circulating tumor cell enrichment process.

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

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

[0036] The step of detecting the activity of circulating tumor cells enriched in the blood after the aforementioned treatment, in order to determine the drug sensitivity of the corresponding organism to the chemotherapy drug, includes:

[0037] Immunofluorescence staining and bioorthogonal group fluorescence staining were performed on the circulating tumor cells enriched in the treated blood sample and the control blood sample.

[0038] For both types of blood samples, n stained circulating tumor cells were randomly selected. The cell area of ​​each corresponding cell and the luminescence intensity of the bioorthogonal group of each corresponding cell were calculated. The average luminescence intensity (RFI) per unit perimeter of each corresponding cell was also calculated. The average RFI of the n stained circulating tumor cells from the two types of blood samples was calculated, and the ratio (RCV) between the two was calculated. This yielded the relative activity of the circulating tumor cells enriched in the blood after the treatment.

[0039] The drug sensitivity of the corresponding organism to the chemotherapy drug is determined based on the ratio RCV.

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

[0041] Further, the step of determining the drug sensitivity of the corresponding organism to the chemotherapy drug based on the ratio RCV includes:

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

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

[0044] In some embodiments, the chemotherapy drug includes at least one of doxorubicin, paclitaxel, and cisplatin. Attached Figure Description

[0045] Figure 1 shows the blood cell / CTC detection results of the existing scheme and the scheme of this application;

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

[0047] Figure 3 shows the effect curves of different concentrations of DOX, CDDP, and PTX on the activity of MCF-7 cells in Example 1.

[0048] Figure 4 shows the results of drug susceptibility testing on artificial CTC samples (spike-in);

[0049] Figure 5 shows the results of drug sensitivity testing on clinical samples. Detailed Implementation

[0050] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a more complete understanding of the present application. It should be understood that the present 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 commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in this application's description is for descriptive purposes only and is not intended to be limiting of the application. Terminology

[0052] Unless otherwise stated or in case of conflict, the terms or phrases used in this application shall have the following meanings:

[0053] The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These 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 using 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 connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: 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, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0054] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0055] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0056] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

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

[0058] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0059] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this application, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed in this application should be understood to include any and all subranges included therein.

[0060] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0061] In this 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 references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0063] It is understood that the only existing real-time pre-evaluation method for cancer treatment regimens using CTCs is the "Detection of Cellular Glucose Metabolic Activity Using 2-NBDG as a Probe" protocol developed by Polaris Biotechnology Co., Ltd. (https: / / polarisbiology.com / home), CN202110153914.1, a device for monitoring 2-NBDG-labeled circulating tumor cells in vivo. However, ordinary blood cells also have normal glucose metabolism, resulting in significant background noise and low data reliability in this protocol. Furthermore, this protocol requires whole blood to be treated with a treatment regimen before glucose detection using the 2-NBDG probe, demanding that CTCs retain cell viability during laboratory testing, thus imposing stringent sample requirements.

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

[0065] S110. Trace cells are treated with the drug to be tested, and bioorthogonal groups are introduced onto the trace cells through glucose metabolism engineering to obtain treated cells.

[0066] S120. Analyze the characteristics of the bioorthogonal groups on the treated cells to determine the effect of the test drug on the activity of the trace cells.

[0067] Bioorthogonal groups are chemical groups capable of undergoing chemical reactions under specific conditions within a living organism. They exhibit high inertness to other biomolecules in vivo, reacting only with specific paired groups. This reaction is highly selective and efficient, without interfering with normal biological processes. In the detection method described in this application, bioorthogonal groups are introduced onto trace cells through glycolysis engineering, enabling the quantitative detection of the drug's effect on trace cell activity by analyzing the characteristics (e.g., quantity) of the bioorthogonal groups on the trace cells.

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

[0069] The trace cells, the test drug, and sugar molecules modified with the bioorthogonal group are mixed and left to stand 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 the following: 2-aminomannose unit, 2-aminogalactose unit, 2-glucosamine unit, and 6-azido-L-fucose unit. Specifically, the 2-aminomannose unit, 2-aminogalactose unit, 2-glucosamine unit, and 6-azido-L-fucose unit each have the structure shown in the following formula, where X = (CH2)n: n is any number between 1 and 20:

[0072] Derivatives (i.e., molecules in which all or part of the acetyl group has been deprotected or replaced with other protecting groups). The aforementioned deprotection refers to molecules with exposed hydroxyl groups obtained by removing all or part of the acetyl group; the aforementioned other protecting groups refer to other acyl groups, including propionyl, butyryl, etc.

[0073] It should be noted that the bioorthogonal group is not limited to the azide group, but can also be other bioorthogonal groups.

[0074] The metabolic pathways involved in this application 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, and are not limited to the sugar molecules mentioned above.

[0075] In a specific example, the sugar molecule is Ac4ManNAz, or 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.

[0076] The step of placing the item for the predetermined time includes placing it at 2-8℃ for 24 hours.

[0077] In some embodiments, the detection method further includes the following step: introducing bioorthogonal groups into the trace cells that have not been treated with the test drug through glucose metabolism engineering to obtain control cells.

[0078] By using control cells, the effect of the drug on trace amounts of tumor cells can be determined through relative cell activity.

[0079] The process of obtaining control cells is basically similar to that of obtaining treated cells. The main difference is whether the cells are treated with the test drug. Other operational descriptions can be found above and will not be repeated here.

[0080] Furthermore, the step of analyzing the characteristics of the bioorthogonal groups on the treated cells to determine the effect of the test drug on the trace cell activity includes S121-S123:

[0081] S121. Fluorescent staining is performed on the biological orthogonal groups on the treated cells and the control cells.

[0082] Fluorescent staining of biological orthogonal groups facilitates the analysis of their characteristics.

[0083] 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.

[0084] Where 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.

[0085] The average luminous intensity (RFI) per unit perimeter of each corresponding cell is calculated using Formula 1, which is as follows:

[0086] RFI = FI / {2π[(S / π)^0.5]}

[0087] Where S is the cell area of ​​each corresponding cell, and FI is the luminescence intensity of the bioorthogonal group of each corresponding cell.

[0088] S123. Calculate the average RFI of n treated cells and the average RFI of m control cells, and calculate the ratio tRFI between the two. 实验组 / tRFI 对照组 According to the ratio tRFI 实验组 / tRFI 对照组 To determine the effect of the test drug on the activity of the trace cells.

[0089] Wherein, according to the ratio tRFI 实验组 / tRFI 对照组 The steps for determining the effect of the test drug on the trace cell activity include:

[0090] If the ratio approaches 1, then the drug to be tested has no effect on the activity of the trace cells.

[0091] If the ratio approaches 0, the drug to be tested has an infinitely large effect on the activity of the trace cells.

[0092] In some embodiments, the bioorthogonal group includes an azide group;

[0093] And / or, the number of cells in the trace cells is less than 100;

[0094] And / or, the trace cells are trace tumor cells, and the drug to be tested is a chemotherapy drug or an antitumor drug.

[0095] This application utilizes the activity of abnormal glucose metabolism pathways inherent in tumor cells for cell viability evaluation. When tumor cells exhibit high activity, this pathway is metabolically active; when tumor cells die, this pathway is completely inactivated. The advantage of this pathway is that, due to abnormal tumor cell metabolism, certain chemical groups (such as azides) can be anchored to the cell membrane via this pathway and quantitatively measured through fluorescent staining, thus providing a method for measuring trace cell viability.

[0096] Based on the aforementioned methods for detecting the effects of drugs on trace cell activity, this application further develops a "real-time" chemotherapy drug sensitivity testing technique using CTCs.

[0097] The second aspect of this application provides a method for detecting chemotherapy drug sensitivity, comprising the following steps S210-S240:

[0098] S210. The detection method of the first aspect 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 plotted. Then, the test drug concentration of the chemotherapy drug is obtained according to the activity effect curve. The test drug concentration is the standard drug concentration used for chemotherapy drug sensitivity testing.

[0099] The detection methods for the first aspect are detailed above and will not be repeated here.

[0100] In some specific examples, the tumor cells were MCF-7 cells, and the corresponding test drug concentrations were as follows: DOX (doxorubicin) at 624 nM, CDDP (cisplatin) at 139 μM, and PTX (paclitaxel) at 20 nM.

[0101] S220. The blood sample to be tested is processed with the corresponding chemotherapy drug at the concentration of the test drug, and the blood sample to be tested is subjected to glucose metabolism engineering to obtain the processed blood sample.

[0102] The operation of performing glucose metabolism engineering on the blood sample to be tested is similar to the operation of introducing bioorthogonal groups onto the trace cells through glucose metabolism engineering in the detection method of the first aspect. For specific details, please refer to the above text, and it will not be repeated here.

[0103] The blood samples to be tested are fresh blood samples.

[0104] In a specific example, step S220 includes: pre-filling a blood collection tube with 100 μMAc4ManNAz (i.e., 0.5 μmol per 5 ml of blood) and the corresponding drug, mixing the blood after collection, and placing it at 2-8 degrees Celsius for 24 hours to obtain the processed blood sample.

[0105] S230. The treated blood is subjected to circulating tumor cell enrichment treatment.

[0106] The methods for enriching circulating tumor cells include, but are not limited to, the methods described in patent ZL 2021 1 1494317.1.

[0107] S240. Detect the activity of circulating tumor cells enriched in the blood after the treatment to determine the drug sensitivity of the corresponding body to the chemotherapy drug.

[0108] In some embodiments, the chemotherapy drug sensitivity detection method further includes the following steps: performing glucose metabolism engineering on the blood sample to be tested that has not been treated with the chemotherapy drug to obtain a control blood sample; and performing circulating tumor cell enrichment treatment on the control blood sample;

[0109] Steps S240 include S241-S243:

[0110] S241. Immunofluorescence staining and bioorthogonal group fluorescence staining are performed on the circulating tumor cells enriched in the treated blood sample and the control blood sample.

[0111] S242. For both types of blood samples, n stained circulating tumor cells are randomly selected. The cell area of ​​each corresponding cell and the luminescence intensity of the bioorthogonal group of each corresponding cell are calculated. The average luminescence intensity (RFI) per unit perimeter of each corresponding cell is calculated. The average RFI of the n stained circulating tumor cells of the two types of blood samples is calculated and the ratio (RCV) between the two is calculated. This gives the relative activity of the circulating tumor cells enriched in the blood after the treatment.

[0112] S243. Determine the drug sensitivity of the body corresponding to the blood sample to be tested to the chemotherapy drug based on the ratio RCV.

[0113] 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%;

[0114] Further, the step of determining the drug sensitivity of the corresponding organism to the chemotherapy drug based on the ratio RCV includes:

[0115] If the ratio RCV is higher than 70%, then the drug sensitivity to the chemotherapy drug is drug resistance.

[0116] If the ratio RCV is less than 60%, then the drug sensitivity to the chemotherapy drug is considered sensitive.

[0117] In some embodiments, the chemotherapy drug includes at least one of doxorubicin, paclitaxel, and cisplatin.

[0118] The main technical solution of this application is the development of a quantitative testing method for the effect of a drug on the activity of trace amounts (in this solution, "trace amount" refers to less than 100 cells) of tumor cells, and based on this method, a "real-time" chemotherapy drug sensitivity testing technology using CTCs has been further developed. It mainly includes:

[0119] 1. A quantitative method for detecting the effect of drugs based on bioorthogonal metabolic glycoengineering on the activity of trace tumor cells;

[0120] 2. Based on the above method, use CTC to perform "real-time" chemotherapy drug sensitivity testing technology.

[0121] Figure 1 shows a comparison of blood cell / CTC detection results between the existing method and the method proposed in this application. In Figure 1, the left image is a promotional color image collected from Chenan Biotechnology Co., Ltd., and the right image is a fluorescence detection image of the method proposed in this application. As can be seen from Figure 1, the noise of this application is very low (green channel).

[0122] Figure 2 compares the technical routes of existing solutions with those of this application. In Figure 2, a) represents the technical route of Chenan Biotechnology Co., Ltd., and b) represents the technical route of this application. As can be seen from Figure 2, when performing laboratory fluorescent staining, the sample of this application does not require CTCs to be in a viable state, thus reducing the requirements for sample storage / transportation and making it more convenient and economical.

[0123] Therefore, the above-mentioned scheme of this application is used to evaluate the cell activity of abnormal glucose metabolism pathways in tumor cells, and is less affected by normal blood cells. At the same time, the cell activity evaluation process begins as soon as the blood enters the blood collection tube. Even if the CTCs in the blood are completely inactivated when the blood reaches the laboratory, it will not affect the test results, and the requirements for the sample are lenient.

[0124] The following are specific examples.

[0125] Unless otherwise specified, the drugs and instruments used in the examples are conventional choices in the art. Experimental methods not specifying particular conditions in the examples are typically performed under standard conditions, such as those described in literature, books, or methods recommended by the reagent kit manufacturer.

[0126] Example 1

[0127] 1. Quantitative testing of the effect of drugs on the activity of trace tumor cells

[0128] A quantitative assay method for the effect of drugs on the activity of trace tumor cells specifically refers to the simultaneous treatment of trace cells with chemotherapy drugs and metabolic glycoengineering using 100 μM Ac4ManNAz. After 24 hours, the final cell viability is quantitatively evaluated by measuring the number of azide groups introduced during metabolic glycoengineering on the cell membrane surface. The specific procedure is as follows:

[0129] (1) The experiment was divided into an experimental group and a control group. The cell treatment process was as follows:

[0130] Cell treatment in the experimental group: Trace amounts of cells (i.e., MCF-7 cells, 100 cells per cell), 100 μM Ac4ManNAz, and different concentrations of corresponding chemotherapy drugs were mixed and incubated at 2-8 degrees Celsius for 24 hours. The chemotherapy drugs were DOX (doxacin), CDDP (cisplatin), and PTX (paclitaxel). The different concentrations of DOX were 0 nM, 20 nM, 100 nM, 200 nM, 500 nM, 1000 nM, and 1500 nM. The different concentrations of CDDP were 0, 5 μM, 10 μM, 20 μM, 100 μM, 200 μM, and 300 μM. The different concentrations of PTX were 0 nM, 1 nM, 2 nM, 5 nM, 10 nM, 20 nM, and 40 nM.

[0131] Cell treatment for the control group: Trace cells (i.e., MCF-7 cells, 100 cells) were mixed with 100 μM Ac4ManNAz and placed at 2-8 degrees Celsius for 24 hours.

[0132] (2) The cells after the above two treatments were stained with azide groups. The azide group specific staining protocol used was as follows: the cells did not need to be fixed or treated. They were treated with DBCO-biotin (100μM, 1 hour), washed with PBS 3 times, treated with Alexa Fluor 488-labeled streptavidin (50μM, 15 minutes), washed with PBS 3 times, and centrifuged with 450g for each step.

[0133] (3) The azide-specific fluorescence intensity (FI) and cell area (S) on the membrane of each tumor cell were statistically analyzed using software such as ImageJ. The average fluorescence intensity per unit perimeter of each tumor cell was obtained using the formula RFI = FI / {2π[(S / π)^0.5]}. The RFI of 50 randomly selected cells was then averaged to obtain the total RFI (tRFI) for the drug-treated group and the control group. Finally, the ratio tRFI was calculated. 实验组 / tRFI对照组 via tRFI 实 验组 / tRFI 对照组 The ratio is used to obtain the RCV (relative cell activity) parameter, which represents the effect of the drug on trace tumor cells. If there is no effect at all, the IR approaches 1; if the effect is infinitely large, the IR approaches 0. Curves were plotted using the RCV values ​​obtained from different drug concentrations. The drug concentration corresponding to RCV = 65% was selected as the standard parameter for the next step of chemotherapy susceptibility testing (see Figure 3). Figure 3 shows the effect curves of different concentrations of DOX, CDDP, and PTX on the activity of MCF-7 cells in Example 1.

[0134] The drug concentrations of the chemotherapy regimens obtained by screening MCF-7 cells were: DOX 624 nM, CDDP 139 μM, and PTX 20 nM.

[0135] 2. Treatment plan pretreatment of CTCs

[0136] By plotting the effect curves of different chemotherapy drug treatment regimens on the activity of trace tumor cells, suitable drug concentrations for chemotherapy drug sensitivity testing were selected. As shown in Figure 3, the drug concentrations of the chemotherapy regimens obtained using MCF-7 cells were: DOX 624 nM, CDDP 139 μM, and PTX 20 nM.

[0137] While using the above steps to screen and process fresh blood samples, metabolic sugar engineering was also performed. Specifically, 100 μM Ac4ManNAz (0.5 μmol per 5 mL of blood) and the corresponding drug were pre-placed in the blood collection tube (no drug was placed in the control group). After collecting blood, 5 mL of blood sample was added to the above blood collection tube, mixed well, and placed at 2-8 degrees Celsius for 24 hours.

[0138] The fresh blood samples were from 10 patients (stage 3 breast cancer patients). The 10 patients were numbered P1-P10, with P1-P7 being randomized patients and P8-P10 being patients who had received paclitaxel and cisplatin combination therapy.

[0139] 3. CTC enrichment

[0140] The blood obtained from 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 1 1494317.1.

[0141] 4. Individualized drug sensitivity testing for patients can be conducted by detecting the activity of CTCs.

[0142] The obtained CTCs were subjected to immunofluorescence staining and chemiluminescence staining. Data from the drug-treated group and the control group were statistically analyzed using the method described in "1. Quantitative Test of the Effect of Drugs on the Activity of Trace Tumor Cells," and the RCV of each drug on the sample was calculated. The detection results are shown in Figures 4-5 and Table 1.

[0143] As shown in Figure 4, in artificial CTC samples (tumor cell lines mixed into the blood of healthy individuals), doxorubicin (DOX) resistant tumor cell samples are clearly more resistant to doxorubicin treatment.

[0144] As shown in Figure 5 and Table 1, the clinical population that had received combination therapy with paclitaxel and cisplatin (P8-P10) showed 100% complete resistance to both drugs.

[0145] 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 relatively scattered random resistance / sensitivity, but patients P8-P10 who had received paclitaxel + cisplatin combination therapy generally showed 100% resistance to paclitaxel and cisplatin.

[0146] It is evident that this method possesses extremely high reliability. Currently, this application considers the standard drug concentration (i.e., the aforementioned tRFI) to be... 实验组 / tRFI 对照组 Under treatment with 65% of the drug concentration, clinical samples with an RCV higher than 70% are considered resistant, and those with an RCV lower than 60% are considered sensitive.

[0147] Table 1. Statistical results of blood samples from 10 patients (P1-P10) to different drugs.

[0148] In summary, this application presents a method for quantitative testing of trace cell activity based on bioorthogonal metabolic sugar engineering. Building upon this method, a novel technique for real-time (less than 2 days) chemotherapy drug sensitivity testing using CTCs has been developed. This enables personalized screening of chemotherapy drugs, thereby achieving precision cancer treatment.

[0149] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0150] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for detecting the effect of a drug on trace cell activity, characterized in that, Includes the following steps: Trace cells were treated with the drug to be tested, and bioorthogonal groups were introduced onto the trace cells through glucose metabolism engineering to obtain treated cells; The characteristics of the bioorthogonal groups on the treated cells were analyzed to determine the effect of the test drug on the trace cell activity.

2. The detection method according to claim 1, characterized in that, The step of treating trace cells with the drug to be tested and introducing bioorthogonal groups onto the trace cells through glucose metabolism engineering includes: The trace cells, the test drug, and sugar molecules modified with the bioorthogonal group are mixed and left to stand 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 molecule includes sugar units and their derivatives; Optionally, the sugar unit includes any one of 2-aminomannose, 2-aminogalactose, 2-glucosamine, and 6-azido-L-fucose; further, the 2-aminomannose, 2-aminogalactose, 2-glucosamine, and 6-azido-L-fucose units each have the structure shown in the following formula, where 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 includes the following step: introducing bioorthogonal groups onto the trace cells that have not been treated with the test drug through glucose 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 test drug on the trace cell activity includes: Fluorescent staining was performed 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 of n treated cells and the average RFI of m control cells, and calculate the ratio tRFI between the two. 实验组 / tRFI 对照组 According to the ratio tRFI 实验组 / tRFI 对照组 To determine the effect of the test drug on the activity of the trace cells.

5. The detection method according to claim 4, characterized in that, The average luminescence intensity (RFI) per unit perimeter of each corresponding cell was calculated using Formula 1, which is as follows: RFI=FI / {2π[(S / π)^0.5]} Where 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 steps for determining the effect of the test drug on the trace cell activity include: If the ratio approaches 1, then 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 infinitely large effect on the activity of the trace cells.

7. The detection method according to any one of claims 1-6, characterized in that, The bioorthogonal group includes an azide group; And / or, the number of cells in 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 antitumor drug.

8. A method for detecting the sensitivity of chemotherapy drugs, characterized in that, Includes the following steps: The detection method according to any one of claims 1-7 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 plotted. Then, the test drug concentration of the chemotherapy drug is obtained according to the activity effect curve. The test drug concentration is the standard drug concentration used for chemotherapy drug sensitivity testing. The blood sample to be tested was processed with the corresponding chemotherapy drug at the test drug concentration, and the blood sample to be tested was subjected to glucose metabolism engineering to obtain the processed blood sample. The treated blood was then subjected to a circulating tumor cell enrichment process. The activity of circulating tumor cells enriched in the blood after the treatment is detected to determine the drug sensitivity of the corresponding body to the chemotherapy drug.

9. The method for detecting chemotherapeutic drug sensitivity according to claim 8, characterized in that, The chemotherapy drug sensitivity detection method further includes the following steps: performing glucose metabolism engineering on the blood sample to be tested that has not been treated with the chemotherapy drug to obtain a control blood sample; and performing circulating tumor cell enrichment treatment on the control blood sample. The step of detecting the activity of circulating tumor cells enriched in the blood after the aforementioned treatment, in order to determine the drug sensitivity of the corresponding organism to the chemotherapy drug, includes: Immunofluorescence staining and bioorthogonal group fluorescence staining were performed on the circulating tumor cells enriched in the treated blood sample and the control blood sample. For both types of blood samples, n stained circulating tumor cells were randomly selected. The cell area of ​​each corresponding cell and the luminescence intensity of the bioorthogonal groups in each corresponding cell were calculated. The average luminescence intensity (RFI) per unit perimeter of each corresponding cell was also calculated. The values ​​of n stained cells from both types of blood samples were then calculated. The average RFI of the circulating tumor cells and the ratio RCV are calculated to obtain the relative activity of the circulating tumor cells enriched in the blood after the treatment. The drug sensitivity of the corresponding organism to the chemotherapy drug is determined based on the ratio RCV.

10. The method for detecting chemotherapeutic drug sensitivity according to claim 9, characterized in that, The test concentration of the chemotherapy drug is the ratio tRFI. 实验组 / tRFI 对照组 The drug concentration corresponding to 65%; Further, the step of determining the drug sensitivity of the corresponding organism to the chemotherapy drug based on the ratio RCV includes: If the ratio RCV is higher than 70%, then the drug sensitivity to the chemotherapy drug is drug resistance. If the ratio RCV is less than 60%, then the drug sensitivity to the chemotherapy drug is considered sensitive.

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

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