Method for extracting circulating tumor cells

Abstract

Disclosed in the present invention is a method for extracting circulating tumor cells, comprising the following steps: labeling circulating tumor cells in a sample to be extracted by means of metabolic glycoengineering, to obtain a sample to be extracted containing the labeled circulating tumor cells; using a biotin-streptavidin magnetic bead system to capture the labeled circulating tumor cells from the sample to be extracted; and using a reducing reagent to reduce the captured labeled circulating tumor cells, to obtain circulating tumor cells.

Description

Methods for extracting circulating tumor cells Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for extracting circulating tumor cells. Background Art

[0002] In 1869, Australian physician Ashworth first discovered and proposed the concept of circulating tumor cells (CTCs) in the blood of breast cancer patients. CTCs are believed to be closely associated with tumor metastasis and lethality, and are the cell structure currently available that most closely reflects a patient's true disease progression. Therefore, the isolation and purification of CTCs is crucial for understanding a patient's real-time disease progression.

[0003] The biggest technical bottleneck in using CTCs for drug susceptibility testing is purification technology. The primary challenge is the extremely low abundance of CTCs. Theoretically, each milliliter of blood contains billions of red blood cells and millions of white blood cells, while CTCs number only a few to dozens. Extracting high-purity CTCs from such a large, mixed cell population requires precise differentiation of CTCs from other blood cells.

[0004] Currently, there are four main methods for CTC isolation and purification: ISET membrane separation, microfluidics, immune-recognition-based magnetic bead separation, and combined separation methods. ISET membrane separation and immune-recognition-based magnetic bead separation methods are highly damaging to cells, and the resulting CTCs can only be counted and are not suitable for downstream applications. Therefore, an ideal CTC extraction process must overcome the damage to the CTCs themselves and maintain their metabolic activity.

[0005] Currently, only microfluidic chip technology can be used to isolate active CTCs on the market. This technology primarily targets the physical differences between CTCs and blood cells, including size, density, and morphology, and separates CTC-containing microdroplets using microelectrodes or micropumps. However, the main limitation is that the separation principle is physical recognition, which poses challenges in broad spectrum analysis and metabolic uniformity.

[0006] Patent CN113358865A proposes a CTC detection method based on bioorthogonal metabolic glycoengineering markers, enabling accurate and non-destructive detection of CTCs. However, the long detection time (approximately one hour) leads to a small amount of nonspecific adhesion and cell death, which is not conducive to downstream applications of CTCs.

[0007] Summary of the Invention

[0008] Based on this, the present application provides a method for extracting circulating tumor cells, which can extract circulating tumor cells in a shorter time, and is beneficial to reducing nonspecific adhesion of cells and damage to cells.

[0009] A method for extracting circulating tumor cells comprises the following steps:

[0010] labeling circulating tumor cells in the sample to be extracted by metabolic glycoengineering to obtain the sample to be extracted containing labeled circulating tumor cells;

[0011] capturing the labeled circulating tumor cells from the sample using a biotin-streptavidin magnetic bead system;

[0012] The captured labeled circulating tumor cells are reduced using a reducing agent to obtain the circulating tumor cells.

[0013] In the above-mentioned circulating tumor cell extraction method, a biotin-streptavidin magnetic bead system is used to capture labeled circulating tumor cells from the sample to be extracted. The identification process is shortened from 60 minutes to 10 minutes, thereby reducing the cell damage that may be caused by nonspecific adhesion and surface interface contact, thereby improving the purity, viability and non-destructiveness of CTCs.

[0014] In one embodiment, the step of capturing the labeled circulating tumor cells from the sample to be extracted using a biotin-streptavidin magnetic bead system comprises:

[0015] reacting dibenzocyclooctyne-disulfide bond-biotin with the sample to be extracted containing the labeled circulating tumor cells to obtain biotinylated cells;

[0016] The biotinylated cells are reacted with magnetic particles linked to streptavidin to capture the labeled circulating tumor cells.

[0017] In one embodiment, before the step of reacting dibenzocyclooctyne-disulfide-biotin with the sample to be extracted containing the labeled circulating tumor cells, the step of preparing the dibenzocyclooctyne-disulfide-biotin is further included:

[0018] Biotin is reacted with cystamine and then condensed with dibenzocyclooctyne-carboxyl to obtain the dibenzocyclooctyne-disulfide-biotin.

[0019] In one embodiment, before the step of reacting the biotinylated cells with magnetic particles linked to streptavidin, the step further comprises preparing the magnetic particles linked to streptavidin:

[0020] The magnetic particles connected with the carboxyl group are subjected to a condensation reaction with streptavidin to obtain the magnetic particles connected with the streptavidin.

[0021] In one embodiment, before the step of capturing the labeled circulating tumor cells from the sample using a biotin-streptavidin magnetic bead system, the following step is further included:

[0022] The labeled sample is treated with a red blood cell lysis solution to lyse the red blood cells in the sample.

[0023] In one embodiment, after the step of treating the labeled sample with a red blood cell lysis solution, the method further includes the following steps:

[0024] The cell suspension obtained by lysis is adsorbed with a leukocyte adsorbent to remove leukocytes from the sample to be extracted.

[0025] In one embodiment, the leukocyte adsorption agent includes CD45 magnetic beads.

[0026] In one embodiment, the step of labeling circulating tumor cells in the sample to be extracted by metabolic glycoengineering includes:

[0027] The circulating tumor cells in the sample to be extracted are labeled with sugar molecules, wherein the sugar molecules include at least one of 2-azidomannosamine, 2-azidoglucosamine, 2-azidogalactosamine and 6-azidofucose.

[0028] In one embodiment, the step of reducing the captured labeled circulating tumor cells using the reducing reagent comprises:

[0029] The sample to be extracted with the labeled circulating tumor cells captured is treated with the reducing reagent for more than 40 minutes, and the circulating tumor cells released between the 10th minute and the 40th minute are collected and processed.

[0030] In one embodiment, the reducing agent comprises at least one of dithiothreitol, β-mercaptoethanol, and tris(2-carboxyethyl)phosphine hydrochloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a synthetic route diagram of molecule B;

[0032] Figure 2 is a synthetic route diagram of material C;

[0033] FIG3 is a schematic diagram of the process of extracting circulating tumor cells of the present application;

[0034] FIG4 is a graph showing the chromatographic detection results of molecule B;

[0035] FIG5 is a graph showing the chromatographic detection results of material C;

[0036] FIG6 is a scanning electron microscope image of material C;

[0037] FIG7 is a diagram showing the metabolic homogeneity of CTCs in Example 3 of the present application. DETAILED DESCRIPTION

[0038] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, the following detailed description of the specific embodiments of the present invention is provided in conjunction with specific embodiments and accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0039] One embodiment of the present application provides a method for extracting circulating tumor cells, comprising the following steps S110-S130:

[0040] S110, labeling circulating tumor cells in the sample to be extracted by metabolic glycoengineering to obtain a sample to be extracted containing labeled circulating tumor cells;

[0041] S120, capturing the labeled circulating tumor cells from the sample to be extracted using a biotin-streptavidin magnetic bead system;

[0042] S130, reducing the captured labeled circulating tumor cells with a reducing reagent to obtain circulating tumor cells.

[0043] In the above-mentioned circulating tumor cell extraction method, a biotin-streptavidin magnetic bead system is used to capture labeled circulating tumor cells from the sample to be extracted. The identification process is shortened from 60 minutes to 10 minutes, thereby reducing the cell damage that may be caused by nonspecific adhesion and surface interface contact, thereby improving the purity, viability and non-destructiveness of CTCs.

[0044] In one embodiment, the steps of S120 include S121-S123:

[0045] S121. Reacting dibenzocyclooctyne-disulfide-biotin with a sample to be extracted containing labeled circulating tumor cells to obtain biotinylated cells.

[0046] S123. The biotinylated cells are reacted with magnetic particles linked to streptavidin to capture the labeled circulating tumor cells.

[0047] In one embodiment, before S121, the step of preparing dibenzocyclooctyne-disulfide-biotin is further included: biotin is reacted with cystamine, and then condensed with dibenzocyclooctyne-carboxyl (i.e., DBCO-COOH) to obtain dibenzocyclooctyne-disulfide-biotin (i.e., DBCO-SS-biotin, referred to as molecule B).

[0048] Specifically, the synthetic route of molecule B is shown in Figure 1. The preparation process of molecule B includes: dissolving biotin and cystine hydrochloride in a methanol solution containing triethylamine in a 1:1 molar ratio, stirring for half an hour to completely dissolve them. 1.2 equivalents and 1.5 equivalents of NHS and EDC are added in sequence, and stirred at room temperature overnight. After removing the solution, the intermediate is separated using a chromatographic column. The intermediate is dissolved in methanol, and 1 equivalent of DBCO-COOH, 1.2 equivalents of NHS, and 1.5 equivalents of EDC are added in sequence, and stirred at room temperature overnight. After removing the solution, the product DBCO-SS-biotin is separated using a chromatographic column (total yield 43%, purity >96%). It is prepared into a 30mM DMSO stock solution and frozen at -20°C for use.

[0049] In one embodiment, before S123, the method further includes preparing magnetic particles connected to streptavidin: condensing the magnetic particles connected to carboxyl groups with streptavidin to obtain magnetic particles connected to streptavidin (referred to as Material C).

[0050] Specifically, the synthetic route of Material C is shown in Figure 2. The preparation process of Material C includes: using an ultrasonicator to evenly disperse 5 mL of carboxyl nanoparticles (2.8 micron diameter, commercially available), adding 10 mL of a PBS solution containing 500 μM NHS and 1 mM EDC, and sonicating for half an hour. Slowly add 10 mL of a PBS solution containing 100 μM streptavidin. Shake overnight at 2-8°C. The resulting mixture is washed three times with PBS and three times with sterile double-distilled water, and finally dispersed in sterile double-distilled water.

[0051] In one embodiment, before S120, the following step is further included: treating the labeled sample to be extracted with a red blood cell lysing solution to lyse the red blood cells in the sample to be extracted. The red blood cell lysing solution can be a conventional red blood cell lysing solution.

[0052] Furthermore, after treating the labeled sample to be extracted with a red blood cell lysis solution, the method further includes treating the cell suspension obtained by lysis with a leukocyte adsorbent to remove leukocytes from the sample to be extracted. Treatment with a leukocyte adsorbent can remove most leukocytes to obtain a primary enriched CTC suspension, thereby improving CTC purity.

[0053] Among them, the leukocyte adsorbent includes CD45 magnetic beads.

[0054] In one embodiment, S110 includes labeling circulating tumor cells in the sample to be extracted with sugar molecules, wherein the sugar molecules include at least one of 2-azidomannosamine, 2-azidoglucosamine, 2-azidogalactosamine, and 6-azidofucose.

[0055] Specifically, the steps of labeling circulating tumor cells in a sample to be extracted with sugar molecules include:

[0056] Commercially purchased 3D-printed extracellular matrix was soaked in DPBS containing 100 μM Ac4ManNAz overnight. The mixture was quickly transferred to an EDTA blood collection tube and refrigerated at 2-8°C.

[0057] Fresh patient blood is drawn and stored in tube A, shaken repeatedly ten times, and transported to a designated location at 2-8 degrees Celsius. Storage, transportation, and preservation time must be between 9-24 hours.

[0058] In one embodiment, S130 includes: using the reducing reagent to treat the sample to be extracted with the captured labeled circulating tumor cells for more than 40 minutes, and collecting the circulating tumor cells released between the 10th minute and the 40th minute.

[0059] Wherein, the reducing agent includes at least one of dithiothreitol, β-mercaptoethanol and tris(2-carboxyethyl)phosphine hydrochloride.

[0060] As shown in Figure 3, in the method for extracting circulating tumor cells of the present application, first, a special extracellular matrix-like tube impregnated with oligosaccharides is used for blood collection and blood transport / metabolic sugar engineering labeling process; secondly, red blood cells are removed using red blood cell lysis solution; then, CD45 nanomagnetic beads are used for negative enrichment to remove most white blood cells; then, a bioorthogonal reaction / biotin-streptavidin magnetic bead system is used for positive enrichment to further remove white blood cells and other blood components, and CTCs are purified for a second time; finally, the nanomagnetic beads adhered to the CTCs are released to obtain high-purity CTCs.

[0061] The circulating tumor cell extraction method of the present application incorporates CD45 magnetic bead negative enrichment to improve CTC purity; the true positive enrichment process is biotin-streptavidin recognition, which shortens the recognition process from 60 minutes to 10 minutes, thereby reducing cell damage that may be caused by nonspecific adhesion and surface interface contact, thereby improving CTC purity, viability, and non-destructiveness.

[0062] Compared with existing microfluidic technology, the extraction method of this application extracts CTCs with higher purity, overcomes CTC heterogeneity, and has better metabolic uniformity of the obtained CTCs. In addition, the extraction of CTCs in this application is based on the abnormal sugar metabolism pathway of tumors. Therefore, compared with CTCs obtained by microfluidic technology based on physical property separation, the CTCs extracted in this application have good metabolic uniformity, all CTCs have high activity, and when used in downstream physiological and biochemical experiments, the error is also smaller.

[0063] The following are specific examples.

[0064] Unless otherwise specified, the drugs and instruments used in the examples are all conventionally selected in the art. Experimental methods without specific conditions specified in the examples are generally carried out under conventional conditions, such as those described in literature or books, or methods recommended by the kit manufacturer.

[0065] Example 1

[0066] The preparation process of molecule B includes:

[0067] Biotin and cystine hydrochloride were dissolved in a methanol solution containing triethylamine at a 1:1 molar ratio and stirred for half an hour to completely dissolve. 1.2 equivalents of NHS and 1.5 equivalents of EDC were added in sequence, and the mixture was stirred at room temperature overnight. After removing the solvent, the intermediate was isolated using a chromatographic column. The intermediate was dissolved in methanol, and 1 equivalent of DBCO-COOH, 1.2 equivalents of NHS, and 1.5 equivalents of EDC were added in sequence, and the mixture was stirred at room temperature overnight. After removing the solvent, the product DBCO-SS-biotin was isolated using a chromatographic column (total yield 43%, purity >96%). This product was prepared into a 30 mM DMSO stock solution and frozen at -20°C until use. DBCO-SS-biotin is molecule B. The chromatographic analysis results of molecule B are shown in Figure 4.

[0068] Example 2

[0069] The preparation process of Material C includes:

[0070] Use an ultrasonicator to evenly disperse 5 mL of carboxyl nanoparticles (2.8 μm diameter, commercially available). Add 10 mL of a PBS solution containing 500 μM NHS and 1 mM EDC, and sonicate for half an hour. Slowly add 10 mL of a PBS solution containing 100 μM streptavidin. Shake overnight at 2-8°C. The resulting mixture is washed three times with PBS and three times with sterile double-distilled water, and finally dispersed in sterile double-distilled water to obtain Material C. The chromatographic analysis results of Material C are shown in Figure 5. The scanning electron microscopy results of Material C are shown in Figure 6.

[0071] Example 3

[0072] 1. Blood extraction, storage and selective labeling

[0073] Fresh patient blood was drawn and stored in tube A. The blood was shaken repeatedly ten times and transported to a designated location at 2-8 degrees Celsius. Storage, transportation, and preservation were ensured to last between 9 and 24 hours. The patient's condition is shown in Table 1. Detailed labeling procedures are described in patent CN113358865A.

[0074] 2. Red blood cell removal

[0075] The blood sample was treated with a commercially available red blood cell lysis buffer to obtain a cell suspension containing CTCs.

[0076] 3. CD45 magnetic beads negative enrichment to remove most white blood cells

[0077] The cell suspension containing CTCs was treated with CD45 magnetic beads. Each milliliter of blood was mixed with 200 μL of magnetic beads and incubated at 4°C for 30 minutes to remove most of the white blood cells and obtain the primary enriched CTC suspension.

[0078] 4. CTC surface biotinylation and biotin magnetic bead positive enrichment, secondary purification of CTC

[0079] A biotinylation working solution was prepared at a 1:1000 ratio using the Molecule B stock solution. The cell surface of the primary enriched CTC suspension was selectively biotinylated (biotinylation procedure: 1 mL of the working solution was added to each mL of blood-separated cell suspension, incubated at 4°C for 60 minutes, centrifuged, and washed once with PBS containing 2% FBS). Material C was then used for secondary enrichment of CTCs (secondary enrichment procedure: 30 μL of magnetic beads C was added to each mL of blood-separated cell suspension, incubated at 4°C for 15 minutes, and after adsorption, the beads and adsorbed cells were separated using a magnetic stand). This yielded a secondary enriched CTC-magnetic bead suspension. The Molecule B stock solution contained 30 mM Molecule B.

[0080] 5. Time gradient release of CTCs and three-step purification of CTCs

[0081] The obtained CTC-magnetic bead suspension was treated with a 10 mM dithiothreitol (DTT) PBS solution (specific treatment steps: use 1 mL of DTT working solution for each milliliter of CTC-magnetic bead suspension obtained by blood separation, gently shake for 40 minutes, use a magnetic rack to remove the detached magnetic beads, and retain the released CTC suspension). The cells released in the first ten minutes were discarded. After 10-40 minutes, the detached magnetic beads were removed by a magnetic rack, the obtained cells were released, and the suspension was retained to obtain the final enriched CTC suspension.

[0082] 6. CTC purity and quantity detection:

[0083] 5mL of patient blood was extracted through steps 1-5 of this example, and the resulting CTC suspension was fixed on a glass slide. Immunomicroscopy staining was performed using pCK, CD45, and nuclear dyes. Cells with pCK+CD45-nuclei+ were identified as CTCs, and other nuclei+ cells were identified as non-CTC cells. The test results are shown in Table 1. As can be seen from Table 1, the purity of the CTCs extracted by this application is between 3-48%. The purity of the CTCs obtained in patent CN113358865A ranges from 0.1% to 1%. The purity of the CTCs extracted by this application is increased to 3%-48%. The extraction method of this application is more conducive to obtaining CTCs with higher purity.

[0084] Table 1 The number and purity of CTCs extracted from the blood of different patients using the extraction method implemented in this paper

[0085] 7. CTC metabolic homogeneity detection

[0086] 5 mL of patient (i.e., patient AD) blood was extracted using steps 1-5 of this example, and the resulting CTC suspension was distributed in a 96-well plate containing a cell slide. The cells were incubated with a glycolysis probe, a pCK probe, a nuclear dye, and a CD45 probe. After 2 hours, the supernatant was carefully aspirated. After fixing the cells, they were carefully washed three times and the fluorescence intensity was determined using a Z2 fully automated fluorescence scanning microscope. The coefficient of variation of the fluorescence intensity was calculated. The test results are shown in Figure 7.

[0087] As shown in Figure 7, pCK+, nucleus+, and CD45- cells were identified as CTCs. Analysis of their glucose metabolism activity, as measured by a glycolysis probe, revealed coefficients of variation for AD data of 4.6%, 4.2%, 4.0%, and 6.2%, respectively, demonstrating good metabolic homogeneity. The coefficient of variation for CTC metabolic homogeneity obtained in patent CN113358865A ranged from 15.2% to 27.1%. The coefficient of variation for CTC metabolic homogeneity obtained in the present application's extraction method was reduced to 4.2% to 6.2%, demonstrating that the present application's extraction method is more conducive to improving CTC metabolic homogeneity.

[0088] In summary, the circulating tumor cell extraction method of the present application incorporates CD45 magnetic bead negative enrichment, thereby improving CTC purity; the true positive enrichment process is biotin-streptavidin recognition, which shortens the recognition process from 60 minutes to 10 minutes, thereby reducing cell damage that may be caused by nonspecific adhesion and surface interface contact, thereby improving CTC purity and viability, and non-destructiveness, overcoming CTC heterogeneity, and achieving better metabolic homogeneity of the resulting CTCs, resulting in smaller errors when applied to downstream physiological and biochemical experiments.

[0089] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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.

[0090] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for extracting circulating tumor cells, characterized in that, Comprising the following steps: Performing a labeling treatment on circulating tumor cells in a sample to be extracted by metabolic glycoengineering to obtain the sample to be extracted containing the labeled circulating tumor cells; Capturing the labeled circulating tumor cells from the sample to be extracted by using a biotin-streptavidin magnetic bead system; Reducing the captured labeled circulating tumor cells by using a reducing agent to obtain the circulating tumor cells.

2. The extraction method according to claim 1, characterized in that, The step of capturing the labeled circulating tumor cells from the sample to be extracted by using a biotin-streptavidin magnetic bead system includes: Reacting dibenzocyclooctyne-disulfide-biotin with the sample to be extracted containing the labeled circulating tumor cells to obtain biotinylated cells; Reacting the biotinylated cells with magnetic microparticles conjugated with streptavidin to capture the labeled circulating tumor cells.

3. The extraction method according to claim 2, characterized in that, Before the step of reacting dibenzocyclooctyne-disulfide-biotin with the sample to be extracted containing the labeled circulating tumor cells, the step of preparing the dibenzocyclooctyne-disulfide-biotin is further included: Reacting biotin with cystamine, and then performing a condensation reaction with dibenzocyclooctyne-carboxyl to obtain the dibenzocyclooctyne-disulfide-biotin.

4. The extraction method according to claim 2, characterized in that, Before the step of reacting the biotinylated cells with magnetic microparticles conjugated with streptavidin, the preparation of the magnetic microparticles conjugated with streptavidin is further included: Performing a condensation reaction on magnetic microparticles conjugated with carboxyl and streptavidin to obtain the magnetic microparticles conjugated with streptavidin.

5. The extraction method according to any one of claims 1-4, characterized in that, Before the step of capturing the labeled circulating tumor cells from the sample to be extracted by using a biotin-streptavidin magnetic bead system, the following steps are further included: Treating the sample to be extracted after the labeling treatment with a red blood cell lysate to lyse red blood cells in the sample to be extracted.

6. The extraction method according to claim 5, characterized in that, After the step of treating the sample to be extracted after the labeling treatment with a red blood cell lysate, the following steps are further included: Adsorbing the cell suspension obtained by lysis with a white blood cell adsorbent to remove white blood cells in the sample to be extracted.

7. The extraction method according to claim 6, characterized in that, The adsorbent includes CD45 magnetic beads.

8. The extraction method according to claim 1, characterized in that, The step of performing a labeling treatment on circulating tumor cells in a sample to be extracted by metabolic glycoengineering includes: Performing a labeling treatment on the circulating tumor cells in the sample to be extracted by using a sugar molecule, and the sugar molecule includes at least one of 2-azidomannosamine, 2-azido-glucosamine, 2-azidogalactosamine, and 6-azido-fucose.

9. The extraction method according to claim 1, characterized in that, The step of reducing the captured labeled circulating tumor cells by using the reducing agent includes: Treating the sample to be extracted capturing the labeled circulating tumor cells with the reducing agent for more than 40 minutes, and collecting the circulating tumor cells released during the period from the 10th minute to the 40th minute of the treatment.

10. The extraction method according to any one of claims 1-4, 6-9, characterized in that, The reducing agent includes at least one of dithiothreitol, β-mercaptoethanol, and tris(2-carboxyethyl)phosphine hydrochloride.

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