Staining solution for live circulating tumor-related rare cell and use thereof

Abstract

The present application relates to the technical field of biomedicine, and provides a staining solution for a live circulating tumor-related rare cell and a use thereof. The present application provides a use of a graphene-based tumor cell nuclear targeting fluorescent nanoprobe in the preparation of a live circulating tumor-related rare cell product, and the staining solution for a live circulating tumor-related rare cell, wherein the staining solution comprises the graphene-based tumor cell nuclear targeting fluorescent nanoprobe and a diluent. The present application further provides a kit for testing a live circulating tumor-related rare cell.

Description

An active circulating tumor-associated rare cell staining solution and its application Technical Field

[0001] This application belongs to the field of biomedical technology, and in particular relates to an active circulating tumor-associated rare cell staining solution and its application. Background Technology

[0002] Circulating tumor cells (CTCs) are tumor cells that detach from the primary or metastatic tumor and enter the bloodstream. They are typically closely associated with hematogenous metastasis and poor prognosis. CTC detection plays an important role in the auxiliary diagnosis of tumors, monitoring of recurrence and metastasis, evaluation of treatment efficacy, monitoring of drug resistance, and early screening. However, most circulating tumor cells (CTCs) lose their biological activity in the bloodstream due to factors such as apoptosis, dormancy, hypoxia, or exposure to blood shear stress, drugs, and immune system attacks. Only a very small percentage (<0.01%) of CTCs with specific genetic and phenotypic advantages can maintain treatment resistance and have a high metastatic potential, thus leading to distant metastasis. Therefore, a single CTC test may not be able to comprehensively and accurately reveal the biological characteristics of a patient's tumor.

[0003] Circulating tumor-associated rare cells (LCTRCs) are biologically active rare cells that enter the peripheral bloodstream and are closely associated with tumorigenesis, progression, treatment resistance, metastasis, and tumor burden. LCTRCs include circulating tumor cells (CTCs), tumor-associated white blood cells (WBCs) such as macrophages (TAMs) and neutrophils (TANs), tumor-associated stromal cells such as cancer-associated fibroblasts (CAFs), tumor-associated endothelial cells (CECs), and cell clusters (CTMs) composed of tumor-associated cells. These cells play a crucial role in tumor biology, reflecting the complex interactions between tumor cells and their microenvironment, and providing valuable insights into metastasis mechanisms, treatment resistance, and disease progression. Compared to individual CTCs, LTCRCs can more comprehensively and accurately reflect the biological characteristics of a patient's tumor, including tumor invasiveness, recurrence and metastatic potential, treatment resistance, and adaptation. Therefore, LTCRCs have greater clinical value in revealing tumor biological behavior. However, the detection and application of LCTRCs, including CTCs, are greatly limited. Furthermore, due to the scarcity and high heterogeneity of tumor cells in human peripheral blood, as well as the complexity of the metastatic process, current methods for detecting CTCs and tumor-associated variant white blood cells (WBCs) in peripheral blood suffer from low sensitivity, low specificity, and low accuracy. Therefore, there is an urgent need in this field to provide a method that can detect active circulating tumor-associated rare cells in peripheral blood with high sensitivity, high specificity, and high accuracy. Summary of the Invention

[0004] Therefore, one of the purposes of this application is to provide the application of graphene-based tumor cell nuclear-targeting fluorescent nanoprobes (GTTN) in the preparation of products for detecting active circulating tumor-associated rare cells.

[0005] The second objective of this application is to provide a staining solution for active circulating tumor-associated rare cells that can stain active circulating tumor-associated rare cells with high specificity and high sensitivity.

[0006] The third objective of this application is to provide a kit for detecting active circulating tumor-associated rare cells (CMRs) with high sensitivity, high specificity, and high accuracy.

[0007] To achieve the above-mentioned objectives, this application provides the following technical solutions:

[0008] This application provides the application of graphene-based tumor cell nucleus-targeting fluorescent nanoprobes in the preparation of products for detecting active circulating tumor-associated rare cells.

[0009] This application provides an active circulating tumor-associated rare cell staining solution, which includes a graphene-based tumor cell nuclear-targeting fluorescent nanoprobe and a diluent.

[0010] This application also provides the application of the above-mentioned staining solution in the preparation of products for detecting active circulating tumor-associated rare cells.

[0011] This application also provides a kit for detecting active circulating tumor-associated rare cells, the kit comprising the staining solution described above.

[0012] This application also provides a method for detecting active circulating tumor-associated rare cells. Attached Figure Description

[0013] Figure 1 shows the results of Example 5. In Figure 1, a) images of the lungs of mice injected with 4T1 cells via the tail vein at different times and subcutaneously, from left to right: normal control group, tail vein injection for 5 days, 10 days, 15 days, and subcutaneous injection for 15 days; b) histopathological staining images of various tissues from mice injected with 4T1 cells via the tail vein at different times and subcutaneously, from left to right: normal control group, tail vein injection for 5 days, 10 days, 15 days, and subcutaneous injection, with green arrows indicating tumor cells in the lung tissue; c) the number of labeled tumor cells in the blood of mice injected with 4T1 cells via the tail vein at different times and subcutaneously; d) the number of labeled tumor cells in the blood of mice after tail vein injection of different tumor cells.

[0014] Figure 2 shows the fluorescence imaging results of labeled active circulating tumor-associated rare cells in clinical samples using the staining solution and CK of this application, where CK is a tumor-specific marker and CD45 is used to label leukocytes;

[0015] Figure 3 shows the results of identifying the number of active circulating tumor-associated rare cells in the peripheral blood of healthy individuals and patients with different cancers using the staining solution described in this application. Detailed Implementation

[0016] This application provides the application of graphene-based tumor cell nuclear-targeting fluorescent nanoprobes in the preparation of products for detecting active circulating tumor-associated rare cells (CTAs). In some embodiments of this application, the product is a reagent or kit. In some embodiments of this application, the active CTAs include circulating tumor cells and / or circulating tumor cell-associated leukocytes (CTCs). This application is the first to propose that GTTN can accurately identify and label active CTAs in peripheral blood, and can detect the presence of active CTAs in peripheral blood with high sensitivity, high specificity, and high accuracy.

[0017] In this application, the preparation method of the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe (GTTN) is described in the authorized patents published by CN 111467510A and application number 202010278513.4. Specifically, the preparation method of GTTN includes the following steps: 0.5 g of pyrene powder is added to 25 mL of nitric acid (concentration 65-68 wt%) at 80 °C and reacted for 24 h; after the reaction is completed, the mixture is cooled, washed with 150 mL of deionized water, and filtered through a 0.22 μm filter membrane; the filtrate is added to 50 mL of Na2SO3 aqueous solution (concentration 0.5 mol / L) and stirred for 0.5 h, then transferred to a 150 mL ceramic autoclave and heated at 130 °C for 12 h; after cooling to room temperature, the obtained material is transferred to an autoclave lined with polytetrafluoroethylene and placed in a vacuum drying chamber at 200 °C for 12 h; after the reaction is completed, the mixture is cooled, and the obtained system is filtered. The filtrate contains GTTN, and this filtrate is referred to as GTTN stock solution.

[0018] GTTN is an amphiphilic fluorescent probe with a graphene-like single-crystal structure that can specifically target tumor cells. However, because tumor cells and active circulating tumor-associated rare cells (TARFCs) differ significantly in terms of both their environment and heterogeneity, a probe that specifically targets tumor cells is not necessarily applicable to the specific targeting of TRFCs in peripheral blood.

[0019] This application provides an active circulating tumor-associated rare cell staining solution, which includes a graphene-based tumor cell nuclear-targeting fluorescent nanoprobe and a diluent.

[0020] In some embodiments of this application, the diluent is composed of NaCl, KCl, Na₂HPO₄, KH₂PO₄, and distilled water. In some embodiments, the amounts of each ingredient are 8g NaCl, 0.2g KCl, 1.44g Na₂HPO₄, 0.24g KH₂PO₄, and 800mL distilled water. This application does not specifically limit the source of each ingredient in the diluent; commercially available products commonly used in the art can be used. In some embodiments of this application, the concentration of GTTN in the staining solution is 200μg / mL. In this application, if the peripheral blood contains active circulating tumor-associated rare cells (TARFCs), then GTTN can stain these TRFCs. The concentration design in this application ensures that the staining will not be washed away within a short period after staining.

[0021] This application also provides the use of the above-mentioned staining solution in the preparation of products for detecting active circulating tumor-associated rare cells. In some embodiments of this application, the product is a kit.

[0022] This application also provides a kit for detecting active circulating tumor-associated rare cells, the kit comprising the staining solution described above.

[0023] This application also provides a method for detecting active circulating tumor-associated rare cells, comprising the following steps: obtaining peripheral blood from the individual to be tested, dividing it into control peripheral blood and test peripheral blood; lysing the erythrocytes in the control peripheral blood and test peripheral blood, centrifuging and discarding the supernatant to obtain control cell precipitate and test cell precipitate; adding the above-mentioned staining solution to the test cell precipitate for incubation, centrifuging and discarding the supernatant, washing and centrifuging and discarding the supernatant, and resuspending to obtain a stained cell suspension; resuspending the control cell precipitate to obtain an unstained cell suspension; using flow cytometry at an excitation wavelength of 405 nm to detect the fluorescence values ​​of the stained cell suspension and the unstained cell suspension respectively; if the stained cell suspension contains cells with fluorescence values ​​higher than those of the unstained cell suspension, then the cells are active circulating tumor-associated rare cells; if the stained cell suspension does not contain cells with fluorescence values ​​higher than those of the unstained cell suspension, it indicates that there are no active circulating tumor-associated rare cells in the peripheral blood.

[0024] In some embodiments of this application, erythrocyte lysis is performed using erythrocyte lysis buffer. This application does not specifically limit the source of the erythrocyte lysis buffer; commercially available products commonly used in the art are acceptable. In some embodiments of this application, the volume ratio of staining solution to peripheral blood is 1:5, 1:6, 1:7, 1:8, or 1:9. In some embodiments of this application, the incubation temperature is 37°C, and the incubation time is 30 min. In some embodiments of this application, the centrifugation conditions are 1500 r / min for 3 min. In some embodiments of this application, the solution used for washing and resuspension is a diluent, preferably composed of NaCl, KCl, Na₂HPO₄, KH₂PO₄, and distilled water, wherein the preferred amounts of each raw material are 8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄, and 800 mL distilled water.

[0025] The active circulating tumor-associated rare cell (CMA) staining solution, kit, or method provided in this application can sensitively and accurately stain active CMAs in peripheral blood, thereby enabling rapid detection of active CMAs in peripheral blood. Compared with existing methods for detecting active CMAs, the operation process is simpler and faster when using the staining solution or kit provided in this application, which is helpful for early cancer screening and clinical auxiliary diagnosis.

[0026] The technical solutions provided in this application will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of this application.

[0027] Unless otherwise specified, the following embodiments are all conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] The diluent used in the following examples consisted of 8g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4 and 800mL distilled water.

[0030] Example 1

[0031] A staining solution for active circulating tumor-associated rare cells, comprising a diluent and GTTN, wherein the concentration of GTTN in the staining solution is 200 μg / mL.

[0032] Example 2

[0033] A kit for detecting active circulating tumor-associated rare cells, comprising the staining solution, red blood cell lysis buffer, and diluent of Example 1.

[0034] Example 3

[0035] A method for detecting active circulating tumor-associated rare cells comprises the following steps:

[0036] (1) 10 mL of human peripheral blood was drawn and placed in two anticoagulant tubes, with 5 mL of peripheral blood in each anticoagulant tube. These tubes were designated as control peripheral blood and test peripheral blood, respectively. The tubes were placed in a 4°C refrigerator and processed within 6 hours.

[0037] (2) Add red blood cell lysis buffer to the control peripheral blood and test peripheral blood samples at a volume ratio of 1:10, incubate at 4℃ for 10 min, centrifuge at 1500 r / min for 3 min and discard the supernatant; add 5 mL PBS buffer to wash, centrifuge at 1500 r / min for 3 min and discard the supernatant to obtain control cell pellet and test cell pellet.

[0038] (3) Add 1 mL of the staining solution from Example 1 to the cell pellet to be tested, incubate at 37°C for 30 min, centrifuge at 1500 r / min for 3 min and discard the supernatant, wash with 5 mL of PBS buffer, centrifuge at 1500 r / min for 3 min and discard the supernatant, resuspend in 1 mL of PBS buffer to obtain the stained cell suspension. Add 1 mL of PBS buffer to the control cell pellet and resuspend to obtain the unstained cell suspension.

[0039] (4) The fluorescence values ​​of stained cell suspension and unstained cell suspension were detected by flow cytometry at an excitation wavelength of 405 nm. If the stained cell suspension contained cells with fluorescence values ​​higher than those in the unstained cell suspension, the cells were active circulating tumor-associated rare cells. If the stained cell suspension did not contain cells with fluorescence values ​​higher than those in the unstained cell suspension, it indicated that there were no active circulating tumor-associated rare cells in the peripheral blood.

[0040] Example 4

[0041] A method for detecting active circulating tumor-associated rare cells comprises the following steps:

[0042] (1) 20 mL of human peripheral blood was drawn and placed in two anticoagulant tubes, with 10 mL of peripheral blood in each tube. These tubes were designated as control peripheral blood and test peripheral blood, respectively. The tubes were placed in a 4°C refrigerator and processed within 6 hours.

[0043] (2) Add red blood cell lysis buffer to the control peripheral blood and the test peripheral blood at a volume ratio of 1:10, incubate at 4℃ for 10 min, centrifuge at 1500 r / min for 3 min and discard the supernatant; add 5 mL PBS buffer to wash, centrifuge at 1500 r / min for 3 min and discard the supernatant to obtain control cell precipitate and test cell precipitate.

[0044] (3) Add 1 mL of the staining solution from Example 1 to the cell pellet to be tested, incubate at 37°C for 30 min, centrifuge at 1500 r / min for 3 min and discard the supernatant, wash with 5 mL of PBS buffer, centrifuge at 1500 r / min for 3 min and discard the supernatant, resuspend in 1 mL of PBS buffer to obtain the stained cell suspension. Add 1 mL of PBS buffer to the control cell pellet and resuspend to obtain the unstained cell suspension.

[0045] (4) The fluorescence values ​​of stained cell suspension and unstained cell suspension were detected by flow cytometry at an excitation wavelength of 405 nm. If the stained cell suspension contained cells with fluorescence values ​​higher than those in the unstained cell suspension, the cells were active circulating tumor-associated rare cells. If the stained cell suspension did not contain cells with fluorescence values ​​higher than those in the unstained cell suspension, it indicated that there were no active circulating tumor-associated rare cells in the peripheral blood.

[0046] Example 5

[0047] Thirty mice were purchased and randomly divided into a normal control group (CON) and a tumor model group. The tumor model group was further randomly divided into five subgroups: breast cancer model (4T1), colon adenocarcinoma model (SW620), leukemia model (C1498), melanoma model (B16), and liver cancer model (HepG2), with five mice in each subgroup. The normal control group was injected with 200 μL of physiological saline, while the tumor model groups were injected via tail vein with 1 × 10⁻⁶ mice. 6 Tumor cells per 200 μL were used to model the breast cancer in 5 mice, except for those induced by tail vein injection. The other mice were induced by subcutaneous injection. Subcutaneous tumor modeling involved subcutaneous injection of 1 × 10⁻⁶ cells. 7 4 T1 cells per 200 μL.

[0048] After the above models were successfully constructed, lung tissues were taken from mice injected via the tail vein in the breast cancer model group at 5d, 10d, and 15d, as well as from mice injected subcutaneously at 15d and the normal control group for observation. The results are shown in Figure 1a. The heart, liver, spleen, lung, and kidney were obtained and subjected to HE pathological staining to observe the pathological results of the major organs of the mice. The results are shown in Figure 1b, which shows that cancer cell metastases can be clearly seen on the surface of the lung tissue of mice injected with tumor cells.

[0049] The method of Example 3 was used to detect whether there were active circulating tumor-associated rare cells in the peripheral blood of mice in the normal control group and each tumor model group. The results are shown in Figures 1c and 1d, indicating that the staining solution provided in this application can accurately detect whether there are active circulating tumor-associated rare cells in blood samples.

[0050] Example 6

[0051] Fluorescence imaging of labeled active circulating tumor-associated rare cells in clinical samples was performed using the method of Example 3 and cytokeratin (CK, a tumor-specific marker), and the results are shown in Figure 2. The specific methods are as follows:

[0052] Take 5 mL of patient blood, add erythrocyte lysis buffer at a volume ratio of 1:10, incubate at 4°C for 10 min, centrifuge at 1500 r / min for 3 min and discard the supernatant, wash with PBS and centrifuge and discard the supernatant; add 1 mL of GTTN staining solution (200 mg / L) to the cell pellet, incubate at 37°C for 30 min, centrifuge at 1500 r / min for 3 min and discard the supernatant, wash with PBS and centrifuge and discard the supernatant; add 1 mL of fresh 4% paraformaldehyde to the cell pellet and fix for 10 min, centrifuge at 1500 r / min for 3 min and discard the supernatant, wash with PBS and centrifuge and discard the supernatant, resuspend in 1 mL of ultrapure water and count the cells, take 0.1 mL to prepare a cell smear.

[0053] Draw a closed hydrophobic zone around the tissue using a hydrophobic pen, permeate with 0.5% Triton X-100 for 10 min, wash cells twice with PBS for 5 min each time; block cells with 10% goat serum for 30 min, and aspirate excess liquid; add primary antibody (prepared with 3% BSA, CK-pan mouse monoclonal / polyclonal antibody; CD45 rabbit monoclonal / polyclonal antibody—dilution ratio according to the instructions), and incubate in a humidified chamber for 60 min; wash three times with PBST (1 mL Tween-20 added to 1 L of 1× dilution buffer), 5 min each time; add secondary antibody (prepared with 3% BSA, Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L); Alexa Fluor 555-labeled goat anti-mouse IgG (H+L)—dilution ratio according to the instructions), and incubate in a humidified chamber for 60 min; wash three times with PBST, 5 min each time; wash once with ultrapure water, and tilt to dry the smear. The prepared cell smears were observed under a laser confocal microscope (the excitation wavelengths for GTTN staining, CD45 (labeled leukocytes), and CK were 405 nm, 488 nm, and 555 nm, respectively).

[0054] Peripheral blood samples from healthy individuals, lung cancer patients, gastric cancer patients, colon cancer patients, liver cancer patients, and breast cancer patients were tested using the method described in Example 3. These samples were sourced from Shanghai Pudong Gongli Hospital, Shanghai Changzheng Hospital, and the Affiliated Hospital of Hainan Medical University, among others. The results, shown in Figure 3, demonstrate that the staining solution and detection method provided in this application can accurately detect active circulating tumor-associated rare cells, with a sensitivity exceeding 90% and a specificity exceeding 95%. Sensitivity = detected positive samples / all positive samples; specificity = detected negative samples / all negative samples.

[0055] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. Application of graphene-based tumor cell nucleus-targeting fluorescent nanoprobes in the preparation of products for detecting active circulating tumor-associated rare cells.

2. The application according to claim 1, characterized in that, The products include reagents or kits.

3. The application according to claim 1, characterized in that, The active circulating tumor-associated rare cells include circulating tumor cells and / or circulating tumor cell-associated leukocytes.

4. A staining solution for active circulating tumor-related rare cells, characterized by, The staining solution includes graphene-based tumor cell nuclear-targeting fluorescent nanoprobes and a diluent.

5. The dyeing solution according to claim 4, characterized in that, The concentration of the graphene-based tumor cell nuclear-targeting fluorescent nanoprobe in the staining solution was 200 μg / mL.

6. The use of the staining solution of claim 4 or 5 in the preparation of products for detecting active circulating tumor-associated rare cells.

7. Use according to claim 6, characterized in that, The products include reagents or kits.

8. A kit for detecting actively cycling tumor-associated rare cells, comprising, The kit includes the staining solution as described in claim 4 or 5.

9. A method for detecting active circulating tumor-associated rare cells, characterized in that, The process includes the following steps: obtaining peripheral blood from the individual to be tested, dividing it into control peripheral blood and test peripheral blood; lysing the red blood cells in the control peripheral blood and test peripheral blood, centrifuging and discarding the supernatant to obtain control cell precipitate and test cell precipitate; adding the staining solution described in claim 4 or 5 to the test cell precipitate for incubation, centrifuging and discarding the supernatant, washing and centrifuging again to discard the supernatant, and resuspending to obtain a stained cell suspension; The control cell pellet was resuspended to obtain an unstained cell suspension; the fluorescence values ​​of the stained and unstained cell suspensions were detected by flow cytometry at an excitation wavelength of 405 nm; if the stained cell suspension contained cells with fluorescence values ​​higher than those of the unstained cell suspension, the cells were considered to be active circulating tumor-associated rare cells. If no cells in the stained cell suspension have a fluorescence value higher than that in the unstained cell suspension, it indicates that there are no active circulating tumor-associated rare cells in the peripheral blood.

10. The method according to claim 9, characterized in that, The volume ratio of staining solution to peripheral blood tested is 1:5 to 10.

11. The method according to claim 9, characterized in that, The centrifugation conditions were 1500 r / min for 3 min.

12. The method according to claim 9, characterized in that, The solution used for washing and resuspension is a diluent.

13. The method according to claim 12, characterized in that, The diluent consists of NaCl, KCl, Na2HPO4, KH2PO4, and distilled water.

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