Active circulating tumor related rare cell staining solution and application thereof

Abstract

The invention provides an active circulating tumor related rare cell staining solution and application thereof, and belongs to the technical field of biomedicine. The invention firstly proposes that the graphene-based tumor cell nucleus targeting fluorescent nanoprobe can accurately identify and mark the active circulating tumor related rare cells in peripheral blood, and can detect whether the peripheral blood contains the active circulating tumor related rare cells or not with high sensitivity, high specificity and high accuracy. The staining solution provided by the invention comprises a graphene-based tumor cell nucleus targeting fluorescent nanoprobe and a diluent. The staining solution or the kit provided by the invention can sensitively and accurately stain active circulating tumor related rare cells in peripheral blood, and compared with the existing detection method, the staining solution or the kit provided by the invention has the advantages of simpler and quicker operation process, and contributes to early screening and clinical auxiliary diagnosis of cancers.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a staining solution for active circulating tumor-related rare cells and its application. Background Art

[0002] Circulating tumor cells (CTCs) refer to tumor cells that shed from the primary or metastatic foci of a tumor and enter the bloodstream, and are usually closely related to hematogenous metastasis and poor prognosis of the tumor. CTC detection has important applications in the auxiliary diagnosis of tumors, recurrence and metastasis monitoring, treatment effect evaluation, drug resistance monitoring, and early screening. However, most circulating tumor cells (CTCs) in the bloodstream will lose their biological activity due to natural apoptosis, dormancy, hypoxia, or factors such as blood shear stress, drugs, and immune system attacks. Only a very small number (<0.01%) of CTCs with special genetic and phenotypic advantages can maintain treatment resistance and have a high metastatic potential, thus causing distant metastasis. Therefore, single CTC detection may not comprehensively and accurately reveal the biological characteristics of a patient's tumor.

[0003] Active circulating tumor-related rare cells (LCTRCs) refer to rare cells with biological activity that enter the peripheral bloodstream and are closely related to tumor occurrence, progression, treatment resistance, metastasis, and tumor burden. LCTRCs include CTCs, tumor-related white blood cells (WBCs), such as tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs), tumor-related stromal cells, such as cancer-associated fibroblasts (CAFs), tumor-associated endothelial cells (CECs), and cell clusters (CTMs) composed of tumor-related cells. These cells play a key role in tumor biology, reflecting the complex interactions between tumor cells and their microenvironment, and providing valuable insights into metastatic mechanisms, treatment resistance, and disease progression. Compared with individual CTCs, LTCRCs can more comprehensively and accurately reflect the biological characteristics of a patient's tumor, including tumor invasiveness, recurrence and metastasis potential, treatment resistance, and adaptability. Therefore, LTCRCs have higher clinical value in revealing tumor biological behavior. However, the detection and application of LCTRCs, including CTCs, are greatly restricted, and due to reasons such as the scarcity of tumor cells in human peripheral blood, high heterogeneity, and the complexity of the metastasis process, the current detection of CTCs and tumor-related variant WBCs in peripheral blood has defects of low sensitivity, low specificity, and low accuracy. Therefore, there is an urgent need in the art to provide a method for highly sensitive, highly specific, and highly accurate detection of active circulating tumor-related rare cells in peripheral blood. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide the application of graphene-based tumor cell nucleus-targeted fluorescent nanoprobe (GTTN) in the preparation of products for detecting rare cells related to active circulating tumors.

[0005] Another objective of the present invention is to provide a staining solution for rare cells related to active circulating tumors, which can stain rare cells related to active circulating tumors with high specificity and high sensitivity.

[0006] A third objective of the present invention is to provide a kit for rare cells related to active circulating tumors, which can detect whether there are rare cells related to active circulating tumors in peripheral blood with high sensitivity, high specificity and high accuracy.

[0007] In order to achieve the above-mentioned invention objectives, the present invention provides the following technical solutions:

[0008] The present invention provides the application of graphene-based tumor cell nucleus-targeted fluorescent nanoprobe in the preparation of products for detecting rare cells related to active circulating tumors.

[0009] The present invention provides a staining solution for rare cells related to active circulating tumors, and the staining solution includes graphene-based tumor cell nucleus-targeted fluorescent nanoprobe and diluent.

[0010] Preferably, the concentration of graphene-based tumor cell nucleus-targeted fluorescent nanoprobe in the staining solution is 200 μg / mL.

[0011] The present invention also provides the application of the above-mentioned staining solution in the preparation of products for detecting rare cells related to active circulating tumors.

[0012] The present invention also provides a kit for detecting rare cells related to active circulating tumors, and the kit includes the above-mentioned staining solution.

[0013] Preferably, when using the kit for detection, the following steps are included: obtaining the peripheral blood of 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 above-mentioned staining solution to the test cell precipitate for incubation, centrifuging and discarding the supernatant, washing, centrifuging and discarding the supernatant, and resuspending to obtain a cell suspension after staining; resuspending the control cell precipitate to obtain an unstained cell suspension; using a flow cytometer to detect the fluorescence values of the cell suspension after staining and the unstained cell suspension respectively at an excitation wavelength of 405 nm; if there are cells in the cell suspension after staining with fluorescence values higher than those of the unstained cell suspension, then these cells are rare cells related to active circulating tumors; if there are no cells in the cell suspension after staining with fluorescence values higher than those of the unstained cell suspension, it indicates that there are no rare cells related to active circulating tumors in the peripheral blood.

[0014] Preferably, the volume ratio of the staining solution to the peripheral blood for detection is 1:5 to 10.

[0015] Preferably, the centrifugation conditions are centrifugation at 1500 r / min for 3 min.

[0016] Preferably, the solution used for washing and resuspending is a diluent.

[0017] Advantages of the present invention:

[0018] The present invention firstly proposes that GTTN can accurately identify and label rare active circulating tumor-related cells in peripheral blood, and can detect with high sensitivity, high specificity and high accuracy whether there are rare active circulating tumor-related cells in peripheral blood.

[0019] The staining solution or kit for rare active circulating tumor-related cells provided by the present invention can sensitively and accurately stain rare active circulating tumor-related cells in peripheral blood, and then be used for rapid detection of rare active circulating tumor-related cells in peripheral blood. Compared with the existing detection methods for rare active circulating tumor-related cells, when using the staining solution or kit provided by the present invention for detecting rare active circulating tumor-related cells, the operation process is simpler and faster, which is helpful for early cancer screening and clinical auxiliary diagnosis. Description of the Drawings

[0020] Figure 1 The results of Example 5, where a is the lung pictures of mice with 4T1 cells injected into the tail vein at different times and 4T1 cells injected subcutaneously. From left to right are the results of the normal control group, tail vein injection for 5 d, 10 d, 15 d and subcutaneous injection for 15 d; b is the pathological staining pictures of various tissues of mice with 4T1 cells injected into the tail vein at different times and 4T1 cells injected subcutaneously. From left to right are the results of the normal control group, tail vein injection for 5 d, 10 d, 15 d and subcutaneous injection. The green arrows in the lung tissue are tumor cells; c is the number of tumor cells labeled in the blood of mice with 4T1 cells injected into the tail vein at different times and 4T1 cells injected subcutaneously; d is the number of tumor cells labeled in the blood after different tumor cells are injected into the tail vein of mice;

[0021] Figure 2 The results of fluorescence imaging of rare active circulating tumor-related cells labeled in clinical samples using the staining solution of the present invention and CK. CK is a tumor-specific marker, and CD45 labels white blood cells;

[0022] Figure 3 The results of using the staining solution of the present invention to identify the number of rare active circulating tumor-related cells in the peripheral blood of healthy people and different cancer patients. Detailed Embodiments

[0023] The present invention provides an application of a graphene-based tumor cell nucleus-targeting fluorescent nanoprobe in the preparation of a product for detecting rare cells related to active circulating tumors. In the present invention, the product preferably includes a reagent and a kit. In the present invention, the rare cells related to active circulating tumors include circulating tumor cells and circulating tumor cell-related white blood cells.

[0024] In the present invention, for the preparation method of the graphene-based tumor cell nucleus-targeting fluorescent nanoprobe (GTTN), reference can be made to the authorized patent with the publication number CN 111467510A and the application number 202010278513.4. Specifically, the preparation method of the GTTN includes the following steps: adding pyrene powder (0.5 g) to nitric acid (25 mL, with a concentration of 65 - 68 wt%) at 80 °C and reacting for 24 h; after the reaction is completed, cooling, washing the obtained system with 150 mL of deionized water, and filtering with a 0.22 μm filter membrane; adding the obtained filtrate to an aqueous solution of Na 2 SO 3 (50 mL, with a concentration of 0.5 mol / L), stirring for 0.5 h, then transferring to a 150 mL ceramic autoclave and heating at 130 °C for 12 h; then cooling to room temperature, transferring the obtained material to an autoclave with a polytetrafluoroethylene lining, and reacting in a 200 °C vacuum drying chamber for 12 h; after the reaction is completed, cooling, filtering the obtained system, and the obtained filtrate contains GTTN, and this filtrate is denoted as the GTTN stock solution.

[0025] GTTN is an amphiphilic fluorescent probe with a graphene-like single crystal structure and can specifically target tumor cells. However, since there are significant differences between tumor cells and rare cells related to active circulating tumors in terms of both the existing environment and heterogeneity, etc., a probe that specifically targets tumor cells does not necessarily mean it is also suitable for specifically targeting rare cells related to active circulating tumors in peripheral blood.

[0026] The present invention provides a staining solution for rare cells related to active circulating tumors, and the staining solution includes a graphene-based tumor cell nucleus-targeting fluorescent nanoprobe and a diluent.

[0027] In the present invention, the diluent preferably consists of NaCl, KCl, Na 2 HPO 4 , KH 2 PO 4 and distilled water, and the dosage of each raw material is preferably 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na 2 HPO 4 , 0.24 g of KH 2 PO 4And 800 mL of distilled water. The present invention has no special limitation on the specific sources of the raw materials in the diluent, and conventional commercially available products in the art can be used. In the staining solution of the present invention, the concentration of GTTN is preferably 200 μg / mL. In the present invention, if the peripheral blood contains active circulating tumor-related rare cells, then GTTN can enter the active circulating tumor-related rare cells for staining, and the concentration design of the present invention will ensure that it will not be washed off in a short time after staining.

[0028] The present invention also provides the application of the above staining solution in the preparation of products for detecting active circulating tumor-related rare cells. In the present invention, the type of the product preferably includes a kit.

[0029] The present invention also provides a kit for detecting active circulating tumor-related rare cells, and the kit includes the above staining solution.

[0030] In the present invention, when using the kit for detection, the following steps are included: obtaining the peripheral blood of 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 to discard the supernatant to obtain control cell precipitate and test cell precipitate; adding the staining solution to the test cell precipitate for incubation, centrifuging to discard the supernatant, washing and centrifuging to discard the supernatant, and resuspending to obtain a stained cell suspension; resuspending the control cell precipitate to obtain an unstained cell suspension; using a flow cytometer to detect the fluorescence values of the stained cell suspension and the unstained cell suspension at an excitation wavelength of 405 nm respectively; if there are cells in the stained cell suspension with fluorescence values higher than those of the unstained cell suspension, then the cells are active circulating tumor-related rare cells; if there are no cells in the stained cell suspension with fluorescence values higher than those of the unstained cell suspension, it indicates that there are no active circulating tumor-related rare cells in the peripheral blood.

[0031] In the present invention, preferably, red blood cell lysate is used to lyse red blood cells. The present invention has no special limitation on the specific sources of the red blood cell lysate, and conventional commercially available products in the art can be used. In the present invention, the volume ratio of the staining solution to the test peripheral blood is preferably 1:5 to 10, more preferably 1:6 to 9. In the present invention, the temperature of the incubation is preferably 37 °C, and the time of the incubation is preferably 30 min. In the present invention, the centrifugation conditions are preferably centrifugation at 1500 r / min for 3 min; the solution used for washing and resuspending is preferably a diluent, and the diluent preferably consists of NaCl, KCl, Na 2 HPO 4 、KH 2 PO 4 and distilled water, and the dosage of each raw material is preferably 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na 2 HPO 4, 0.24 g KH 2 PO 4 and 800 mL of distilled water.

[0032] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] In the following embodiments, unless otherwise specified, all are conventional methods.

[0034] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0035] The dilution solution used in the following embodiments is composed of 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na 2 HPO 4 , 0.24 g KH 2 PO 4 and 800 mL of distilled water.

[0036] Example 1

[0037] An active circulating tumor-associated rare cell staining solution, which consists of a dilution solution and GTTN, and the concentration of GTTN in the staining solution is 200 μg / mL.

[0038] Example 2

[0039] A kit for detecting active circulating tumor-associated rare cells, which includes the staining solution, red blood cell lysate, and dilution solution of Example 1.

[0040] Example 3

[0041] A method for detecting active circulating tumor-associated rare cells, which consists of the following steps:

[0042] (1) Draw 10 mL of human peripheral blood and place it evenly in two anticoagulant tubes, with 5 mL of peripheral blood in each anticoagulant tube, which are respectively recorded as control peripheral blood and test peripheral blood, place them in a 4°C refrigerator, and process them within 6 hours.

[0043] (2) Add red blood cell lysate to the blood samples of the control peripheral blood and the test peripheral blood 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; add 5 mL of PBS buffer solution to wash and then centrifuge at 1500 r / min for 3 min and discard the supernatant to obtain the control cell precipitate and the 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, discard the supernatant, add 5 mL of PBS buffer to wash, then centrifuge at 1500 r / min for 3 min and discard the supernatant. Add 1 mL of PBS buffer to resuspend and obtain the stained cell suspension. Add 1 mL of PBS buffer to resuspend the control cell pellet to obtain the unstained cell suspension.

[0045] (4) Use a flow cytometer to detect the fluorescence values of the stained cell suspension and the unstained cell suspension at an excitation wavelength of 405 nm. If there are cells in the stained cell suspension with fluorescence values higher than those of the unstained cell suspension, then these cells are active circulating tumor - associated rare cells. If there are no cells in the stained cell suspension 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.

[0046] Example 4

[0047] A method for detecting active circulating tumor - associated rare cells, which consists of the following steps:

[0048] (1) Draw 20 mL of human peripheral blood and evenly place it in two anticoagulation tubes, with 10 mL of peripheral blood in each anticoagulation tube, which are respectively marked as control peripheral blood and test peripheral blood, place them in a 4 °C refrigerator and process them within 6 h.

[0049] (2) Add erythrocyte lysate to the blood samples of the control peripheral blood and the test peripheral blood 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. Add 5 mL of PBS buffer to wash, then centrifuge at 1500 r / min for 3 min and discard the supernatant to obtain the control cell pellet and the cell pellet to be tested.

[0050] (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, discard the supernatant, add 5 mL of PBS buffer to wash, then centrifuge at 1500 r / min for 3 min and discard the supernatant. Add 1 mL of PBS buffer to resuspend and obtain the stained cell suspension. Add 1 mL of PBS buffer to resuspend the control cell pellet to obtain the unstained cell suspension.

[0051] (4) Use a flow cytometer to detect the fluorescence values of the stained cell suspension and the unstained cell suspension at an excitation wavelength of 405 nm. If there are cells in the stained cell suspension with fluorescence values higher than those of the unstained cell suspension, then these cells are active circulating tumor - associated rare cells. If there are no cells in the stained cell suspension 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.

[0052] Example 5

[0053] 30 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 a breast cancer model group (4T1), a colon adenocarcinoma model group (SW620), a leukemia model group (C1498), a melanoma model group (B16), and a liver cancer model group (HepG2), with 5 mice in each group. Mice in the normal control group were injected with 200 μL of normal saline, and mice in the tumor model group were injected with 1×10 6 cells / 200 μL of the corresponding tumor cells via the tail vein. Among the 5 mice in the breast cancer model group, except for those injected via the tail vein for modeling, the remaining mice were subcutaneously injected for modeling. The subcutaneous injection for modeling (subcutaneous tumor) was: subcutaneous injection of 1×10 7 cells / 200 μL of 4T1 cells.

[0054] After the successful construction of each of the above models, the lung tissues of the mice in the breast cancer model group that were injected via the tail vein on the 5th, 10th, and 15th days and those subcutaneously injected on the 15th day, as well as the lung tissues of the normal control group, were observed. The results are as shown in Figure 1 a, and their hearts, livers, spleens, lungs, and kidneys were obtained for HE pathological staining to observe the pathological results of the main organs of the mice. The results are as shown in Figure 1 b, which shows that cancer cell metastasis foci can be clearly seen on the surface of the lung tissues of the mice injected with tumor cells.

[0055] The method of Example 3 was used to detect whether there were active circulating tumor-related rare cells in the peripheral blood of mice in the normal control group and each tumor model group. The results are as shown in Figure 1 c and d, indicating that the staining solution provided by the present invention can accurately detect whether the blood sample contains active circulating tumor-related rare cells.

[0056] Example 6

[0057] The method of Example 3 and cytokeratin (CK, a tumor-specific marker) were respectively used for fluorescence imaging of the labeled active circulating tumor-related rare cells in clinical samples. The results are as shown in Figure 2 .

[0058] The specific method is as follows:

[0059] Take 5 mL of the patient's blood, add erythrocyte lysate at a volume ratio of 1:10, incubate at 4 °C for 10 min, centrifuge at 1500 r / min for 3 min, discard the supernatant, wash with PBS and then centrifuge to discard the supernatant; add 1 ml of staining solution GTTN (200 mg / L) to the cell pellet, incubate at 37 °C for 30 min, centrifuge at 1500 r / min for 3 min, discard the supernatant, wash with PBS and then centrifuge to discard the supernatant; add 1 mL of fresh 4% paraformaldehyde to the cell pellet to fix for 10 min, centrifuge at 1500 r / min for 3 min, discard the supernatant, wash with PBS and then centrifuge to discard the supernatant, resuspend with 1 mL of ultrapure water and count, take 0.1 mL to make a cell smear.

[0060] Draw a closed hydrophobic circle around the tissue with a hydrophobic pen, permeabilize with 0.5% Triton X-100 for 10 min, wash the cells twice with PBS for 5 min each time; block the cells with 10% goat serum for 30 min, aspirate the excess liquid; add the primary antibody (prepared with 3% BSA, CK-pan mouse monoclonal / polyclonal antibody; CD45 rabbit monoclonal / polyclonal antibody - the dilution ratio refers to the instruction manual), incubate in a humid box for 60 min; wash with PBST (add 1 mL of Tween-20 to 1 L of 1× dilution solution) 3 times for 5 min each time; add the 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) - the dilution ratio refers to the instruction manual), incubate in a humid box for 60 min; wash with PBST 3 times for 5 min each time; wash once with ultrapure water, tilt to dry the smear. Place the processed cell smear under a laser confocal microscope for observation (the excitation wavelengths of GTTN staining solution, CD45 (labeling white blood cells) and CK are 405 nm, 488 nm and 555 nm respectively).

[0061] The methods of Example 3 were used to detect the peripheral blood samples of healthy people, lung cancer patients, gastric cancer patients, colon cancer patients, liver cancer patients and breast cancer patients respectively. The above samples were from Shanghai Pudong Gongli Hospital, Shanghai Changzheng Hospital, Affiliated Hospital of Hainan Medical University, etc. The results are as Figure 3 shown, indicating that the staining solution and detection method provided by the present invention can accurately detect active circulating tumor-related rare cells, and the detection sensitivity reaches more than 90%, and the specificity reaches more than 95%. Among them, sensitivity = detected positive samples / all positive samples; specificity = detected negative samples / all negative samples.

[0062] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

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

2. A staining solution for active circulating tumor-associated rare cells, characterized in that: The staining solution comprises a graphene-based tumor cell nucleus-targeted fluorescent nanoprobe and a diluent.

3. The dyeing solution according to claim 2, characterized in that The concentration of the graphene-based tumor cell nucleus-targeted fluorescent nanoprobe in the staining solution is 200 μg / mL.

4. Use of the staining solution according to claim 2 or 3 in the preparation of a product for detecting active circulating tumor-associated rare cells.

5. A kit for detecting active circulating tumor-associated rare cells, characterized in that: The kit comprises the staining solution according to claim 2 or 3.

6. The kit according to claim 5, characterized in that When the kit is used for detection, the following steps are included: 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 the test peripheral blood, centrifuging and discarding the supernatant to obtain control cell sediment and test cell sediment; adding the staining solution to the test cell sediment for incubation, centrifuging and discarding the supernatant, washing and centrifuging and discarding the supernatant, and resuspending to obtain a stained cell suspension; The control cell pellet is resuspended to obtain an unstained cell suspension; the fluorescence values ​​of the stained cell suspension and the unstained cell suspension are respectively detected by flow cytometry at an excitation wavelength of 405 nm; if the stained cell suspension contains cells with a fluorescence value higher than that of the unstained cell suspension, the cells are active circulating tumor-associated rare cells; If there are no cells in the stained cell suspension whose fluorescence value is higher than that of the unstained cell suspension, it indicates that there are no active circulating tumor-associated rare cells in the peripheral blood.

7. The kit according to claim 6, characterized in that The volume ratio of staining solution to peripheral blood to be tested is 1:5-10.

8. The kit according to claim 6, characterized in that The centrifugal condition is 1500r / min for 3min.

9. The kit according to claim 6, characterized in that The solution used for washing and resuspension is the diluent.

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