阅读说明：本技术 一种提高血液循环肿瘤细胞俘获的方法 (Method for improving blood circulation tumor cell capture ) 是由 鲍伟胜 鲍岳洋 王春雷 于 2021-09-24 设计创作，主要内容包括：本发明公开了一种提高血液循环肿瘤细胞俘获的方法,包括如下步骤：S1、将待检测血液通过膜芯片的过滤,将CTC留在膜上；S2、加入荧光标记的rVAR2/anti-CD45荧光抗体/DAPI到膜上；S3、荧光显微镜CTC计数。本发明应用rVAR2荧光染色俘获后的CTC,俘获/识别CTC不依赖于CTC表面表达单一蛋白,尽可能的把上皮细胞来源的CTC和间质细胞性质的CTC一起俘获；这样提高了俘获的效率和准确性,而且俘获方法经济、简便。(The invention discloses a method for improving blood circulation tumor cell capture, which comprises the following steps: s1, filtering the blood to be detected through a membrane chip, and keeping CTC on the membrane; s2, adding fluorescent-labeled rVAR2/anti-CD45 fluorescent antibody/DAPI to the membrane; s3, fluorescence microscopy CTC count. According to the invention, the captured CTC is stained by rVAR2 fluorescence, the CTC is captured/identified without depending on the single protein expressed on the surface of the CTC, and the CTC from epithelial cells and the CTC with the characteristics of mesenchymal cells are captured together as far as possible; thus, the efficiency and accuracy of the capture are improved, and the capture method is economical and simple.)

1. A method of increasing blood circulation tumor cell capture, comprising the steps of:

s1, filtering the blood to be detected through a membrane chip, and keeping CTC on the membrane;

s2, adding fluorescent-labeled rVAR2/anti-CD45 fluorescent antibody/DAPI to the membrane;

s3, fluorescence microscopy CTC count.

2. The method for improving blood circulation tumor cell capture according to claim 1, wherein the CTCs in the blood are captured and enriched by wrapping rVAR2 on the surface of magnetic beads to make the rVAR2 magnetic beads, and then identifying the CTCs with Pan-CK or EpCAM fluorescent antibodies.

3. The method of claim 1, wherein the capture rate of cells in S1 is > 85%.

Technical Field

The invention relates to a method for improving blood circulation tumor cell capture.

Background

CTC refers to tumor cells that enter the blood circulation. CTC assays are an assay for the determination of mobile cancer cells in the blood. The ultra-early stage micro-cancer which cannot be found by the image examination can be identified by the CTC examination, the tumor can be found earlier than the traditional diagnosis method in clinic, and the incidence of the ultra-early stage primary cancer and the ultra-early stage cancer metastasis or recurrence can be known. CTC of patients with tumors such as lung cancer, rectal cancer, breast cancer, prostate cancer, pancreatic cancer and the like is an important biomarker for observing the prognosis treatment effect of the tumors in blood. The greater the number of CTCs in the blood of a patient with a tumor, the poorer the prognosis of the patient.

Currently, there are two broad categories of CTC capture technologies based on principles-physical and chemical methods. The chemical method is comparatively represented byThe U.S. FDA's only approved CTC capture technology. The key principle of the technology is to capture tumor cells by using antigen-antibody binding. Antigen and antibody based chemistry has some disadvantages: a) binding of antigen to the antibody readily causes apoptosis of CTCs, resulting in a reduction in the number of captured CTCs. This has been confirmed by a number of studies; b) EMT cells-cells transformed from epithelial to mesenchymal cells that do not express traditional epithelial cell surface antigen markers and are therefore not recognized and captured by antibodies-cannot be captured. The most malignant of these cells is the intension of tumor metastasis.

The physical method depends on the morphological size and density of the cells. The difference in size according to morphology is the main method. The method is simple and convenient, does not easily cause apoptosis of CTC, and can capture EMT cells. After CTC capture based on tumor cell size technology, captured tumor cells need to be identified and counted, and enriched tumor cells are identified with fluorescent antibodies. CTC was fluorescently identified as CK +, CD45-, and DAPI +, which are also current gold standards for fluorescence diagnosis of CTC. But CK also shows down-regulation, even disappearance, during growth. Also, cancer stem cells lack the expression of CK. Thus, detection with CK when capturing CTCs will lose certain specific CTCs, resulting in a reduction in the number of cells detected.

For EMT cells, fluorescent antibodies to vimentin may be used in the fluorescent antibody cocktail. However, the multiple antibodies should increase the cost and complexity of the assay on the one hand, and increase non-specifically bound leukocytes on the other hand. Also, EMT cells are not present in the blood of all tumor patients.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defect of inaccurate detection number of CTC in blood in the prior art, provide a method for capturing CTC by fluorescence with rVAR2 by utilizing the characteristics that ofCS (carcinoembryonic chondroitin sulfate) specificity is expressed on the surface of almost all tumor cells and VAR2 specificity is combined with ofCS, and improve the detection number and sensitivity of CTC.

In order to solve the technical problems, the invention provides the following technical scheme:

a method of increasing blood circulation tumor cell capture, comprising the steps of:

s1, filtering the blood to be detected through a membrane chip, and keeping CTC on the membrane;

s2, adding fluorescent-labeled rVAR2/anti-CD45 fluorescent antibody/DAPI to the membrane;

s3, fluorescence microscopy CTC count.

Further, rVAR2 is wrapped on the surface of the magnetic beads to make rVAR2 magnetic beads to capture and enrich CTC in blood, and then Pan-CK or EpCAM fluorescent antibody is used for recognizing the CTC.

Fluorescent labeling of recombinant VAR2 protein (rVAR2) commercially available fluorescent dyes for antibody and protein labeling mainly include CFTM series, AlexaSeries, DyLight series, Cy series, IR Dye series, etc. Most used is AlexaSeries and CFTM series. The excitation light is classified into blue, green, orange-red, far infrared, near infrared, and the like. The function of the fluorescently labeled rVAR2 was equivalent to that of a fluorescent antibody to ofCS, specifically binding to ofCS. Its fluorescent dye is chosen to distinguish it from the fluorescent dye of the CD45 antibody and the fluorescent dye of DAPI.

CD45 fluorescent antibody: its fluorescent dye is chosen to be distinguishable from the fluorescent dyes of rVAR2 and DAPI.

DAPI: DAPI is a fluorescent dye that binds strongly to DNA. Because DAPI is permeable to intact cell membranes, it can be used for staining of both living and fixed cells. It binds to the AT base pair of the minor groove of double-stranded DNA, and the fluorescence intensity of the DAPI molecule bound to the double-stranded DNA is increased by about 20 times, which is commonly observed with a fluorescence microscope. DAPI dyes are excited with light at the wavelength of ultraviolet light, whose light-emitting wavelength range covers blue to cyan.

The invention is based on a CTC capture method based on tumor cell morphology size, and the CTC is captured and retained on a membrane chip and identified by using rVAR2 fluorescence. There are also many technical solutions which do not use membrane chips as the carrier for the CTC entrapment, but the application of rVAR2 fluorescent staining is protected by the patent.

The rvAR2 can be coated on the surface of magnetic beads to make rVAR2 magnetic beads, the magnetic beads are used for capturing and enriching CTC in blood, and then Pan-CK or EpCAM fluorescent antibody is used for recognizing the CTC. This method is also the protection scope of this patent.

Currently, the capture of CTCs is mainly related to the capture of circulating tumor cells in the blood, and the capture of CTCs in other body fluids is the same as the capture of CTCs in the blood. Therefore, it is within the scope of this patent to identify CTCs wherever capture of CTCs outside the blood is applied to rVAR 2.

The effective effect is as follows:

according to the invention, the captured CTC is subjected to fluorescent staining by rVAR2, an antibody fluorescent staining method based on a CTC surface protein marker, such as CK and EpCAM, is replaced, the CTC is captured/identified without depending on a single protein expressed on the surface of the CTC, and the CTC from epithelial cells and the CTC with characteristics of mesenchymal cells are captured together as far as possible; this improves the efficiency and accuracy of capture. The use of rVAR2 fluorescence capture is more economical and simpler than the two CK + Vimentin fluorescence capture.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 is a microscopic image showing the retention of lung cancer epithelial cells A549 on a membrane chip in example;

FIG. 2 is a microscopic picture of rVAR2 fluorescent stained CTC (rVAR2+/CD45-/DAPI +);

FIG. 3 is a microscopic image of CTC captured by CK fluorescent antibody (Pan-CK +/CD45-/DAPI +);

fig. 4 shows the result of the ctc number distribution of the example.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Examples

A method of increasing blood circulation tumor cell capture, comprising the steps of:

s1, filtering the blood to be detected through a membrane chip, such as:FR1 detection system, leaving CTCs on the membrane;the FR1 detection system (Wanhe combination device No. 20200336) is an ultra-low pressure system, can capture tumor cells in human blood with high efficiency and high purity, and is used for early diagnosis of tumors and accurate treatment of tumors. Blood is passed through the membrane chip under low pressure. Since tumor cells are larger in volume than normal leukocytes, they cannot pass through the pores of the membrane and remain on the membrane. The cells remaining on the membrane are mainly leukocytes and a small number of tumor cells. The red blood cells were almost completely filtered out due to their volume. Gently cleaning the membrane, and removing some red blood cells to ensure that the surface of the membrane is cleaner;

s2, adding fluorescent-labeled rVAR2/anti-CD45 fluorescent antibody/DAPI to the membrane; specifically, a fluorescence-labeled rVAR2 protein, a CD45 fluorescent antibody and DAPI are added to the membrane; wherein DAPI can be added separately after incubation of fluorescent rVAR2 and CD45 fluorescent antibody for incubation;

s3, fluorescence microscopy CTC counts (VAR2+/CD45-/DAPI +). Specifically, the membrane is washed to remove excess fluorescent dye. The tumor cells were observed under a fluorescence microscope and counted.

Verification process

Calculation of CTC Capture Rate of Rarechtcap FR1 detection System- -detection of tumor cell retention rate (spiked-in)

1) Take 2.4X10⁶Perml lung carcinoma epithelial cell A549200ul, 100ul of 2mM Calcein-AM was added and stained for 15 minutes at room temperature. Centrifuge 200g for 5 minutes, pour off the supernatant, wash the cells twice with PBS to remove excess dye. The cells were then resuspended in PBS containing 2mM EDTA and 0.5% BSA.

2) Cells were diluted to the desired concentration of 10-100 cells/50 μ L by statistical sampling of serial dilutions. Subsequently, 50 μ L of the diluted cell suspension was transferred to a 96-well microtiter plate using a micropipette, and the number of cells in the transplant suspension was counted under a microscope. The count was repeated three times to determine the actual number of cells in suspension. After repeating these operations, the volume of the cell suspension used for adding 1ml of whole blood was adjusted to prepare a tumor cell-labeled sample.

3) A certain amount of tumor cells was added to 1ml of whole blood of a healthy person and mixed. Blood passing throughDetection by FR1 detection system (wan combination equipment No. 20200336); the size of the membrane pores in this experiment was 6x40uM, the total membrane area was 6x6mm, the vacuum pump pressure was 2.0 ± 0.1Kpa, and the flow rate was 500 ul/min.

4) Count of viable tumor cells on the membrane. Since Calcein-AM binds only to the nucleus of living cells, it emits green light under excitation of blue light of a fluorescence microscope, and is observed and counted.

5) The experimental results are as follows:

as shown in FIG. 1, the number of captured tumor cells was determined by observing and counting the number of green fluorescent cells under a microscope under a 10X objective. The capture rate of tumor cells A549 was 90. + -. 5%.

The high (> 85%) tumor cell capture efficiency of the FR1 detection system is critical to ensure its clinical utility.

Enumeration of CTCs after spiked in tumor cell capture by rVAR2

1) About 100 lung cancer epithelial cells a549 obtained by serial dilution were added to 1ml of whole blood of a healthy person and mixed well. Blood passing throughFR1 detection system (Anhui Hejie No. 20200336). The size of the membrane pores in this experiment was 6x40uM, the total membrane area was 6x6mm, the vacuum pump pressure was 2.0 ± 0.1Kpa, and the flow rate was 500 ul/min.

2) Two blood samples with A549 lung cancer epithelial cells trapped were prepared, and passed throughFR1 detection system, two membrane chips were obtained, with A549 cells trapped on the membrane. The counts of captured tumor cells were used for rVAR2 and Pan-CK fluorescent solutions, respectively.

3) The membrane is treated by cell fixation, membrane permeability increase, etc.

4) For rVAR2 fluorescent staining: mixing the following components in parts by weight: 100-1: 1000rVAR2-Alexa Fluor 568 and 1: the mixed solution of 100anti-CD 45-FITC antibody was incubated with the cells on the membrane for 1 hour in the dark. After washing, the mixture was washed with 1: incubating the 1000DAPI solution and the membrane for 10 minutes in a dark place, and cleaning; CTCs were observed and counted by fluorescence microscopy. The fluorescent-stained CTC (rVAR2+/CD45-/DAPI +) of rVAR2 is shown in FIG. 2.

5) For pan-CK fluorescent staining: mixing the following components in parts by weight: 100-500 anti-Pan-CK-efluor antibody and 1: the mixed solution of 100anti-CD 45-FITC antibody was incubated with the cells on the membrane for 1 hour in the dark. After washing, the mixture was washed with 1: incubating the 1000DAPI solution and the membrane for 10 minutes in a dark place, and cleaning; CTCs were observed and counted by fluorescence microscopy. CTC captured by CK fluorescent antibody (Pan-CK +/CD45-/DAPI +) is shown in FIG. 3.

6) Cellular images of CTCs were observed under a fluorescent microscope.

7) Results

a) The cells fluorescently stained with rVAR2 appeared as: rVAR2+/CD45-/DAPI +; the number is 89; CTC capture rate 89% (89/100);

b) Pan-CK fluorescently stained cells appeared as: Pan-CK +/CD45-/DAPI +; the number is 86; CTC capture rate 86% (86/100);

the expression of rVAR2+/CD45-/DAPI + from the rVAR2 fluorescent stained cells indicates that the stained cells are tumor cells CTC rather than leukocytes. Leukocytes were positive for CD45, and FITC conjugated with anti-CD45 antibody emitted green light; also, the number of leukocytes in blood is large. Although CS is also expressed on the surface of leukocytes, it cannot bind specifically to VAR 2. ofCS is abundantly expressed on solid tumor cells and binds uniquely to rVAR 2. ofCS is linked to the cell membrane by CSPG (proteoglycan of chondroitin sulfate). Therefore, the rVAR2+/CD45-/DAPI + cells must be tumor cells, namely the A549 lung cancer epithelial cells of the experiment.

The Pan-CK protein is expressed on the surface of most tumor cells, and CTC can be marked by using a fluorescent antibody of Pan-CK. Pan-CK +/CD45-/DAPI + is the fluorescent staining characteristic of A549 lung cancer epithelial cells. The number of the CTCs fluorescently labeled by Pan-CK is almost the same as that of the CTCs fluorescently labeled by rVAR2, and the fact that the CTCs fluorescently labeled by rVAR2 are A549 lung cancer epithelial cells is proved from another aspect.

Enumeration of CTC Capture in blood of Lung cancer patients by rVAR2

1. Three lung cancer patients were treated with peripheral blood three times, 4mL each time. 4ml of peripheral blood are respectively passed throughFR1 detection system (Wanhe equipment No. 20200336) for blood filtration, and the CTC in blood is retained on membrane chip;

2. according to the standard operation procedure of cellular fluorescence immunochemistry, the corresponding membrane chip is respectively stained and counted by rVAR2, Pan-CK and Vimentin;

3. fluorescent staining with rVAR 2: CTCs captured on membrane chips were fluorescently stained with rVAR2-Alexa Fluor 568/anti-CD45-FITC antibody/DAPI, and cells exhibiting the appearance of rVAR2+/CD45-/DAPI + fluorescence were designated as rVAR2 fluorescence-positive CTCs;

4. Pan-CK fluorescent staining: the CTC captured on the membrane chip is subjected to fluorescent staining by using anti-Pan-CK-effector antibody/anti-CD 45-FITC antibody/DAPI, and if cells show a Pan-CK +/CD45-/DAPI + fluorescent appearance, the cells are called the CTC with Pan-CK fluorescent staining positive;

5. vimentin fluorescent staining: CTCs captured on membrane chips were fluorescently stained with anti-Vimentin-efour antibody/anti-CD 45-FITC antibody/DAPI, and cells exhibiting the Vimentin +/CD45-/DAPI + fluorescent appearance were designated as Vimentin fluorescent-positive CTCs.

The results are shown in FIG. 4. As can be seen from fig. 4:

a) the number of CTCs contained in 4ml of peripheral blood was not the same in 3 patients with lung cancer; vimentin +/CD45-/DAPI + tumor cells were found in blood of patients 1 and 3, respectively, and Vimentin + suggests that this is EMT cells (epithelial to mesenchymal transformed cells) that lack the protein markers of epithelial cells such as EpCAM, Pan-CK, etc., but express Vimentin-Vimentin in the cytoplasm.

b) The number of fluorescently labeled CTCs of rVAR2 was significantly greater than those of Pan-Ck, even with the addition of EMT cells. Suggesting that the double negative CTC of Pan-CK-/CD45-/DAPI + still exists in the blood of the tumor patients. During the development of tumors, proteins on the cell surface are variously changed. CK. EpCAM may down-regulate its expression, or even disappear. This double negative cell fraction is converted to EMT cells, the remainder remaining in the cell state pan-CK-/CD45-/DAPI +. The Pan-CK-/CD45-/DAPI + CTC was not capable of being labeled with Pan-CK fluorescent antibody and Vimentin fluorescent antibody. CTCs that cannot be labeled in this way will affect the outcome of the CTC assay.

c) Therefore, the fluorescent marked captured CTC by the rVAR2 does not depend on the surface expression of the CTC to a single marker protein, and the CTC can capture both CK positive CTC and CK negative CTC, so that the CTC captured by the membrane chip can be more comprehensively identified, the detection sensitivity is improved, and the application of the technology in early screening of tumors and accurate treatment of tumors is facilitated.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.