CN113774026A

CN113774026A - Method for sorting circulating tumor cells by combining optomechanical force with particles

Info

Publication number: CN113774026A
Application number: CN202111059920.7A
Authority: CN
Inventors: 刘威; 陈贝
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2021-09-10
Filing date: 2021-09-10
Publication date: 2021-12-10

Abstract

The invention discloses a method for sorting circulating tumor cells by combining optomechanical force with particles, and belongs to the technical field of biological medicines. The method comprises the steps of incubating functionalized particles and samples containing CTCs and White Blood Cells (WBCs), and separating the CTCs and the white blood cells combined with the functionalized particles by light force to achieve the purpose of sorting circulating tumor cells; the functionalized particles are modified with specific molecules targeting CTCs. According to the invention, the CTCs are combined with the functionalized particles, so that the refractive index of the CTCs is obviously increased, the CTCs are obviously different from leukocytes, and the CTCs are separated from the leukocytes under the action of light. The invention can efficiently capture and identify the CTCs, and can rapidly identify the CTCs while capturing the CTCs; the invention utilizes the light force to separate CTCs from leukocytes in a non-contact and non-destructive manner, thereby ensuring the purity of sorted cells and the activity of the cells.

Description

Method for sorting circulating tumor cells by combining optomechanical force with particles

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a method for sorting circulating tumor cells by combining optomechanical force with particles.

Background

Circulating Tumor Cells (CTCs) are shed from solid tumor foci (primary foci, metastatic foci) and spread to other tissue sites of the human body with peripheral blood, causing the spread and proliferation of tumors. Thus, CTCs are considered seeds for tumor metastasis and are a significant cause of death in cancer patients. CTC is one of the important means of liquid biopsy, and dynamic detection, treatment effect evaluation and personalized treatment of tumors can be realized by detecting the quantity change of CTC in peripheral blood.

CTCs carrying genetic information of cancer patients are rare in blood (about 1-10 per ml of blood), and thus it is very difficult to efficiently isolate CTCs from blood. Most of the methods developed today for separating CTCs are based on the properties (size, density, dielectric constant, etc.) of CTCs, however, because CTCs and leukocytes have many overlapping properties in these respects, leukocytes are often collected while the CTCs are separated, which results in a decrease in the purity of sorted CTCs. In recent years, many nanoparticles and nanostructures have been developed for sorting CTCs, and despite good sorting efficiency, cell sorting purity on the nanoscale is still not desirable due to the problem of significant non-specific adsorption of leukocytes by nanoparticles and nanostructures. Therefore, the separation of CTCs with high efficiency and high purity is a problem which is urgently to be overcome at present.

The optical force is an effective means for identifying and separating trace substances, and can realize non-contact and non-destructive precise control on cells. Therefore, the optical force has great application potential in cell sorting. However, CTCs and leukocytes have similar optical constants, and thus, it remains a challenge to accurately separate and rapidly identify CTCs and leukocytes by optometry.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a method for sorting circulating tumor cells by combining optomechanical force with particles.

The purpose of the invention is realized by the following technical scheme:

a method for sorting circulating tumor cells by combining light with particles is characterized in that functionalized particles are incubated with samples containing CTCs and White Blood Cells (WBCs), and the CTCs and the white blood cells combined with the functionalized particles are separated by light, so that the aim of sorting the circulating tumor cells is fulfilled. The functionalized particles are modified with specific molecules targeting CTCs. The principle of the method is as follows: due to the fact that the functionalized particles are modified on the surfaces of the functionalized particles with specific molecules targeting CTCs, specific binding can be generated between the functionalized particles and CTCs in a sample. The optical constants (refractive indexes) of the CTCs combined with the functionalized particles are obviously increased, and the aim of obviously different from the refractive index of white blood cells is fulfilled. Under laser irradiation, the CTCs bound to the functionalized microparticles are subjected to a significantly greater optical force than the leukocytes, and the CTCs bound to the functionalized microparticles move toward a location remote from the laser and are thus separated from the leukocytes.

The specific molecules of the target CTCs are biomarkers capable of specifically recognizing and binding the surfaces of the CTCs, and the biomarkers on the surfaces of the CTCs comprise epithelial marker cytokeratin, epithelial cell adhesion molecules, tumor embryo antigens, human epidermal growth factor receptor 2, venous endothelial cell molecules, periderm proteins, sialylated Lewis oligosaccharide-X, acetaldehyde dehydrogenase 1, vimentin, urokinase receptor, heparanase, prostate specific membrane antigen, CD44, CK18, CD133, CD90, CD45 and CD 146. Preferably, the specific molecules targeting the CTCs are antibodies capable of specifically recognizing and binding biomarkers on the surfaces of the CTCs, and the antibodies comprise epithelial marker cytokeratin antibodies, epithelial cell adhesion molecule antibodies, tumor embryo antibodies, human epidermal growth factor receptor 2 antibodies, venous endothelial cell molecule antibodies, peridermin antibodies, sialylated Lewis oligosaccharide-X antibodies, acetaldehyde dehydrogenase 1 antibodies, vimentin antibodies, urokinase receptor antibodies, heparanase antibodies, prostate specific membrane antibodies, anti-CD44, anti-CK18, anti-CD133, anti-CD90, anti-CD45 and anti-CD 146.

Preferably, the fine particles have a refractive index of more than 1.4, and the particle size of the fine particles is 4-8 μm; the surface of the particle is modified with active groups, and the active groups comprise carboxyl, amino or sulfydryl and other groups. The particles can be fluorescent particles with light of different wave bands, and the aim of identifying CTCs can be fulfilled by combining the fluorescent particles with circulating tumor cells.

Preferably, the functionalized microparticles, i.e., the microparticles modified with specific molecules targeting CTCs, are prepared by a method comprising the following steps: firstly, activating active groups modified on the surfaces of the particles, then combining the particles with streptavidin (SA for short), and modifying specific molecules of biotinylated target CTCs on the basis of SA by the specific combination mode of SA and biotin.

Preferably, the sample containing CTCs and leukocytes is obtained by a method comprising the steps of: obtaining a blood sample of a cancer patient, and obtaining a lymphocyte layer by percoll cell separation fluid to obtain a sample containing CTCs and white blood cells. Further, the clinical cancer patients are advanced cancer patients with postoperative metastasis and high recurrence degree; the cancer comprises colon cancer, breast cancer, liver cancer, prostate cancer, gastric cancer, lung cancer and the like, and blood samples of cancer patients after operation or chemotherapy are preferably obtained.

Preferably, the method for sorting the circulating tumor cells by the optical force combined particles is performed in a microfluidic chip, and the schematic diagram of the principle is shown in fig. 1. Under the action of the fluid, the CTC combined with the functionalized particles and the white blood cells move along different paths, and the aim of separating the CTC from the white blood cells is fulfilled. More preferably, the method for sorting circulating tumor cells by using the light force combined particles comprises the following steps: (1) the functionalized microparticles are added to a sample containing CTCs and leukocytes, and incubated to allow the functionalized microparticles to specifically bind to the CTCs. (2) And (2) introducing the mixed sample obtained in the step (1) into a microfluidic chip, and opening laser to separate the CTCs combined with the functionalized particles. The incubation condition is preferably incubation at 37 ℃ for 2 h. Wherein the laser is generated by a laser, which couples the optical fiber to the microfluidic channel.

When the particles are fluorescent particles, the CTC separated by the method is combined with the fluorescent particles, and the leucocytes do not have fluorescence, so that the CTC can be rapidly identified. Based on the method, the invention also provides application of the method in separating and identifying the CTCs.

The invention also provides application of the functionalized particles used in the method in preparation of a kit for identifying, capturing or enriching CTCs.

A method for sorting target cells by combining optical force with particles is characterized in that functionalized particles and a sample containing target cells and interfering cells are incubated, and the target cells combined with the functionalized particles and the interfering cells are separated by the optical force, so that the aim of sorting the target cells is fulfilled. The functionalized particles are modified with specific molecules targeting target cells.

The invention has the advantages and beneficial effects that:

(1) the invention provides a way for efficiently capturing and identifying CTCs, which is expected to play an important role in the field of early warning and prevention of cancer metastasis.

(2) The fluorescent particles modified with specific molecules targeting CTCs can capture CTCs and rapidly identify CTCs.

(3) After the fluorescent particles modified with specific molecules of the target CTCs are combined with CTCs, the CTCs can be separated from leukocytes in a non-contact and non-destructive manner by using light force, so that the purity of sorted cells and the activity of the cells are ensured.

Drawings

FIG. 1 is a schematic diagram of light force combined with fluorescent particle separation and identification of CTCs.

FIG. 2 is an SEM image of fluorescent PS microparticles targeting MCF-7 cells.

FIG. 3 is a fluorescence image of fluorescent PS microparticles capturing MCF-7 expressing Epcam and Hela cells not expressing Epcam.

FIG. 4 is a graph of the displacement of PS-CTCs in microfluidic channels.

FIG. 5 is a graph showing the results of example 3. (a) A relationship between recovery efficiency and flow rate of PS-CTCs, (b) a relationship between collection purity and flow rate of PS-CTCs, (c) cellular activity of collected PS-CTCs, and (d) a fluorescence map of cellular activity of collected PS-CTCs.

Detailed Description

The features and advantages of the present invention will be further understood with reference to the following examples and accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.

Example 1: use of PS microparticle modified antibodies for capturing CTCs

200 μ L (10mg/mL) of orange fluorescent PS beads (available from Tianjin Bileshi, particle size 5 μm, refractive index about 1.6) with carboxyl groups on the surface were centrifuged at 10000rpm for 10 minutes. The PS beads were then rinsed thoroughly with MES solution and then soaked in 200 μ LEDC/NHS solution (4mg/mL EDC and 6mg/mL NHS in 0.1M MES solution) for 30 min. After centrifugation, mix with 50. mu.g/mL SA solution at 4 ℃ for 10 h. Followed by 3 washes with PBS and incubation with 20. mu.g/mL biotinylated anti-EpCAM antibody for 2h at room temperature. After centrifugal washing, the mixture was washed with 1mL of MCF-7 (10)⁵) Thin and thinCells were incubated for 2 h. Then fixed with 2.5% glutaraldehyde. Samples were dehydrated by 15%, 30%, 50%, 70%, 80%, 90%, 100% alcohol concentration, and dried by supercritical carbon dioxide. The scanning electron microscope observation result shows in fig. 2, the surface of the cancer cell is attached with a PS sphere, which indicates that the PS sphere modified antibody successfully targets the target cell.

The antibody-modified PS beads were used to capture different cells (MCF-7 cells and Hela cells), and then the cells were stained with DAPI, and the capture was observed under a fluorescent microscope, as shown in FIG. 3. The MCF-7 cell surface of the positive EpCAM expression has a plurality of microspheres, while the Hela cell surface of the negative EpCAM expression has no microspheres, which indicates that the modified PS spheres have the specific targeting capability. The MCF-7 cell surface showed orange fluorescence and the Hela cell surface did not show fluorescence, which indicated that the cells could be identified by fluorescence.

Example 2: microfluidic system sorting of PS-CTCs

The microfluidic sorting system consists of two main components, a microfluidic device and an optical sorting system (1064 nm laser coupled optical fiber). The microfluidic device is made of PDMS, is manufactured by using a standard soft lithography technology, and has a structure shown in figure 1; the overall height of the channel was 100 microns, the width was 150 microns, the sample inlet width was 75 microns, the other 2 PBS inlets were 50 microns, the width of the targeted cell outlet was 50 microns, and the width of the background cell outlet was 100 microns. The sample and PBS flows were delivered to the channel by glass syringes at a flow rate of 30. mu.L/h, the syringes being driven by syringe pumps. The sorting process was then observed under a microscope using an optical sorting system, turning on the laser, with the power of the laser-coupled fiber at 1W. The optical sorting system consists of a laser and an optical fiber, and 1064nm infrared laser is generated by a solid-state laser; light is coupled to the microfluidic channel by a laser.

Following the above procedure, the PS-ball trapped CTC (MCF-7) from example 1 was flowed into the microfluidic system, the fiber-linked laser was turned on, the fiber power was measured to be 1W, and the movement of the laser-applied PS-CTC within the microchannel was observed, with the results shown in FIG. 4. And the moving state of the cell in the channel is increased along with the increase of time, the PS-CTC moves along the direction far away from the optical fiber under the action of light, and simultaneously, the PS-CTC moves at the upper end of the microfluidic channel under the thrust of the injection pump.

Example 3:

(1) relationship between flow rate and CTC recovery efficiency: 10000 breast cancer cells (MCF-7) were added to 300. mu.L DMEM containing the PS bead-modified antibody of example 1 (4mg/mL), incubated at 37 ℃ for 2 hours, and collected by the microfluidic sorting system of example 2 at different flow rates; the collected cells were counted under a microscope, and the recovery efficiency was 100% per collected cell/input cell. The results are shown in fig. 5a, where the recovery efficiency gradually decreases with increasing flow rate.

(2) Relationship between flow rate and CTC recovery purity: 10000 breast cancer cells (MCF-7) were added to 300. mu.L DMEM containing the PS sphere-modified antibody of example 1 (4mg/mL) and incubated at 37 ℃ for 2 hours, followed by addition of 10⁵One FDA stained leukocyte, and a mixed sample was collected by the microfluidic sorting system in example 2 at different flow rates, and the collected cells were counted under a microscope to recover the purity of PS-CTC cells/(number of PS-CTC cells + number of leukocytes) × 100%. The results are shown in fig. 5b, with increasing flow rate, the purity of the recovered CTC cells gradually decreased.

(3) Cell viability was assessed using propidium iodide PI and fluorescein diacetate FDA. PI is a red fluorescent nucleic acid dye, staining dead cells, while FDA is a green fluorescent nucleic acid dye, staining live cells. PI and FDA solution 5. mu.g/mL, for cell viability testing. To test cell viability, 10000 breast cancer cells (MCF-7) were added to 300 μ L DMEM containing PS sphere modified antibody of example 1 (4mg/mL), incubated at 37 ℃ for 2 hours, and collected by microfluidic sorting system set at 100 μ L/h in example 2; freshly passaged MCF-7 cells served as a control group. Live and dead cells were stained with PI and FDA, respectively, and then counted under a fluorescent microscope. The results are shown in fig. 5c and 5d, where the cellular activity of the PS-CTCs is about 95%, indicating that the whole microfluidic separation system does not affect the cellular activity.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A method for sorting circulating tumor cells by combining optoforce with microparticles, which is characterized in that: the method comprises the steps of incubating the functionalized particles and a sample containing circulating tumor cells and white blood cells, and separating the circulating tumor cells and the white blood cells which are combined with the functionalized particles by light force; the functionalized particles are modified with specific molecules targeting circulating tumor cells.

2. The method of claim 1, wherein the light-force binding microparticles are selected from the group consisting of: the specific molecules targeting the CTCs are biomarkers capable of specifically recognizing and binding the surfaces of the CTCs, and the biomarkers on the surfaces of the CTCs comprise epithelial marker cytokeratin, epithelial cell adhesion molecules, tumor embryo antigens, human epidermal growth factor receptor 2, venous endothelial cell molecules, periderm proteins, sialylated Lewis oligosaccharide-X, acetaldehyde dehydrogenase 1, vimentin, urokinase receptor, heparanase, prostate specific membrane antigen, CD44, CK18, CD133, CD90, CD45 and CD 146;

the specific molecule of the target CTCs is an antibody which can specifically recognize and combine with the biomarkers on the surfaces of the CTCs.

3. The method of claim 1, wherein the light-force binding microparticles are selected from the group consisting of: the particle has a refractive index of more than 1.4 and a particle size of 4-8 μm; the surface of the particle is modified with active groups.

4. The method of claim 1, wherein the light-force binding microparticles are selected from the group consisting of: the functionalized particles are prepared by a method comprising the following steps: firstly, activating active groups modified on the surfaces of the particles, then combining the particles with streptavidin, and modifying specific molecules of biotinylated target circulating tumor cells on the basis of the streptavidin in a specific combination mode of the streptavidin and biotin.

5. The method of claim 1, wherein the light-force binding microparticles are selected from the group consisting of: the sample containing the circulating tumor cells and the white blood cells is obtained by a method comprising the following steps: obtaining a blood sample of a cancer patient, and obtaining a lymphocyte layer by percoll cell separation fluid, wherein the obtained lymphocyte layer is a sample containing circulating tumor cells and white blood cells.

6. The method of sorting circulating tumor cells with the optical binding microparticles of any one of claims 1-5, wherein: the method is carried out in a microfluidic chip.

7. The method of claim 6, wherein the light-force binding particles are selected from the group consisting of: the method comprises the following steps:

(1) adding the functionalized particles to a sample containing circulating tumor cells and white blood cells, and incubating to ensure that the functionalized particles are specifically combined with the circulating tumor cells;

(2) and (2) introducing the mixed sample obtained in the step (1) into a microfluidic chip, turning on laser, and separating out the circulating tumor cells combined with the functionalized particles.

8. Use of the method of sorting circulating tumor cells using optical binding particles according to any one of claims 1 to 7 for the isolation and identification of circulating tumor cells, wherein: the particles are fluorescent particles.

9. Use of the functionalized microparticle of claim 1 in the preparation of a kit for identifying, capturing or enriching CTCs.

10. A method of sorting target cells using optokinetic binding particles, comprising: the method comprises the steps of incubating functionalized particles and a sample containing target cells and interfering cells, and separating the target cells and the interfering cells combined with the functionalized particles by light force to achieve the purpose of sorting the target cells; the functionalized particles are modified with specific molecules targeting target cells.