A method for detecting circulating tumor cells based on the expression amount of sialic acid on the cell membrane surface and single cell detection technology

Abstract

The application discloses a method for detecting circulating tumor cells based on cell membrane surface sialic acid expression quantity and single cell detection technology, and belongs to the field of biomedical technology. The method comprises the following steps: using a boron acid group modified probe to label cell surface sialic acid in a blood sample after erythrocyte lysis; passing the labeled sample through a phase transfer flow focusing chip to enrich and purify circulating tumor cells based on cell size; and detecting the enriched and purified cells by a single cell analysis method. The application solves the problems of high cost, long time consumption and high missed detection rate of the current circulating tumor cell detection method applied in clinical practice, and provides a method with simple operation and higher universality for circulating tumor cell detection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for detecting circulating tumor cells based on the expression level of sialic acid on the cell membrane surface and single-cell detection technology. Background Technology

[0002] Currently, tumor diagnosis primarily relies on imaging, making early diagnosis of cancer difficult. Most cancer patients are diagnosed at middle or late stages, often with metastasis, making effective treatment challenging and resulting in extremely low cure rates. Furthermore, the main sources of genetic testing samples for cancer patients are surgical pathology slides or biopsy samples. However, obtaining these samples is difficult, invasive, and carries certain risks for cancer patients. Moreover, biopsy pathology cannot dynamically track disease progression. Therefore, liquid biopsy methods based on tumor markers have attracted significant attention.

[0003] Circulating tumor cells (CTCs) refer to tumor cells that detach from tumor tissue and enter the bloodstream. They have received increasing attention as tumor markers that can be used in liquid biopsies. CTCs can form new tumors under certain conditions, leading to cancer spread and metastasis. The presence of CTCs indicates the presence of a tumor and the possibility of metastasis. Studies have shown that tumor metastasis can occur even in the early stages of cancer, indicating that detecting CTCs in peripheral blood is one method for achieving early cancer diagnosis. However, in the early stages of cancer, the concentration of CTCs in peripheral blood is very low, generally only a few per milliliter, while blood cells are abundant. Therefore, achieving early cancer diagnosis places high demands on the analytical methods for CTCs.

[0004] Currently, existing CTC detection technologies are based on Johnson & Johnson's CellSearch detection technology. It is currently the only product approved by both the FDA (approved in 2004) and CFDA (approved in 2012) for the management of malignant tumor diseases in the detection of circulating tumor cells. The basic principle of CellSearch for detecting circulating tumor cells is as follows: Magnetic nanoparticles modified with antibodies against epithelial cell adhesion molecule (EpCAM), which is highly expressed on the surface of various circulating tumor cells, are used to capture EpCAM-highly expressed CTCs from the blood through an antigen-antibody reaction and separate them from the blood matrix based on magnetic force. The captured cells are then subjected to various fluorescent staining methods (anti-CD45, anti-CK8, DAPI staining, etc.) to jointly identify the CTCs. Currently, the CellSearch system and its modified methods are not performing well in clinical sample testing, mainly due to technical limitations of the CellSearch system. One major reason is the significant differences in surface antigen expression among CTCs from different sources. CellSearch only captures CTCs based on EpCAM, which is highly expressed on the surface of circulating tumor cells from various epithelial sources. However, there are cell lines with low or even almost no EpCAM expression (such as mesodermal and a small number of epithelial tumor cells), resulting in a high rate of missed detection. Therefore, there is a need to develop biomarkers with greater versatility and higher cell expression levels for CTC detection. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and provide a method for detecting circulating tumor cells based on the expression level of sialic acid on the cell membrane surface and single-cell detection technology.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for detecting circulating tumor cells based on the expression level of sialic acid on the cell membrane surface and single-cell detection technology includes the following steps:

[0008] (1) A probe modified with boric acid group was used to label the sialic acid on the cell surface of blood samples after erythrocyte lysis.

[0009] (2) The labeled sample was enriched and purified by phase transfer flow focusing chip based on cell size.

[0010] (3) The enriched and purified cells are detected by single-cell analysis to achieve the purpose of quantitative or semi-quantitative analysis of the number of circulating tumor cells and the expression level of surface sialic acid.

[0011] In step (1), the boric acid group-modified probe includes boric acid or phenylboronic acid-functionalized small molecules, polymers, self-assembled molecules, micro / nanoparticles, gold nanoparticles, etc. Preferably, the boric acid group-modified probe is a phenylboronic acid-modified gold nanoparticle probe, which is preferably prepared using gold nanoparticles (AuNPs) and 4-mercaptophenylboronic acid.

[0012] In step (2), the phase transfer flow focusing chip has a central channel, with one end being an inner inlet and the other end being an inner outlet. Two channels connecting to the outer inlet are located on either side of the central channel near the inner inlet, and two channels connecting to the outer outlet are located on either side of the central channel near the inner outlet. The inner and outer inlets are respectively accommodating buffer solution (preferably PBS) and the sample. The inner and outer outlets are respectively connected to a single-cell analysis and detection device and a waste collection container. The width of the central outlet channel (the section from the connection point between the central outlet and the outer outlet to the inner outlet) is preferably 100–200 μm and less than or equal to the width of the other channels. The width of the other channels is preferably 100–200 μm, and the channel height is preferably 40–60 μm. The sum of the flow rates of the inner and outer inlets is preferably 140–210 μL / min, and the ratio of their flow rates is preferably 6–9:1.

[0013] In step (3), the single-cell analysis method includes mass spectrometry detection, optical signal detection, electrical signal detection, etc.

[0014] In some implementation methods, the method for detecting circulating tumor cells based on the expression level of sialic acid on the cell membrane surface and single-cell detection technology, wherein the boric acid group-modified probe in step (1) is preferably phenylboronic acid-modified gold nanoparticles, the inner outlet of the phase transfer flow focusing chip in step (2) is connected to the nebulizer of inductively coupled plasma-mass spectrometry (ICP-MS), and the enriched and purified cells in step (3) are introduced into the inductively coupled plasma-mass spectrometer for detection in single-cell mode.

[0015] The method for detecting circulating tumor cells based on the expression level of sialic acid on the cell membrane surface and single-cell detection technology, wherein the primary tumors of circulating tumor cells include those from different sources such as epithelium and mesoderm.

[0016] This invention uses phenylboronic acid-modified gold nanoparticles (AuNP-PBA) as an Au-containing probe, utilizing the selective recognition of phenylboronic acid and sialic acid to label sialic acid on the cell surface of blood samples after erythrocyte lysis. The labeled cell samples are processed using a phase-transfer flow focusing (CP-MS) chip. Under optimized chip channel width and flow rate, the sample introduced through the outer inlet and the buffer solution introduced through the inner inlet form three laminar flows on both sides and in the middle of the central channel, respectively. Smaller red blood cells, white blood cells, and platelets remain in the laminar flow on both sides with almost no phase transfer and flow out from the side channels and are discarded. Larger CTCs undergo phase transfer under the influence of inertial lift and flow towards the central channel, where they are collected at the inner outlet, achieving a certain degree of purification. The central channel outlet is connected to the nebulizer of an ICP-MS, and the collected cells are introduced into the instrument for detection in single-cell mode. The more sialic acid on the surface of a cell, the higher the amount of Au labeled, and the stronger the pulse signal generated in the ICP-MS. CTCs generally have significantly higher sialic acid expression levels than blood cells, resulting in higher pulse signal intensity generated by CTCs in ICP-MS. By optimizing the detection threshold and filtering the signal, the signals of blood cells (mainly white blood cells) can be further filtered out, leaving only the signals of CTCs. These signals can then be used to count CTCs and quantify their surface sialic acid.

[0017] This invention solves the problems of high cost, long processing time, and high false negative rate in current circulating tumor cell (CTC) detection methods applied clinically, providing a simpler and more versatile method for CTC detection. Compared with existing technologies, this invention has the following advantages:

[0018] (1) Using sialic acid as a marker, its absolute expression level on the surface of CTCs is significantly higher than that of the commonly used EpCAM. For CTCs that can be detected by the CellSearch system, it can largely avoid the missed detection of cell subpopulations with low EpCAM expression caused by cell heterogeneity, and improve the detection rate of CTCs.

[0019] (2) Using sialic acid as a marker, it also shows a significantly higher expression level in cell lines with low or even almost no EpCAM expression compared to blood cells, enabling the detection of CTCs in a wider range and improving the detection rate of CTCs.

[0020] (3) Using the boric acid-sialic acid recognition system to label CTCs is more cost-effective, has wider applicability, faster reaction kinetics, shorter labeling reaction time, and shorter sample detection time compared to antigen-antibody, aptamer-aptamer and other recognition systems.

[0021] (4) The expression of sialic acid on the surface of blood cells leads to more significant interference from blood cells in methods that use sialic acid as a marker. Single-cell detection methods can identify CTCs from a large number of blood cells based on signal intensity information, have a higher tolerance to blood cell interference, and have lower requirements for the performance of pretreatment methods.

[0022] (5) The online single-cell detection method is simpler to operate in terms of material preparation and sample preparation compared with the method based on chemical affinity capture followed by immunofluorescence staining, and has a higher sample detection throughput. Attached Figure Description

[0023] Figure 1 The diagram shows the chip design (a), physical image (b), and schematic diagram of the cell purification working principle (c).

[0024] Figure 2 A schematic diagram of single-cell ICP-MS assay (a) and data processing methods (b). Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0026] Example 1: Preparation of AuNP-PBA probe

[0027] The synthesis of small-particle-size (5 nm) AuNPs followed the method described in the literature (Talanta, 2018, 176:40-46). The pH of the synthesized AuNPs was adjusted to 11 using 0.5 M KOH. 10 mL of the above AuNPs (50 mg / L as Au) was taken, and 200 μL of 0.1 mM 4-mercaptophenylboronic acid (dissolved in 5 mM KOH) was added dropwise while stirring. After stirring at room temperature for 2 hours, the mixture was stored at 4 °C and designated as AuNP-PBA. Take 2 mL of the above AuNP-PBA and centrifuge at 13000 rpm for 10 minutes to remove aggregated nanoparticles. Take 1.5 mL of the supernatant and add 150 μL of 1% bovine serum albumin (BSA) solution. Mix well and let stand at room temperature for 30 minutes. Purify using ultrafiltration, changing the solvent as needed (using a 0.5 mL, 10 KD ultrafiltration tube; centrifugation conditions: 13000 rpm, 10 min, 6 times; washing solution: PBS). The Au concentration of the obtained AuNP-PBA was determined by ICP-MS and diluted to 100 mg / L (calculated as Au) and stored at 4 °C.

[0028] Example 2: Fabrication of a Phase Transfer Flow Focusing Chip

[0029] The design of the phase-transfer flow focusing chip referenced and optimized the method described in the literature (Microsystems & Nanoengineering, 2019, 5:8). Figure 1 The chip has a central channel with an inner inlet at one end and an inner outlet at the other. Near the inner inlet, two channels connect to the outer inlet on either side of the central channel, and near the inner outlet, two channels connect to the outer outlet on either side. Except for the central outlet channel (the section from the central channel connecting the outer outlet to the inner outlet), which has a width of 120μm, all other channels are 200μm wide. The template is fabricated using soft photolithography based on SU-8 2050 photoresist, with a spin-coating thickness (i.e., the final channel height) of 50μm. The PDMS prepolymer is processed through mixing, pouring, peeling, dicing, drilling, and bonding to form the chip.

[0030] Example 3 Cell Culture and Labeling Methods

[0031] All cell lines used in this invention were cultured in a complete culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin dual antibiotics, in an incubator with saturated humidity, 37°C, and 5% CO2. Specifically, MCF-7 (human breast cancer cell line) and HeLa (human cervical cancer cell line) were cultured in DMEM; HepG2 (human liver cancer cell line), MG63 (human osteosarcoma cell line), and HOS (human osteosarcoma cell line) were cultured in MEM; and 6T-CEM (human T-cell leukemia cell line) and 143b (human osteosarcoma cell line) were cultured in RPMI 1640.

[0032] The cell suspension obtained after trypsin digestion was centrifuged (1500 rpm, 3 min) to remove the culture medium, redispersed in PBS (pH 7.2, containing 1% BSA), and then counted using a hemocytometer. After centrifugation (1500 rpm, 3 min) to remove BSA, the suspension was redispersed in PBS (pH 5.5) and diluted to 6 × 10⁻⁶. 5 cell / mL. Take 0.6 mL of the cell suspension at the above density, add 15 μL of 100 mg / L AuNP-PBA from Example 1, mix well, let stand at room temperature for 15 minutes, centrifuge (1500 rpm, 3 min) to remove excess probe, resuspend, and dilute with PBS (pH = 5.5) to different cell densities as test samples (see subsequent examples for specific densities).

[0033] Example 4: Clinical Blood Sample Processing and Labeling Methods

[0034] Peripheral blood samples were collected using K2EDTA anticoagulant blood collection tubes and processed within 48 hours to ensure optimal test results. 0.6 mL of blood was placed in an EP tube, 1.8 mL of erythrocyte lysis buffer was added, and the sample was shaken well and allowed to stand for 10 min. Then, it was centrifuged at 2500 rpm for 5 min, and the supernatant was discarded. The collected white plaques were washed once with 0.6 mL of PBS (pH 7.2, containing 1% BSA), then redispersed with 0.6 mL of PBS (pH 5.5). 15 μL of 100 mg / L AuNP-PBA from Example 1 was added, and the sample was mixed well, allowed to stand at room temperature for 15 min, and then centrifuged (1500 rpm, 3 min) to remove excess probe. The sample was resuspended in 0.6 mL of PBS (pH 5.5) as the test sample.

[0035] Example 5: Chip-based cell sample processing and online single-cell ICP-MS detection method

[0036] PBS (pH = 5.5) was introduced into the chip's internal inlet at a flow rate of 126 μL / min, and the sample was introduced into the external inlet at a flow rate of 14 μL / min. The internal outlet was connected to the ICP-MS nebulizer via a Tygon tube, and the external outlet was connected to a waste cup. The ICP-MS parameters are shown in Table 1, and the acquisition time for each sample signal was 8 min. Based on the acquired time-resolved data ( Figure 2 a) The instrument's data processing software can provide the corresponding cell pulse signal frequency-signal intensity distribution map. Figure 2 b). First, based on white blood cell count (1×10⁻⁶) 6 (cells / mL) and tumor cells (1×10) 5 The measurement results of a mixed sample (cells / mL) can be used to determine the signal threshold between hemorrhagic cells and CTCs. This threshold is then applied to the detection results of other samples; signals above the threshold are interpreted as signals generated by CTCs. In a white blood cell background (1×10⁻⁶), the signal threshold is lowered. 6 In the presence of (cells / mL), the cell size and signal intensity (density of 1×10⁻⁶) of different tumor cell lines were compared. 5 The detection limit (the measured value at 1 / mL) and the detection limit are shown in Table 2. The detection performance is at a medium level compared with similar works. However, this method can detect HeLa cells that are negative for EpCAM expression and osteosarcoma cells from three types of mesodermal origin. Compared with the Cellsearch system, it has a wider range of applications and is superior in terms of sample preparation, detection time, and cost.

[0037] Table 1. Operating parameters for inductively coupled plasma mass spectrometry

[0038]

[0039] Table 2. Detection limit of CTCs by this method

[0040]

[0041] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for detecting circulating tumor cells based on the expression level of sialic acid on the cell membrane surface and single-cell detection technology, characterized in that: Includes the following steps: (1) Use a borate group-modified probe to label sialic acid on the cell surface of blood samples after erythrocyte lysis; (2) The labeled sample was enriched and purified by phase transfer flow focusing chip based on cell size; (3) The enriched and purified cells are detected by single-cell analysis to achieve the purpose of quantitative or semi-quantitative analysis of the number of circulating tumor cells and the expression level of surface sialic acid.

2. The method according to claim 1, characterized in that: In step (1), the boric acid group-modified probe is a phenylboronic acid-modified gold nanoprobe.

3. The method according to claim 1, characterized in that: In step (2), the phase transfer flow focusing chip is provided with a central channel, one end of which is an inner inlet and the other end is an inner outlet. Two channels connecting to the outer inlet are provided on both sides of the central channel near the inner inlet, and two channels connecting to the outer outlet are provided on both sides of the central channel near the inner outlet.

4. The method according to claim 3, characterized in that: The internal and external inlets are respectively used to introduce buffer solution and sample, while the internal and external outlets are respectively connected to the single-cell analysis and detection equipment and the waste liquid collection container.

5. The method according to claim 3, characterized in that: The section from the outer exit to the inner exit of the central channel is the central exit channel. The width of the central exit channel is 100-200μm and is less than or equal to the width of other channels. The width of other channels is 100-200μm, and the height of the channels is 40-60μm.

6. The method according to claim 3, characterized in that: The sum of the flow velocities at the inner and outer inlets is 140–210 μL / min, and the flow velocity ratio between the inner and outer inlets is 6–9:

1.

7. The method according to claim 1, characterized in that: In step (3), the single-cell analysis method includes mass spectrometry detection, optical signal detection, and electrical signal detection.

8. The method according to claim 1, characterized in that: In step (1), the boric acid group-modified probe is phenylboronic acid-modified gold nanoparticles. In step (2), the inner outlet of the phase transfer flow focusing chip is connected to the nebulizer of the inductively coupled plasma mass spectrometer. In step (3), the enriched and purified cells are introduced into the inductively coupled plasma mass spectrometer for detection in single-cell mode.

9. The method according to any one of claims 1-8, characterized in that: Primary tumors containing circulating tumor cells include those of epithelial and mesodermal origin.

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