CN116397003A - Method for detecting and recovering circulating tumor cells - Google Patents

Abstract

The invention discloses a method for detecting and recovering circulating tumor cells, which utilizes a super-hydrophilic-super-hydrophobic array microchip to divide liquid drops to obtain sub-liquid drops of encapsulated cells and MMP9 probes, and realizes the detection and recovery of CTC cells according to fluorescence conditions reflecting the cell functional activity. The invention utilizes micro-droplets in SDAM to measure MMP enzyme on single cells, and can identify CTC with different phenotypes by setting a proper threshold value, or distinguish tumor cells and white blood cells and mutant cells from unmutated cells, and meanwhile, the CTC detected and recovered by the invention has obvious advantages in terms of cell state, activity and cell function due to the advantages in terms of detection speed and physiological environment simulation.

Description

Method for detecting and recovering circulating tumor cells

Technical Field

The invention relates to a method for detecting and recovering circulating tumor cells, and belongs to the technical field of biology.

Background

Circulating Tumor Cells (CTCs) are defined as a class of tumor cells that escape from primary or metastatic lesions and circulate in the peripheral blood, with the origin traced back to 1869, thomas Ashworth first reporting that tumor cells similar to primary lesions were found in the peripheral blood of breast cancer patients. Today, researchers want to improve cancer treatment patterns through research on CTCs, including early detection, real-time monitoring of therapeutic response, screening for new therapeutic targets, elucidating drug resistance mechanisms, improving patient prognosis, etc.

CTC detection is mainly accomplished by direct immunostaining of cell membranes or cytoplasmic antigens, including epithelial, mesenchymal, tissue-specific and tumor-associated proteins. Most CTC assays currently use the same identification procedure as the FDA approved CellSearch system: tumor cells were identified with fluorescent-labeled keratin antibodies, while CD45 staining was used for leukocyte depletion. Prostate Specific Antigen (PSA) or breast specific galactophore globulin (mammaglobin) can also be used for CTC detection of specific cancer types. CTCs can also be detected using nucleic acid-based strategies. Detection of CTCs at the mRNA or DNA level requires the design of PCR reactions with primers for tissue-specific, organ-specific or tumor-specific transcripts or tumor-specific gene mutations, translocations or methylation patterns. However, other current methods based on N-cadherein or vimentin are interfered by white blood cells, which express these proteins after activation, and contamination of non-tumor associated cells may further complicate downstream molecular analysis of the gene mutation or transcription profile; in breast cancer, CK19 transcripts are commonly used in clinical studies. However, many transcripts (e.g., CK18, CK19, PSMA, etc.) are expressed at low levels in leukocytes.

In addition, epithelial-mesenchymal transition occurs dynamically during tumor metastasis. Methods based on typical CTC biomarkers, including epithelial cell adhesion molecule (EpCAM) or Pan-cytokeratins, tend to miss mesenchymal CTCs. The methods of detection of mesenchymal cells based on N-cadherein or vimentin are interfered with by blood and bone marrow cells that also express these proteins, and contamination of these non-tumor-associated cells may further complicate downstream analysis of gene mutations or transcriptomes, etc. There is thus still a need to find a method that is able to distinguish between different subtypes of CTCs, including mesenchymal CTCs.

Furthermore, only a small fraction of CTCs are able to maintain the ability to transfer remotely, CTCs have an in vitro half-life estimated to be 1.0-2.4 hours, and the processing time required for conventional CTC detection techniques is long, resulting in reduced CTC activity.

In summary, a new CTC detection method is needed.

Disclosure of Invention

In order to solve the problems, the invention provides a novel CTC detection method which can rapidly and accurately detect single CTC secretion protein MMP9 and can distinguish tumor cells from normal cells, CTCs of different subtypes and mutated and unmutated cells.

A first object of the present invention is to provide a method for detecting circulating tumor cells, comprising the steps of:

s1, preparing an array microchip with a superhydrophilic-superhydrophobic alternating region on the surface;

s2, preparing a sample to be detected into a cell suspension, and adding the cell suspension and a probe solution to the surface of the array microchip to prepare liquid drops which simultaneously wrap cells and probes; the probe comprises a sequence specifically cut by matrix metalloproteinase 9, and fluorescent groups and quenching groups are respectively modified at two ends of the sequence;

and S3, after incubation for a period of time, detecting fluorescence change on the array microchip under the wet conditions such as cell suspension or adherence, and the like, so as to realize the function and activity detection of the circulating tumor cells.

Further, the detection method can be used to distinguish between different subtypes of circulating tumor cells.

Further, the detection method can be used to distinguish between circulating tumor cells and leukocytes.

Further, the contact angle of the super-hydrophilic region on the array microchip is 1-10 degrees.

Further, on the array microchip, the contact angle of the superhydrophobic region is 150-160 degrees.

Further, the preparation method of the array microchip with the superhydrophilic-superhydrophobic alternating regions on the surface comprises the following steps: forming a candle ash layer on the surface of a chip substrate, forming a carbon-silicon dioxide layer through deposition of tetraethoxysilane, performing calcination and surface activation treatment, performing hydrophobic modification through octadecyltrichlorosilane to obtain a super-hydrophobic surface, designing a mask (the thickness of the mask is negligible) according to the shape of the array, and performing ultraviolet irradiation to obtain the array microchip with the super-hydrophilic-super-hydrophobic alternating area on the surface.

Further, the super-hydrophilic region can be in any shape such as a circle, a square and the like, and the area can be set as required, and the super-hydrophilic region is circular in the invention and has a diameter of 250 μm.

Further, the cell concentration in the cell suspension was 1X 10 ⁶ ～5×10 ⁶ Individual cells/ml, preferably 1X 10 ⁶ Individual cells/ml.

Further, the final concentration of the probe after mixing the cell suspension with the probe is 1 to 30. Mu.M, in the present embodiment 10. Mu.M.

Further, when the cell suspension and the probe solution are added, the two solutions may be added to the chip separately, or may be mixed first and then added dropwise to the chip, specifically: 1) Sequentially adding the probe solution and the cell suspension to an array microchip to slide to obtain micro-droplets, and mixing the probe and the cells; 2) Or directly sliding the mixed solution of the cell suspension and the probe solution on the surface of the array microchip to obtain the micro-droplet biochemical reactor separated into small units.

Further, in step S2, the number of cells wrapped by the droplet is one or several, preferably one.

Further, the amino acid sequence of the matrix metalloproteinase 9 specific cleavage sequence is GPLGMWSRKC.

Further, fluorescent reporter groups and quencher groups include, but are not limited to, FITC and DABCYL; QSY7 and 5-TAMRA, etc.

Further, in step S3, the incubation is performed for at least 10 minutes, preferably 10-60 minutes.

A second object of the present invention is to provide a test kit for differentiating between different subtypes of circulating tumor cells, the test kit comprising:

an array microchip having superhydrophilic-superhydrophobic alternating regions on a surface thereof;

and the probe comprises a sequence specifically cut by the matrix metalloproteinase 9, and fluorescent groups and quenching groups are respectively modified at two ends of the sequence.

The invention innovatively utilizes the microchip with the super-hydrophilic-super-hydrophobic structure to obtain micro-droplets arranged in an array manner, and can measure MMP9 enzyme activity of single cells. Since CTCs of the epithelial-like subtype or CTCs of the mesenchymal subtype are located differently in EMT transition, have different cell characteristics and functions, and the secreted MMP enzyme activities are also different, it is possible to identify or distinguish CTCs of different subtypes at the level of single cell analysis by the method of the present invention. Of course, those skilled in the art will appreciate that the methods of the present invention may also be used to study disease progression by identifying or differentiating other types of CTC subtypes.

A third object of the present invention is to provide an assay kit for distinguishing circulating tumor cells from leukocytes, comprising:

an array microchip having superhydrophilic-superhydrophobic alternating regions on a surface thereof;

and the probe comprises a sequence specifically cut by the matrix metalloproteinase 9, and fluorescent groups and quenching groups are respectively modified at two ends of the sequence.

In the present invention, a strict threshold line is set to distinguish between normal cells (white blood cells) and tumor cells by utilizing the significant secretion difference of MMP 9. In this case, the isolated CTCs are subjected to downstream applications such as immunostaining or molecular analysis, etc., to maximize contamination with leukocytes.

A fourth object of the present invention is to provide a method for recovering circulating tumor cells, comprising the steps of:

(1) Preparing an array microchip with superhydrophilic-superhydrophobic alternating regions on the surface;

(2) Preparing a sample containing circulating tumor cells into a cell suspension, and adding the cell suspension and a probe solution to the surface of the array microchip to prepare droplets which simultaneously encapsulate the cells and the probes; the probe comprises a sequence specifically cut by matrix metalloproteinase 9, and fluorescent groups and quenching groups are respectively modified at two ends of the sequence;

(3) After incubation for a period of time, the circulating tumor cells are precisely positioned and recovered based on the coordinates of the droplet array according to the fluorescence on the array microchip.

Further, in step (3), two recovery modes are included: (1) negative pressure recovery based on capillary operation; (2) based on the blending/re-splitting of the cell-free blank solution droplet and the chip droplet unit, the cell is recovered in the cell-free blank droplet in a mode of re-distributing.

Regarding recycling of circulating tumor cells, methods such as chamber 3D culture and microfluidic control exist at present, but due to the long culture period and harsh culture conditions of the traditional culture method and the long time consumption of specialized operations and intelligent response type introduction of additional biological and chemical reagents in the microfluidic control, the activity, state and reality of the recycled circulating tumor cells are greatly different, so that the subsequent research is not facilitated. In the invention, the array-type arranged and physically addressable micro-droplets are adopted to recycle the CTC, more importantly, the micro-droplets of the invention do not fix the captured CTC as in the traditional mode, but adopt non-fixed capturing, and single CTC cells can freely move in the droplets, so that the state of the CTC in blood circulation can be better simulated, the activity of the collected cells is higher, and the detection of the CTC related index is closer to the actual data. Of course, those skilled in the art will appreciate that the strategy presented in the present invention may be extended to the detection of other living cells.

A fifth object of the present invention is to provide an assay kit for distinguishing between mutated tumor cells and unmutated cells, the assay kit comprising:

an array microchip having superhydrophilic-superhydrophobic alternating regions on a surface thereof;

and the probe comprises a sequence specifically cut by the matrix metalloproteinase 9, and fluorescent groups and quenching groups are respectively modified at two ends of the sequence.

In order to distinguish mutated tumor cells from non-mutated normal cells, the invention utilizes the property that KRAS gene mutation is a key node of MMP9 enzyme secretion regulation, takes A549 cells as mutated tumor cells, detects that A549 secretion MMP is different from other cells by an array microchip method, further confirms the KRAS mutation of A549 by a gene detection method, and verifies the feasibility of the method. In practical application, the method can be used for monitoring and early warning abnormal mutation in vivo, drug resistance screening, pathogenic mutation screening and the like.

The invention has the beneficial effects that:

(1) The invention can complete detection in a short time (less than 1 hour), can better simulate the environment of circulating tumor cells in vivo, and has better maintenance effect on the activity of the cells.

(2) Because of the dehumidification effect, the micro-droplets and the cells contained in the micro-droplets are separated in a designated hydrophilic area, and the single-cell separation mode does not need covalent modification and fixation, so that the cells can be extracted as required later; by moisturizing, micro-droplets avoid evaporation, and the biochemical reaction and detection are well maintained; the liquid drop reacts through free diffusion, combines with a specific probe, is free of washing, reduces loss of a detection object and cells, and has functional reaction of living cells.

(3) In the invention, the tumor cells and the normal cells have obvious fluorescence difference, so that the CTC and the normal cells can be distinguished at the single cell level; simultaneously, single cells are subjected to MMP9 enzyme measurement by utilizing micro-droplets in SDAM, and CTCs with different phenotypes can be identified by setting a proper threshold.

(4) The open interface in the present invention allows for the localization, recovery and release of any cell of interest by micromanipulation for downstream analysis, such as immunofluorescent staining, genomic sequencing, transcriptome sequencing, and the like.

Drawings

FIG. 1 is a schematic illustration of the preparation of a super-wetted drop array microchip SDAM;

FIG. 2 is a schematic diagram of the structure of a super-wetted drop array microchip SDAM;

FIG. 3 is a schematic diagram of MMP9 enzymatic reaction;

FIG. 4 shows the effect of different cell concentrations on the detection effect;

FIG. 5 shows the detection effect of droplets coating different cell numbers;

FIG. 6 is the detection of tumor cells and normal cells, and the detection of mesenchymal cells;

fig. 7 is an effect advantage of the SDAM method.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

EXAMPLE 1 preparation of super-wetted drop array microchip (SDAM)

The preparation flow is shown in figure 1. Slide with 98% H ₂ SO ₄ And 30% H ₂ O ₂ The mixed solution of the solutions (7:3 v/v) was washed for 1 hour, rinsed with ultra pure water and dried with nitrogen. The solution was deeply washed with acetone, ethanol and ultrapure water at room temperature for 30 minutes, respectively. The slide was placed on a stable burning candle flame at a rate of 5 cm/sec to form a uniform candle soot. NH in closed vessel ₃ ·H ₂ Chemical Vapor Deposition (CVD) of O and TEOS reacted for 24 hours to produce carbon/silica micro-nano structures on the slides. The carbon layer was removed by calcination at 550℃for 2 h. The calcined slide was treated with O ₂ Plasma treatment for 3 minutes, the surface of which generates more hydroxyl groups. Immediately, the slide glass was immersed in an anhydrous toluene solution containing 1vol% ots, and reacted at room temperature for 8 minutes. Then the glass slide is washed by toluene and ethanol respectively,curing is carried out at 100℃for 15 minutes. The superhydrophobic slide was fastened with a self-made mask and was inspected in a high pressure mercury lamp (about 250mW/cm ² ) Exposing for 40 minutes. Upon cooling, the slide exposed areas became superhydrophilic, while the non-exposed areas remained superhydrophobic, thereby making a Superwetted Drop Array Microchip (SDAM). The structure obtained is shown in figure 2.

Example 2 detection of CTC cells

The micro-droplets generated by the method provide a micro-reactor with definite volume for detecting MMP9 enzyme activity of single tumor cells or single heterogeneous CTC. The single cells and probes were packed in the same droplet and incubated in serum-free and phenol red-free medium at 37 ℃. Once a single cell begins to secrete MMP9 enzyme, the enzymatic reaction is immediately initiated, cleaving the peptide sequence specifically cleaved by MMP9 enzyme, breaking the fluorescent group (FITC) and the quenching group (DABCYL), thereby releasing fluorescence (schematic see fig. 3).

Due to the discontinuous dewetting effect of the SDAM surface, the droplets are slid at a specified sliding rate (0.5 cm/s) at the pipette tip, and the sub-droplets are rapidly split by alternating superhydrophilic and superhydrophobic regions. Cell suspensions of different concentrations were applied to determine the specific cell number on a single droplet. Shown in FIG. 4 at 1X 10 per milliliter ⁶ The proportion of droplets surrounding individual tumor cells was greatest (about 38%) at the loading concentration of individual cells.

Example 3

Figure 5A shows that the fluorescence values of MMP9 enzymes secreted by individual tumor cells reached saturation within 1 hour. FIG. 5B shows that single tumor cells (A2, B2) pre-stained with Hoechst are readily distinguishable from background signals (A1, B1; A2, B1; A1, B2) with approximately 3-fold fluorescence differences. Fig. 5C shows the scenario where a larger area is scanned, with droplets encapsulating different numbers of tumor cells, and their MMP9 enzyme secretion can be observed simultaneously. Briefly, there are 20 empty droplets, 30 droplets surrounding one cell, 15 droplets containing two cells, and 5 droplets capturing three cells. Semi-quantitative analysis of the images showed that each droplet containing 2-3 cells fluoresced significantly higher than the empty droplet. Even droplets containing only one cell can be distinguished from those empty droplets, with fluorescence differences approaching 3-fold.

Example 4

Figure 6A shows that we validated method selectivity by mixing a549 tumor cells with White Blood Cells (WBCs) provided by healthy humans. Leukocytes were enriched with anti-CD 45 conjugated immunomagnetic beads and pre-stained with DIL dye. A549 tumor cells were pre-stained with Hoechst. Individual a549 tumor cells (Hoechst staining identification) were isolated in the upper left corner drop of the 2 x 2 array, and 1 or 2 white blood cells (DIL staining identification) were isolated in the remaining drops. The green fluorescence corresponding to the secretion of MMP9 in the former is obviously stronger than that in the latter. Figure 56 after statistical analysis of droplets isolated from individual a549 cells or individual WBCs (360 total, half from a549 cells, and the other half from 3 healthy people), we were able to determine the threshold fluorescence intensity (50 a.u.) of MMP9 secretion for selective identification of tumor cells.

We used TGF- β as an inducer to transform a549 cells from epithelial cells to mesenchymal cells, and used EpCAM (epithelial biomarker) and vimentin (mesenchymal biomarker) antibodies to test the extent of transformation, respectively, by fluorescent staining of cells. Significant reversal of fluorescence intensity based on EpCAM and vimentin immunostaining indicated that TGF- β treatment of a549 cells resulted in efficient EMT (fig. 6C-D). Flow cytometry data also validated the trend of TGF- β induction of the mesenchymal (+)/epithelial (-) characteristics of a549 cells (fig. 6E). A549 cells before and after TGF- β treatment were subjected to Hoechst pre-staining, loaded on SDAM, respectively, and MMP9 secretion thereof was measured at the single cell level (FIG. 6F). Statistical analysis showed that MMP9 secretion was distinguishable in both groups of cells with or without TGF- β treatment (.x.p < 0.01) (fig. 6F).

The SDAM experiments of the present invention for the first time found that tumor cells with more mesenchymal characteristics (+tgf- β) have higher MMP9 secretion than epithelial tumor cells (-TGF- β) at the single cell level, which may be related to their different ability to adapt/modify the tumor microenvironment.

Example 5

The SDAM method of the present invention has two advantages (1) cells separated in the droplet are not forcedly immobilized on the substrate surface; (2) Droplets on the SDAM can be contacted through the glass capillary tip. Thus, target cells can be rapidly located, extracted, and recovered from the droplet array of the SDAM by standard micromanipulation (fig. 7A). After mixing A549 and healthy WBCs, fluorescence detection was performed, cells were divided into two groups according to MMP9 secretion threshold (50 a.u.), and then drug resistance gene KRAS mutation detection was performed. MMP9 can be demonstrated as a basis for distinguishing between mutated and unmutated based on the pathways associated with secretion of MMP9 by KRAS gene (Table 1). As shown in FIG. 7B, DNA sequencing results demonstrated that there was a point mutation (G.fwdarw.A) at the G12S site of KRAS gene in cells with higher secretion activity of MMP 9. Fig. 7C shows a map of SDAM fluorescence images obtained from scanning a lung cancer patient in a double-blind test of clinical specimens. Referring to the enlarged view in fig. 7C, there are 5 single cell droplets wrapped on the SDAM that produce significantly higher fluorescence than the other droplets. This suggests that these droplet-encapsulated cells have higher secretion of MMP9 and are therefore candidates for CTCs. Cells in the droplets were then recovered from the SDAM, respectively, and then immunofluorescent stained to identify their epithelial and mesenchymal characteristics. The staining results confirmed that all 5 cells recovered from SDAM were indeed CTCs (fig. 7D). Our SDAM analysis allows screening and recovery of cytokeratain+/vimentin-/CD 45-/DAPI+ epithelial CTCs (FIG. 7D) and cytokeratain-/vimentin+/CD 45-/DAPI+ mesenchymal CTCs (FIG. 7D below). Unlike traditional CTC detection methods in the literature, our SDAM method can provide an index of related MMP9 secretion of individual CTCs, which is valuable for prognostic research and development of cancer personalized therapies. After our SDAM method classifies MMP9 secretion, whole transcriptome sequencing was used to analyze gene expression of epithelial-like or mesenchymal-like CTCs. FIG. 7E shows 10 EMT-related classical gene expression profiles in which CDH23, MUC1, CDH1, KRT18 and other genes were down-regulated and Smad2, CDH2, SPARC, SNAI1, twist1 were up-regulated. We performed classical KEGG pathway analysis, ranking the first 20 pathways in order of transcriptome differential weights. Some of which are considered in the literature to be relevant to the EMT process, are marked in the blue font in fig. 7F. Therefore, our whole transcriptome sequencing data combined with the SDAM method reveals a strong correlation between MMP9 secretion activity of CTCs and their EMT regulation, which can provide new opportunities for discovery of other tumor biology.

TABLE 1

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (10)

1. A method for detecting circulating tumor cells, comprising the steps of:

s1, preparing an array microchip with a superhydrophilic-superhydrophobic alternating region on the surface;

s2, preparing a sample to be detected into a cell suspension, and adding the cell suspension and a probe solution to the surface of the array microchip to prepare liquid drops which simultaneously wrap cells and probes; the probe comprises a sequence specifically cut by matrix metalloproteinase 9, and fluorescent groups and quenching groups are respectively modified at two ends of the sequence;

and S3, after incubation for a period of time, detecting fluorescence change on the array microchip under the condition of cell suspension or adherence, so as to realize the function and activity detection of the circulating tumor cells.

2. The method of claim 1, wherein: the contact angle of the super-hydrophilic area on the array microchip is 1-10 degrees.

3. The method of claim 1, wherein: the contact angle of the super-hydrophobic area on the array microchip is 150-160 degrees.

4. The method of claim 1, wherein the method of preparing an array microchip having superhydrophilic-superhydrophobic alternating regions on the surface comprises the steps of: forming a candle ash layer on the surface of a chip substrate, forming a carbon-silicon dioxide layer through deposition of tetraethoxysilane, performing calcination and surface activation treatment, performing hydrophobic modification through octadecyl trichlorosilane to obtain a super-hydrophobic surface, designing a mask according to the shape of the array, and performing ultraviolet irradiation to obtain the array microchip with the super-hydrophilic-super-hydrophobic alternating area on the surface.

5. The method of claim 1, wherein: in step S2, the probe solution and the cell suspension are respectively added to an array microchip to slide, so as to obtain micro-droplets, and the probe and the cell are mixed; or sliding the mixed solution of the cell suspension and the probe solution on the surface of the array microchip to obtain the micro-droplet biochemical reactor separated into small units.

6. The method of claim 1, wherein: the amino acid sequence of the matrix metalloproteinase 9 specific cleavage sequence is GPLGMWSRKC.

7. A test kit for differentiating between different subtypes of circulating tumor cells, the test kit comprising:

an array microchip having superhydrophilic-superhydrophobic alternating regions on a surface thereof;

and the probe comprises a sequence specifically cut by the matrix metalloproteinase 9, and fluorescent groups and quenching groups are respectively modified at two ends of the sequence.

8. A test kit for distinguishing between circulating tumor cells and leukocytes, the test kit comprising:

an array microchip having superhydrophilic-superhydrophobic alternating regions on a surface thereof;

and the probe comprises a sequence specifically cut by the matrix metalloproteinase 9, and fluorescent groups and quenching groups are respectively modified at two ends of the sequence.

9. A method for recovering circulating tumor cells, comprising the steps of:

(1) Preparing an array microchip with superhydrophilic-superhydrophobic alternating regions on the surface;

(2) Preparing a sample containing circulating tumor cells into a cell suspension, and adding the cell suspension and a probe solution to the surface of the array microchip to prepare droplets which simultaneously encapsulate the cells and the probes; the probe comprises a sequence specifically cut by matrix metalloproteinase 9, and fluorescent groups and quenching groups are respectively modified at two ends of the sequence;

(3) After incubation for a period of time, the circulating tumor cells are positioned and recovered based on the coordinates of the droplet array according to the fluorescence on the array microchip.

10. A test kit for distinguishing between mutated tumor cells and unmutated cells, the test kit comprising:

an array microchip having superhydrophilic-superhydrophobic alternating regions on a surface thereof;

and the probe comprises a sequence specifically cut by the matrix metalloproteinase 9, and fluorescent groups and quenching groups are respectively modified at two ends of the sequence.

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