CN111443197A - Method for analyzing phenotype of circulating tumor cells of liver cancer - Google Patents
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Abstract
The invention discloses a phenotype analysis method of circulating tumor cells of liver cancer, which comprises the following steps of 1) constructing a microfluidic chip comprising two processing units, wherein each processing unit comprises: the micro-column micro-sampling device comprises a micro-array consisting of a plurality of micro-columns with triangular cross sections, two sample inlets positioned on one side of an inlet of the triangular micro-array and two sample outlets positioned on one side of an outlet of the triangular micro-array; two processing units respectively modify epithelial cell adhesion molecule EpCAM antibody and asialoglycoprotein receptor ASGPR antibody 2) samples respectively enter from inlets of the two processing units and flow out from outlets; 3) circulating tumor cells after capture by the two processing units were identified by immunostaining for epithelial and non-epithelial phenotypes. The method can improve the capture efficiency, purity and detection rate of the circulating tumor cells of the liver cancer, and can perform phenotypic analysis on the circulating tumor cells of the liver cancer.
Description
Technical Field
The invention relates to a phenotype analysis method of circulating tumor cells of liver cancer.
Background
The incidence of hepatocellular carcinoma ranks worldwide fifth in all cancers and is the third leading cause of cancer-related death. The gold standard for diagnosis of hepatocellular carcinoma is tissue biopsy, however this method involves the risk of difficult sampling, insufficient representativeness, complications, etc. Early screening for hepatocellular carcinoma mainly relies on imaging methods and serum Alpha Fetoprotein (AFP) detection. The imaging method relies on subjective judgment of an operator, and the sensitivity is insufficient. The AFP detection sensitivity and specificity are respectively only 39-64% and 76-91%, and the false positive and false negative rates are high. Therefore, there is an urgent need to find a highly accurate, highly sensitive, noninvasive diagnosis method for hepatocellular carcinoma.
Circulating Tumor Cells (CTCs) refer to Tumor cells that fall from a Tumor lesion and enter the blood circulation, which are important causes of Tumor spread and metastasis. Research has shown that hepatocellular carcinoma circulating tumor cells are related to prognosis and malignant progression of hepatocytes, so detection of hepatocellular carcinoma CTCs can be used in the fields of early diagnosis of hepatocellular carcinoma, recurrence metastasis monitoring, efficacy evaluation and the like. The current method for isolating hepatocellular carcinoma CTCs based on the affinity method relies mainly on Epithelial cell adhesion molecule (EpCAM) recognition, the most classical method being the CellSearch method, which is currently the only CTC isolation method approved by the us FDA, and which isolates CTCs from whole blood via magnetic beads coupled with EpCAM antibodies. Research shows that the EpCAM positive rate of hepatocellular carcinoma tissues is only 20% -35%, so that separation through identification of a single epithelial marker inevitably causes omission, and is difficult to provide meaningful clinical guidance. The occurrence of Epithelial-mesenchymal transition (EMT) processes in the circulatory system by CTCs leads to heterogeneity of CTCs. The process can increase epithelial cell transfer and invasion capacity, is accompanied by disappearance of epithelial-like characteristics of CTC and acquisition of mesenchymal characteristics, plays an important role in the process that CTC falls off from a primary focus, enters a blood circulation system and forms a tumor metastasis focus, and is closely inseparable from the tumor metastasis and recurrence processes. Multiple studies show that overexpression of the interstitial marker vimentin has strong correlation with late-stage hepatocellular carcinoma and worse prognosis, and therefore, the problems of low capture efficiency, single capture phenotype and limited clinical application are caused by the fact that only EpCAM identification is used for capturing liver cancer circulating tumor cells. The simultaneous separation of epithelial-type and mesenchymal-type hepatocellular carcinoma CTCs is of great significance for exploring the formation mechanism of cancer, understanding the invasion process of metastasis and the like.
Disclosure of Invention
The invention mainly aims to provide a method for analyzing the phenotype of circulating tumor cells of liver cancer.
The technical scheme adopted by the invention for solving the technical problem is as follows:
a method for analyzing the phenotype of circulating tumor cells of liver cancer comprises the following steps:
1) constructing a capture chip, said capture chip comprising two processing units, wherein each processing unit comprises:
the micro-array is composed of a plurality of micro-columns with triangular cross sections, and a sample inlet positioned on one side of an inlet of the triangular micro-array and a sample outlet positioned on one side of an outlet of the triangular micro-array;
wherein, the microarray adopts the following arrangement mode: dc 1.4G (Delta lambda/lambda)0.48Wherein G refers to the horizontal spacing between the microcolumns, λ is the distance between the centers of two microcolumns in the same column, Δ λ refers to the lateral offset value of the second triangle relative to the first triangle in the same row, and Dc is the microfluidic whole critical dimension;
the microarray of the first processing unit is modified with an epithelial cell adhesion molecule antibody, and the microarray of the second processing unit is provided with a asialoglycoprotein receptor antibody;
2) the samples respectively enter from the inlets of the two processing units and flow out from the outlets;
3) circulating tumor cells after capture by the two processing units were identified by immunostaining for epithelial and non-epithelial phenotypes.
In the present invention, G is 38-50 μm, λ is 110-130 μm, Δ λ is 2-5 μm, and Dc is 11-13 μm. More preferably, G is 50 μm, λ is 120 μm, Δ λ is 3.8 μm, and Dc is 13 μm.
In the present invention, the flow rate of the sample in each treatment unit is 0.5 to 0.7m L/h.
In the present invention, the flow rate of the sample in each treatment unit was 0.6m L/h.
In a preferred embodiment of the invention, in step 3), the immunostaining method is to use DAPI as a nuclear dye, ASGPR to identify hepatocellular carcinoma CTCs, EpCAM to identify epithelial-type CTCs, and CD45 as a marker for leukocytes.
In a preferred embodiment of the invention, the identification criteria for epithelial CTCs are DAPI +, EpCAM +, ASGPR +/-, CD45-, the identification criteria for non-epithelial CTCs are DAPI +, ASGPR +, EpCAM-, CD45-, and the identification criteria for leukocytes are DAPI +, EpCAM-, ASGPR1-, CD45 +.
In a preferred embodiment of the invention, the microarray is arranged in a rectangular pattern with an entrance formed by a row of bar-shaped posts on the left side.
In a preferred embodiment of the present invention, the microarray is divided into two microarray units by the middle partition, each microarray unit corresponding to one sample outlet.
In a preferred embodiment of the invention, the chip is provided with a glass slide, a PDMS lower layer, a PDMS channel layer and a PDMS upper layer from bottom to top, wherein two sample inlets and two sample outlets are positioned on the PDMS upper layer, and the microarray is positioned on the PDMS channel layer.
In a preferred embodiment of the present invention, the PDMS channel layer is provided with a circular sample inlet at a position corresponding to the first sample inlet of the PDMS upper layer, the circular sample inlet is connected to the microarray through a first connecting arm, and the right end of the first connecting arm is located in the middle of the left side of the microarray; the PDMS channel layer is provided with a circular sample inlet at a position corresponding to the second sample inlet on the upper PDMS layer, a second connecting arm is arranged on the right side of the circular sample inlet and is further bifurcated into two annular arms and a microarray inlet connection, wherein the joint of the upper annular arm is positioned on the upper side of the joint of the first connecting arm and the microarray, and the joint of the lower annular arm is positioned on the lower side of the joint of the first connecting arm and the microarray.
Compared with the background technology, the technical scheme has the following advantages:
1. the method is provided with two processing units, the hepatocellular carcinoma CTCs are captured by adopting a liver parenchymal cell marker ASGPR and an epithelial cancer cell marker EpCAM, epithelial CTCs and non-epithelial CTCs are distinguished by an immunostaining method, the problems of low capture efficiency and interstitial CTCs omission caused by dependence on epithelial markers in the existing method are solved, the analysis of various phenotypic hepatocellular carcinoma CTCs is realized, and the problem of heterogeneity of the CTCs is solved.
2. The microarray adopts the following arrangement mode: dc 1.4G (Delta lambda/lambda)0.48By adopting the micro-fluidic chip with the critical dimension collision effect, different fluid paths are formed between cancer cells and blood cells in a fluid, the cancer cells can be efficiently captured, the nonspecific adsorption of the blood cells is low, and the purity is improved.
G is 38-50 μm, λ is 110-130 μm, Δ λ is 2-5 μm, Dc is 11-13 μm. Is an optimal arrangement mode suitable for capturing liver cancer cells;
4. the average capture efficiency of the EpCAM channel and the ASGPR channel is high at the flow rate of 0.5-0.7m L/h, the maximum capture efficiency can reach 90% and 87%, respectively, and the nonspecific adsorption rate of the CCRF-CEM is 4% and 11%, respectively.
5. The microarray is divided into an upper mirror symmetry unit and a lower mirror symmetry unit by the middle parting strip, which can increase the cell dispersion degree in the sample and improve the separation efficiency.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 is a schematic cross-sectional view of a liver cancer circulating tumor cell capture chip of the present invention.
Fig. 2 is a top view of the present invention.
Fig. 3 is a partial enlargement of fig. 2 at a.
Fig. 4 is a partial enlargement at B of fig. 3.
FIG. 5 is a schematic diagram showing the relationship between the columns.
FIG. 6 is one of the result graphs, wherein: flow cytogram of huh-7 mode cells; flow cytogram of sk-HEP-1 mode cells; flow cytogram of ccrf-CEM negative control cells; size distribution of HUH-7, SK-HEP-1, CCPF-CEM, WBCs
Figure 7 is a second graph of the results, wherein a. optimize the flow rate of the EpCAM channel; B. optimizing the flow rate of the ASGPR channel; C. linear range of buffer environment EpCAM channel; D. linear range of buffer environment ASGPR channel; E. capture efficiency of whole blood ambient EpCAM channel (20 or 100 cells selected); F. capture efficiency of the Whole blood Environment ASGPR channel (selection of 20 or 100 cells)
FIG. 8 is a graph showing the results of blood sample analysis.
FIG. 9 statistical analysis of total number of CTCs for EpCAM alone, (B) ASGPR alone, and (C) a combination of EpCAM-ASGPR.
FIG. 10 clinical diagnostic value study-in comparison to AFP. Receiver operating characteristic curve (ROC) analysis was performed on three capture methods and the clinically common early-screening marker AFP with benign liver disease patients as a control group and hepatocellular carcinoma patients as an experimental group (FIG. 10A); total number of CTCs in patients at different AFP levels was analyzed (fig. 10B).
FIG. 11 correlation of CTC phenotype with hepatocellular carcinoma staging. The EpCAM channel failed to distinguish between early and late stage patients (P > 0.05), whereas addition of ASGPR allowed early and late stage patients (P < 0.05) (fig. 11A); correlation analysis of different phenotypic CTCs with staging (fig. 11B).
Detailed Description
Referring to fig. 1 to 5, a liver cancer circulating tumor cell capturing chip includes four layers from bottom to top, which are: the glass slide 1, the PDMS lower layer 2, the PDMS channel layer 3 and the PDMS upper layer 4.
Wherein, be equipped with four introduction ports and four outlet ports on PDMS upper layer 4, be respectively: a first sample inlet 41, a second sample inlet 42, a third sample inlet 43, a fourth sample inlet 44, a first sample outlet 45, a second sample outlet 46, a third sample outlet 47 and a fourth sample outlet 48.
Two identical processing units, namely a first processing unit 3A and a second processing unit 3B, are arranged in parallel on the PDMS channel layer 3. Wherein the content of the first and second substances,
the first processing unit 3A includes: a microarray composed of a plurality of microcolumns 35 having a triangular cross section, and a first inlet 41 and a second inlet 42 located at the left side of the triangular microarray, and a first outlet 45 and a second outlet 46 located at the right side of the triangular microarray.
The second processing unit 3B includes: a microarray composed of a plurality of microcolumns 35 having a triangular cross section, and a third sample inlet 43, a fourth sample inlet 44 located at the left side of the triangular microarray, and a third sample outlet 47 and a fourth sample outlet 48 located at the right side of the triangular microarray.
Referring to fig. 2 and 3, taking the first processing unit 3A as an example, the microarray is arranged in a rectangle, the left side is provided with an inlet formed by a row of strip-shaped pillars 38, after the sample enters the microarray at the inlet, the sample is divided into an upper microarray unit and a lower microarray unit through a middle partition 36 for processing, wherein the upper microarray unit corresponds to the first sample outlet 45, and the lower microarray unit corresponds to the second sample outlet 46.
The PDMS channel layer 3 is provided with a circular sample inlet 31 at a position corresponding to the first sample inlet 41 of the PDMS upper layer 4, the circular sample inlet 31 is connected with the microarray through a connecting arm 32, and the right end of the connecting arm 32 is located in the middle of the left side of the microarray. At the position corresponding to the second injection port 42 of the PDMS upper layer 4, the PDMS channel layer 3 is provided with a circular injection port 33, the connecting arm 34 is connected to the right side of the circular injection port 33, the connecting arm 34 is further bifurcated into two annular arms 37 connected to the microarray inlet, wherein the connecting position of the upper annular arm is located on the upper side of the connecting arm 34 and the microarray connecting position, and the connecting position of the lower annular arm is located on the lower side of the connecting arm 34 and the microarray connecting position.
Referring to fig. 4 and 5, the microarray arrangement is as follows: g refers to the horizontal distance between the columns, lambda is the distance between the centers of two columns in the same column, delta lambda refers to the offset value of the second triangle in the same row relative to the first triangle, the microfluidic whole critical dimension Dc, fluid flows in the microfluidic chip in a laminar flow mode, liver cancer cells larger than the critical dimension break through the original laminar flow in the microfluidic chip and frequently contact with the microarray, the capture rate can be improved, blood cells smaller than the critical dimension flow in the original laminar flow, the probability of collision with the microarray is low, and the purity is improved; from the formula Dc ═ 1.4G: (delta lambda/lambda)0.48And obtaining the critical dimension. In the present embodiment, the first and second electrodes are,g was 50 μm, λ was 120 μm, and Δ λ was 3.8 μm, giving a Dc of 13 μm.
After the above chip is prepared, the conjugated antibody is chemically modified, 4.75% (V/V) (3-mercaptopropyl) trimethoxysilane prepared by absolute ethyl alcohol is added from an inlet, the chip is placed at room temperature for 1h, washed by absolute ethyl alcohol and placed in a 100 ℃ oven for 1h, when the chip is cooled to room temperature, 0.5mg/m L GMBS prepared by absolute ethyl alcohol is added from the inlet, the chip is placed at room temperature for 30min and washed by PBS, 20 mu g/m L streptavidin prepared by PBS is added from the inlet, the chip is placed at room temperature for 1h or 4 ℃ overnight, PBS is added from the inlet for washing, 20 mu g/m L biotinylated antibody prepared by PBS is added, the chip is placed at room temperature for 1h, and finally the chip is washed by PBS.
Thereafter, 20 μ g/m L EpCAM and 20 μ g/m L ASGPR (asialoglycoprotein receptor) antibodies were added from first processing unit 3A and second processing unit 3B, respectively, and incubated at room temperature for 1 h.
And simulating the capture and characterization of the liver cancer cells.
Thereafter, flow cytometric analysis was performed using fluorescent pre-stained target cells (HUH-7& SK-HEP-1) and control cells (CCRF-CEM or WBC). Fig. 6A shows that HUH-7 can be used as a model cell of a liver cancer cell highly expressing EpCAM, fig. 6B shows that SK-HEP-1 can be used as a model cell of a liver cancer cell highly expressing ASGPR, fig. 6CCCRF-CEM is used as a negative control, and fig. 6D shows that the critical dimension of the chip is 13 μm, i.e., the model cell is larger than the critical dimension, and the negative control cell is smaller than the critical dimension.
Optimization of flow rate and cell capture efficiency.
FIG. 7 is a graph of the average capture efficiency of EpCAM and ASGPR channels at 90% and 87% for PBS and 4% and 11% for CCRF-CEM, respectively, at lower flow rates, with less non-specific adsorption of target cells, higher non-specific adsorption, and lower flow rates, with less capture efficiency of target cells, thus 0.6m L/h was chosen as the optimal capture conditions, and a linear range examination (FIG. 7B) shows that 5-200 hepatoma cells are suspended in PBS and injected into the chip at 0.6m L/h, the EpCAM (FIG. 7B, top) and GPR channels (FIG. 7B, bottom) have better linearity, with higher capture efficiency and lower adsorption efficiency, as shown in FIG. 7B, with less non-specific adsorption of HERPMA and HERPE cells, respectively, and the results in 0.6m 89% for the experimental and 7% for the patient, 7C-7C, and 7C, respectively, with less residual adsorption, and 7C, respectively, and 7C, showing the results in the following experimental data of the results in the results.
And (4) analyzing a clinical sample.
(1) The identification criteria of the epithelial CTCs are DAPI +, EpCAM +, ASGPR +/-, CD45-, the identification criteria of the non-epithelial CTCs are DAPI +, ASGPR +, EpCAM, CD45-, the identification criteria of the leukocytes are DAPI +, EpCAM-, ASGPR1-, CD45 +. as shown in FIG. 8, the results show that the epithelial and non-epithelial CTCs are successfully captured.
(2) The clinical analysis-ability to distinguish cancer patients from healthy and benign patients for liver disease-the number of CTCs in both EpCAM and ASGPR channels was 1m L for each loading format-the final number of CTCs in both EpCAM and ASGPR channels was converted to a sum of the number of CTCs in both channels/2 m L-ASGPR combined use-the results of CTC detection in 45 cases of hepatocellular carcinoma patients demonstrated 66.7% detection, 93% detection in ASGPR channel, 100% detection in EpCAM-ASGPR combined use, and omission was avoided using diabody combination.
(3) The sensitivity of the EpCAM-ASGPR channel is 66.7% under the condition that the optimal cutoff value is 1, the specificity is 100%, the sensitivity of the ASGPR channel is 91.1% under the condition that the area under the curve is 0.667. cutoff is 3, the specificity is 100%, the area under the curve is 0.911. EpCAM-ASGPR combination is used, the sensitivity is 97.8% under the condition that the optimal cutoff value is 1.5, the specificity is 100%, the area under the curve is 0.978. the area under the AFP curve is only 0.477, the sensitivity is 6.44%, the specificity is 83.3%, the AFP diagnostic standard of hepatocellular carcinoma is more than or equal to 400 μ g/L, the diagnostic standard of liver disease is more than or equal to 8.1 μ g/L, the clinical diagnostic range of AFP S specificity is found to 83.3%, the combined AFP-AFP diagnostic value of AFP-ASGPR-AFP combined detection is more than or equal to 400 μ g/3679%, the combined detection value of all patients, thus the combined detection results in the combined detection of the combined detection result in the combined detection of the AFP detection of the combined detection of the AFP detection results in the combined detection of the AFP detection of the combined detection of the.
(4) Correlation of CTC phenotype with hepatocellular carcinoma stage. The correlation of the total number of CTCs and the number of CTCs of different phenotypes with the stage was examined, and the results are shown in FIG. 11. The EpCAM channel failed to distinguish between early and late stage patients (P > 0.05), whereas addition of ASGPR allowed early and late stage patients (P < 0.05) (fig. 11A). Since the diagnostic value of ASGPR in combination with EpCAM was highest, the association of CTCs of different phenotypes with staging was analyzed for this result (fig. 11B), which indicates that non-epithelial CTCs are significantly higher in late stage patients than in early stage patients and thus may be associated with a poorer prognosis. Therefore, the analysis of the total number of the hepatocellular carcinoma CTCs and the number of the CTCs with different phenotypes has application potential in diagnosis and prognosis evaluation of the hepatocellular carcinoma stage.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.
Claims (10)
1. A method for analyzing the phenotype of circulating tumor cells of liver cancer comprises the following steps:
1) constructing a capture chip, said capture chip comprising two processing units, wherein each processing unit comprises:
the micro-array is composed of a plurality of micro-columns with triangular cross sections, and a sample inlet positioned on one side of an inlet of the triangular micro-array and a sample outlet positioned on one side of an outlet of the triangular micro-array;
wherein, the microarray adopts the following arrangement mode: dc 1.4G (Delta lambda/lambda)0.48Wherein G refers to the horizontal spacing between the microcolumns, λ is the distance between the centers of two microcolumns in the same column, Δ λ refers to the lateral offset value of the second triangle relative to the first triangle in the same row, and Dc is the microfluidic whole critical dimension;
the microarray of the first processing unit is modified with an EpCAM antibody provided with an epithelial cell adhesion molecule, and the microarray of the second processing unit is provided with an ASGPR antibody provided with a asialoglycoprotein receptor;
2) the samples respectively enter from the inlets of the two processing units and flow out from the outlets;
3) circulating tumor cells after capture by the two processing units were identified by immunostaining for epithelial and non-epithelial phenotypes.
2. The method of claim 1, wherein the method comprises the steps of: g is 38-50 μm, λ is 110-130 μm, Δ λ is 2-5 μm, Dc is 11-13 μm.
3. The method of claim 2, wherein the flow rate of the sample in each processing unit is 0.5-0.7m L/h.
4. The method of claim 2, wherein the flow rate of the sample in each processing unit is 0.6m L/h.
5. The method of claim 1, wherein the method comprises the steps of: in the step 3), the immunostaining method comprises the steps of using DAPI as a nuclear dye, identifying hepatocellular carcinoma CTC by ASGPR, identifying epithelial hepatocellular carcinoma CTC by EpCAM, and using CD45 as a leukocyte marker.
6. The method of claim 5, wherein the method comprises the steps of: the identification criteria for epithelial CTCs are DAPI +, EpCAM +, ASGPR +/-, CD45-, and the identification criteria for non-epithelial CTCs are DAPI +, ASGPR +, EpCAM-, CD45-, and the identification criteria for leukocytes are DAPI +, EpCAM-, ASGPR1-, CD45 +.
7. The method of claim 1, wherein the method comprises the steps of: the microarray is arranged in a rectangle, and the left side of the microarray is provided with an inlet consisting of a row of strip-shaped columns.
8. The method of claim 7, wherein the method comprises the steps of: the microarray is divided into an upper microarray unit and a lower microarray unit by a middle division bar, and each microarray unit corresponds to one sample outlet.
9. The method of any one of claims 1 to 8, wherein the method comprises the steps of: the chip is provided with a glass slide, a PDMS lower layer, a PDMS channel layer and a PDMS upper layer from bottom to top, wherein two sample inlets and two sample outlets are positioned on the PDMS upper layer, and the microarray is positioned on the PDMS channel layer.
10. The method of claim 9, wherein the method comprises the steps of: the PDMS channel layer is provided with a circular sample inlet at a position corresponding to the first sample inlet of the PDMS upper layer, the circular sample inlet is connected with the microarray through a first connecting arm, and the right end of the first connecting arm is positioned in the middle of the left side of the microarray; the PDMS channel layer is provided with a circular sample inlet at a position corresponding to a second sample inlet on the upper layer of PDMS, the right side of the circular sample inlet is provided with a second connecting arm, the second connecting arm is bifurcated into two annular arms to be connected with the microarray inlet, the connecting part of the upper annular arm is positioned on the upper side of the connecting part of the first connecting arm and the microarray, and the connecting part of the lower annular arm is positioned on the lower side of the connecting part of the first connecting arm and the microarray; the first sample inlet is a sample inlet, and the second sample inlet is a buffer solution inlet.
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