CN110389219A - A kind of enrichment detecting method of Epithelial and stromal mixed type and PD-L1 positive circulating tumor cell - Google Patents
A kind of enrichment detecting method of Epithelial and stromal mixed type and PD-L1 positive circulating tumor cell Download PDFInfo
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Abstract
The invention discloses a kind of Epithelial and stromal mixed type and the enrichment detecting methods of PD-L1 positive circulating tumor cell, comprising: the Epithelial and stromal mixed antibody of biotin labeling (using the EpCAM antibody of biotin labeling and the anti-CSV antibody of biotin labeling)；The separation and concentration of Epithelial and stromal mixed type CTCs；Using fluorescein-labeled Epithelial and stromal antibody cocktail (using fluorescein-labeled anti-PanCK antibody, fluorescein-labeled anti-Vimentin antibody, fluorescein-labeled anti-CD45 antibody) to the immune detection of different subtype CTCs；Using PD-L1 primary antibody with fluorescein-labeled PD-L1 secondary antibody to the PD-L1 Phenotypic examination of the different subtype CTCs of capture.The method of the present invention not only can be with simple and direct efficiently specifically enrichment detection Epithelial and stromal mixed type CTCs, but also can be enriched with detection epitheliated type and interstitial type CTCs.
Description
Technical field
The present invention relates to the enrichment of circulating tumor cell hypotype and its detection method fields, and in particular to a kind of Epithelial and stromal
The enrichment detecting method of mixed type and PD-L1 positive circulating tumor cell.
Background technique
Circulating tumor cell (Circulating Tumor Cells, CTCs) is spontaneous or because operation of diagnosis and treatment is by solid tumor
Or transfer stove discharges into the tumour cell of Peripheral Circulation, is the general designation for all kinds of tumour cells being present in peripheral blood.
Mainly by epitheliated type CTCs, interstitial type CTCs and Epithelial and stromal mixed type, (Epithelial and stromal transformant and mesenchymal epithelium turn CTCs
Change type) CTCs subgroup and above-mentioned subgroup CTCsPD-L1 positive or negative hypotype composition a plurality of types of heterogeneous populations.
Metastases diffusion is the main reason for leading to cancer progression and associated death.CTCs forms far-end transfer in tumour
During play important role.If containing greater number of CTCs in Blood From Cancer Patienties, prognosis is implied
The bad probability with metastases increases.The metastases that CTCs induces relate generally to Epithelial and stromal conversion (Epithelial to
Mesenchymal Transition, EMT) and mesenchymal epithelium conversion (Mesenchymal to Epithelial
Transition, MET) two dynamic processes, wherein EMT process is related to tumour cell and falls off to be formed to have into blood from primary tumor to turn
Epithelial and stromal mixed type CTCs, the MET process for moving invasion takes part in formation, stabilization and the breeding of far-end transfer stove.In
Along between epitheliated type, Epithelial and stromal mixed type and interstitial type difference phenotype during above-mentioned CTCs generation EMT and MET
CTCs transformation, and the different phase of tumor development (early stage, progressive stage with shift the phase) in for above-mentioned different subgroups
CTCs detection has different clinical values: the early stage auxiliary that the detection and analysis of epitheliated type CTCs can be applied to tumour identifies
The directions such as diagnosis and prognosis evaluation；Epithelial and stromal mixed type and interstitial type CTCs, which are tested and analyzed, can instruct progressive stage and shift the phase
The transfer and relapse monitoring of tumor patient and curative effect monitoring etc..Therefore, it is obtained in a manner of noninvasive and detection blood of cancer patients
In different subgroups CTCs, the especially CTCs of Epithelial and stromal mixed type (EMT and MET) is to tumor patient clinical tumor early stage
Auxiliary diagnosis, Index for diagnosis, oncotherapy curative effect monitoring and evaluation, tumour metastasis and recurrence monitoring and early warning, individuation
The survival condition for treating guidance and improvement tumor patient suffers from important clinical value.
Programmed cell formula death ligand 1 (Programmedcelldeath1ligand1, PD-L1), also referred to as surface antigen
Break up cluster 274 (clusterofdifferentiation274, CD274), is a kind of intracorporal protein of the mankind, CTCs can lead to
It is overexpressed PD-L1, the tumor microenvironment for the induced synthesis inhibitive ability of immunity that makes to transfer under T cell effector function escapes the anti-of body
Tumor immunity makes tumour cell from the monitoring and removing of body immune system.By detecting the PD-L1 expression of tumour,
Selection PD-L1 positive individuals can help to block the immune tolerance of tumour cell using PD-L1 immunosuppressor, and it is lasting to generate
Antineoplaston effect.The detection method of PD-L1 mainly uses immunohistochemical method at present.This method is considered as PD-
The goldstandard of L1 detection, but surgical indication or there are the cancer patients of surgical contraindication is lacked for part, due to tumor tissues
It can not cut off, fine needle puncture tissue be easy to cause tumour spread again, and PD-L1 status assessment becomes difficult.Therefore, it clinically needs
A kind of noninvasive liquid biopsy method detected by PD-L1 on CTCs assesses the expression status of patient PD-L1.
Quantity of the CTCs in peripheral blood is very rare, only accounts for the 1/10 of peripheral white blood cells6~1/107, therefore for not
Effective enrichment of same type CTCs with detection technique bottleneck is higher, difficulty is bigger, always affect the practical application of CTCs.At present
The enrichment detection of CTCs mainly uses following 2 kinds of forms: 1. relying on anti-EpCAM enrichment capture CTCs and anti-cell keratin
(CK8, CK18 and CK19 etc.) antibody and CD45 identify the method (being represented as CellSearch system) of detection CTCs, such method
Only for the enrichment and detection of epitheliated type CTCs, and blood using amount is big (7.5mL), and can not be enriched with detection has important clinical value
The CTCs of interstitial phenotype and Epithelial and stromal mixed type；2. not depending on capture marker enrichment CTCs (membrane filter method, gradient centrifugation
Deng), although overcoming the limitation of enrichment capture mark object, the enrichment specificity of such mode is not high, and blood using amount is big
(10mL), meeting missing inspection has the CTCs of special biophysical properties, and there are still the CTCs for being only capable of the single subgroup of enrichment detection
Defect, it is most important that still can not be enriched with detection Epithelial and stromal mixed type CTCs.Therefore the enrichment and inspection of routine CTCs
It is big to survey not only blood using amount, and is often confined to the enrichment and detection of the CTCs of single monoid, it is mixed that detection Epithelial and stromal can not be enriched with
The CTCs of mould assembly is influenced due to heterogeneity, dynamic evolution of CTCs etc., the enrichment of single type and detection method very great Cheng
Degree limits the clinical application effect of CTCs.
To overcome existing CTCs enrichment detection technique detection blood using amount big, it is only capable of enrichment detection epitheliated type or interstitial type is a kind of
Table is immunized in the CTCs of single type, the PD-L1 that can not be enriched with the CTCs and its different type CTCs of detection Epithelial and stromal mixed type
The technological deficiency of type is limited to, and the present invention is captured by Epithelial and stromal specificity and detects antibody marlcers, specific immunity inspection
Point antibody marlcers combines with capture dyeing enhancement solution in conjunction with microflow control technique, provide a kind of blood using amount it is small, not only can height
The CTCs of sensitive specifically enrichment detection epitheliated type, interstitial type is imitated, and the detection prior art can be enriched with to the maximum extent not
The Epithelial and stromal mixed type CTCs and epitheliated type PD-L1 positive phenotypes, Epithelial and stromal mixed type PD-L1 sun of detection can be enriched with
Property phenotype and interstitial type PD-L1 positive phenotypes CTCs method, for tumour accurate treatment clinical application provide it is relatively reliable
Diagnosis, treatment and Index for diagnosis evidence.
Summary of the invention
It is big to solve existing CTCs enrichment detection technique detection blood using amount, it is only limitted to enrichment detection epitheliated type or interstitial type list
One monoid CTCs, and the skill of CTCs and different type CTCs the PD-L1 immunophenotype of detection Epithelial and stromal mixed type cannot be enriched with
Art limitation, it is an object of the invention to capture by Epithelial and stromal specificity and detect antibody marlcers, specific immunity inspection
Point antibody marlcers combines with capture dyeing enhancement solution provides a kind of Epithelial and stromal mixed type in conjunction with microflow control technique and PD-L1 is positive
Property circulating tumor cell enrichment detecting method, specially a kind of blood using amount is small, can the special enrichment detection of simple and direct efficient and sensible
Epitheliated type, interstitial type, Epithelial and stromal mixed type CTCs and epitheliated type PD-L1 positive phenotypes, Epithelial and stromal mixed type PD-L1 are positive
The method of phenotype and interstitial type PD-L1 positive phenotypes CTCs.
In addition, Multiple Antibodies will affect the joint efficiency of target cell and antibody when combining, lead to the capture and inspection of CTCs
Surveying specificity reduces.To solve this problem, the present invention also aims to, by capture enhancement solution with dye enhancement solution combine,
The sufficiently antigenic determinant of exposure CTCs cell membrane surface makes the capture of Epithelial and stromal specificity and detection antibody and specific immunity
Checkpoint antibody is easier on the antigen for being incorporated in CTCs cell membrane surface, to improve the combination of capture and detection antibody to CTCs
Specificity and sensibility, while enhancing detection antibody in the fluorescence intensity of CTCs cell membrane surface, make the fluorescence developing shape of CTCs
The more typical complete display of state.
Specific technical solution used by the present invention solves the above problems is as follows:
A kind of enrichment detecting method of Epithelial and stromal mixed type and PD-L1 positive circulating tumor cell, comprising the following steps:
1) preparation of the micro-fluidic immunocapture carrier of the Epithelial and stromal mixed antibody of biotin labeling；
The Epithelial and stromal mixed antibody of the biotin labeling uses the EpCAM (Epithelial Cell Adhesion of biotin labeling
Molecule) antibody and biotin labeling anti-CSV (cell surface vimentin) antibody；
2) separation and concentration of Epithelial and stromal mixed type CTCs；
3) using fluorescein-labeled Epithelial and stromal antibody cocktail to the immune detection of different subtype CTCs；
The fluorescein-labeled Epithelial and stromal antibody cocktail uses fluorescein-labeled anti-PanCK antibody, fluorescence
Anti- Vimentin antibody, the fluorescein-labeled anti-CD45 antibody of element label；
4) using PD-L1 primary antibody and fluorescein-labeled PD-L1 secondary antibody to the different subtype CTCs's captured in step 3)
PD-L1 Phenotypic examination.
In the present invention, is captured using Epithelial and stromal specificity and detect antibody marlcers, specific immunity checkpoint antibody
Marker is combined with capture dyeing enhancement solution to be combined between microflow control technique enrichment detection epitheliated type CTCs, interstitial type CTCs and epithelium
The PD-L1 immunophenotype of matter mixed type CTCs and its different type CTCs；Wherein Epithelial and stromal specificity capture and inspection
Antibody marlcers are surveyed to include: anti-EpCAM (epithelial cell adhesion molecule) antibody, anti-CSV (cell surface vimentin) antibody, resist
PanCK antibody, anti-Vimentin (vimentin) antibody, anti-CD45 antibody；The specific immunity checkpoint antibody labels
Object includes: anti-PD-L1 primary antibody and anti-PD-L1 secondary antibody；This method using Epithelial and stromal specificity capture with detect antibody marlcers,
Specific immunity checkpoint antibody marlcers, which are combined with capture enhancement solution with dyeing enhancement solution, combines microflow control technique not only to solve
Existing CTCs detection technique blood using amount big problem, improves the detection sensitivity of CTCs, and solve simultaneously on this basis
Existing CTCs detection technique of having determined cannot detect the technological deficiency that cannot be enriched with detection Epithelial and stromal mixed type CTCs, not only can be with
Simple and direct efficiently specifically enrichment detection Epithelial and stromal mixed type CTCs, and detection epitheliated type and interstitial type CTCs can be enriched with.
It is used as the preferred technical solution of the present invention below:
In step 1), (epithelium is thin by the EpCAM of biotin labeling in the Epithelial and stromal mixed antibody of the biotin labeling
Intercellular adhesion molecule) antibody mass percent be 35%~60%；In the Epithelial and stromal mixed antibody of the biotin labeling
The mass percent of anti-CSV (cell surface vimentin) antibody of biotin labeling is 25%~50%.
Preferably, microflow control technique is micro-fluidic carrier chip technology；It is furthermore preferred that being to include containing PDMS (poly- diformazan
Radical siloxane) etching herringbone microvovtex rotary fluid path top layer and the micro-fluidic dress containing nanometer substrate coating structure substrate
It sets.
The preparation of the micro-fluidic immunocapture carrier of the Epithelial and stromal mixed antibody of the biotin labeling specifically includes:
Solution of streptavidin is drawn with pipettor, micro-fluidic carrier is added, is placed after being incubated for 3~5h at 10~35 DEG C
In the dry 20~28h of hothouse of the ambient humidity lower than 30%, until no liquid residual in micro-fluidic carrier, micro- with alcohol treatment
Flow control carrier rinses micro-fluidic carrier with PBS buffer solution immediately after；
By anti-CSV (the cell table of anti-EpCAM (epithelial cell adhesion molecule) antibody of biotin labeling and biotin labeling
Face vimentin) antibody mixed liquor, be added in micro-fluidic carrier after 35~39 DEG C of 25~35min of incubation, rushed with PBS buffer solution
Micro-fluidic carrier is washed, is added in capture enhancement solution to micro-fluidic carrier, 10~35 DEG C of 0.5~1.5h of incubation are rushed with PBS buffer solution
Micro-fluidic carrier is washed, the preparation of the micro-fluidic immunocapture carrier of the Epithelial and stromal mixed antibody of biotin labeling is completed, by it
It is spare to be placed in wet box.
The capture enhancement solution be containing mass percentage concentration be 0.2%~2% surfactant, quality percentage it is dense
The BSA (bovine serum albumin(BSA)) and mass percentage concentration that degree is 0.5%~10% are the mixing of 88%~99.3% closing serum
Solution；
In step 2), the separation and concentration of Epithelial and stromal mixed type CTCs is specifically included:
Peripheral blood is diluted with PBS buffer solution, is added to the porous of the gradient centrifugation pipe containing monocyte separation medium later
Above barrier, after centrifugation, PBMCs (monocyte) layer is drawn, and transfer them in sterile centrifugation tube, use cell cleaning solution
It washing PBMCs (monocyte), centrifugation, reject supernatant adds cell cleaning solution and PBMCs (monocyte) is softly resuspended,
Complete the preparation of the rough segmentation chaotropic of CTCs；
The rough segmentation chaotropic of CTCs is drawn, using miniflow pumping system with the slow at the uniform velocity injection step of the injection rate of 3~4mL/h
1) in the micro-fluidic immunocapture carrier of the Epithelial and stromal mixed antibody of the biotin labeling prepared in, it is slow then to draw PBS
Fliud flushing rinses micro-fluidic immunocapture carrier, completes the enrichment of Epithelial and stromal mixed type CTCs, obtains being enriched with Epithelial and stromal mixed
The micro-fluidic carrier of mould assembly CTCs.
In step 3), using fluorescein-labeled Epithelial and stromal antibody cocktail to the immune detection of different subtype CTCs,
It specifically includes:
Take cell fixer, injection step 2) in be enriched in the micro-fluidic carrier of Epithelial and stromal mixed type CTCs, 10~
It after 35 DEG C of 15~25min of reaction, draws PBS buffer solution and rinses micro-fluidic carrier, complete cell and fix；
Cell-permeant liquid is drawn with pipettor, injection is completed in the fixed micro-fluidic carrier of cell, is placed in and is protected from light wet box
In after 10~35 DEG C of 8~12min of effect, draw PBS buffer solution and rinse micro-fluidic carrier, complete cell-permeant；
By fluorescein-labeled anti-PanCK antibody, fluorescein-labeled anti-Vimentin antibody, fluorescein-labeled anti-
CD45 antibody, dyeing block liquid A, dyeing that the mixed liquor of liquid B and antibody diluent is blocked to be added to and the micro- of cell-permeant are completed
In flow control carrier 10~35 DEG C be protected from light 1.5~2.5h of incubation after, with the slow micro-fluidic carrier of PBS buffer solution, then draw nucleic acid
Dyeing liquor injects micro-fluidic carrier, is placed in and is protected from light 10~35 DEG C of 10~20min of incubation in wet box, rinses miniflow with PBS buffer solution
Carrier is controlled, epitheliated type CTCs, Epithelial and stromal mixed type CTCs, the immune detection with interstitial type CTCs are completed, it is aobvious with fluorescence is inverted
Micro mirror observes fluorescence developing situation.
It is further preferred that the fluorescein-labeled anti-PanCK antibody, fluorescein-labeled anti-Vimentin are anti-
The volume ratio that body, fluorescein-labeled anti-CD45 antibody, dyeing block liquid A to block liquid B to mix with dyeing is 1:1:1:1:1.
It is the solution containing FC receptor blocking pharmacon that the dyeing, which blocks liquid A,；It is to contain mouse blood that the dyeing, which blocks liquid B,
Clearly, the solution of one or more of rabbit anteserum, sheep blood serum (including one kind) ingredient；The antibody diluent includes containing quality
Percentage concentration is 2%~10%BSA (bovine serum albumin(BSA)) and mass percentage concentration is 0.3%~5% protein protection stabilizer
Solution.
Preferably, the protein protection stabilizer is the mixture of glycerol and chemical inertness high molecular polymer.It is more excellent
Choosing, chemical inertness high molecular polymer is PEG.
In step 4), using PD-L1 primary antibody and fluorescein-labeled PD-L1 secondary antibody to the different subtype captured in step 3)
The PD-L1 Phenotypic examination of CTCs, specifically includes:
Block liquid A, dyeing that liquid B and antibody diluent mixed liquor is blocked to be added in step 3) anti-PD-L1 primary antibody, dyeing
It in the micro-fluidic carrier that should be completed, is placed in wet box after being protected from light 10~35 DEG C of 1.5~2.5h of incubation, is rinsed with PBS buffer solution micro-
Flow control carrier；
Fluorescein-labeled PD-L1 secondary antibody is slowly added to the miniflow of PD-L1 primary antibody reaction is completed with antibody diluent
It controls in carrier, is placed in wet box after being protected from light 10~35 DEG C of 1.5~2.5h of incubation, rinse micro-fluidic carrier with PBS buffer solution, complete
The PD-L1 Phenotypic examination of different subtype CTCs observes coloration result with inverted fluorescence microscope.
The PD-L1 primary antibody is 1:2:2 with the volume ratio that dyeing blocks liquid A, dyeing that liquid B is blocked to mix；The fluorescence
The percentage by volume of fluorescein label PD-L1 secondary antibody is 2%~5% in the PD-L1 secondary antibody antibody diluent of element label.
The anti-PanCK antibody, anti-Vimentin antibody, anti-CD45 antibody and anti-PD-L1 secondary antibody are respectively marked can
Fluorescein with the different emission being mutually distinguishable.According to well known to a person skilled in the art different special in order to distinguish
Property detection antibody, different fluoresceins that the present invention uses label can be under different optical filters by fluorescence microscope
It distinguishes completely.The fluorescent dye that mark fluorescent element can use this field any commonly employed, it is furthermore preferred that including FITC (different sulphur
Cyanic acid fluorescein), PE (phycoerythrin), Alexa Fluor Series Molecules, (perdinin-chlorophyll-protein is multiple by PerCP
Close object), APC (other phycocyanin), TRITC (tetramethylisothiocyanate rhodamine) etc., fluorescence developing is different can mutual area
Not；
A kind of enrichment detecting method of Epithelial and stromal mixed type and PD-L1 positive circulating tumor cell, comprising the following steps:
1) using the preparation of the micro-fluidic immunocapture carrier of the Epithelial and stromal mixed antibody of biotin labeling
300 μ L solution of streptavidin are drawn with pipettor, micro-fluidic carrier is added, are put after being incubated for 4h at 25 DEG C of room temperature
It is dry for 24 hours to set the hothouse for being lower than 30% in ambient humidity, until no liquid residual in micro-fluidic carrier.With 300 μ L's 95%
The micro-fluidic carrier of alcohol treatment 1 time rinses micro-fluidic carrier twice with PBS buffer solution immediately after.
By biotin labeling anti-EpCAM (epithelial cell adhesion molecule) antibody, biotin labeling anti-CSV (cell surface wave
Shape albumen) 200 μ L of antibody mixed liquor, it is added in micro-fluidic carrier after 37 DEG C of incubation 30min, it is slow with 300 μ L PBS buffer solution
It rinses micro-fluidic carrier twice, is added in 200 μ L capture enhancement solution to micro-fluidic carrier, is incubated at room temperature 1h, it is slow with 300 μ L PBS
Fliud flushing slowly rinses micro-fluidic carrier twice, completes the preparation that Epithelial and stromal mixture captures micro-fluidic carrier, places it in wet
Box is spare.
2) separation and concentration of Epithelial and stromal mixed type CTCs
2mL peripheral blood is diluted with PBS buffer solution 1:1, adds it to the gradient centrifugation containing 3mL monocyte separation medium
It is careful slowly to draw PBMCs (monocyte) layer after 1000 × g of room temperature is centrifuged 10min above the porous barrier of pipe, and by its
It is transferred in sterile centrifugation tube and slowly washs PBMCs using 15mL cell cleaning solution, 300 × g of room temperature is centrifuged 10min, careful to abandon
Except supernatant, adds 1mL cell cleaning solution and PBMCs is softly resuspended, complete the preparation of the rough segmentation chaotropic of CTCs.
Above-mentioned 1mL CTCs rough segmentation chaotropic is drawn using miniflow pumping system, it is slow with the injection rate of 3.6mL/h at room temperature
Slowly at the uniform velocity injection step 1) in the micro-fluidic carrier for preparing, then draw 300 μ LPBS buffers and slowly rinse micro-fluidic load
Body twice, completes the enrichment of Epithelial and stromal mixed type CTCs.
3) immune detection of Epithelial and stromal mixed type CTCs
Take 200 μ L cell fixers, injection step 2) in be enriched in the micro-fluidic carrier of Epithelial and stromal mixed type CTCs,
It after reacting at room temperature 20min, draws 300 μ L PBS buffer solution and rinses micro-fluidic carrier twice, complete cell and fix.
200 μ L cell-permeant liquid are drawn with pipettor, injection is completed in the fixed micro-fluidic carrier of cell, is placed in and is protected from light
In wet box after room temperature effect 10min, draws 300 μ L PBS buffer solution and rinse micro-fluidic carrier three times, complete cell-permeant.
By fluorescein-labeled anti-PanCK antibody, fluorescein-labeled anti-Vimentin antibody, fluorescein-labeled anti-
CD45 antibody, dyeing block liquid A, dyeing to block the 250 μ L of mixed liquor of liquid B and antibody diluent that room temperature in micro-fluidic carrier is added
It is protected from light after being incubated for 2h, slowly rinses micro-fluidic carrier twice with 300 μ L PBS buffer solution, then draw 150 μ L nucleic acid staining liquid
It is slowly injected into above-mentioned micro-fluidic carrier, is placed in be protected from light in wet box and is incubated at room temperature 15min, is slowly rinsed with 300 μ L PBS buffer solution
Micro-fluidic carrier twice, completes epitheliated type CTCs, Epithelial and stromal mixed type CTCs, the immune detection with interstitial type CTCs, with
Set fluorescence microscope fluorescence developing situation.
4) the PD-L1 Phenotypic examination of different subtype CTCs
Block liquid A, dyeing that liquid B and 150 μ L of antibody diluent mixed liquor is blocked to be slowly added to walk PD-L1 primary antibody, dyeing
It is rapid 3) in reacted in the micro-fluidic carrier of completion, be placed in wet box after being protected from light incubation at room temperature 2h, it is slow with 300 μ L PBS buffer solution
Slowly micro-fluidic carrier is rinsed three times.
It is slowly added to above-mentioned PD- be completed after fluorescein-labeled PD-L1 secondary antibody is diluted to 300 μ L with antibody diluent
In the micro-fluidic carrier of L1 primary antibody reaction, it is placed in wet box after being protected from light incubation at room temperature 2h, is slowly rinsed with 300 μ L PBS buffer solution
Micro-fluidic carrier three times, completes the PD-L1 Phenotypic examination of different subtype CTCs, observes coloration result with inverted fluorescence microscope.
Preferably, monocyte separation medium described in step 2) is to contain ficoll (Ficoll) and Angiografin
Human lymphocyte separating liquid, density be 1.077 ± 0.001g/mL；The gradient centrifugation pipe is internal porous containing one
The centrifuge tube of barrier partition can assist the separation of PBMCs, can effectively maintain PBMCs stratification state, convenient for CTCs separation behaviour
Make；The cell cleaning solution is preferably the mixed liquor containing cell culture medium and 2% fetal calf serum.The miniflow pumping system
For what is combined containing changeover valve with pipeline, the micro-fluid pump of minute fluid can be manipulated by control software constant speed quantitative.It is more excellent
Choosing, the maximum volume of the micro-fluid pump is 500 microlitres.
Preferably, cell fixer described in step 3) is preferably the aqueous solution for containing 2%PFA (paraformaldehyde)；It is described
Cell-permeant liquid be preferably the aqueous solution containing 0.4%Triton X-100；The nucleic acid staining liquid is usual cell core
Dye liquor is preferably one of Hoechst33342 or DAPI (4', 6- diamidino -2-phenylindone).
Compared with prior art, the present invention has the following advantages and effects:
This method is captured using Epithelial and stromal specificity and detects antibody marlcers, specific immunity checkpoint antibody labels
Object, which is combined with capture enhancement solution with dyeing enhancement solution, combines microflow control technique to not only solve existing CTCs detection technique blood using amount
Big problem improves the detection sensitivity of CTCs, and solving existing CTCs detection technique simultaneously on this basis cannot
Detection cannot be enriched with the technological deficiency of detection Epithelial and stromal mixed type CTCs, not only can be in simple and direct efficiently specifically enrichment detection
Skin interstitial mixed type CTCs, and detection epitheliated type and interstitial type CTCs can be enriched with.Meanwhile the present invention is mentioned in a manner of noninvasive
A kind of detection method of the PD-L1 immunophenotype of different type CTCs is supplied, simpler and more direct economy compensates for prior art detection
The defect of PD-L1.
In addition, the present invention is combined by capture dyeing enhancement solution, the antigenic determinant of abundant exposure CTCs cell membrane surface,
Sensitivity and specificity of the CTCs capture with detection are increased, CTCs fluorescence developing form is preferably presented, is conducive to cytopathy
Manage form and phenotypic analysis.The present invention is that the liquid biopsy based on CTCs is sentenced in noninvasive accurate diagnosis, treatment and the prognosis of tumour
It is disconnected to provide a kind of relatively reliable economical and practical clinical application mode.
Detailed description of the invention
Fig. 1 is the colour developing schematic diagram of epitheliated type circulating tumor cell；
Fig. 2 is the colour developing schematic diagram of interstitial type circulating tumor cell；
Fig. 3 is the colour developing schematic diagram of Epithelial and stromal mixed type circulating tumor cell；
Fig. 4 is the colour developing schematic diagram of epitheliated type circulating tumor cell PD-L1 positive expression；
Fig. 5 is the colour developing schematic diagram of interstitial type circulating tumor cell PD-L1 positive expression；
Fig. 6 is Epithelial and stromal mixed type circulating tumor cell PD-L1 positive expression colour developing schematic diagram；
In figure: the circulating tumor cell of the above Different groups is all from clinical lung cancer sample, and white arrow indicates not in figure
The CTCs of deme, A are to merge image, and B is Hoechst33342 labeling CT Cs nucleus colour developing image (practical is blue), C
It develops the color image (practical is orange) for CD45-PE, D is PanCk-PerCP colour developing image (practical is peony), and E is
Vimentin-FITC develops the color image (practical for green), and F is that (far infrared, micro- scarnning mirror are assigned to Huang to PD-L1-Alexa 647
Color).
Specific embodiment
Combined with specific embodiments below and attached drawing the invention will be further described.
The detection of expression of the detection of 1 lung cancer circulating tumor cell parting of embodiment and its PD-L1
Material: micro-fluidic carrier uses micro-fluidic chip, and miniflow pumping system uses circulating tumor cell assay instrument (WY-
C3000, the production of Hangzhou Hua get Sen Bioisystech Co., Ltd)
1. the preparation of the micro-fluidic immunocapture carrier of the Epithelial and stromal mixed antibody of biotin labeling
1.1. drawing 300 μ L concentration with pipettor is 5 μ g/mL solution of streptavidin, is added in micro-fluidic chip, normal
Hothouse drying of the ambient humidity lower than 30% is placed on for 24 hours after being incubated for 4h at 25 DEG C of temperature, until no liquid in micro-fluidic chip
Residual.
1.2. 300 μ L volume basis are drawn to state in 95% ethanol injection micro-fluidic chip, it is slow with 1 × PBS immediately after
Fliud flushing is rinsed twice.
1.3. by biotin labeling anti-EpCAM (epithelial cell adhesion molecule) antibody, biotin labeling anti-CSV (cell table
Face vimentin) 200 μ L of antibody mixed liquor (two kinds of mixed mass percents of antibody are as follows: the anti-EpCAM of biotin labeling is anti-
Body 50%；The anti-CSV antibody 50% of biotin labeling), it is added in micro-fluidic carrier after 37 DEG C of incubation 30min, it is slow with 300 μ L PBS
Fliud flushing slowly rinses micro-fluidic chip twice.
1.4. it is drawn in 200 μ L capture enhancement solution injection micro-fluidic chip with pipettor, 25 DEG C of incubation 1h of room temperature.Capture increases
Strong liquid be containing mass percent be 0.4% Triton X-100, mass percent be 3% BSA (bovine serum albumin(BSA))
It is the mixed solution of 87% closing serum with mass percent, surplus is water.
1.5. micro-fluidic carrier is slowly rinsed twice with 300 μ L PBS buffer solution, complete the capture of Epithelial and stromal mixed antibody
The preparation of micro-fluidic chip, it is spare to place it in wet box.
2. the separation and concentration of Epithelial and stromal mixed type CTCs
2.1 dilute 2mL peripheral blood with 2mL PBS buffer solution, and by 4mL dilution blood sample, it is added to containing 3mL monocyte
Separating liquid- 1077 density gradient centrifugation liquidAbove the porous barrier of gradient centrifugation pipe,
25 DEG C of 1000 × g of room temperature are centrifuged 10min.Monocyte separation medium is the people containing ficoll (Ficoll) and Angiografin
Lymphocyte separation medium, density are 1.077 ± 0.001g/mL；Gradient centrifugation pipe is internal containing a porous screen phragma plate
Centrifuge tube can assist the separation of PBMCs, can effectively maintain PBMCs stratification state, be convenient for CTCs lock out operation；
2.2 careful slowly absorption PBMCs (monocyte) layers, and transfer them in sterile centrifugation tube.
2.3 15mL cell cleaning solution is added into centrifuge tube slowly washs PBMCs, and 25 DEG C of 300 × g of room temperature are centrifuged 10min.
Cell cleaning solution is the mixing containing mass percent 98% cell culture medium RPMI1640 and 2% fetal calf serum of mass percent
Liquid.
2.4 with the careful reject supernatant of pipettor, adds 1mL cell cleaning solution and PBMCs is softly resuspended, complete CTCs's
The preparation of rough segmentation chaotropic.
2.5 draw above-mentioned 1mLCTCs rough segmentation chaotropic with miniflow pumping system [circulating tumor cell assay instrument (WY-C3000)],
In the micro-fluidic chip slowly at the uniform velocity prepared in injection step 1 at 25 DEG C of room temperature with the injection rate of 3.6mL/h, then inhale
It takes 300 1 × PBS buffer solution of μ L slowly to rinse micro-fluidic chip twice, completes the enrichment of Epithelial and stromal mixed type CTCs.Miniflow
Pumping system is to combine containing changeover valve with pipeline, and the microfluid of minute fluid can be manipulated by control software constant speed quantitative
Pump.The maximum volume of micro-fluid pump is 500 microlitres.
3. the immune detection of Epithelial and stromal mixed type CTCs
3.1. pipettor takes 200 μ L cell fixers, and the miniflow of Epithelial and stromal mixed type CTCs is enriched in injection step 2
It controls in chip, after 25 DEG C of reaction 20min of room temperature, draws 300 μ 1 × PBS buffer solution of L and rinse micro-fluidic chip twice, complete thin
Born of the same parents fix.Cell fixer uses the aqueous solution containing mass percent 2%PFA (paraformaldehyde)；
3.2. 200 μ L cell-permeant liquid are drawn with pipettor, injection is completed in the fixed micro-fluidic chip of cell, is placed in
It is protected from light in wet box after room temperature effect 10min, draws 300 μ 1 × PBS buffer solution of L and rinse micro-fluidic chip three times, it is logical to complete cell
Thoroughly.Cell-permeant liquid uses the aqueous solution containing mass percent 0.4%TritonX-100；
3.3. the anti-Vimentin antibody of anti-10 μ L of PanCK antibody, fluorescein FITC label fluorescein PerCP marked
Anti- 10 μ L of CD45 antibody of 10 μ L, fluorescein PE label, dyeing block 10 μ L of liquid A, dyeing that 10 μ L of liquid B and antibody is blocked to dilute
200 μ L of liquid is added in micro-fluidic chip after mixing, and 25 DEG C of room temperature are protected from light incubation 2h.It is to contain FC receptor blocking that dyeing, which blocks liquid A,
The solution of agent；It is the solution containing mouse serum and rabbit anteserum that dyeing, which blocks liquid B,；Antibody diluent is to be containing mass percent
2%BSA and mass percent are the solution of 0.5% protein protection stabilizer.Protein protection stabilizer is glycerol and and PEG.
3.4. 300 1 × PBS buffer solution of μ L are drawn with pipettor and slowly rinses micro-fluidic chip twice.
3.5. 150 μ L nucleic acid staining liquid Hoechst33342 are then drawn to be slowly injected into above-mentioned micro-fluidic chip, are placed in
25 DEG C of incubation 15min of room temperature in wet box are protected from light, slowly rinse micro-fluidic carrier twice with 300 μ L PBS buffer solution, complete epithelium
Type CTCs, Epithelial and stromal mixed type CTCs, the immune detection with interstitial type CTCs observe fluorescence developing with inverted fluorescence microscope
Situation.
4. the inverted fluorescence microscope of CTCs parting observes result interpretation
4.1. epitheliated type CTCs phenotype, Fig. 1: Hoechst33342+, PanCK+, Vimentin-, CD45-；
4.2. interstitial type CTCs phenotype, Fig. 2: Hoechst33342+, PanCK-, Vimentin+, CD45-；
4.3. Epithelial and stromal mixed type CTCs phenotype, Fig. 3: Hoechst33342+, PanCK+, Vimentin+, CD45-
5. the PD-L1 Phenotypic examination of different subtype CTCs
5.1. 6 μ L of PD-L1 primary antibody is drawn respectively with pipettor, dyeing blocks 12 μ L of liquid A, dyeing blocks 12 μ of liquid B and anti-
120 μ L of body dilution after mixing, is slowly added in the micro-fluidic chip for having reacted completion in step 3, is placed in wet box and keeps away
Light is incubated at room temperature 2h
5.2. micro-fluidic chip is slowly rinsed three times with 300 1 × PBS buffer solution of μ L.
5.3. the 6 μ L of PD-L1 secondary antibody that fluorescein Alexa 647 is marked is diluted to 300 μ L with 294 μ L antibody diluents
Afterwards, it is slowly added in the micro-fluidic chip that the reaction of PD-L1 primary antibody is completed in 5.2, is placed in wet box and is protected from light 25 DEG C of room temperature incubations
2h；
5.4. micro-fluidic carrier is slowly rinsed three times with 300 μ L PBS buffer solution, complete the PD-L1 table of different subtype CTCs
Type detection, with the coloration result of inverted fluorescence microscope observation PD-L1.
6. the inverted fluorescence microscope of CTCs parting observes result interpretation
6.1. the epitheliated type CTCs of PD-L1 positive phenotypes, Fig. 4: Hoechst33342+, PanCK+, Vimentin-,
CD45-,PD-L1+；
6.2. the interstitial type CTCs of PD-L1 positive phenotypes, Fig. 5: Hoechst33342+, PanCK-, Vimentin+,
CD45-,PD-L1+；
6.3. the Epithelial and stromal mixed type CTCs of PD-L1 positive phenotypes, Fig. 6: Hoechst33342+, PanCK+,
Vimentin+,CD45-,PD-L1+；
2 CTCs of embodiment enrichment detection Effectiveness Comparison experiment
Comparative example: the difference from embodiment 1 is that, conventionally method setting a group (captures antibody in the present embodiment
Anti- PanCK antibody and anti-CD45 antibody is used only using only the detection of anti-EpCAM antibody capture) (capture antibody is used only anti-with b group
Anti- CSV antibody and anti-CD45 antibody is used only in CSV antibody test antibody) comparative experiments is done with the technical program.
Material: 1) breast cancer cell line BT474, MCF7, SKBR3 and MDAMB231 of different times；2) 6 parts of 2ml health
Human peripheral.
BT474 150, MCF7 150, SKBR3 150 are mixed into MDAMB231150 cell suspension and contained
There are 6 parts of suspension of 600 or so cells, in the healthy human peripheral blood for mixing 6 parts of 2ml respectively, is prepared as simulation blood sample.
According to the method in embodiment 1: contrast groups a group 200 μ L biotin labeling anti-EpCAMs (Epithelial Cell Adhesion point
Son) the micro-fluidic immunocapture carrier of Antibody preparation, detects the mass percent that antibody uses anti-PanCK and anti-CD45 antibody 1:1；
The contrast groups b group micro-fluidic immunocapture carrier of 200 μ L biotin labeling anti-CSV (cell surface vimentin) Antibody preparation,
Detect the mass percent that antibody uses anti-CSV and anti-CD45 antibody 1:1；Experimental group c group is using the method preparation in embodiment 1
The micro-fluidic immunocapture carrier of Epithelial and stromal mixed antibody；Separation and concentration, detection and the inversion fluorescence microscopy of above-mentioned 3 groups of CTCs
Sem observation result interpretation is all made of the method in embodiment 1, and each experiment is repeated 1 times, and as a result calculates the capture of epitheliated type CTCs
The capture rate of efficiency, the capture rate of interstitial type CTCs and Epithelial and stromal mixed type CTCs, on capture rate %=chip
Cell quantity/investment cell quantity.
Concrete outcome is as shown in table 1, the results showed that c group completely catches different type CTCs using the method for embodiment 1
It obtains efficiency and is totally higher than the method that tradition single enrichment and detection antibody is respectively adopted in control methods a group and b group.
Table 1
Note: average capture rate+interstitial type CTCs average capture of the overall capture rate %=epitheliated type CTCs of *, CTCs
Rate+Epithelial and stromal mixed type CTCs average capture rate
The part being not described in this specification is all made of structure and principle known in those skilled in the art,
For the prior art.
By foregoing description, those skilled in the art can implement.
In addition, it should be noted that, above-mentioned embodiment is only a preferred solution of the present invention, not to this
Invention makes any form of restriction, it will be understood by those skilled in the art that being subject to technical solution of the present invention and inventive concept
Equivalent substitution or change all should fall within the scope of protection of the appended claims of the present invention.
Claims (11)
1. the enrichment detecting method of a kind of Epithelial and stromal mixed type and PD-L1 positive circulating tumor cell, which is characterized in that including
Following steps:
1) preparation of the micro-fluidic immunocapture carrier of the Epithelial and stromal mixed antibody of biotin labeling；
The Epithelial and stromal mixed antibody of the biotin labeling using biotin labeling EpCAM antibody and biotin labeling
Anti- CSV antibody；
2) separation and concentration of Epithelial and stromal mixed type CTCs；
3) using fluorescein-labeled Epithelial and stromal antibody cocktail to the immune detection of different subtype CTCs；
The fluorescein-labeled Epithelial and stromal antibody cocktail uses fluorescein-labeled anti-PanCK antibody, fluorescein mark
Anti- Vimentin antibody, the fluorescein-labeled anti-CD45 antibody of note；
4) PD-L1 using PD-L1 primary antibody and fluorescein-labeled PD-L1 secondary antibody to the different subtype CTCs captured in step 3)
Phenotypic examination.
2. enrichment detecting method according to claim 1, which is characterized in that in step 1), the biotin labeling
The mass percent of the EpCAM antibody of biotin labeling is 35%~60% in Epithelial and stromal mixed antibody；The biotin
The mass percent of the anti-CSV antibody of biotin labeling is 25%~50% in the Epithelial and stromal mixed antibody of label.
3. enrichment detecting method according to claim 1, which is characterized in that in step 1), the biotin labeling
The preparation of the micro-fluidic immunocapture carrier of Epithelial and stromal mixed antibody specifically includes:
Solution of streptavidin is drawn with pipettor, micro-fluidic carrier is added, is placed on ring after being incubated for 3~5h at 10~35 DEG C
Dry 20~the 28h of hothouse of the border humidity lower than 30%, until no liquid residual in micro-fluidic carrier, micro-fluidic with alcohol treatment
Carrier rinses micro-fluidic carrier with PBS buffer solution immediately after；
The mixed liquor of the anti-EpCAM antibody of biotin labeling and the anti-CSV antibody of biotin labeling is added in micro-fluidic carrier
After 35~39 DEG C of 25~35min of incubation, micro-fluidic carrier is rinsed with PBS buffer solution, capture enhancement solution is added to micro-fluidic carrier
Interior, 10~35 DEG C of 0.5~1.5h of incubation rinse micro-fluidic carrier with PBS buffer solution, and the Epithelial and stromal for completing biotin labeling is mixed
The preparation for closing the micro-fluidic immunocapture carrier of antibody, it is spare to place it in wet box.
4. enrichment detecting method according to claim 3, which is characterized in that the capture enhancement solution is to contain quality hundred
The bovine serum albumin(BSA) and quality that surfactant that point concentration is 0.2%~2%, mass percentage concentration are 0.5%~10%
Percentage concentration is the mixed solution of 88%~99.3% closing serum.
5. enrichment detecting method according to claim 1, which is characterized in that in step 2), Epithelial and stromal mixed type CTCs
Separation and concentration specifically include:
Peripheral blood is diluted with PBS buffer solution, is added to the porous barrier of the gradient centrifugation pipe containing monocyte separation medium later
Mononuclear cell layer after centrifugation, is drawn, and transfer them in sterile centrifugation tube in top, thin using cell cleaning solution washing monokaryon
Born of the same parents, centrifugation, reject supernatant add cell cleaning solution and monocyte are softly resuspended, and complete the preparation of the rough segmentation chaotropic of CTCs；
The rough segmentation chaotropic of CTCs is drawn, using miniflow pumping system with the slow at the uniform velocity injection step 1 of the injection rate of 3~4mL/h) in
In the micro-fluidic immunocapture carrier of the Epithelial and stromal mixed antibody of the biotin labeling prepared, PBS buffer solution is then drawn
Micro-fluidic immunocapture carrier is rinsed, the enrichment of Epithelial and stromal mixed type CTCs is completed, obtains being enriched with Epithelial and stromal mixed type
The micro-fluidic carrier of CTCs.
6. enrichment detecting method according to claim 1, which is characterized in that in step 3), on fluorescein-labeled
Skin interstitial antibody cocktail specifically includes the immune detection of different subtype CTCs:
Take cell fixer, injection step 2) in be enriched in the micro-fluidic carrier of Epithelial and stromal mixed type CTCs, 10~35 DEG C
It after reacting 15~25min, draws PBS buffer solution and rinses micro-fluidic carrier, complete cell and fix；
Cell-permeant liquid is drawn with pipettor, injection is completed in the fixed micro-fluidic carrier of cell, is placed in and is protected from light 10 in wet box
After~35 DEG C of 8~12min of effect, draws PBS buffer solution and rinse micro-fluidic carrier, complete cell-permeant；
Fluorescein-labeled anti-PanCK antibody, fluorescein-labeled anti-Vimentin antibody, fluorescein-labeled anti-CD45 are resisted
Body, dyeing block liquid A, dyeing that the mixed liquor of liquid B and antibody diluent is blocked to be added to the micro-fluidic load that cell-permeant is completed
After internal 10~35 DEG C are protected from light 1.5~2.5h of incubation, with the slow micro-fluidic carrier of PBS buffer solution, nucleic acid staining liquid is then drawn
Micro-fluidic carrier is injected, is placed in and is protected from light 10~35 DEG C of 10~20min of incubation in wet box, rinses micro-fluidic carrier with PBS buffer solution,
Epitheliated type CTCs, Epithelial and stromal mixed type CTCs, the immune detection with interstitial type CTCs are completed, is observed with inverted fluorescence microscope
Fluorescence developing situation.
7. enrichment detecting method according to claim 6, which is characterized in that in step 3), described is fluorescein-labeled
Anti- PanCK antibody, fluorescein-labeled anti-Vimentin antibody, fluorescein-labeled anti-CD45 antibody, dyeing block liquid A and dye
The volume ratio of the disconnected liquid B mixing of color blocking is 1:1:1:1:1.
8. enrichment detecting method according to claim 6, which is characterized in that in step 3), the dyeing blocking liquid A is
Solution containing FC receptor blocking pharmacon；The dyeing block liquid B be containing one of mouse serum, rabbit anteserum, sheep blood serum with
The solution of upper ingredient；It is 2%~10% bovine serum albumin(BSA) and matter that the antibody diluent, which includes containing mass percentage concentration,
Measure the solution that percentage concentration is 0.3%~5% protein protection stabilizer.
9. enrichment detecting method according to claim 1, which is characterized in that in step 4), using PD-L1 primary antibody and fluorescence
PD-L1 Phenotypic examination of the PD-L1 secondary antibody of element label to the different subtype CTCs captured in step 3), specifically includes:
It blocks liquid A, dyeing that liquid B is blocked to be added in step 3) with antibody diluent mixed liquor PD-L1 primary antibody, dyeing to have reacted
At micro-fluidic carrier in, be placed in wet box after being protected from light 10~35 DEG C of 1.5~2.5h of incubation, with PBS buffer solution rinse it is micro-fluidic
Carrier；
Fluorescein-labeled PD-L1 secondary antibody is slowly added to the micro-fluidic load of PD-L1 primary antibody reaction is completed with antibody diluent
It in body, is placed in wet box after being protected from light 10~35 DEG C of 1.5~2.5h of incubation, rinses micro-fluidic carrier with PBS buffer solution, complete different
The PD-L1 Phenotypic examination of subtype C TCs observes coloration result with inverted fluorescence microscope.
10. enrichment detecting method according to claim 9, which is characterized in that in step 4), the PD-L1 primary antibody and dye
Color blocking break liquid A, dyeing block liquid B mixing volume ratio be 1:2:2.
11. enrichment detecting method according to claim 9, which is characterized in that in step 4), described is fluorescein-labeled
The percentage by volume of fluorescein label PD-L1 secondary antibody is 2%~5% in PD-L1 secondary antibody antibody diluent.
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