CN110029141A - A kind of method of testing drug to the breeder reaction for the tumour cell for being originated from patient - Google Patents
A kind of method of testing drug to the breeder reaction for the tumour cell for being originated from patient Download PDFInfo
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- CN110029141A CN110029141A CN201910148989.3A CN201910148989A CN110029141A CN 110029141 A CN110029141 A CN 110029141A CN 201910148989 A CN201910148989 A CN 201910148989A CN 110029141 A CN110029141 A CN 110029141A
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
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Abstract
The present invention relates to use of the 3D cell culture technology in vitro drug testing method, more particularly to a kind of testing drug to the method for the breeder reaction for the tumour cell for being originated from patient, it can reliably can screen drug candidate by predictive proliferation assay, be used for effect and/or mechanism of action；The following steps are included: (1) obtains cell from biopsy or tumor resection material；(2) cell of acquisition is cultivated on 3D extracellular matrix；(3) tumour cell that processing is cultivated on 3D extracellular matrix is cured with medicine；(4) tumour cell for crossing step process is subjected to high-content imaging；(5) the high-content imaging for the tumour cell that assessment processing is crossed, thus breeder reaction of the testing drug to the tumour cell for being originated from patient.
Description
Technical field
The present invention relates to use of the 3D cell culture technology in vitro drug testing method, more particularly to a kind of survey
Method of the reagent object to the breeder reaction for the tumour cell for being originated from patient.
Background technique
Currently, the trend of drug development and disease correlation studies is to progressively disengage making for non-human primate model
With recently, in NIH research, the use of chimpanzee is significantly reduced, this trend has more been highlighted, in addition, being based on 3D cell
The method of cultivation platform can make cell obtain natural 3D phenotype, and can enhance cell Proliferation, differentiation and function, and know
Traditional 2D (two dimension) cell culture processes can not accurately indicate the progression of disease in the true world 3D, drug test and/or
Biochemistry and Physiologic Studies.Therefore, people already and will the advanced technology based on vitro platform of continual exploitation, utilize
Complicated bracket and extracellular matrix carrys out sertoli cell growth.
However, certain limitation that current 3D cell culture technology has, including undesirable reproducibility and stability,
The complexity of component, the difficulty zoomed in or out, and/or improve the demand of physiological medium.Specifically, in 3D growth ring
The antiproliferative of oncology drug candidate being tested in border, using the tumour cell for being originated from patient predicts measurement, can be because of height
Degree is heterogeneous, the low availability of low proliferation and/or sufficiently characterization sample, and generates uncertain as a result, therefore, this field
It needs to overcome such limitation.
3D cell culture processes and the relevant technologies and device can be in Publication No. 20110143960 and 20120309016
United States Patent (USP) in find；But the embodiment in the undisclosed present invention of these bibliography and/or corresponding document.
Cited all documents and bibliography be herein by reference herein and in referenced patent document
It is integrally incorporated herein.
Summary of the invention
In order to solve the above technical problems, the present invention provides one kind can reliably can be sieved by predictive proliferation assay
Drug candidate is selected, for the testing drug of effect and/or mechanism of action to the side of the breeder reaction for the tumour cell for being originated from patient
Method.
The present invention provides a kind of highly sensitive spherical originally culture platforms of 3D, including are covered on extracellular matrix
3D globuli cell, drug therapy example, vitro proliferation terminal, and high-content cell imaging, number for feasibility opinion
It according to analysis and explains, wherein 3D globuli cell is primary tumor or the tumour cell from patient.Specifically, of the invention
A kind of innovative approach is provided, the sample and/or Tumor biopsy samples biology of retrospective annotation and perspective acquisition is utilized
Library and relevant molecule and treatment data, the optimal conditions for making sample grown 3D orbicule, the high-content as reading
Imaging, and for the optimization label of subspecies cluster analysis.
A kind of method of testing drug of the invention to the breeder reaction for the tumour cell for being originated from patient, including following step
It is rapid:
(1) cell is obtained from biopsy or tumor resection material；
(2) cell of acquisition is cultivated on 3D extracellular matrix；
(3) tumour cell that processing is cultivated on 3D extracellular matrix is cured with medicine；
(4) tumour cell for crossing step process is subjected to high-content imaging；
(5) the high-content imaging for the tumour cell that assessment processing is crossed, so that testing drug is to the tumour cell for being originated from patient
Breeder reaction.
A kind of method of testing drug of the invention to the breeder reaction for the tumour cell for being originated from patient, the step (1)
Further include by the cell xenograft of acquisition into mouse to form tumour, and tumour cell is obtained from mouse.
A kind of method of testing drug of the invention to the breeder reaction for the tumour cell for being originated from patient, the living tissue
It checks or tumor resection material comes from biological library.
A kind of testing drug of the invention is described processed to the method for the breeder reaction for the tumour cell for being originated from patient
Tumour cell be in the formation of tumour sphere.
A kind of method of testing drug of the invention to the breeder reaction for the tumour cell for being originated from patient, the drug choosing
From small-molecule drug, kinase inhibitor, macromolecular and combinations thereof.
A kind of testing drug of the invention to be originated from patient tumour cell breeder reaction method, it is described be originated from suffer from
The tumour cell of person is primary tumors cells.
A kind of testing drug of the invention to be originated from patient tumour cell breeder reaction method, it is described be originated from suffer from
It is thin that the tumour cell of person is selected from breast cancer cell, prostate gland cancer cell, non-small cell lung cancer cell, ovarian cancer cell, melanoma
Born of the same parents and pancreatic cancer cell.
A kind of testing drug of the invention is to the method for the breeder reaction for the tumour cell for being originated from patient, and the proliferation is instead
EdU incorporation, work-dead cell counts, Colony forming and combinations thereof should be selected from.
A kind of testing drug of the invention can pass through the method for the breeder reaction for the tumour cell for being originated from patient
Proliferation, colony form, Apoptosis and combinations thereof the processed tumour cell of technology evaluation.
Compared with prior art the invention has the benefit that
The access in the primary tumo(u)r library to high quality and unique quantization is utilized in method of the invention, to try in clinic
It tests in design and obtains high-caliber professional technique, knowledge and information, the sample of retrospective annotation and perspective acquisition is utilized
And/or Tumor biopsy samples biology library and relevant molecule and treatment data, the optimization for making sample grown 3D orbicule
Condition, the high-content imaging as reading, and for the optimization label of subspecies cluster analysis.
Method of the invention provides a kind of resisting for measurement anticancer agent in physiology relevant environment for vitro proliferation measurement
The effective ways of proliferation activity.By in biopsy tumour cell or excision material be used as and single cell suspension or be used for
Cell culture, and adjust form orbicule in vitro, to simulate tumor environment.In treatment end, it is imaged and is surveyed using high-content
Drug candidate is measured, to measure the antiproliferative potentiality of drug candidate, to measure the proliferation of individual cells.
By using method of the invention, tumour sphere can reliable simulation Tumor Microstructure, and it is specific by dyeing
Label can detect and analyze foreign cell group.In addition, primary tumors cells (PTC) sample is attractive model,
The reason is that the sample can reflect the heterogeneity of cancer, and it can preferably predict the reaction to anticancer agent.
Detailed description of the invention
Figure 1A-B, which is shown using the xenograft (PDX) for being originated from patient, obtains cell, carries out vitro proliferation measurement
Mode；
Fig. 2A-C: Fig. 2A shows that the z- of orbicule is stacked, and carries out maximal projection, then to capture cell image；Fig. 2 B
It shows through the channel DAPI and EdU captured image；Fig. 2 C shows the drug candidate of the EdU MFI on multiple plates
Compare data；
A-B: Fig. 3 A of Fig. 3 is shown using 2 kinds of chemotherapy drug candidates, the image of the spherical volume morphing of capture；Fig. 3 B
Show the sphere size data of processed cell；
Fig. 4 shows the image and data for carrying out apoptosis induction to the processed PTC of neural spore rhzomorph；
A-C: Fig. 5 A of Fig. 5 shows the image of DAPI, EdU and CK dyeing；Fig. 5 B-C shows processed cell subsets
In Proliferation data.
Specific embodiment
With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is used for
Illustrate the present invention, but is not intended to limit the scope of the invention.
Embodiment 1
Vitro proliferation analysis
The tumour cell from patient is set to thaw in 37 DEG C of water-baths, until the cell suspending liquid of freezing sufficiently melts, with
Mud solution is formed, cell suspending liquid is slowly transferred in 50ml conical pipe by cell filter, and is filled out into conical pipe
Completely it is warmed to 37 DEG C of conditioned medium；Conical pipe 5min is rotated with 800RPM at 4 DEG C, makes cell precipitation；Pass through suction
The method of supernatant carefully removes supernatant without interfering cell precipitation, then by cell precipitation and is resuspended in 1-2ml warm
In conditioned medium, cell is counted using TC10,10 μ l cell suspending liquids is finally taken out and is transferred to and surveyed for trypan blue exclusion
In fixed conduit, to assess the general health of cell in cell density and bottle, work-dead cell is counted.
With(ECM from Trevigen) coats 6 orifice plates, with the ice for being free of growth factor and replenishers
Cold conditioned medium dilutes ECM to 1:20, and the diluted Cultrex solution of 1ml or every 100mm training is added in every hole in 6 orifice plates
It supports ware and the diluted Cultrex solution of 3ml is added, 6 orifice plates are incubated into 2h at 37 DEG C, the 6 orifice plates of coating are placed at room temperature
Coating Solution is carefully sucked out in 5-10min in cell culture hood, immediately inoculating cell immediately, by with cell flask or
Culture dish is transferred under microscope from incubator, and observes cell under the microscope, to determine the percentage converged, assessment
The rough valuation of cell percentages in region without cell covering in flask or culture dish, after cell, which reaches 70%, to be converged,
It is separated/is passed on.
Transport medium, trypsin-EDTA solutions and sterile PBS are warmed to 37 DEG C in a water bath, and incubate 15-
Division/passage cell supernatant is transferred in the conical pipe of 15-50ml by 30min, and it is sterile that 1ml is added in the every hole of 6 orifice plates
The sterile PBS of 5ml is added in PBS or every 100mm culture dish, to cover single layer, by tilting flask or culture dish in two directions
2-3 times, the cleaning solution in rinsing liquid is transferred in the 15-50ml conical pipe containing supernatant, 6 orifice plates by Lai Chongxi single layer
1ml trypsin-EDTA solutions are added in every hole or 5ml trypsin-EDTA solutions are added in every 100mm culture dish, to cover list
Layer.By flask or culture dish in CO25-7min is incubated in incubator, the PTC grown on the ECM of 1:20 is needed and trypsase
It incubates together and is up to 10min, observe cell under the microscope, and gently beat the side of flask or culture dish, observe cell
It is separated into unicellular, after cell separates and floats, that is, isometric culture medium containing FBS (fetal calf serum) is added, also referred to as
For chemosensitivity culture medium, (FBS can make trypsin inactivation, and reduce the injury to cell to the maximum extent) obtains thin
The entire contents of flask or culture dish are transferred in 50ml conical pipe by born of the same parents, by conical pipe with 200XG RPM centrifugation
Culture medium is carefully sucked out in the case where not interference particle in 5min, the conditioned medium of 1-2ml is added, on lightly
Under aspirate 5 times, suspend precipitating again, and counts cell.
Cell is subjected to secondary culture on the ECM of 1:20 in a manner of 3D, supernatant is removed, tilts plate, and does not have to move
The bottom of liquid pipe contact plate, obtains cell from ECM, rinses cell with PBS, 1ml trypsase-is added in the every hole of 6 orifice plates
5ml trypsase-EDTA is added in EDTA or every 10cm culture dish, and cell is incubated 5min at 37 DEG C；Using scraper from hole
Cell is proposed, and using the cell in chemosensitivity medium washes scraper, so that trypsin inactivation, and by flask or training
Feeding ware is put back in incubator.
By the chemical entities of the medicament in measurement use, to prepare certain density drug stock solution, according to label
The information that (such as obtaining from suppliers) or sponsor provide collects information.Before opening bottle, if it is in lower temperature
Lower storage, then incubate 30min for drug at room temperature, weighs the desired amount of drug, and the desired amount of DMSO is added, and (dimethyl is sub-
Sulfone), and it is mixed sufficiently so that dissolution, it is carried out according to 50-250 μ l by equal part according to mixed total volume, and mark storage
Pipe, and memotron is divided into four parts, wherein two parts are stored at -80 DEG C；Portion is stored at -20 DEG C；Portion is stored in 2-8
At DEG C.
It prepares the working stock of drug solution: stock solution being diluted in suitable cell culture medium, obtains 10ml's
The working stock of 1mM, by 0.22 μm of syringe filter by filter sterilizing solution.
By the 1mM working stock or 1 μM of stoste (10ml: by the medium that dilute drug in cell culture medium
It is prepared by dilution 1mM stoste 1000X) prepare drug dilution liquid.The phase in Nunc deep-well plates is added in 1ml drug dilution liquid
Position is answered, and corresponding position is added in suitable drug.
Method by handling cell 48h with 10 μM of EdU is marked thin with EdU (5- acetenyl -2'- BrdU)
Born of the same parents.By the volume (every 100 μ l of hole) of the fixed solution (37% formaldehyde in PBS) needed for measuring, to fix cell.Passing through will
37% formalin is mixed with the ratio of 1:10 with daily freshly prepared 1X PBS, to prepare fixed solution.From incubator
It is middle to take out the plate for having cell, and by after 45-90 ° of plate inclination, 200 μ l culture mediums carefully are taken out from side, in room
The PBS of 100 μ l is added under temperature into every hole, the liquid of 80-90% in every hole is removed using 200 μ l pipettes, 200 μ are added
LPBS removes the liquid of 80-90% in every hole.100 μ l fixed solutions are added, plate is incubated into 15- in the dark at room temperature
20min.100 μ lPBS are added, the liquid of every hole 80-90% is removed using 200 μ l pipettes.Other 200 μ lPBS is added, it will
Plate incubates 5-10min in the dark.Remove the liquid of 80-90%.100 μ lPBS are added.
By the volume (every 100 μ l of hole) of the permeabilization solution (the 0.1%Triton X 100 in PBS) needed for measuring, make
Cell permeabilization by mixing the Triton X 100 of 10 μ l with the daily freshly prepared 1X PBS of 10ml, and passes through 1 μm
Syringe filter filtering, to prepare permeabilization solution.After last time is washed, 100 μ l permeabilization buffers are added, by plate in room
Temperature is lower to incubate 15-20min, and 100 μ lPBS are added, remove the liquid of 80-90% from every hole using 200 μ l pipettes, are added
Other 200 μ lPBS, incubates 5-10min for plate, removes the liquid of 80-90% in every hole.
By measuring Click-The total volume of HCS (high-content screening) mixture carries out Click- to cell
EdU dyeing, by the way that 2ml distilled water is added in labeled as the bottle of component E and is sufficiently mixed, to prepare Click-EdU is slow
Additive is rushed, for the first time after use, buffer additive is stored at -20 DEG C；By in distilled water with the dilution proportion of 1:10
10X solution prepares the buffer additive of 1X concentration, by distilled water with the dilution proportion 10X solution (component of 1:10
C) Click- of 1X concentration is preparedEdU reaction buffer；Alex Fluor Azide (component B) is thawed, in measurement
Before, component is mixed to prepare mixture in the following order: 1X Click-EdU reaction buffer (component A), copper sulphate
Solution (component D), Alexa Fluor azide (component B) and 1X Click-EdU buffer additive (component E)；
According to following amount:
1X Click-EdU reaction buffer: 12 holes (640 μ l), 24 holes (1.28ml), 48 holes (2.55ml), 96 holes
(5.1ml), 144 holes (7.7ml), 192 holes (10.2ml)；Copper-bath: 12 holes (30 μ l), 24 holes (60 μ l), 48 holes (120
μ l), 96 holes (240 μ l), 144 holes (360 μ l), 192 holes (480 μ l)；The hole Alexa Fluor Azide:12 (1.9 μ l), 24
Hole (3.75 μ l), 48 holes (7.5 μ l), 96 holes (15 μ l), 144 holes (22.5 μ l), 192 holes (30 μ l)；Click-EdU is slow
Rush additive: 12 holes (75 μ l), 24 holes (150 μ l), 48 holes (300 μ l), 96 holes (600 μ l), 144 holes (900 μ l), 192 holes
(1.2ml)。
Withdrawing plate simultaneously removes last cleaning solution.45-50 μ l mixture is added in every hole, at room temperature in the dark by plate
30-60min is incubated, reaction mixture, and 100 μ lLisod- being added under being stored at 4 DEG C are removedReaction rinsing buffering
Plate is incubated 5-10min by liquid in the dark.It removes wash buffer and 200 μ lPBS is simultaneously added, by plate Incubation in dark at room temperature
5-10min。
By the volume (every 50 μ l of hole) of the DAPI solution needed for measuring, DAPI (4', 6'- diamidino-is carried out to cell
2-phenylindone, dihydrochloride) (core) dyeing, by mixing 10 μ l stock solutions in 10mlPBS, to prepare 1X DAPI
Solution.50 μ lDAPI solution are added into every hole, by cell Incubation in dark 30min at room temperature, pass through 100 μ lPBS of addition
Excessive DAPI is washed, and removes the liquid of 80-90% in every hole using 200 μ l pipettes.Other 200 μ lPBS is added,
The liquid that 200 μ l pipettes remove 80-90% in every hole is reused, 50 μ lPBS are added before analysis, is imaged and is read by HC
Hole is taken, and calculates antiproliferative effect of drug.
Embodiment 2
The use of xenograft (PDX) from patient
By fresh or inventory the tumor cell injection from patient into the mouse of immunologic inadequacy, obtains and spread
Plate cell, using chemotherapy drug candidate, cis-platinum and Doxorubicin, carries out in vitro drug to orbicule and controls to form orbicule
It treats, is imaged by the HC of EdU incorporation measurement, measures cell Proliferation.(Figure 1A-B)
Using many algorithms for corresponding to experiment, capture and processing cell image.DAPI is selected on channel 1, with identification
Effective object selects EdU on channel 2, to identify the cell mass of incorporation DNA, records and integrate each pixel in each channel
Intensity, by overall strength divided by total pixel, obtain the average fluorescent strength (MFI) in the channel, Duo Gexi to obtain overall strength
The integral mean fluorescence intensity of born of the same parents obtains the average value of MFI divided by the total cell number in every hole, in order to make cell number between hole
Changeability standardization assesses external drug effect, EdU MFI is for more multiple flat using the average value of the MFI of EdU signal
Drug effect on plate.(Fig. 2A-C)
Use spherical morphology as to grown with orbicule, that the tumour cell from patient plays is pharmaceutically-active
Measurement carries out nucleus (DAPI), EdU (proliferative cell) and cytokeratin (CK) (epithelial cell) dyeing and HC imaging,
The size of sphere or tumour growth can be used for assessing the effect of the drug candidate as chemotherapeutant.(Fig. 3 A-B)
Other terminals (for example, apoptosis-inducing), it can also be used to assess drug candidate (for example, cephalosporin) in head
Effect in neck, lung cancer, breast cancer and ovarian cancer cell.Apoptosis: CellEvent is usedTMMeasurement mean intensity lures
It leads, and is quantified using HC imaging.Amount.(Fig. 4)
Carry out the subgroup analysis of tumor tissues.By the way that EdU incorporation to be measured to the dyeing group with tumour-specific markers object
It closes, assesses effect of the drug candidate in specific cells group, use CK as the label of epithelial cell (mainly tumour cell)
Object.The fluorescent image of method analysis orbicule is figured, using image cytometry to observe the proliferation in cell subsets and analyze drug
Target effect.(Fig. 5 A-C)
The present invention provides one kind to be used for the culture of 3D globuli cell and/or vitro proliferation method for measuring.By extracellular
Matrix (ECM) copying and saving or fresh primary tumors cells (PTC) or the tumour cell from patient, to increase again
Into the number of vegetative state.With the combination of single medicine or drug candidate, medicament and/or drug candidate or medicament, to 3D
Globuli cell culture is handled.High sensitivity and the imaging of accurate high-content illustrate multiple terminals in the prior art,
For example, vegetative state, Apoptosis, form and/or surface marker.
3D cell culture technology is well known, and technical staff is easily mastered.Such technology is continuously developed, and just
It is more and more related to human and animal's physiology.For example, Haycock, JW have looked back the 3D technology in " molecular biology method "
And method.2011；695:1-15.
In one embodiment of the present of invention, develops and exclusively obtained DNA, RNA to match in Cell Cryopreservation
The adjoint biological library (retrospective annotation and perspective acquisition) being characterized with protein.From the tumor biopsy of patient or
Then its freezen protective is biological integrity sample living to generate and constantly expand biological library by the cell obtained in resection
Product.The biological sample data of tumour cell include but is not limited to DNA (mutation, duplication number, transposition), RNA (gene expression,
Spliced variants) and the related data of protein (antigen detection and size variation).Based on being taken to DNA, RNA and/or protein
Sample probability, patient select example, cell screening, Phenotype typing and/or novel marker exploitation, such data to can provide high level
Quality assurance.Therefore, biological library provides a whole set of molecular testing ability, has the swollen of target molecule feature to screen
Tumor.
Method of the invention provides a kind of resisting for measurement anticancer agent in physiology relevant environment for vitro proliferation measurement
The effective ways of proliferation activity.By in biopsy tumour cell or excision material be used as and single cell suspension or be used for
Cell culture, and adjust form orbicule in vitro, to simulate tumor environment.In treatment end, it is imaged and is surveyed using high-content
Drug candidate is measured, to measure the antiproliferative potentiality of drug candidate, to measure the proliferation of individual cells.
In another embodiment, this method includes using animal xenograft object (for example, mouse animal xenograft object).
For example, being originated from patient's by fresh or inventory the tumor cell injection from patient into the mouse of immunocompromised host to develop
Xenograft (PDX).It is inoculated with the cell obtained, to form the orbicule for being used in vitro drug therapy.It is mixed and is surveyed by EdU
Method measurement cell Proliferation is determined, to assess drug candidate or medicament (for example, chemotherapy (for example, cis-platinum, Doxorubicin))
Effect.
In embodiment, this method include transhipment culture medium, enzymic digestion, cryoprotector, defreezing method, liquidating plan,
Vitality assessment, regulation technology, control fibroblast, culture medium selection, ECM, orbicule type, imaging technique, segmentation, in advance
Survey any one or more of analysis and/or subgroup analysis.
In embodiment, this method include using freezen protective or fresh primary tumors cells (PTC), bring back to life and/
Or the combination of vegetative state, single medicine and/or drug therapy example is reentered, and/or for analyzing proliferation, apoptosis, shape
The imaging of the high-content of state and/or surface marker.
In embodiment, this method includes the proliferation assay of tumour sphere, subgroup analysis and/or colony somatometry of physique.Increase
Cell colonization mixes EdU, usesMicro (the U.S. paddy molecule instrument positioned at the California city Sen Niweier
Device company) cell is imaged and is quantified.By using method of the invention, tumour sphere being capable of reliable simulation tumour
Micro-structure, and by dyeing specific markers, it can detect and analyze foreign cell group.In addition, primary tumors cells (PTC) sample
Product are attractive models, the reason is that the sample can reflect the heterogeneity of cancer, and can preferably be predicted to anticancer agent
Reaction.
Primary tumors cells (PTC) can form orbicule.Orbicule is complicated structure, needs z- to stack, then leads to
Maximal projection is crossed, capture cell image is imaged by high-content.After z- stacking, characterized using various algorithm process images
Cell.DAPI (channel 1) is for identifying effective object.Edu (channel 2) is used to identify the cell mass of incorporation DNA.It is specific at this
In the case of, the present inventor is assigned with white mask to identify negative (-) cell of EdU and red mask and identify the EdU positive
(+) cell.But representative mask/door can be distributed to positive and negative cells, to carry out population analysis.It records and integrates every
The intensity of each pixel in a channel, to obtain overall strength.Overall strength show that the mean fluorecence in the channel is strong divided by total pixel
It spends (MFI).The integral mean fluorescence intensity of multiple cells obtains being averaged for average fluorescent strength divided by the total cell number in every hole
Value.In order to standardize the changeability of cell number between hole, the average value of the average fluorescent strength of EdU signal can be used to comment
Estimate external drug effect.EdU average fluorescent strength is for the drug effect on more multiple plates.
Spherical-like morphology or colony form can be used as to the drug grown with orbicule, the tumour cell from patient plays
The measurement of effect.In this way, the size or tumour growth of orbicule are used to assess the effect of chemotherapeutant.Other ends
Select the effect that can also be used for assessment drug candidate.For example, using the apoptosis induction in CellEventTM measurement cell, and use
High-content imaging is quantitative.
The subgroup analysis of tumor tissues is related to many methods.By the way that EdU incorporation is measured and tumour-specific markers object
Dyeing composition can assess effect of the drug candidate in specific cells group.It is (main that cytokeratin (CK) is used as epithelial cell
Tumour cell) marker.The fluorescent image of method analysis orbicule is figured, using image cytometry to observe cell subsets
In proliferation, and analyze influence of the drug to cell population of interest.
The access in the primary tumo(u)r library to high quality and unique quantization is utilized in method of the invention, to try in clinic
It tests in design and obtains high-caliber professional technique, knowledge and information.Assuming that the researching and designing of driving is related to vitro treatment research,
Marker frequency (for example, marker (+) and marker (-) tumour) is assessed by the combination of single medicine or drug example.
Assuming that developmental research design can determine the marker exploitation of respondent and non-response person group and/or clinical test.Therefore, originally
Method includes subgroup analysis, which can provide the steady sensitivity of native tumoral cell and accurate quantitative analysis, the 3D in 3D matrix
Matrix can be by having multichannel and multi-endpoint (for example, nuclear staining (DAPI, Hoechst), epithelial cell, thin in the prior art
The open channel of born of the same parents' keratin (CK), proliferative cell (EdU) and/or other markers) high-content imaging measure.
It (does not include exit orifice that 3D cell covering on extracellular matrix, which includes with the processing that 60 hole formats carry out in triplicate,；
Culture medium in exit orifice).The researching and designing carried out using the 3D cell covering with primary tumors cells or PTC, including increase
Grow terminal.Research variable include but is not limited to dosage range, administration frequency, exposure duration, measurement the duration, pharmaceutical agent combinations,
Addition sequence, addition time, relative effectivenes and/or concentration.Researching and designing variable includes but is not limited to mechanism of drug action
(MOA), protein kinase (PK), compound stability, clinical program, target expression and/or cell activity.
This method includes the image analysis of 3D sphere originally culture model platform.In some embodiments, the method makes
WithMicro (the Mei Gu molecule instrument company positioned at California) capture PTC and/or3.1.0.81 high-content image, to analyze image.Custom pack " multi-wavelength cell score " can be used for really
The EdU signal in DAPI signal and channel 2 (channel Cy5) in routing 1.The algorithm includes (channel 1) DAPI, for knowing
Ineffective object (denominator) and EdU (channel 2), to identify the cell mass for including DNA (molecule).It records and integrates each pixel
With the intensity in each channel, to obtain overall strength.Overall strength obtains the average fluorescent strength in the channel divided by total pixel
(MFI).The synthesis MFI of multiple cells obtains the average value of MFI divided by every hole total cell number.In order to make cell number between hole
Changeability standardization, can be used the MFI average value of EdU signal.
Method of the invention should make together with the tumour cell (for example, primary tumors cells (PTC)) for being originated from patient
With.Tumour cell from patient can for any tumor type cell (for example, breast cancer cell, prostate gland cancer cell, non-
Small cell lung cancer cell, ovarian cancer cell, melanoma cells and pancreatic cancer cell).
Method of the invention can measure and assessment breeder reaction, including but not limited to EdU incorporation, work-dead cell counts,
Colony forming and/or combination thereof.
Method of the invention also uses assessment technology, including but not limited to proliferation, colony form, Apoptosis and/or
A combination thereof.
Clinical test is constantly subjected to the puzzlement of unselected PATIENT POPULATION, these PATIENT POPULATIONs do not benefit from any given survey
Try therapy.It is especially true in cancer field.Therefore, people are increasingly ready to diagnose and/or select or screening test, come
Measurement and/or analysis and/or discovery biomarker and/or other terminals.Therefore, The inventive process provides a kind of machines
System, by the mechanism, clinical test therapy can benefit from more patients for selectively depending on target therapeutic agent.Therefore,
Patient's selection scheme can make the clinical benefit of people's improvement target therapeutic agent, and excluding can not be from the treatment of target therapeutic agent
Benefited patient.
Drug and/or drug candidate and/or drug agent include but is not limited to biomolecule, for example, nucleic acid and amino acid are big
Molecule, kinase inhibitor, chemically synthesized active material small molecule, the synthesizing amino acid macromolecular being connect with small molecule and/or
A combination thereof.Commonly known small molecule is low molecular weight compound, is typically used as enzyme matrix or the regulator as bioprocess.
In certain embodiments, lesser macromolecular (for example, RNA interfering (RNAi) and Microrna (miRNA) molecule) can be used as waiting
Select drug and/or medicament.In embodiment, the macromolecular of including but not limited to protein, peptide and fusion protein can be employed as medicine
Object and/or drug candidate and/or pharmaceutical agent.
Chemotherapy drug candidate is the important drug candidate of one kind and/or pharmaceutical agent in disclosed method.Chemo-Therapy
Treat drug candidates include but is not limited to carboplatin,(gemcitabine),(taxol), melbine, support
Pool for health,It is (pemetrexed), albumin mating type taxol, Barasertib, everolimus, cis-platinum, how soft
Than star, Elesclomol, Irinotecan, camptothecine, olaparib, Etoposide, vinorelbine and/or combination thereof.
Kinase inhibitor is for the important drug candidate of one kind and/or pharmaceutical agent in method of the invention.Kinases suppression
Preparation may interfere with the connection of phosphorylation site on phosphoric acid molecules and target protein.Many kinase inhibitors, which can be commercially available, (to be criticized
It is quasi-), wherein several be in clinical experimental stage.In addition, the micromolecular inhibitor of particular types protein kinase activity has become
Important anticancer drug.(Ventura et al. (2006) " clinical and conversion oncology " 8:153-60).The kinases of approval presses down
Preparation cancer drug includes but is not limited to Wei Mofeini, Lapatinib, Tarceva, Imatinib.Such kinase inhibitor can
Target the kinases expressed in the cell.Kinase inhibitor includes but is not limited to Wei Mofeini, Lapatinib, Gefitinib, Da Lu
House replaces, Barasertib, gram azoles are beautiful for Buddhist nun, monoclonal antibody for Buddhist nun, Pimasertib, linatinib, Sorafenib, department
(for example, easily Puli's nurse Ma), DTIC, 10-deacetyltaxol (for example, albumin mating type taxol,Docetaxel)
And/or combination thereof.
Biological agent is a kind of synthesis or the drug from organism, can be used as drug candidate or pharmaceutical preparation.For example,
Antibody preparation such as Trastuzumab (Herceptin) can be considered biological agent.
Other small-molecule drugs include but is not limited to protease inhibitors bortezomib, PARP (Poly ADP-ribose polymerase)
Inhibitor Rucaparib and olaparib, low hypomethylation agent Da Kejin (Decitabine), DNA damage inducer
Methazolastone, alkylating agent ifosfamide and/or combination thereof.
The technology and program for being described herein or quoting usually can be by those skilled in the art by using conventional method, very
Understand and use well, for example, method is widely used in Sambrook et al. description, " molecular cloning: laboratory manual the 3rd edition
(2001), positioned at the CSH Press of York Cold Spring Harbor.Modern agreement 1N molecular biology (F.M.Ausubel
Et al., a1 editions (2003))；A kind of Enzymology method series (Co., Ltd, academic press): PCR2: " practical approach "
(M.J.MacPherson, B.D.Hames and G.R.Taylor editions (1995)), Harlow and Lane editions (1988) are " anti-
Body, laboratory manual and animal cell culture " (R.I.Freshney editions (1987)).
In described above: " small molecule " is defined as molecular weight less than 10kDa (usually less than 2kDa, preferably smaller than 1kDa)
Molecule.Small molecule includes but is not limited to inorganic molecule, organic molecule, the organic molecule containing inorganic component, includes radioactivity
Molecule, synthetic molecules, peptide mimics and the antibody analog of atom.As therapeutic agent, small molecule is easier to permeation cell, no
It is degradable, and be less susceptible to cause immune response compared to macromolecular.This document describes small molecules (for example, antibody and cell factor
Peptide mimics and small molecule toxins).See, e.g., Casset et al. (2003), " biochemistry is ground with biophysics
Study carefully communication " 307:198-205；Muyldermans (2001) " biotechnology magazine " 74:277-302；Li (2000) " from
Right biotechnology " 18:1251-1256；Apostolopoulos et al. (2002) " Modern Pharmaceutical Chemistry " 9:411-420；
Monfardini et al. (2002) " modern medicines design " 8:2185-2199；Domingues et al. (1999) " is tied naturally
Structure and biology " 6:652-656；Sato and Sone (2003) " Biochemical Journal " 371:603-608；Number is 6,
326,482 United States Patent (USP).
" macromolecular " refers to big biopolymer, including but not limited to nucleic acid, protein, carbohydrate and lipid.
" cell ", " cell line " and " cell culture " is used interchangeably, this all class name include offspring.Therefore,
Word " transformant " and " transformed cells " include main subject cell and culture as derived from it, without regard to transfer
Quantity.It should also be understood that the DNA content of all offsprings may be not exactly the same due to artificially or being unintentionally mutated.Including it is prominent
Becoming the cell screened in offspring and initial transformed cells has identical function or bioactivity.It from the context can be clearly
Find out different titles.
" antibody " refers to any type of antibody for showing required bioactivity.Therefore, antibody can most widely anticipate
Justice use, specifically cover monoclonal antibody (including full length monoclonal antibodies), polyclonal antibody, multi-specific antibody (for example,
Bispecific antibody), chimeric antibody, humanized antibody, fully human antibodies etc., as long as they to can express required biology living
Property.
Suitable for animal, the mankind, experimental subjects, cell, tissue, organ or biofluid " administration " and " treatment " or
" therapy " refers to external source drug, treatment, diagnosticum or component and animal, the mankind, subject, cell, tissue, organ or biology
The contact of liquid." administration " and " treatment " or " therapy " can refer to treatment, pharmacokinetics, diagnosis, research and experimental method etc..
The processing of cell includes contact and reagent contact with fluid of the reagent with cell, and wherein fluid is contacted with cell.It " gives
Medicine " and " treatment " also refer to the external and vitro treatment of cell, reagent, diagnosis, binding component or another cell etc..It is suitable for
" treatment " or " therapy " of the mankind, animal doctor or research object refer to for studying and the therapeutic treatment of diagnostic application, prevention
Property or preventive measure." treatment " or " therapy " suitable for the mankind, animal doctor or research object or cell, tissue or organ is wrapped
Include the contact of medicament with animal subjects, cell, tissue, physiological compartment or physiological fluid." treatment of cell " further includes medicament
The case where (for example, in liquid phase or gel phase) contacting with receptor (or heterodimer) and agonist or antagonist do not connect
The case where touch cells or receptor.
" patient " includes people and non-human mammal (for example, monkey, dog, cat, rabbit, ox, horse, sheep, goat, pig etc.).
" inhibition ", which refers to, measurably slows down, reduces, interferes or prevents or block enzymatic activity.It is desirable that enzymatic activity subtracts
Slow or be reduced at least 20%, 30%, 50%, 70%, 90% or even 100%, this is to measure enzyme using suitable measuring method
Determined by activity.
" isolated nucleic acid molecules " or " isolated protein " or " isolated antibody " refer to from usually with natural origin phase
The nucleic acid molecules or protein or antibody being identified and isolated from least one contaminated nucleic acid, protein or the antibody molecule closed.Point
From nucleic acid molecules or protein or antibody be different from the form or environment that it finds in nature.Therefore, isolated nucleic acid
Molecule is different from nucleic acid molecules present in n cell.
" pharmaceutically acceptable ", which refers to, can be used for preparing usually safe and nontoxic, both abiology nor undesirable drug
Component also includes veterinary purpose and the acceptable drug component of human pharmaceutical use.
" nucleic acid molecules " refer to DNA or RNA.DNA molecular is separated from sequence (or nucleotide sequence), is in by sequence
Close continuous (in the direction 5' and 3').For example, " nucleic acid molecules " may include that insertion is carrier (such as plasmid or viral vectors) or whole
Close the DNA molecular in prokaryotes or Eukaryotic genomic DNA." isolated nucleic acid molecules " also may include cDNA (mutual
Mend DNA) molecule.Isolated nucleic acid molecule after manipulating including other nucleic acid sequences is commonly referred to as recombinant molecule.RNA molecule by
Nucleotide (ribonucleotide) composition, and it is usually single-stranded.RNA is encoded by DNA molecular, or DNA molecular is used to turn as template
Record, so that mRNA (mRNA) can translate into its corresponding amino acid sequence.Short interfering rna is that length is about 20-25 alkali
Base typically serves to the effect for interfering one or more gene expressions to the double-stranded RNA of (or nucleotide).Microrna (miRNA)
It is minimum RNA segment, length is about 22 nucleotide, and is usually adjusted after the transcription or transcription of one or more genes
In work.
" amino acid molecular " refers to the protein or polypeptide encoded by DNA molecular.Protein is by one or more amino acid
Sequence chain composition, has multiple functions in living cells and organism.
All publications referred in description above are expressly incorporated herein by reference herein.Not
Depart from the scope of the present invention and spirit in the case where, those skilled in the art will be clearly understood that the method various modifications and
Variation.Being directed to particular aspects and/or other embodiments herein, the present invention is described, it is to be understood that according to right
It is required that the present invention should not be unduly limited to these aspects and/or embodiment.It should also be understood that for implementing the present invention
Described in mode various modifications, for cell research or those skilled in the relevant art, can by available information be easy
Ground understands and/or obtains, and is included in the scope of the claims.
Claims (9)
1. a kind of testing drug is to the method for the breeder reaction for the tumour cell for being originated from patient, which is characterized in that including following step
It is rapid:
(1) cell is obtained from biopsy or tumor resection material；
(2) cell of acquisition is cultivated on 3D extracellular matrix；
(3) tumour cell that processing is cultivated on 3D extracellular matrix is cured with medicine；
(4) tumour cell for crossing step process is subjected to high-content imaging；
(5) the high-content imaging for the tumour cell that assessment processing is crossed, thus increasing of the testing drug to the tumour cell for being originated from patient
Grow reaction.
2. a kind of testing drug as described in claim 1 is to the method for the breeder reaction for the tumour cell for being originated from patient, special
Sign is, the step (1) further include by the cell xenograft of acquisition into mouse to form tumour, and obtained from mouse
Tumour cell.
3. a kind of testing drug as described in claim 1 is to the method for the breeder reaction for the tumour cell for being originated from patient, special
Sign is that the biopsy or tumor resection material come from biological library.
4. a kind of testing drug as described in claim 1 is to the method for the breeder reaction for the tumour cell for being originated from patient, special
Sign is that the treated tumour cell is in the formation of tumour sphere.
5. a kind of testing drug as described in claim 1 is to the method for the breeder reaction for the tumour cell for being originated from patient, special
Sign is that the drug is selected from small-molecule drug, kinase inhibitor, macromolecular and combinations thereof.
6. a kind of testing drug as described in claim 1 is to the method for the breeder reaction for the tumour cell for being originated from patient, special
Sign is that the tumour cell from patient is primary tumors cells.
7. a kind of testing drug as claimed in claim 6 is to the method for the breeder reaction for the tumour cell for being originated from patient, special
Sign is that the tumour cell from patient is selected from breast cancer cell, prostate gland cancer cell, non-small cell lung cancer cell, ovum
Nest cancer cell, melanoma cells and pancreatic cancer cell.
8. a kind of testing drug as described in claim 1 is to the method for the breeder reaction for the tumour cell for being originated from patient, special
Sign is that the breeder reaction is selected from EdU incorporation, work-dead cell counts, Colony forming and combinations thereof.
9. a kind of testing drug as described in claim 1 is to the method for the breeder reaction for the tumour cell for being originated from patient, special
Sign is, can pass through proliferation, colony form, Apoptosis and combinations thereof the processed tumour cell of technology evaluation.
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