CN105287632A - Method for constructing non-small-cell lung cancer gefitinib drug-resistance PDX model - Google Patents
Method for constructing non-small-cell lung cancer gefitinib drug-resistance PDX model Download PDFInfo
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Abstract
The invention belongs to the technical field of lung cancer gefitinib drug-resistance model construction, and particularly relates to a method for constructing a non-small-cell lung cancer gefitinib drug-resistance PDX model. The method includes the steps of constructing a non-small-cell lung cancer PDX model, screening a gefitinib-sensitive non-small-cell lung cancer PDX model and induction constructing of the non-small-cell lung cancer gefitinib drug-resistance PDX model. The model can be used as a substitution of the PDX model which is directly established by non-small-cell lung cancer gefitinib drug-resistance tumor issue. The good research basis is provided for studying gefitinib drug-resistance mechanisms, screening target spots and small molecule drugs in the gefitinib drug-resistance process and hence delaying or interfering of the gefitinib drug-resistance process, and good application and popularization value is achieved.
Description
Technical field
The invention belongs to pulmonary carcinoma gefitinib resistant models constructing technology field, be specifically related to a kind of patent application of construction method of nonsmall-cell lung cancer gefitinib drug resistance PDX model.
Background technology
Pulmonary carcinoma occupies first in global cancer mortality, and nonsmall-cell lung cancer (Non-small-celllungcancer, NSCLC) accounts for 80% of pulmonary carcinoma.Nonsmall-cell lung cancer 5 years survival rates are less than 15%, main cause be about 70% patient be in late period when being diagnosed as lung cancer in non-cellule type first, be associated with metastasis more.Pulmonary carcinoma is one of the cancer species taken the lead at clinical middle application targeted therapy.In the past ten years, micromolecular compound tyrosine kinase inhibitor is widely used in the targeted therapy of NSCLC patient clinically, and its target spot is EGF-R ELISA (EpidermalGrowthFactorReceptor, EGFR).Gefitinib is one of targeted drug of the NSCLC patient being applied to EGFR sudden change clinically.Achieve significant curative effect in analysis of gefitinib for treat NSCLC, but after gefitinib targeted therapy life-time service, nearly all patient can produce drug resistance.Therefore, NSCLC pulmonary carcinoma gefitinib drug resistance process is simulated and to go deep into clear and definite NSCLC gefitinib resistance mechanism significant.
For in the NSCLC patient treatment of gefitinib, the appearance of gefitinib resistant seriously limits its clinical efficacy.In order to study gefitinib acquired drug-resistance mechanism, much Non-small cell lung carcinoma cell line is induced as gefitinib medicine-resistant cell line, such as HCC827, PC-9, A549 are exposed to gefitinib for a long time, finally will induce and become gefitinib medicine-resistant cell line HCC827GR, PC9/ZD and A549GR.The gefitinib medicine-resistant cell line that profit is set up in this way, the research of NSCLC gefitinib resistance mechanism has been carried out in domestic and international much research.Disclose 3 kinds of different resistance mechanisms: the reason that 60% nonsmall-cell lung cancer produces acquired gefitinib drug resistance is because specific EGFR there occurs sudden change, mainly T790M sudden change; 22% is the amplification of some gene, such as, and MET, HER2, BRAF, AXL, MAPK1 or PIK3CA etc.; 4% is the structural transformation from nonsmall-cell lung cancer to epithelial cell pulmonary carcinoma in addition, or has stem cell properties; 10% resistance mechanism is also had to be then unclear.But, because these cells are through too much having homogeneity for In vitro culture, thus cause it fully can not reflect Tumor Heterogeneity in clinical patient tumor tissues thus to there is larger limitation.Therefore, find effective Therapeutic Method to intervene the process that gefitinib drug resistance occurs NSCLC patient, further investigate the mechanism of acquired gefitinib drug resistance, truly can reflect NSCLC patient tumors feature in the urgent need to one, and effectively simulate the animal model of NSCLC patient's gefitinib drug resistance generating process.
Transplanted tumor (the patient-derivedxenografts that the people constructed in immunodeficient mouse by people's tumor tissues direct inoculation is tissue-derived, PDX) model fully remains specimens heterogeneity, therefore, various tumor PDX Transplanted tumor model is extensively set up in the past ten years, and is extensively approved in histology, gene level and pharmacological action level.The foundation of PDX model provides the platform in early stage of a clinical research application, and is widely used in evaluating new clinical treatment and screening molecular target.The PDX model of current nonsmall-cell lung cancer gefitinib drug resistance fails structure always.But the PDX model setting up gefitinib drug resistance has two methods, one is directly the Non-Small Cell Lung Carcinoma of gefitinib drug resistance is carried out inoculation to construct, and one is that the NSCLCPDX model filtering out gefitinib gives gefitinib and induces.The deficiency of the first construction method: the nonsmall-cell lung cancer patient of 1 gefitinib drug resistance is in cancer of late stage usually, have no chance to perform the operation, usually again diagnosed by puncture, and puncture acquired by tissue mass few, be therefore difficult to obtain abundant drug resistance tissue and carry out PDX model construction.2 gefitinib drug resistances are, after taking gefitinib for a long time, drug resistance occurs, and gefitinib generation drug resistance is cannot carry out the tissue in puncture acquisition gefitinib drug resistance process to patient stage by stage before occurring.Therefore, we take second method namely to build NSCLCPDX model, filter out the model group to gefitinib, then the method induction NSCLC transplanted tumor generation gefitinib drug resistance progressively increasing gefitinib dosage is adopted, fully simulate NSCLC patient's gefitinib drug resistance generating process, provide clinical front platform for studying gefitinib drug resistance mechanism further and screening intervention drug resistance Procedures Drug.
Summary of the invention
The object of the invention is to provide a kind of construction method of nonsmall-cell lung cancer gefitinib drug resistance PDX model, constructed nonsmall-cell lung cancer gefitinib drug resistance PDX model can as the succedaneum of the direct PDX model of use gefitinib drug resistance tissue construction, and simulate NSCLC patient gefitinib drug resistance process occurs, for research gefitinib resistance mechanism, the target spot of screening gefitinib drug resistance process and small-molecule drug, and then delay or intervene gefitinib drug resistance process to have great importance and practical value.
Below detailed technology scheme of the present invention is described below.
A construction method for nonsmall-cell lung cancer gefitinib drug resistance PDX model, comprises the following steps:
(1) nonsmall-cell lung cancer (NSCLC) PDX model is first built, be specially:
First, obtain the tissue samples of nonsmall-cell lung cancer patient, be specially: clinical definite is nonsmall-cell lung cancer and the preoperative patient all not accepting radiation and chemotherapy, in acquisition, it is agreed to and after handling corresponding entries, get its tumor tissues as sample, i.e. the nonsmall-cell lung cancer tumor sample in people source;
Secondly, after the nonsmall-cell lung cancer tumor sample process of originate obtained people, routine operation is implanted, and the tumor formed is transplanted tumor;
Monitoring is implanted people and to be originated the SCID mouse growth situation of non-small cell tumor (transplanted tumor), and when tumor has grown to necrosis or liquefaction phenomenon, (now gross tumor volume is generally at 500 ~ 1500mm
3left and right), should go down to posterity in time;
When by during stable being passaged to more than three generations or three generations, tumor (transplanted tumor) is then thought that this nonsmall-cell lung cancer (NSCLC) PDX model construction is successful;
(2) nonsmall-cell lung cancer (NSCLC) PDX model constructed in step (1) is evaluated, filter out the nonsmall-cell lung cancer PDX model of gefitinib;be specially:
First, record tumor growth boundary in constructed nonsmall-cell lung cancer (NSCLC) PDX model and judge, described tumor growth boundary and transplanted tumor is necrosis or gross tumor volume marginal value when there is liquefaction phenomenon when mice tumor growth;
Secondly, pathology and immunohistochemical staining evaluation are carried out to tumor in constructed nonsmall-cell lung cancer (NSCLC) PDX model, described immunohistochemical staining evaluation adopts if label CK5/6, P63 and P40(CK5/6 of lung squamous cancer, P63 and P40 are the scale cancer labelling indexs of generally acknowledging clinically), label TTF-1, CK8/18 and NapsinA(TTF-1 of adenocarcinoma of lung, CK8/18 and NapsinA are the adenocarcinoma labelling indexs of generally acknowledging clinically) etc. evaluate, to determine that transplanted tumor type is typical non-small cell carcinoma transplanted tumor;
Finally, abrupt climatic change is carried out to the EGFR gene of tumor in constructed nonsmall-cell lung cancer (NSCLC) PDX model, can adopt during detection existing detection kit (as Beijing Yakangbo Biotechnology Co., Ltd. " Human epidermal growth factor receptor gene mutation detection kit), determine EGFR gene type by fluorescent PCR is legal; The non-small cell carcinoma PDX model of gefitinib is filtered out according to EGFR catastrophe,
Concrete as L858R, L861Q, G719C, Exon19 disappearance etc. to the EGFR genetic mutation type in the nonsmall-cell lung cancer PDX model of gefitinib;
Situation is changed for genotype after the nonsmall-cell lung cancer PDX model generation drug resistance of gefitinib, occurs EGFRT790M gene mutation, c-Met, HER2, BRAF, AXL, MAPK1 or PIK3CA gene amplification etc.
(3) nonsmall-cell lung cancer gefitinib drug resistance PDX model is built, be specially:
To the nonsmall-cell lung cancer PDX model of the gefitinib that screening in step (2) obtains, give the process of gefitinib gavage, dosage progressively increases administration from 10 ~ 100mg/kg, thus inducing adaptive nonsmall-cell lung cancer gefitinib drug resistance PDX model;
In Induction Process, gefitinib inductive dose first rises to 50mg/kg from 10mg/kg, then rises to 100mg/kg from 50mg/kg;
For ease of detecting, can when each dosage produce drug resistance, cryopreserved tissue sample before going down to posterity, so that the molecular changes after later stage recovery in research drug resistance process;
Gefitinib drug resistance Induction Process totally needs about 7 ~ 10 months time.
At present, PDX model has functional substrate and blood vessel, tumor microenvironment can reflect the feature of the downright bad and Tumor Differentiation of tumor center, is therefore considered to the best model of the truth reflecting people's tumor growth in vivo.These PDX models more can reflect the heterogeneity of the true molecular characteristic sum tumor of patient's in-vivo tumour than the transplanted tumor deriving from cell line.The growth of tumor and the effect of medicine all can be subject to the impact of angiogenesis, somatomedin, interstitial and tumor microenvironment, and therefore, the transplanted tumor that the result based on the acquisition of PDX model obtains than simple cell system more has conviction power.
Although there is the successful structure of fraction of nonsmall cell pulmonary carcinoma gefitinib medicine-resistant cell line model in prior art, but based on complexity and the particularity of clinical research application, add the limitation of cell line model, thus build nonsmall-cell lung cancer gefitinib drug resistance PDX model still tool be of great significance.Adopt general thinking, from nonsmall-cell lung cancer patient, obtain the tumor tissues of gefitinib drug resistance and build PDX model, although have the probability of theoretical operation, but in actual practice, due to the non-availability of tumor tissues in nonsmall-cell lung cancer gefitinib drug resistance process, this thinking is thus made to realize.
The present invention is first by building nonsmall-cell lung cancer PDX model, filter out the PDX model for gefitinib drug resistance sensitivity further, and adopt gefitinib induction to obtain the PDX model of nonsmall-cell lung cancer gefitinib drug resistance based on this, can as the succedaneum of the PDX model of the tumor tissues direct construction by nonsmall-cell lung cancer gefitinib drug resistance, for research gefitinib resistance mechanism, the target spot of screening gefitinib drug resistance process and small-molecule drug, and then delay or intervene gefitinib drug resistance process to provide good Research foundation, there is good application value.
Accompanying drawing explanation
Fig. 1 is the tumor growth curve figure of a wherein routine nonsmall-cell lung cancer Transplanted tumor model in embodiment 1, the wherein P1(first generation), the P2(second filial generation) and the P3(third generation) growth time be respectively 100 days, 84 days and 48 days;
Fig. 2 is the tumor growth curve figure of an other routine nonsmall-cell lung cancer Transplanted tumor model in embodiment 1, the wherein P1(first generation), the P2(second filial generation) and the P3(third generation) growth time be respectively 133 days, 92 days and 61 days;
Fig. 3 is that non-small cell carcinoma patient tumor tissues (P0) and transplanted tumor tissue (P1 and P3) HE dye and immunohistochemical staining figure; Wherein first row P0 transplanted tumor tissue, second is classified as P1 transplanted tumor tissue, 3rd is classified as P3 transplanted tumor tissue, and the first behavior is respectively dyeed (X200) for the HE of transplanted tumor, and second is respectively CK5/6, p40, p63 positive expression (X200) in each generation transplanted tumor to four lines;
Fig. 4 is growth of xenografted curve before and after gefitinib drug resistance, and before gefitinib drug resistance, transplanted tumor growth cycle is 28 days, and after gefitinib drug resistance, the growth of xenografted cycle is 21 days;
Fig. 5 is gefitinib transplanted tumor positive controls (gefitinib 100mg/kg) tumor growth curve;
Fig. 6 is transplanted tumor HE dyeing and immunohistochemical staining figure before and after gefitinib drug resistance; Wherein transplanted tumor tissue after the first behavior gefitinib drug resistance, tumor tissue is transplanted before second behavior gefitinib drug resistance, first HE dyeing (X200) being classified as each generation transplanted tumor, the second to four row are respectively CK8/18, NapsinA, TTF1 positive expression (X200) in each generation transplanted tumor;
Fig. 7 is transplanted tumor EGFR catastrophe before and after gefitinib drug resistance; Wherein left side is transplanted tumor EGFR catastrophe before gefitinib drug resistance, is L858R sudden change; After the gefitinib drug resistance of right side, transplanted tumor EGFR is without sudden change, is wild type;
Fig. 8 is transplanted tumor c-Met gene amplification situation before and after gefitinib drug resistance; Wherein left figure is c-Met amplification after gefitinib drug resistance, and the Lycoperdon polymorphum Vitt place shown in arrow is amplification region, and right figure is without c-Met gene amplification before gefitinib drug resistance.
Detailed description of the invention
Below in conjunction with embodiment, explanation is further explained to the application, before introducing specific embodiment, briefly introduces as follows to partial material used in following embodiment.
material and reagent
CB17-SCID mice, female, 6 ~ 8 week age, 18 ~ 20g, Beijing zoopery company of dimension tonneau China; It should be noted that mouse growth environment is constant temperature and humidity, gnotobasis;
Aseptic PRMI1640 culture medium, hyclone, phosphate buffer (PBS), Hyclone;
Dimethyl sulfoxide, penicillin/streptomycin solution 100x, Sigma;
L-15 solution, Gibco;
Gefitinib tablet (Iressa), Britain's AstraZeneca;
Pentobarbital sodium, traditional Chinese medicines group;
Human epidermal growth factor receptor gene mutation detection kit, Beijing Yakangbo Biotechnology Co., Ltd.;
In addition, in following embodiment serum-free culture medium in be added with 1% antibiotic (100U/ml penicillin, 100U/ml streptomycin); While it is noted that aseptic culture medium should keep fresh, should 4 DEG C of storages when not using, and finished using in 3 weeks.
About the acquisition of the nonsmall-cell lung cancer tumor tissues of people, it should be noted that, patient is all the non-small cell carcinoma patients through prior art clinical definite, patient did not all accept radiation and chemotherapy treatment in the preoperative, and patient does not also suffer from other infectious diseases, patient also all signs the relevant Informed Consent Form for studying.The tumor tissues of patient is placed into immediately in the culture medium of the PRMI1640 of the serum-free containing penicillin and streptomycin after excision, and is transported to rapidly research place for experimentation.From the tumor tissues of collected patient, select the good tissue part of tumor vigor, after removal hemorrhagic necrosis tissue, to immerse in 4 DEG C of physiological saline solution rinsing 3 times, then carry out subsequent experimental process.
embodiment 1
The present embodiment mainly introduces the building process of nonsmall-cell lung cancer (NSCLC) PDX model.
1, the tissue samples of nonsmall-cell lung cancer patient is obtained
The non-small cell carcinoma tumor tissues sample obtained comes from postoperative flesh tissue specimen, generally speaking, should get edge tumor tissues as far as possible when choosing tumor sample further, to avoid in got tissue containing slough.
As required, final selected tumor tissues sample size is 1cm × 1cm.Selected tumor tissues sample is taken pictures and weighed and after making a record, for subsequent detection, test conveniently, acquired tumor tissues sample can be divided into three parts: a part is fixed with 10% formalin, and makes paraffin specimen to carry out pathologic finding and immunohistochemical analysis; A part is in the extraction and analysis of-80 DEG C of cold preservations for DNA/RNA; A part is implanted in severe combined immunodeficient (SevereCombinedImmunodeficiency, SCID) Mice Body and is carried out PDX model construction.
2, the non-small cell carcinoma tumor sample that obtained people originates is implanted mice
After the non-small cell carcinoma tumor sample process of originate obtained people, routine operation implants SCID mice, and the tumor formed is transplanted tumor; Detailed process is as follows:
First, the non-small cell carcinoma tumor sample that people originates is cut into tumor tissues of the same size, and specification is roughly: diameter about 3mm, weight 0.12 ~ 0.15g;
Secondly, mouse anesthesia is planted tumor, detailed process is: give 0.3mL0.4%(mass/volume by every 20g body weight) pentobarbital sodium mice is anaesthetized, after its anesthesia, by 21G syringe needle broken skin, enter penicillin and streptomycin 0.1mL at subcutaneous injection, then subcutaneous implantation cut after tumor tissues sample;
After nonsmall-cell lung cancer tumor tissues is implanted SCID mice, regular monitoring Mouse Weight and tumor (tumor) growing state, when tumor has grown to necrosis or occurred liquefaction phenomenon, recorded now gross tumor volume size and also gone down to posterity in time;
In the tumour transplatation experiment carried out, statistical result shows, when tumor has grown to necrosis or occurred liquefaction phenomenon, gross tumor volume is generally at 500 ~ 1500mm
3left and right (because of tumor sample difference, these data are also incomplete same);
Require emphasis and illustrate, when carrying out transplanted tumor operation for the tumor sample of particular source in following embodiment, transplanted tumor grows to 500mm in Mice Body
3in time, starts to occur downright bad or liquefaction phenomenon, thus in subsequent embodiment tumor growth all with 500mm
3this numerical value is growth boundary;
When by during stable being passaged to more than three generations or three generations, tumor (transplanted tumor) is then thought that this nonsmall-cell lung cancer (NSCLC) PDX model construction is successful; In three to five generations, then for follow-up experimentation, and carried out freezing kind of a preservation by three generations's to ten generation.
It is to be understood that mouse interior tumor (transplanted tumor) volume is calculated as follows:
V=LD×(SD)
2/2；
Wherein V is gross tumor volume, and LD is the longest diameter of tumor, and SD is the shortest diameter of tumor, adopts vernier caliper measurement when LD, SD measure.
Also need to explain and illustrate, tumor succeeding generations operates routinely, roughly the same with tumor implantation process, is specially:
The mice of going down to posterity needing tumor bestows cervical dislocation, soaks in 75% ethanol, dries rear dissection and takes out tumor (be the first generation tumor tissues during first generation sacrifice, be the second filial generation tumor tissues during second filial generation sacrifice, the like);
Taken out tumor tissues is placed in the PBS containing penicillin and streptomycin, after taking pictures and weighing, as required, got tumor tissues can be divided into three parts, a part puts 10% formalin solution, and makes specimen to evaluate further, and a part is in the extraction and analysis of-80 DEG C of cold preservations for DNA/RNA, another part requires to implant SCID mice according to implant procedure, thus completes the work of going down to posterity.
On said method basis, inventor successfully establishes 40 routine nonsmall-cell lung cancer Transplanted tumor model altogether, and 40 routine transplanted tumoies all can be stablized and go down to posterity.In general, in constructed nonsmall-cell lung cancer Transplanted tumor model, first generation growth of xenografted speed is comparatively slow, but from the third generation and growth of xenografted speed and stable later.Wherein two routine transplanted tumor tumor growth curves (tumor is implanted after in Mice Body, measures a gross tumor volume weekly, draws growth curve according to this) as shown in Figure 1 and Figure 2.
One of feature of PDX model is exactly the hysteresis quality of tumor growth.When people's tumor tissues is implanted after in immunodeficient mouse body, early stage will overcome heteroplastic rejection, and the interstitial completed in people's tumor tissues and blood vessel by the interstitial of mice and blood vessel the process that substitutes, so the lag period of just generation tumor growth.Within the lag period, tumor growth is slow, and in the first generation usually betiding PDX model and the second filial generation, so the first generation and second filial generation tumor growth are usually slow in PDX model, to the third generation and later tumor growth stablize, feature keeps.This feature all obtains and proves preferably in Fig. 1, Fig. 2.
embodiment 2
The present embodiment mainly introduces the process for the evaluation of the nonsmall-cell lung cancer PDX model constructed by embodiment 1 and the nonsmall-cell lung cancer PDX model of screening gefitinib.
Nonsmall-cell lung cancer PDX model constructed in embodiment 1 is evaluated, mainly comprises Histopathology assessment and EGFR gene Molecular Evaluation two aspects, specific as follows.
first tumor growth curve will be drawn in Histopathology assessment, be specially:
The computational methods of gross tumor volume as described in example 1 above, measure weekly and calculate a tumor growth volume, and drawing tumor growth curve accordingly; To record and judge simultaneously the critical size value (when namely going down to posterity gross tumor volume value) of tumor growth;
The critical size value that it is emphasized that the tumor growth in the specific nonsmall-cell lung cancer PDX model small mouse body that adopts in following embodiment is 500mm
3left and right, this numerical value is not only the tumor growth volumetric flow rate dividing value of tumor succeeding generations, is also tumor growth volume stop value when induction stops in the drug resistance process of follow-up gefitinib induction.
in Histopathology assessment its secondary to tumor tissues sample carry out basic pathology evaluate,be specially:
In actual mechanical process, tumor tissue section can be prepared respectively to the tumor tissues sample in the initial tumor tissue samples in people source and each succeeding generations, and wherein tumor cell ratio is judged, to determine the basic condition of tumor tissues; Secondly HE dyeing and immunohistochemical staining are carried out to tumor tissue section, to determine type and other pathological characters of tumor;
Statistical result for tumor cell ratio shows, in each nonsmall-cell lung cancer PDX model constructed by the present invention, in tumor tissues, tumor cell ratio is all greater than 70%, demonstrates growth potential preferably;
HE dyeing and immunohistochemical staining result are shown (wherein a routine staining conditions as shown in Figure 3), there is horny pearl and intercellular bridge in HE dyeing display Primary Tumor tissue, P1 and P3 is basically identical for the pathology differentiation degree of transplanted tumor tissue and Primary Tumor tissue; CK5/6, p40 and p63 are positive expression in P0, P1 and P3 generation, and wherein CK5/6 positive cell dyeing is positioned at cell membrane and Cytoplasm, P40 and P63 positive cell dyeing is positioned at nucleus; Each basically identical for the staining conditions of tumor tissues;
The above results shows that tumor tissues has growth potential and the situation that goes down to posterity is comparatively stable preferably.
eGFR gene Molecular Evaluation aspect, mainly adopt existing " Human epidermal growth factor receptor gene mutation detection kit " to utilize comparatively ripe Fluorescence PCR assay to carry out EGFR(EGF-R ELISA to the DNA of tumor tissues) and gene mutation site analysis.
EGFR gene is positioned at people's No. 7 the short arm of a chromosome (7p12), is about 118kb, is made up of 28 exons; Its mRNA transcribing formation is about 5.6kb, and coding molecule amount is the cross-cell membrane glycoprotein of 170kD, and it is active that intracellular region has tyrosine kinase (tyrosinekinase, TK), is responsible for extracellular signal to be passed in born of the same parents.Abnormal EGFR activation can promote the propagation of tumor cell, migration, differentiation, angiogenesis, and can the apoptosis of inhibition tumor cell.
On front 4 exons that EGFR genetic mutation mainly occurs in TK region in born of the same parents (18 ~ 21 exon), research shows: the curative effect that the patients with lung cancer that EGFR gene 18,19,20,21 exon is undergone mutation takes tyrosine kinase inhibitor (tyrosinekinaseinhibitors, TKIs) class medicine is better.And in lung cancer patients in China, the mutation rate of EGFR is about 30% ~ 50%, the G719S point mutation wherein 18 exons occurred accounts for about 5% of EGFR mutation type, the multiple deletion mutation that 19 exons occur accounts for about 45% of EGFR mutation type, 20 exons occur S768I point mutation accounts for EGFR mutation type about 1%, L858R, L861Q point mutation that 21 exons occur accounts for 40% ~ 45% of EGFR mutation type.
On above-mentioned existing Research foundation, concrete analysis process is:
By prior art, routine operation, carries out extraction operation to the DNA of tumor tissues sample;
According to test kit description, Fluorescence PCR assay is adopted to carry out qualitative to EGFR mutational site; When detecting analysis, specifically can carry out the qualitative analysis in mutational site according to 18,19,21 and 20 exons of following table to EGFR gene:
。
The EGFR mutation type of the non-small cell carcinoma model of known gefitinib has: L858R, L861Q, G719C, Exon19 lack, and can filter out the non-small cell carcinoma PDX model of gefitinib accordingly.
embodiment 3
On above-described embodiment 1,2 basis, it is 500mm that inventor's screening obtains tumor growth marginal value
3, EGFR mutation type is the nonsmall-cell lung cancer PDX model of L858R, the present embodiment is mainly introduced and is carried out gefitinib induction with the process obtaining nonsmall-cell lung cancer gefitinib drug resistance PDX model to this PDX model.
For the nonsmall-cell lung cancer PDX model that screening obtains, use its third generation transplanted tumor mice to induce, build the Transplanted tumor model obtaining gefitinib drug resistance; Detailed process is:
Mice is divided into two groups, one group is matched group, another group is experimental group, positive controls gavage gefitinib solution, every day 100mg/kg, after continuous gavage one week, gross tumor volume can obviously reduce (meaning that positive controls is set up: 1 confirms that selected genotypic transplanted tumor in embodiment 2 is responsive 2 confirm that the transplanted tumor before the induction of gefitinib drug resistances is responsive to gefitinib to gefitinib); The gefitinib solution that experimental group gives, dosage increases successively by low dosage (10mg/kg), middle dosage (50mg/kg), high dose (100mg/kg).
It is to be understood that gefitinib dosage installation warrants is: according to the dosage of people, it is probably 50mg/kg that gefitinib is converted into rat dosage; And in current bibliographical information, in the transplanted tumor experiment of the gefitinib mdr cell of gefitinib induction, it is 100mg/kg that mice gives gefitinib dosage, therefore using the standard dose of 100mg/kg as nonsmall-cell lung cancer gefitinib drug resistance; Get it 1/10 for initial dose and 10mg/kg, increase dosage gradually, thus inducing adaptive nonsmall-cell lung cancer gefitinib drug resistance PDX model.
It should be noted that because experimental mice finally may all can not occur gefitinib drug resistance phenomenon, thus experimental group is often organized the suggestion of mice quantity and is at least no less than 10; Testing in inventor's experimentation and often organizing mice quantity is 12.
In addition it is to be understood that in experimentation gavage gefitinib solution prepare as follows:
Tablet containing 250mg gefitinib is weighed, is designated as A;
Then with mortar by gefitinib tablet grind into powder, collect powder weigh in 50mL centrifuge tube, be designated as B;
Calculate actual gefitinib amount, formula is: (A/B) × 250mg;
Experimentally dosage is required is dissolved in gefitinib in normal saline, and is distributed into the low capacity of enough using dosages; Then frozen in-20 ° of C.
positive controls is tested
To the non-small cell carcinoma PDX model screening the gefitinib obtained, after Mice Body is implanted into tumor tissues sample, routine observation record tumor tissue growth situation, in positive controls body, transplanted tumor tissue grows to 100 ~ 200mm
3time, every day, gavage gave gefitinib (100mg/kg), and the transplanted tumor of matched group can be more and more less, can disappear in 1 ~ 3 week.Tumor growth curve as shown in Figure 5.From Fig. 5, result can illustrate, above-mentioned to screen the non-small cell carcinoma PDX Model Tumor tissue obtained be highstrung for gefitinib.
Gefitinib induction group Detailed Experimental process is described below.
low dosage (10mg/kg) gefitinib Induction Process
To the non-small cell carcinoma PDX model screening the gefitinib obtained, after Mice Body is implanted into tumor tissues sample, routine observation record tumor tissue growth situation, treats that in its body, transplanted tumor tissue grows to 100 ~ 200mm
3time, experimental group starts gavage gefitinib solution every day (10mg/kg).After gavage, normally feed, routine observation record growth of xenografted situation and size, these mices are registered as first generation treated with gefitinib group mice.
For experimental mice, then need according to circumstances to carry out intermittent administration; Administration principle is: after administration, when tumor tissues stops growing, will stop giving gefitinib gavage; But then need when tumor tissues starts to become large restoration ecosystem again to treating with gefitinib gavage.From gefitinib be administered into stop administration being designated as treatment time, and from after gefitinib drug withdrawal to tumor tissues again restoration ecosystem be designated as withdrawal time, a treatment time and a withdrawal time synthesize a treatment cycle.
For experimental mice, in the starting stage, in a treatment cycle, treatment time is approximately 4 ~ 5 days, and withdrawal time is approximately 10 days.But along with the growth of tumor tissues, in each treatment cycle, treatment time can be more and more longer, and withdrawal time can be shorter and shorter.
In the end in one-period, when continuing administration every day, tumor can be grown to and reaches the volume 500mm that goes down to posterity
3time (diameter is about 1cm), whole process probably needs 1 ~ 2 month; Now the Induction Process of low dosage terminates, and namely now can think that the gefitinib of non-small cell carcinoma PDX model to low dosage of the gefitinib that screening obtains produces drug resistance.
It is to be understood that in drug resistance Induction Process, the tumor tissues of separate sources is different to gefitinib-sensitive, and the tumor tissues in part source will drug resistance in 1 ~ 2 treatment cycle, and part is originated then 3 ~ 5 treatment cycle just meeting drug resistance.
Also need to explain, the gross tumor volume size for the treatment of terminal in the present embodiment is 500mm
3, but during different tumor tissues stopped treatment, volume is likely not identical, this is determined by the individual variation of tumor tissues; Such as some tumor tissues reaches 1500mm
3time quality or good, and some tumor tissues does not also reach 500mm
3time will be downright bad; So should in first three growth cycle for close observation tumor tissues and growth characteristic, growing into maximum and not having slough (the growth volume critical point of the tumor applied in this enforcement is for 500mm for volume during stopped treatment with the tumor tissue of the third generation
3).
After low dosage induction terminates, put to death mice and take out tumor tissues, by frozen for Partial tumors tissue (being referred to as first generation gefitinib drug-resistant tumor tissue), and Partial tumors tissue is carried out mice according to succeeding generations go down to posterity, for the induction (this mice be referred to as the mice of second filial generation treated with gefitinib) of follow-up gefitinib drug resistance in generation.
Cryopreservation methods: tissue is cut to the piece of tissue (about 0.2 ~ 0.3g) being cut into diameter 5 ~ 6mm, piece of tissue is put into the cryopreservation tube filling 1mL cryopreserving liquid in advance, make cryopreserving liquid fully cover tissue; Then put into procedural freezing storing box, insert-80 DEG C of refrigerator overnight, then put into liquid nitrogen and preserve.
The preparation of tissue freezing solution: 50% hyclone, 40%L-15 solution, 10%DMSO.
Cryopreserved tissue method for resuscitation: the tumor tissues of liquid nitrogen storage is before mice is implanted in application, need first through organizing revival phase, detailed process is: from liquid nitrogen, take out freezing piece of tissue, after 37 DEG C of waters bath with thermostatic control are melted, piece of tissue is placed in culture dish, and with the PBS cleaning twice containing penicillin and streptomycin, subcutaneous transplantation mode then can be adopted to implant in Mice Body.
middle dosage (50mg/kg) gefitinib Induction Process
After first generation gefitinib drug-resistant tumor tissue is implanted mice, the growth course of routine observation record mouse interior tumor, when second filial generation treated with gefitinib mouse tumor tissue grows to 100 ~ 200mm
3time, within every two days, gavage gives middle dosage gefitinib (50mg/kg) solution, and every day observed and recorded tumor tissues size and growing state.
In administration process, same employing interval administering mode, that is: when tumor tissues stops growing, will stop administration; And when tumor tissues grows to 300 ~ 400mm
3time, then give the process of gefitinib (50mg/kg) gavage.
In middle dosage Induction Process: in first treatment cycle, administration time is approximately that (administering mode was: administration in every two days in 6 ~ 10 days, concrete administration time calculates according to dosing interval, such as two days administering modes, treat 10 days, be then administration in the 2nd, 4,6,8,10 day), withdrawal time is approximately 10 ~ 15 days.
After 2 ~ 4 treatment cycle, the mice of every two days gavage modes has started to produce toleration to the dosage of 50mg/kg, when this kind of administering mode, tumor growth does not affect by dosage, and gross tumor volume starts to increase); Now change administering mode, increase administration number of times, change into every day gavage give gefitinib solution (50mg/kg) treatment.
Every day, gavage gave in gefitinib Solution In The Treatment process, and as previously mentioned, same employing interval administering mode, namely when tumor tissues stops growing, will stop administration; And when tumor tissues restoration ecosystem, then give the treatment of gefitinib (50mg/kg) gavage.
It should be explained that, the tumor tissues of gefitinib is very responsive to gefitinib, if namely the mice containing transplanted tumor gives the gefitinib of high dose in gavage every day at the gefitinib drug resistance induction initial stage, then tumor tissues can reduce rapidly, can not restoration ecosystem fast again, thus when being converted to relative high dose gefitinib by the induction of relative low dose gefitinib and inducing, during initial administration adopt doses at intervals but not every day administering mode, the impact of gefitinib on tumor growth can be reduced like this, be beneficial to it and produce drug resistance fast.
Every day gavage give in gefitinib Solution In The Treatment process, along with the growth of tumor tissue, in each treatment cycle, treatment time can be more and more longer, and withdrawal time can be shorter and shorter.In the end in one-period, under every day, gavage gave mice gefitinib (50mg/kg) situation, tumor tissues still can grow and reach 500mm
3left and right, now, can think that the gefitinib of tumor tissues centering dosage (50mg/kg) of non-small cell carcinoma PDX model of gefitinib creates drug resistance.
Above-mentioned middle dosage (50mg/kg) gefitinib drug resistance Induction Process is roughly about 3-4 month.
After middle dosage (50mg/kg) inducible resistance, put to death mice and take out tumor tissues, by frozen for Partial tumors tissue (being referred to as first generation gefitinib drug-resistant tumor tissue) (frozen and recovery with reference to aforementioned low dosage Induction Process), and Partial tumors tissue is carried out mice according to succeeding generations go down to posterity, for the induction (this mice be referred to as the mice of third generation treated with gefitinib) of follow-up gefitinib drug resistance in generation
high dose (100mg/kg) gefitinib Induction Process
After second filial generation gefitinib drug-resistant tumor tissue is implanted mice, the growth course of routine observation record mouse interior tumor, when third generation treated with gefitinib mouse tumor tissue grows to 100 ~ 200mm
3time, within every three days, gavage gives high dose gefitinib (100mg/kg) solution, and every day observed and recorded tumor tissues size and growing state.
It should be explained that, along with increasing of medicine concentration, administering mode is paid particular attention to when increasing dosage, when should not start and direct administration every day, due to the special role of gefitinib in non-small cell carcinoma treatment, if during beginning administering mode can cause tumor to reduce rapidly direct every day, thus good drug resistance inducing effect cannot be obtained, so drug dose is higher, initial administration interval also should be longer, then after tumor produces toleration to the more rectangular formula in this interval, shorten delivery time, until tumor produces toleration to administering mode every day, thus progressively tumor progressively obtains stable drug resistance.
During every three days administering mode treatment mouse tumors, initial stage mouse tumor can reduce comparatively rapidly, when tumor tissue is less than 200mm
3time, will administration be stopped; But when tumor tissue restoration ecosystem is to 300mm
3time even larger, then need to restart to give gefitinib high-dose therapy.
In every three days administering mode therapeutic processes, in first treatment cycle, administration time is approximately 10 ~ 15 days (in concrete administration time reference dosed administration computational methods), and non-administration time is approximately 15 ~ 20 days.After 1 ~ 3 treatment cycle, mice produces drug resistance (drug resistance criterion is with middle dosage Induction Process) to the high dose gefitinib of every three days administering modes; Now change every two days into the therapeutic modality with high dose gefitinib to mouse stomach process.Equally, after 1 ~ 3 treatment cycle, after mice produces drug resistance to the high dose treated with gefitinib of every two days administering modes, change the high dose treated with gefitinib of administering mode every day into.When mice is when giving with high dose treated with gefitinib every day, gross tumor volume still grows and reaches 500mm
3time, can think that non-small cell carcinoma gefitinib drug resistance PDX model construction completes.
Above-mentioned high dose gefitinib Induction Process needs the time to be approximately 3 ~ 4 months.
By the above-mentioned sacrifice to high dose gefitinib drug resistance, dissect, take out tumor tissues frozen (frozen and recovery reference low dosage Induction Process) or after again implanting mice, namely can be used for the research work of further gefitinib resistance mechanism.
For verifying that whether constructed non-small cell carcinoma gefitinib drug resistance PDX model is successful further, after tumor tissues can being implanted again mice, carry out actual verification.Matched group and gefitinib drug resistance checking group is divided into during checking.Matched group gavage normal saline, periodic measurement tumor tissue size, and draw tumor tissue growth's curve.Gefitinib drug resistance checking group, every day is given and the process of gefitinib gavage according to high dose (100mg/kg) administering mode, periodic measurement tumor tissue size, and draws tumor tissue growth's curve.When matched group is consistent with tumor tissue growth's curve of gefitinib drug resistance checking group, can assert that nonsmall-cell lung cancer gefitinib drug resistance Transplanted tumor model successfully constructs.
For obtaining more stable and reliable Transplanted tumor model, the above-mentioned nonsmall-cell lung cancer gefitinib drug resistance transplanted tumor successfully constructed can be carried out more than three times mices to go down to posterity, nonsmall-cell lung cancer gefitinib drug resistance PDX model obtained like this will be highly stable, may be used for the research of gefitinib resistance mechanism.
inspection example
For confirming the technique effect of the nonsmall-cell lung cancer gefitinib drug resistance PDX model constructed by the present invention further, inventors performed and further check assessment, briefly introducing as follows.
the contrast of the growing state of the parent transplanted tumor of the transplanted tumor after drug resistance and gefitinib (not drug resistance)
To the tumor tissues sample in the people source before the tumor tissues in obtained non-small cell carcinoma gefitinib drug resistance PDX model and its induction, implant SCID mice respectively, periodic measurement tumor growth situation, and draw tumor growth curve.Result as shown in Figure 4.
As can be seen from the figure, the growth cycle of the tumor tissues after drug resistance is shorter than the growth cycle of the not tumor tissues of drug resistance, and tumor tissue growth's speed after drug resistance.
histological type and the immunohistologic evaluation of the parent transplanted tumor of the transplanted tumor after drug resistance and gefitinib (not drug resistance) contrast
To the tumor tissues sample in the people source before the tumor tissues in obtained non-small cell carcinoma gefitinib drug resistance PDX model and its induction, its histological type and immunohistochemical analysis contrast situation are as shown in Figure 6.
As can be seen from Figure 6, both histological types are identical, and immunohistochemistry positive index is identical, illustrate that the tumor tissues after gefitinib drug resistance maintains the pathological characters of primary patient substantially.
the EGFR gene of the parent transplanted tumor of the transplanted tumor after drug resistance and gefitinib (not drug resistance) and the contrast of c-met gene
The EGFR gene of gefitinib drug resistance pre-neoplastic tissue is L858R sudden change, and after gefitinib drug resistance, tumor tissues is then wild type (the PDX model namely constructed by the application), specifically as shown in Figure 7.
C-met gene amplification is negative in transplanted tumor before gefitinib drug resistance, and is positive in transplanting after gefitinib inducible resistance, concrete as Fig. 8.
This genotypic change, illustrates that this transplanted tumor is changed in order to gefitinib resistant transplanted tumor by gefitinib-sensitive transplanted tumor, illustrates that gene mutation there occurs restructuring in generation gefitinib drug resistance process.
Claims (3)
1. a construction method for nonsmall-cell lung cancer gefitinib drug resistance PDX model, it is characterized in that, the method comprises the following steps:
(1) nonsmall-cell lung cancer PDX model is first built, be specially:
First, the tissue samples of nonsmall-cell lung cancer patient is obtained; Secondly, after the non-small cell carcinoma tumor sample process of originate obtained people, SCID mice is implanted; When by during stable being passaged to more than three generations or three generations, tumor is then thought that this nonsmall-cell lung cancer PDX model construction is successful;
(2) nonsmall-cell lung cancer PDX model constructed in step (1) is evaluated, filter out the nonsmall-cell lung cancer PDX model of gefitinib;be specially:
First, record tumor growth boundary in constructed nonsmall-cell lung cancer PDX model and judge, described tumor growth boundary and transplanted tumor is necrosis or gross tumor volume marginal value when there is liquefaction phenomenon when mice tumor growth;
Secondly, pathology and immunohistochemical staining evaluation are carried out to tumor in constructed nonsmall-cell lung cancer PDX model;
Finally, abrupt climatic change is carried out to the EGFR gene of tumor in constructed nonsmall-cell lung cancer PDX model; The non-small cell carcinoma PDX model of gefitinib is filtered out according to EGFR catastrophe,
(3) nonsmall-cell lung cancer gefitinib drug resistance PDX model is built, be specially:
To the nonsmall-cell lung cancer PDX model of the gefitinib that screening in step (2) obtains, give the process of gefitinib gavage, dosage progressively increases administration from 10 ~ 100mg/kg, thus induction obtains nonsmall-cell lung cancer gefitinib drug resistance PDX model.
2. the construction method of nonsmall-cell lung cancer gefitinib drug resistance PDX model as claimed in claim 1, it is characterized in that having the EGFR genetic mutation type in the non-small cell carcinoma PDX model of gefitinib in step (2): L858R, L861Q, G719C or Exon19 lack.
3. the construction method of nonsmall-cell lung cancer gefitinib drug resistance PDX model as claimed in claim 1, it is characterized in that, the gefitinib gavage process drug resistance Induction Process in step (3) comprises that low dosage 10mg/kg induces, middle dosage 50mg/kg induces, high dose 100mg/kg induces three processes.
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