CN116121192A

CN116121192A - Human primary lung cancer cell strain with double drug resistance to oxtinib and erlotinib and application thereof

Info

Publication number: CN116121192A
Application number: CN202211672432.8A
Authority: CN
Inventors: 张永昌; 向思琦; 张星; 曾亮; 杨农; 李敏; 许杰; 宋连喜
Original assignee: Central South University; Hunan Cancer Hospital
Current assignee: Central South University; Hunan Cancer Hospital
Priority date: 2022-12-26
Filing date: 2022-12-26
Publication date: 2023-05-16

Abstract

The invention discloses a human primary lung cancer cell strain with double drug resistance to oxatinib and erlotinib, which is derived from a clinical lung adenocarcinoma hydrothorax sample, wherein the human primary lung cancer cell strain is named Lu-01-1623, and the preservation number is CCTCC NO: c2022168, the preservation date is 2022, 8 and 25, and the preservation unit is China center for type culture Collection. Sequencing results show that the common lung cancer cell signaling pathway such as EGFR and MET is active, EGFR19 is deleted, MET is amplified, and the method can be used for screening lung cancer occurrence and development mechanisms and targeted therapeutic drugs.

Description

Human primary lung cancer cell strain with double drug resistance to oxtinib and erlotinib and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a human primary lung cancer cell strain with double drug resistance to oxtinib and erlotinib and application thereof.

Background

Lung cancer is the cancer species with the first incidence and mortality rate, and seriously threatens the health and life of human beings. Lung cancer is largely divided into Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), of which 80% are NSCLC, which is largely divided into adenocarcinoma, squamous carcinoma and large cell lung cancer. Lung adenocarcinoma accounts for about 50% of non-small cell lung cancers, the most common type of lung cancer, and patients with lung adenocarcinoma have very low survival rates for 5 years. Treatment of lung cancer generally includes chemotherapy, radiation therapy, targeted therapy and immunotherapy, but lung cancer inevitably develops resistance in treatment, so treatment against lung cancer has been advanced in the search. Research on the mechanism of occurrence and development of lung adenocarcinoma is helpful for developing new methods for treating lung adenocarcinoma.

Cells currently used for lung cancer research are from a foreign cell bank, and have been widely put into research by researchers to develop various drugs for lung cancer targeted therapies. However, for the treatment of lung cancer without common mutation targets and drug-resistant lung cancer, a new lung cancer cell strain with characteristic gene change is needed, and the lung cancer cell strain with characteristic gene change can be used as a tool for accurate medical treatment, so that the survival time of a lung cancer patient is prolonged. The lung cancer gene pattern of Chinese people is obviously different from that of western people: high EGFR mutation rate, low KRAS mutation rate, multiple combined HBV infections, and the like. Cells currently used for lung cancer research are from a foreign cell bank, and are widely put into research by researchers to develop various drugs for lung cancer targeted therapies. However, for the treatment of lung cancer and drug-resistant lung cancer with mutations in the Chinese gene characteristics, there is a need for lung cancer cell lines with the Chinese gene characteristics. Therefore, establishing the lung cancer cell strain with the characteristic gene change of Chinese is an essential important function for researching the occurrence and development mechanism of Chinese lung cancer and screening personalized candidate medicaments of primary lung cancer, and meanwhile, the lung cancer cell with the characteristic of Chinese gene can be used as a tool for accurate medical treatment, so that the life cycle of a Chinese lung cancer patient is prolonged.

Disclosure of Invention

The invention aims to solve the technical problem of providing a cell strain Lu-01-1623 with Chinese gene characteristics, and provides good in-vivo and in-vitro models for researching the occurrence, drug resistance mechanism and treatment of tumors in patients, thereby promoting the basic research, prevention and clinical treatment of lung cancer.

In order to solve the technical problems, the invention provides a human primary lung cancer cell strain with double drug resistance to oxatinib and erlotinib, which is derived from a hydrothorax sample of clinical lung adenocarcinoma microenvironment, wherein the human primary lung cancer cell strain is named Lu-01-1623, and the preservation number is CCTCC NO: c2022168, the preservation date is 2022, 8 and 25, and the preservation unit is China center for type culture Collection.

Specifically, the Lu-01-1623 has an EGFR19 deletion and MET amplification.

Specifically, lu-01-1623 has CD276, SETBP1, LRP5, FGFR4, DNMT1, ALK, IL7R, GNA, GATA2, EGFR protooncogene mutation and FAT1, SLX4, FANCA, HLA-B, BRCA1, PTCH1, LRP5, HNF1A, ETAA1, CIITA oncogene mutation.

Specifically, the IC50 value of Lu-01-1623 for resistance to oxatinib in vitro was 8.115. Mu.M.

Specifically, the IC50 value of Lu-01-1623 for resistance to oxatinib in vitro was 2.892. Mu.M.

The invention also provides an application of the human primary lung cancer cell strain Lu-01-1623 in the drug resistance of the oxatinib and/or the erlotinib.

In particular, the human primary lung cancer cell line Lu-01-1623 may be used in drug resistance mechanisms, reversal of drug resistance, development and evaluation of new anti-cancer drugs and/or methods.

The invention also provides an application of the human primary lung cancer cell strain Lu-01-1623 in constructing an in-vivo or in-vitro oxatinib and/or erlotinib resistant tumor model of human non-small cell lung cancer.

Specifically, the primary lung cancer cell strain Lu-01-1623 can be used for researching morphological and biological characteristics of the cells of the non-small cell lung cancer of the drug resistant human of the oxtinib and/or the erlotinib, researching the drug resistant mechanism of the tumor, analyzing the sensitivity of the anti-tumor drug, screening and evaluating the anti-tumor drug, developing the drug resistant reversal drug of the tumor, researching more effective tumor treatment method, and can be used for discussing the drug resistant mechanism of the non-small cell lung cancer and researching related signal paths thereof.

The invention provides a cell strain Lu-01-1623 with Chinese gene characteristics, which can be cultured in vitro in a culture medium, and the in vitro culture growth is fast and stable, and can be continuously passed, thereby being convenient for establishing an in vivo model of the primary lung cancer of human beings; the immunodeficient mice have good subcutaneous tumorigenicity, which provides important guarantee for in vivo experiments of medicines, and further provides basis for preparing, screening and evaluating antitumor medicines. Sequencing results show that the common lung cancer cell signaling pathway such as EGFR and MET is active, has common protooncogene EGFR (exon 19 deletion) and MET amplification, and can be used for screening lung cancer occurrence and development mechanisms and targeted therapeutic drugs.

Drawings

In order to more clearly illustrate the technical solutions of the present invention, the following brief description of the drawings is given for the purpose of the present invention, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings without the need for inventive work for a person skilled in the art.

FIG. 1 is a cell morphology (10X) of the microscopic human primary lung cancer cell line Lu-01-1623;

FIG. 2 is an in vitro growth curve of human primary lung cancer cell line Lu-01-1623;

FIG. 3 is the in vitro sensitivity of cells of human primary lung cancer cell line Lu-01-1623 to oxatinib (A549 cells as a control);

FIG. 4 is the in vitro sensitivity of cells of human primary lung cancer cell line Lu-01-1623 to erlotinib (A549 cells as a control);

FIG. 5 is a growth curve of a mouse subcutaneous tumor model of human primary lung cancer cell line Lu-01-1623;

FIG. 6 is a full exon sequencing (WES) proto-oncogene mutant Top10 of the human primary lung cancer cell strain Lu-01-1623;

FIG. 7 is a full exon sequencing (WES) anti-oncogene mutation Top10 of human primary lung cancer cell strain Lu-01-1623;

FIG. 8 is a transcriptome sequencing-based analysis of active signaling pathways and gene sets of human primary lung cancer cell line Lu-01-1623;

FIG. 9 is an in vivo test of the response of human primary lung cancer cell line Lu-01-1623 to oxatinib and erlotinib.

Detailed Description

The following description of the embodiments of the present invention will be made more apparent and fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1 establishment of an oxatinib-resistant human Primary lung adenocarcinoma cell line Lu-01-1623

1. Drawing materials

Fresh clinical lung adenocarcinoma hydrothorax samples (men, 57 years old, primary lung adenocarcinoma tumor, gene diagnosis EGFR19 deficiency, MET amplification, presence of oxtinib resistance (ethical, and patient consent)) were obtained from the hunan tumor hospital, and the collected hydrothorax samples were immediately poured into 50mL centrifuge tubes under aseptic conditions.

2. Sample processing

Centrifuging the centrifuge tube at 4deg.C and 450xg for 5min, and discarding supernatant; PBS was added for one wash pass, and after centrifugation the cells were resuspended in PBS and counted.

3. In vivo passage of animals

(1) Transplanting the counted cells to the lower part of the forelimb or the dorsal side of the hindlimb of the immunodeficient mouse, wherein each inoculation position is about 5 million cells, the inoculation quantity is determined according to the quantity of the cells, and each mouse is inoculated with 1-4 points;

(2) For tumor-bearing mice, observing at least once a week, and timely passaging and freezing up the tumor reaching a certain volume;

(3) After 4-5 passages, tumor volume is selected to reach 500-800mm ³ Is euthanized and tumor mass is removed for single cell isolation.

4. Single cell preparation and passaging

(1) Tumor mass was washed with PBS containing double antibodies to remove connective tissue and necrotic tissue, and then the tissue was transferred to RPMI 1640 medium containing 10mL of non-fetal bovine serum, and tumor samples were cut into small pieces (1-2 mm using sterile surgical scissors ³ Size of;

(2) Transferring the sheared tissues into 15mL of accumax digestive juice, incubating for 1 hour in a 37 ℃ water bath, filtering the incubated mixture with a 70 mu m filter membrane, collecting filtrate in a 50mL centrifuge tube, flushing the filter membrane with 30mL of RPMI 1640 medium containing 10% fetal bovine serum, centrifuging the filtrate at 1300rpm for 5 minutes, and removing the supernatant;

(3) Cells were resuspended in 5mL of RPMI 1640 medium containing 10% fetal bovine serum and transferred to 25mm3 dishes and isolated tumor cells were cultured in a 37℃incubator.

(4) When the cell density reaches 80-90%, the culture medium is sucked and removed, 0.5% of pancreatin is used for digesting the cells and then the cells are inoculated into a new culture flask for carrying out cell passage until the cell density reaches more than 50 generations, the cell growth is good, and the morphology is uniform.

(5) The culture flask of the cultured cells was placed under an inverted microscope, and the result was photographed under a bright field, as shown in fig. 1 (10X), and it was found that primary cell culture and subculture derived from tumor tissue were epithelialized, the cell morphology was uniform, the cells lost contact inhibition, and malignant growth was observed. The human primary lung cancer cell strain is named as Lu-01-1623 and is preserved in China center for type culture Collection, address: the preservation number of the Chinese university of Wuhan in Wuhan is CCTCC No: c2022168, 25 days of storage at 2022, 8.

Example 2 in vitro kinetics of growth of human Primary Lu cancer cell line Lu-01-1623 cells

Lu-01-1623 cells were seeded at 6000/well in 96-well plates for culture, and the number of viable cells per well was measured using CellTiter Glo kit at 6 hours, 24 hours, 48 hours, 72 hours, 96 hours and 120 hours, respectively, as shown in FIG. 2. The results show that: the cell expansion speed is good.

Example 3 sensitivity of the human Primary Lu cancer cell line Lu-01-1623 to Ocetirizine in vitro

In vitro measuring sensitivity of Lu-01-1623 to oxatinib, taking cells in logarithmic growth phase for plating, adjusting cell concentration, adding 90 μl cell suspension into each well of culture plate, and adding cell-free culture solution into blank control well; the plates were incubated at 37℃with 5% CO ₂ Culturing overnight in an incubator with 100% relative humidity; 10 mu L of the oxatinib working solution with different concentrations is added into the cell culture plate, the final concentration of the oxatinib is 30, 6, 1.2, 0.24, 0.048, 0.0096, 0.00192, 0.000384 and 0.0000768 mu M, 3 compound holes are formed in each group, 10 mu L of DMSO-cell culture solution mixed solution is added into solvent control and blank control, the final concentration of the DMSO is 0.25%, and 96-hole cell plates are put back into an incubator for culturing for 72 hours. Then, adding a cell titer-Glo working solution of 50 mu LPromega cell titer-Glo luminescence method cell activity detection kit (Promega-G7573) into each well, and wrapping a cell plate with aluminum foil paper to avoid light; shaking the plate on an orbital shaker for 2 minutes to induce cell lysis, standing the plate at room temperature for 10 minutes to stabilize the luminescence signal, and detecting the luminescence signal on a 2104EnVision plate reader; the Inhibition Rate (IR) of the test compound was calculated: IR (%) = (1- (RLU compound-RLU blank)/(RLU vehicle control-RLU blank) ×100%. Inhibition ratios of compounds at different concentrations were calculated in Excel, followed by GraphPThe ad Prism software makes a inhibition graph and calculates the relevant parameters (IC 50). A549 cells served as controls.

The results are shown in table 1 and fig. 3: the IC50 value of the in vitro-to-lung adenocarcinoma cells Lu-01-1623 is 8.115 mu M, the IC50 of the in vitro-to-non-small cell lung cancer A549 is 0.805 mu M, and the IC50 of the Lu-01-1623 cells to the oxtinib is about 10 times that of the common non-small cell lung cancer A549 cells, which indicates that the Lu-01-1623 shows a certain drug resistance to the oxtinib.

TABLE 1 half-maximal inhibitory concentration of oxatinib on Lu-01-1623 cells

Names of Compounds	LU-01-1623	A549
			Absolute IC50 value (mu M)	8.115	0.805

Example 4 sensitivity of the human Primary Lu cancer cell line Lu-01-1623 to erlotinib in vitro

In vitro measuring sensitivity of Lu-01-1623 to erlotinib, plating cells in logarithmic growth phase, adjusting cell concentration, adding 90 μl cell suspension into each well of culture plate, and adding cell-free culture solution into blank control well; the plates were incubated at 37℃with 5% CO ₂ Culturing overnight in an incubator with 100% relative humidity; 10. Mu.L of erlotinib working solution with different concentrations was added to the above cell culture plates to give final erlotinib concentration of 10,3.3333,1.1111,0.3704,0.1235,0.0412,0.0137,0 in sequence.0046 and 0.0015. Mu.M, 3 wells per group, 10. Mu.L of DMSO-cell culture medium mixture was added to the vehicle control and the blank control, the final DMSO concentration was 0.25%, and 96 well cell plates were returned to the incubator for 96h. Then, adding a CellTiter-Glo working solution of a cell activity detection kit (Promega-G7573) by a 50 mu L PromegaCellTiter-Glo luminescence method into each hole, and wrapping a cell plate by aluminum foil paper to avoid light; shaking the plate on an orbital shaker for 2 minutes to induce cell lysis, standing the plate at room temperature for 10 minutes to stabilize the luminescence signal, and detecting the luminescence signal on a 2104EnVision plate reader; the Inhibition Rate (IR) of the test compound was calculated: IR (%) = (1- (RLU compound-RLU placebo)/(RLU vehicle control-RLU placebo) ×100%. Inhibition ratios of compounds at different concentrations were calculated in Excel, and then GraphPad Prism software was used as inhibition graph and relevant parameters (IC 50). A549 cells were calculated as control.

The results are shown in table 2 and fig. 4: the IC50 value of erlotinib to lung adenocarcinoma cells Lu-01-1623 in vitro is 2.892 mu M, the IC50 of erlotinib to non-small cell lung cancer A549 in vitro is 0.075 mu M, and the IC50 of Lu-01-1623 to erlotinib is about 39 times of that of common non-small cell lung cancer A549 cells, which indicates that Lu-01-1623 shows a certain drug resistance to erlotinib.

TABLE 2 half inhibition concentration of erlotinib on Lu-01-1623 cells

Names of Compounds	Lu-01-1623	A549
			Absolute IC50 value (mu M)	0.075	2.892

EXAMPLE 5 in vivo model establishment of human Primary Lu-01-1623 cell line

Culturing Lu-01-1623 cells in vitro, collecting cells in logarithmic growth phase, counting and preparing suspension, subcutaneously inoculating Lu-01-1623 cells to the right neck and back of NOD SCID mouse, the inoculation volume is 0.2mL, and the inoculation cell amount is 10×10 ⁶ Cell suspension was PBS plus matrigel (volume ratio 1:1), lu-01-1623 subcutaneous xenograft model was established, body weight and tumor length (L) and width (D) of mice were measured 2 to 3 times per week, tumor volume was calculated according to the tumor volume calculation formula (tv=0.5×l×d), and tumor growth curve was plotted according to tumor volume-time, and the results are shown in fig. 5. The results show that: tumor volume reached 2,104mm 67 days after inoculation ³ 。

EXAMPLE 6 characterization of the human Primary lung cancer cell line Lu-01-1623

Collecting Lu-01-1623 cells, carrying out ultrasonic disruption on genomic DNA of the cells by 100-200 bp, constructing a DNA library, carrying out Whole Exon Sequencing (WES), and detecting mutation condition of genes; cell lysis was harvested to collect all transcribed RNA for mRNA enrichment, reverse transcription to cDNA followed by transcriptome (RNAseq) sequencing to analyze gene mutation and expression.

Based on WES sequencing data analysis, mutation conditions of protooncogenes and oncogenes are counted, and the protooncogenes and oncogenes are led from an ONCOKB database. The results of mutant Top10 of protooncogene are shown in FIG. 6, and it can be seen from the results that CD276, SETBP1, LRP5, FGFR4, DNMT1, ALK, IL7R, GNA, GATA2 and EGFR protooncogene of Lu-01-1623 have a plurality of mutation sites, wherein EGFR, ALK and FGFR4 are very potential cancer therapeutic targets, and EGFR has common exon19 (p.745_75del) deletion.

As shown in FIG. 7, the oncogene mutant Top10 has multi-site mutations in the oncogenes FAT1, SLX4, FANCA, HLA-B, BRCA1, PTCH1, LRP5, HNF1A, ETAA, CIITA.

Based on the gene expression data obtained by transcriptome sequencing, a genome variation analysis (GSVA analysis) is performed on the sample to find a specific signal pathway or genome. The higher the score, the more active the pathway/gene set. The results are shown in fig. 8, and it can be seen from the results that the MET pathway, EGFR pathway, small cell lung cancer and non-small cell lung cancer gene sets were active. Based on the RNAseq data, the relative expression amount FPKM (the number of fragments per 1 kilobase from map to exon per million fragments) of MET gene was 156.15, and MET gene was highly expressed.

Example 7 STR detection of human Primary Lu cancer cell line Lu-01-1623

Short tandem repeats (Short tandem repeat, STR), also known as microsatellite DNA, are typically formed by a core sequence of 2-6 bp in length, arranged in tandem repeats, with a number of repetitions between 10-60, and with a high degree of variability in the number of repetitions of the core sequence between individuals, so that the number of repetitions of a set of STR sequences is almost unique among different individuals and is the primary method of cell biology for identifying cell identity and origin. Fresh cultured human primary lung cancer cells Lu-01-1623 were collected, DNA was extracted using the genome extraction kit of Axygen, amplified using the 21-STR amplification protocol, and STR sites and sex gene Amelogenin were detected on an ABI 3730 XL-type genetic analyzer, and the results are shown in Table 3.

The results show that the above sequences are aligned with databases of cell banks such as ATCC, DSMZ, etc., and the same STR test results are not found, thus proving to be unique and that no cross contamination with other cells occurs during the primary culture.

TABLE 3 STR loci and copy number

Example 8 testing of drug response of oxatinib and erlotinib Using Lu-01-1623 in vivo cell model

In vitro culturing Lu-01-1623 cells, collecting cells in logarithmic phase, counting and preparing suspension, subcutaneously inoculating Lu-01-1623 cells into the right neck and back of NOD SCID mouse, and inoculating at a volume of 0.2mLThe cell mass of the inoculation is 10x10 ⁶ The cell suspension is PBS and matrigel (volume ratio is 1:1), and an LU-01-1623 subcutaneous xenograft model is established.

Conventional Lu-01-1623-transplanted model mice were bred, and tumor sizes were observed and measured to be 62-158mm long ³ At this time, the groups were randomized and olcetirizine (25 mg/kg) and erlotinib (50 mg/kg) were given for 21 days, and tumor volumes were observed and measured.

The results are shown in fig. 9, which shows that oxtinib and erlotinib do not significantly inhibit tumor growth; tumor Growth Inhibition (TGI) and relative tumor proliferation (T/C) calculations as shown in table 4, oxatinib and erlotinib exhibited some in vivo resistance.

TABLE 4 results of tumor growth inhibition and relative tumor proliferation rate

Note that:

tumor growth inhibition rate TGI (%): the calculation formula is as follows: TGI (%) = [1- (T) _i -T ₀ )/(C _i -C ₀ )]X 100%. Wherein T is _i Is the average tumor volume, T, at day 21 after administration of the group of components ₀ Is the average tumor volume at the time of administration of the group of components, C _i Mean tumor volume at day 21 after vehicle control group, C ₀ Is the average tumor volume at the time of vehicle control group.

Relative tumor proliferation rate T/C (%): the calculation formula is as follows: T/C% = T _RTV /C _RTV ×100％(T _RTV : treatment group RTV; c (C) _RTV : vehicle control RTV). Relative tumor volume (relative tumor volume, RTV) is calculated from the result of the tumor measurement, calculated as rtv=v _i /V ₀ Wherein V is ₀ Average tumor volume, V, measured in groups (i.e., PG-D0) _i Mean tumor volume at day 21, T _RTV And C _RTV The same day data was taken.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather to enable any modification, equivalent replacement, improvement or the like to be made within the spirit and principles of the invention.

Claims

1. The human primary lung cancer cell strain with double drug resistance to oxatinib and erlotinib is characterized by being derived from a clinical lung adenocarcinoma hydrothorax sample, wherein the human primary lung cancer cell strain is named Lu-01-1623, and the preservation number is CCTCC NO: c2022168, the preservation date is 2022, 8 and 25, and the preservation unit is China center for type culture Collection.

2. The human primary lung cancer cell line of claim 1, wherein said Lu-01-1623 has an EGFR19 deletion and MET amplification.

3. The human primary lung cancer cell line of claim 1, wherein said Lu-01-1623 has CD276,

SETBP1, LRP5, FGFR4, DNMT1, ALK, IL7R, GNA, GATA2, protooncogene mutation of EGFR and oncogene suppression mutations of FAT1, SLX4, FANCA, HLA-B, BRCA1, PTCH1, LRP5, HNF1A, ETAA1, CIITA.

4. The human primary lung cancer cell line of claim 1, wherein said Lu-01-1623 has an IC50 value of 8.115 μm for resistance to oxatinib in vitro.

5. The human primary lung cancer cell line of claim 1, wherein said Lu-01-1623 has an IC50 value of 2.892 μm for erlotinib resistance in vitro.

6. Use of the human primary lung cancer cell line Lu-01-1623 according to claim 1 for resistance to oxatinib and/or erlotinib.

7. The use according to claim 6, wherein the human primary lung cancer cell line Lu-01-1623 is useful in drug resistance mechanisms, reversal of drug resistance, development and evaluation of new anti-cancer drugs and/or methods.

8. Use of the human primary lung cancer cell line Lu-01-1623 of claim 1 in the construction of an in vivo or in vitro tumor model of resistance to oxatinib and/or erlotinib in human non-small cell lung cancer.

9. The use according to claim 8, wherein said human primary lung cancer cell line Lu-01-1623 is useful for studying the morphological and biological characteristics of the oxacetirizine and/or erlotinib resistant human non-small cell lung cancer cells, studying tumor resistance mechanisms, analyzing anti-tumor drug sensitivity and screening and evaluating anti-tumor drugs, developing tumor resistance reversal drugs, studying more effective tumor treatment methods, and for studying non-small cell lung cancer resistance mechanisms and their related signal pathways.