A human primary lung cancer cell line resistant to crizotinib and its application

Abstract

The present invention discloses a human primary lung cancer cell line resistant to crizotinib, which is derived from a surgically resected sample of clinical lung adenocarcinoma. The human primary lung cancer cell line is named Lu-01-1443, and its preservation number is CCTCC NO: C2022166. The preservation date is August 25, 2022, and the preservation unit is the China Center for Type Culture Collection. Lu-01-1443 has an EML4-ALK fusion mutation, can be cultured in vitro in a medium, and has rapid and stable growth in vitro and can be continuously passaged, thus facilitating the establishment of an in vivo model of human lung cancer; it has good subcutaneous tumorigenicity in immunodeficient mice, providing an important guarantee for in vivo experiments of drugs, and thus providing a basis for the preparation, screening, and evaluation of anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a human primary lung cancer cell line resistant to crizotinib and its application. Background Art

[0002] Lung cancer, as the most common type of cancer in the world currently, is the main cause of the increased mortality of cancer-related diseases, seriously threatening human health and life. According to the clinical histological type, lung cancer can be subdivided into four different types: squamous cell carcinoma, adenocarcinoma, large cell undifferentiated carcinoma, and small cell undifferentiated carcinoma. Among these four different types, small cell lung cancer is different from the other several tumor types in terms of disease clinical manifestations, the degree of metastasis in the middle and late stages, and clinical treatment principles. Therefore, we summarize these three different types of lung cancer other than small cell lung cancer and classify them collectively as non-small cell lung cancer. Lung adenocarcinoma accounts for about 50% of non-small cell lung cancer. The 5-year survival rate of patients with lung adenocarcinoma is very low, and about 75% of the patients have developed and entered the middle and late stages of the tumor when they are clinically first diagnosed with this disease. Therefore, studying the occurrence mechanism of lung adenocarcinoma helps to develop treatment methods for lung adenocarcinoma.

[0003] With the in-depth study of the tumor pathogenesis and its biological behavior, currently people have focused on molecular targeted therapy with high specificity and mild adverse reactions. In recent years, the field of molecular targeted therapy for lung cancer has focused on targets such as EGFR, K-ras, and VEGF. Among them, EGRF targeted therapy has achieved gratifying results. However, with the discovery of EML4-ALK and TGF-ALK fusion genes by Rikova and Soda, and later the discovery of KIF5B-ALK fusion gene by Wong, the ALK (Anaplastic lymphoma kinase) fusion gene has become a research hotspot in lung cancer targeted therapy.

[0004] In cell line experiments and genetically engineered mouse models, EML4-ALK is a powerful carcinogenic driver, and cancer cells with ALK fusion genes are highly sensitive to ALK kinase inhibition. In these cancer cells, ALK is the only regulator that endangers the cell growth and survival pathways, including the PI3K-Akt and MEK-ERK pathways. ALK inhibition can lead to the inhibition of these pathways, inducing the arrest of cell proliferation and apoptosis. The early research and development of Pfizer on the new therapeutic drug crizotinib for the c-MET oncogene has an inhibitory effect on ALK kinase. Recent clinical trials have shown that patients with advanced ALK-positive non-small cell lung cancer are highly sensitive to crizotinib targeted therapy. However, with the in-depth study, although most ALK-positive non-small cell lung cancer patients are sensitive to the ALK tyrosine kinase inhibitor crizotinib, they will inevitably relapse due to drug resistance within one year.

[0005] As a commonly used drug for targeted therapy of non-small cell lung cancer patients, crizotinib has significant efficacy and safety in clinical applications. However, as one of the targeted drugs, many patients develop acquired drug resistance due to various factors such as gene mutations. The mechanisms of acquired drug resistance mainly include drug target variations (including ALK copy number amplification or kinase domain mutations), bypass activation, and other drug resistance mechanisms, and some patients have multiple drug resistance mechanisms coexisting. In addition, the drug resistance mechanisms of about 30% of ALK inhibitor-resistant patients are still unclear.

[0006] To explore the mechanism of acquired resistance to crizotinib, we established a crizotinib-resistant human lung cancer cell line, Lu-01-1443, which has an EML4-ALK fusion mutation. There is no report of such a crizotinib-resistant non-small cell lung cancer cell line at home and abroad. In addition, the cell lines commonly used for studying the mechanism of lung cancer development and drug screening currently come from foreign cell banks such as ATCC, DSMZ, ECACC, and JCRB, and there is no cell line with Chinese gene characteristics. The lung cancer gene map of Chinese people is significantly different from that of Westerners: a high EGFR mutation rate, a low KRAS mutation rate, a high co-infection rate of HBV, etc. The current cells used for lung cancer research all come from foreign cell banks, and these cells have been widely used by researchers in research, and a variety of drugs for lung cancer targeted therapy have been developed. However, there is an urgent need for lung cancer cell lines with Chinese gene characteristics for the treatment of lung cancer with Chinese gene characteristic mutations and drug-resistant lung cancer. Therefore, establishing a lung cancer cell line with characteristic gene changes of Chinese people plays an essential and important role in studying the mechanism of lung cancer development and screening candidate drugs for personalized primary lung cancer in Chinese people. At the same time, lung cancer cells with Chinese gene characteristics can be used as tools for precision medicine to improve the survival period of Chinese lung cancer patients. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a cell line Lu-01-1443 with Chinese gene characteristics, which provides good in vivo and in vitro models for studying the occurrence, drug resistance mechanism, and treatment of tumors in patients, thereby promoting basic research, prevention, and clinical treatment of lung cancer.

[0008] To solve the above technical problems, the present invention provides a crizotinib-resistant primary human lung cancer cell line, which is derived from a pleural effusion sample of the clinical lung adenocarcinoma microenvironment. The primary human lung cancer cell line is named Lu-01-1443, with a preservation number of CCTCC NO: C2022166, a preservation date of August 25, 2022, and the address of the preservation unit is Wuhan University, Wuhan, China. The preservation unit is the China Center for Type Culture Collection.

[0009] Specifically, the Lu-01-1443 has an EML4-ALK fusion mutation.

[0010] Specifically, the Lu-01-1443 has proto-oncogene mutations of ARHGEF28, IL7R, ALK, RPS6KB2, NRG1, GLI1, FGFR4, DNMT1, AURKA, ATXN7 and tumor suppressor gene mutations of KM2TC, NCOR1, HLA-B, FAT1, ATXN2, RECQL4, HNF1A, FANCA, ETAA1, BARD1.

[0011] Specifically, the Lu-01-1443 is resistant to crizotinib (25 mg / kg) in an in vivo model, with a tumor growth inhibition rate of 33.7% and a relative tumor growth rate of 67.7%.

[0012] Specifically, the Lu-01-1443 is sensitive to alectinib (20 mg / kg) in an in vivo model, with a tumor growth inhibition rate of 100.6% and a relative tumor growth rate of 3.4%.

[0013] The present invention also provides the use of the aforementioned human primary lung cancer cell line Lu-01-1443 in crizotinib resistance.

[0014] Specifically, the human primary lung cancer cell line Lu-01-1443 can be used for drug resistance mechanisms, drug resistance reversal, development and evaluation of new anti-cancer drugs and / or methods.

[0015] The present invention also provides the use of the aforementioned human primary lung cancer cell line Lu-01-1443 in constructing an in vivo or in vitro crizotinib-resistant tumor model of human non-small cell lung cancer.

[0016] Specifically, the human primary lung cancer cell line Lu-01-1443 can be used to study the morphology and biological characteristics of crizotinib-resistant human non-small cell lung cancer cells, study tumor drug resistance mechanisms, analyze anti-tumor drug sensitivity and screen and evaluate anti-tumor drugs, develop tumor drug resistance reversal drugs, study more effective tumor treatment methods, and can be used to explore the drug resistance mechanisms of non-small cell lung cancer and the research of its related signaling pathways.

[0017] By providing a cell line Lu-01-1443 with Chinese gene characteristics, the present invention can be used to study the morphology and biological characteristics of crizotinib-resistant human non-small cell lung cancer cells, study tumor drug resistance mechanisms, analyze anti-tumor drug sensitivity and screen and evaluate anti-tumor drugs, develop tumor drug resistance reversal drugs, study more effective tumor treatment methods, etc., and can be used to explore the drug resistance mechanisms of non-small cell lung cancer and the research of its related signaling pathways, with high scientific research and production application value, and is expected to produce good scientific research, economic and social benefits. Brief Description of the Drawings

[0018] To more clearly illustrate the technical solutions of the present invention, the accompanying drawings required for the present invention are briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0019] Figure 1 It is a morphological diagram (10X) of human primary lung cancer cell line Lu-01-1443 under a microscope;

[0020] Figure 2 It is the in vitro growth curve of human primary lung cancer cell line Lu-01-1443;

[0021] Figure 3 It is the growth curve of the subcutaneous tumor model of human primary lung cancer cell line Lu-01-1443 in mice;

[0022] Figure 4 It is the drug reactivity of the in vivo model of human primary lung cancer cell line Lu-01-1443 to crizotinib and alectinib;

[0023] Figure 5 It is the top 10 proto-oncogene mutations of whole exome sequencing (WES) of human primary lung cancer cell line Lu-01-1443;

[0024] Figure 6 It is the top 10 tumor suppressor gene mutations of whole exome sequencing (WES) of human primary lung cancer cell line Lu-01-1443;

[0025] Figure 7 It is the analysis of active signaling pathways and gene sets based on transcriptome sequencing of human primary lung cancer cell line Lu-01-1443. Detailed Embodiments

[0026] The technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments in the present invention belong to the scope of protection of the present invention.

[0027] Example 1. Establishment of a crizotinib-resistant human primary lung adenocarcinoma cell line Lu-01-1443

[0028] 1. Material collection

[0029] Fresh clinical lung adenocarcinoma surgical resection samples were obtained from Hunan Cancer Hospital (male, 49 years old, primary lung adenocarcinoma tumor (in line with ethics and patient consent), ALK+ patient, resistant to crizotinib, with EML4-ALK fusion mutation). Immediately, the tumor tissue was placed in sterile tissue preservation fluid pre-cooled to 4°C under sterile conditions and transferred to a pre-sterilized biosafety cabinet.

[0030] 2. Sample processing

[0031] The tissue was taken out from the centrifuge tube and quickly transferred to a 10 cm culture dish, and washed twice with PBS containing double antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin). Necrotic tissue and surrounding non-tumor tissue were removed, and it was ensured as much as possible that the necrotic part was not used for inoculation.

[0032] 3. In vivo passage in animals

[0033] (1) The tumor tissue was transplanted subcutaneously into the dorsal side of the forelimb or hindlimb of immunodeficient mice using a tumor inoculation needle, and each piece of tissue was about 30 - 50 mm 3 , and the inoculation quantity was determined according to the overall size of the patient's tumor tissue. 1 - 4 points were inoculated into each mouse;

[0034] (2) For tumor-bearing mice, they were observed at least once a week, and tumors reaching a certain volume (400 - 1000 mm 3 ) were passaged in time and cryopreserved;

[0035] (3) After 4 - 5 passages, mice with tumor volumes reaching 500 - 800 mm 3 were euthanized and the tumor masses were dissected for single-cell isolation.

[0036] 4. Single-cell preparation and passage

[0037] (1) The tumor mass was washed with PBS containing double antibiotics to remove connective tissue and necrotic tissue, and then the tissue was transferred to 10 mL of RPMI 1640 medium without fetal bovine serum. The tumor sample was cut into small pieces (1 - 2 mm 3 in size) with sterile surgical scissors;

[0038] (2) The minced tissue was transferred to 15 mL of accumax digestive solution and incubated in a 37°C water bath for 1 hour. The incubated mixture was filtered through a 70 μm filter membrane, and the filtrate was collected in a 50 mL centrifuge tube. The filter membrane was rinsed with 30 mL of RPMI 1640 medium containing 10% fetal bovine serum, and the filtrate was centrifuged at 1300 rpm for 5 minutes to remove the supernatant;

[0039] (3) The cells were resuspended with 5 mL of RPMI 1640 medium containing 10% fetal bovine serum and transferred to 25 mm3 In a culture dish, the isolated tumor cells were cultured in an incubator at 37°C under 5% CO 2 conditions.

[0040] (4) When the cell density reached 80 - 90%, the culture medium was aspirated, the cells were digested with 0.5% trypsin and inoculated into a new culture flask for cell passage. After passage more than 50 times, the cells grew well and had a relatively uniform morphology.

[0041] (5) The culture flask containing the cultured cells was placed under an inverted microscope and photographed under bright field. The results are shown in Figure 1 (10X). It can be seen that the primary cell culture and passage culture derived from the tumor tissue were epithelial-like, with relatively uniform cell morphology. The cells lost contact inhibition and grew malignantly. This human primary lung cancer cell line was named Lu-01-1443, deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, deposit number CCTCC No: C2022166, and the deposit date was August 25, 2022.

[0042] Example 2. STR detection of human primary lung cancer cell line Lu-01-1443

[0043] Short tandem repeat (STR), also known as microsatellite DNA, is generally composed of a core sequence with a length of 2 - 6 bp arranged in tandem multiple times, and the number of repeats is mostly between 10 - 60 times. The number of repeats of the core sequence among individuals shows high variability. Therefore, the number of repeats of a set of STR sequences is almost unique among different individuals, and it is the main method for cell biology to identify the identity and origin of cells. Freshly cultured human primary lung cancer cells Lu-01-1443 were collected, the genomic DNA of the cells was extracted, and PCR amplification was performed using 5'-end labeled STR primers, and the resulting product was sequenced. The copy numbers of STR loci are shown in Table 1.

[0044] The results showed that when the above sequences were compared with the databases of cell banks such as ATCC and DSMZ, no identical STR detection results were found, which proved that it was unique and there was no cross-contamination with other cells during the primary culture process.

[0045] Table 1. Copy numbers of STR loci

[0046]

[0047] Example 3. In vitro growth kinetics of human primary lung cancer cell line Lu-01-1443 cells

[0048] The Lu-01-1443 cells were seeded in 96-well plates at a density of 6000 cells per well and cultured. The number of viable cells in each well was measured using the CellTiter Glo kit at 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, and 120 hours respectively. The results are shown in Figure 2 . The results showed that the cell amplification rate was good.

[0049] Example 4. Establishment of an in vivo model of human primary lung cancer cell line Lu-01-1443

[0050] The Lu-01-1443 cells were cultured in vitro. Cells in the logarithmic growth phase were collected, counted, and resuspended. The Lu-01-1443 cells were subcutaneously inoculated into the right cervical and dorsal regions of BALB / c Nude mice at a volume of 0.2 mL and a cell number of 5x10 6 . The cell suspension was PBS plus Matrigel (volume ratio 1:1). A subcutaneous xenograft model of Lu-01-1443 was established. The body weight of the mice and the length (L) and width (D) of the tumors were measured 2 to 3 times a week. The tumor volume was calculated according to the tumor volume calculation formula (TV = 0.5 * L * D * D), and the tumor growth curve was plotted based on the tumor volume over time. The results are shown in Figure 3 .

[0051] The results showed that 14 days after inoculation, the tumor volume reached 2228 mm 3 .

[0052] Example 5. Testing the drug responses of crizotinib and alectinib using the in vivo model of Lu-01-1443 cells

[0053] The Lu-01-1443 cells were cultured in vitro. Cells in the logarithmic growth phase were collected, counted, and resuspended. The Lu-01-1443 cells were subcutaneously inoculated into the right cervical and dorsal regions of BALB / c nude mice at a volume of 0.2 mL and a cell number of 5x10 6 . The cell suspension was PBS plus Matrigel (volume ratio 1:1). A subcutaneous xenograft model of Lu-01-1443 was established. The mice with the Lu-01-1443 transplantation model were conventionally fed. When the tumor size grew to 121 - 173 mm 3 , the mice were randomly grouped and gavaged with crizotinib (25 mg / kg) and alectinib (20 mg / kg) for 13 days. The tumor volume was observed and measured. The results are shown in Figure 4As shown, the results showed that crizotinib (25 mg / kg) could not significantly inhibit tumor growth, while alectinib (20 mg / kg) could significantly inhibit tumor growth; the tumor growth inhibition rate (TGI) and relative tumor proliferation rate (T / C) were calculated as shown in Table 2, and Lu-01-1443 showed resistance to crizotinib and sensitivity to alectinib.

[0054] Table 2. Results of tumor growth inhibition rate and relative tumor proliferation rate

[0055]

[0056] Note:

[0057] Tumor growth inhibition rate TGI (%) : The calculation formula is as follows: TGI (%) = [1 - (T i - T 0 ) / (C i - C 0 )] × 100%. Where T i is the average tumor volume on the 21st day after grouping in the dosing group, T 0 is the average tumor volume at the time of grouping in the dosing group, C i is the average tumor volume on the 21st day after grouping in the vehicle control group, and C 0 is the average tumor volume at the time of grouping in the vehicle control group.

[0058] Relative tumor proliferation rate T / C (%) : The calculation formula is as follows: T / C% = T RTV / C RTV × 100% (T RTV : RTV of the treatment group; C RTV : RTV of the vehicle control group). According to the results of tumor measurement, the relative tumor volume (relative tumor volume, RTV) was calculated, and the calculation formula was RTV = V i / V 0 , where V 0 is the average tumor volume measured at the time of grouping (i.e., PG-D0), V i is the average tumor volume on the 21st day, and T RTV and C RTV take data on the same day.

[0059] The p-value was analyzed by One-way ANOVA based on the tumor volume (compared with the vehicle control group), and p < 0.05 was considered to have a significant difference.

[0060] Example 6. Analysis of characteristic genes of human primary lung cancer cell line Lu-01-1443 resistant to crizotinib

[0061] Collect Lu-01-1443 cells, ultrasonically fragment the genomic DNA of the cells into 100 - 200 bp, construct a DNA library, perform whole-exome sequencing (WES) to detect gene mutations; collect cell lysates to collect all transcribed RNA for mRNA enrichment, then reverse transcribe it into cDNA and perform transcriptome (RNAseq) sequencing to analyze gene mutations and expression.

[0062] Based on the analysis of WES sequencing data, count the mutation situations of proto-oncogenes and tumor suppressor genes in the samples. The proto-oncogenes and tumor suppressor genes are cited from the ONCOKB database. The top 10 results of proto-oncogene mutations are as Figure 5 shown. It can be seen from the results that the proto-oncogenes ARHGEF28, IL7R, ALK, RPS6KB2, NRG1, GLI1, FGFR4, DNMT1, AURKA, and ATXN7 of Lu-01-1443 have multiple mutation sites, among which ALK, NRG1, and FGFR4 are highly potential cancer treatment targets.

[0063] The top 10 results of tumor suppressor gene mutations are as Figure 6 shown. Tumor suppressor genes such as KM2TC, NCOR1, HLA-B, FAT1, ATXN2, RECQL4, HNF1A, FANCA, ETAA1, and BARD1 have multi-site mutations.

[0064] Based on the gene expression data obtained from transcriptome sequencing, perform gene set variation analysis (GSVA analysis) on the samples to find specific signaling pathways or gene sets. The higher the score, the more active the pathway / gene set. The results are as Figure 7 shown. It can be seen from the results that the activities of the MET pathway, small cell lung cancer, and non-small cell lung cancer gene sets are relatively active.

[0065] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A primary human lung cancer cell line resistant to crizotinib, characterized in that it is derived from a surgically resected sample of clinical lung adenocarcinoma. The primary human lung cancer cell line is named Lu-01-1443, with the deposit number CCTCC NO: C2022166, the deposit date being August 25, 2022, and the deposit unit being the China Center for Type Culture Collection. The Lu-01-1443 has an EML4-ALK fusion mutation.

2. The primary human lung cancer cell line according to claim 1, characterized in that the Lu-01-1443 has proto-oncogene mutations of ARHGEF28, IL7R, ALK, RPS6KB2, NRG1, GLI1, FGFR4, DNMT1, AURKA, ATXN7 and tumor suppressor gene mutations of KMT2C, NCOR1, HLA-B, FAT1, ATXN2, RECQL4, HNF1A, FANCA, ETAA1, BARD1.

3. The primary human lung cancer cell line according to claim 1, characterized in that the Lu-01-1443 is resistant to crizotinib in an in vivo model, with a tumor growth inhibition rate of 33.7% and a relative tumor proliferation rate of 67.7%.

4. The primary human lung cancer cell line according to claim 1, characterized in that the Lu-01-1443 is sensitive to alectinib in an in vivo model, with a tumor growth inhibition rate of 100.6% and a relative tumor proliferation rate of 3.4%.

5. Use of the primary human lung cancer cell line according to claim 1 in constructing an in vivo or in vitro crizotinib-resistant tumor model of human non-small cell lung cancer.

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