CN118879636A - A cell line AlmoR1 and its application - Google Patents

Abstract

The present application discloses a cell line AlmoR1 and its application, which relates to the field of cell biology technology and aims to solve the technical problem of the lack of stable non-small cell lung cancer brain metastasis targeted resistant cell line in the prior art. The cell line AlmoR1 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is May 9, 2024, and the deposit number is CGMCC NO.45858.

Description

A cell line AlmoR1 and its application

Technical Field

The present application relates to the technical field of cell biology, and in particular to a cell line AlmoR1 and applications thereof.

Background Art

Lung cancer is one of the most common tumors in clinical practice, and the incidence of brain metastasis is high. Half of lung cancer patients will develop brain metastasis during the progression of the disease, and the incidence of brain metastasis in lung cancer patients with positive driver genes (such as EGFR mutations and ALK fusions) is as high as 70.3%. Once lung cancer brain metastasis is diagnosed, the prognosis is extremely poor, with an average survival time of only 2-3 months.

In recent years, small molecule TKIs (tyrosine kinase receptor inhibitors) drugs based on driver genes have brought hope for the treatment of brain metastases due to their good blood-brain barrier penetration and high local control rate. Among them, TKIs targeting EGFR and ALK can prolong the median survival time of patients to 10-19 months; and, according to the guidelines, small molecule TKIs have become the first-line drug for patients with brain metastases from driver gene-positive lung cancer. However, clinical studies have found that a large number of patients have intracranial disease progression during long-term use of targeted therapy. At present, the treatment strategy after progression of TKIs treatment is mainly local resection and radiotherapy, which has limited means and poor efficacy. Due to the particularity of the microenvironment of the central nervous system, the treatment methods and drug mechanisms of the primary lesions are different from intracranial resistance. Therefore, there is a lack of effective systemic treatment methods in clinical practice to control intracranial lesions that progress after resistance. However, due to the difficulty in obtaining intracranial resistant lesions, there is still a lack of understanding of the mechanism of TKIs resistance in brain metastases.

At present, the drug resistance models of brain metastases of non-small cell lung cancer reported at home and abroad are mainly divided into in vivo and in vitro. Among them, the in vitro method of inducing cell resistance is separated from the tumor microenvironment, and most of the in vivo drug resistance models lack the intracranial metastasis process and cannot completely simulate the process of targeted drug resistance of brain metastases of non-small cell lung cancer in the human body. Therefore, the establishment of a stable targeted drug-resistant cell line of brain metastases of non-small cell lung cancer can provide a good research tool for the basic research of intracranial targeted therapy resistance and provide new ideas for the treatment of brain metastases of non-small cell lung cancer after targeted drug resistance.

Summary of the invention

The present application provides a cell line AlmoR1 and its application, aiming to solve the technical problem of the lack of stable non-small cell lung cancer brain metastasis targeted resistant cell line in the prior art.

To solve the above technical problems, the embodiments of the present application provide: a cell line AlmoR1, the cell line AlmoR1 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is May 9, 2024, and the deposit number is CGMCC NO. 45858.

As some optional embodiments of the present application, the cell line AlmoR1 has an EGFR E746_A750del (ex19del) mutation.

As some optional embodiments of the present application, the cell line AlmoR1 has 15 SNP mutation sites and 2 indel mutation sites on the EGFR gene.

As some optional embodiments of the present application, the numbers of the SNP mutation sites are rs712830, rs2072454, rs2270247, rs2075109, rs2075110, rs11506105, rs3735059, rs4947987, rs1558544, rs759162, rs17336919, rs6970262, rs1140475, rs139068680 and rs2692456;

The indel mutation sites are numbered rs121913421 and rs34723095, respectively.

As some optional embodiments of the present application, the cell line AlmoR1 has 30 SNP mutation sites and 5 indel mutation sites on the ALK gene.

As some optional embodiments of the present application, the numbers of the SNP mutation sites are rs1728828, rs1881421, rs1881420, rs1670283, rs12619135, rs1728826, rs1670284, rs3738870, rs4666178, rs3820712, rs6748797, rs1625283, rs1534545, rs15691 56, rs3795850, rs4666182, rs2276550, rs12714270, rs2256740, rs4666183, rs4589708, rs2272409, rs4666194, rs11127213, rs11127214, rs11127215, rs2293564, rs13428230, rs2256376, and rs4358080;

The indel mutation sites are numbered rs1391843363, rs397706189, rs10623528, rs3832036 and rs540486879, respectively.

As some optional embodiments of the present application, the cell line AlmoR1 has 4 SNP mutation sites and 1 indel mutation site on the MET gene.

As some optional embodiments of the present application, the SNP mutation sites are numbered rs62469056, rs38860, rs13223756 and rs6947629; the indel mutation site is numbered rs35212357.

As some optional embodiments of the present application, the cell line AlmoR1 is resistant to tyrosine kinase inhibitors, and the tyrosine kinase inhibitors include: osimertinib with an IC50 of 7.142 μM and ametinib with an IC50 of 7.289 μM.

On the other hand, the present application also provides an application of the cell line AlmoR1 as described above, namely, its application in testing the efficacy of tyrosine kinase inhibitors and in studying the molecular mechanism of targeted resistance to EGFR inhibitors.

The present application provides another application of the above-mentioned cell line AlmoR1, namely, application in establishing an animal model of brain metastasis of non-small cell lung cancer.

Compared with the prior art, the cell line AlmoR1 described in the present application is the first targeted drug-resistant cell line with EGFR gene mutation extracted from ex vivo brain tissue. It is of the same species as the source patient of the ex vivo brain tissue. Both primary cells and tumor tissues have EGFR 19 exon deletion mutations and show general resistance to the third-generation EGFR-TKIs. In addition, the AlmoR1 cell line shows good tumorigenicity in the in situ transplantation model in the brain. This primary cell line can be continuously passaged in vitro, and the current generation has exceeded 50 generations, and the in vitro continuous culture time exceeds 9 months. During the passage process, the cell morphology, proliferation dynamics and genetic characteristics are stable. After liquid nitrogen freezing, the cell shape after recovery remains unchanged, and it is a stable cell line. The cell line AlmoR1 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee, and the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is May 9, 2024, and the preservation number is CGMCC NO.45858.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the specific implementation of the present application or the technical solution in the prior art, the following is a brief introduction to the drawings required for the specific implementation or the prior art description. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale.

Figure 1 is a schematic diagram of obtaining brain metastases specimens of human non-small cell lung cancer resistant to the third-generation EGFR-TKIs; Part A represents the diagnosis and treatment timeline of a patient with brain metastases of non-small cell lung cancer carrying EGFR mutations; Part B represents the brain magnetic resonance imaging of the patient at different treatment stages corresponding to Figure A, and the brain metastasis is the part indicated by the red arrow; Part C represents the HE and immunohistochemistry (Ki-67, CK) staining images of the brain metastasis specimens of the last surgery (2023.7.23).

Figure 2 is a schematic diagram of the culture and identification results of the AlmoR1 cell line; Part A shows the morphology of the AlmoR1 cell line, optical microscopy of the 1st, 10th, 20th, 30th, 40th and 50th generations of AlmoR1 cells (magnified 10 times and 20 times, respectively), and the scale bar is 200μm; Part B shows the expression of pan-CK in AlmoR1 cells using immunofluorescence technology. The green fluorescent signal is pan-CK, and the blue fluorescent signal is DAPI; Parts C and D show the short tandem repeat sequence (STR) analysis map of the AlmoR1 cell line, showing the characteristic peaks of each locus.

Figure 3 is a schematic diagram of the AlmoR1 cell line with EGFR exon 19 deletion mutation; wherein, Part A and Part B represent the number and mutation types of single nucleotide variations (SNVs) and insertions and deletions (indels) of well-known lung cancer-related genes (TP53, PTEN, PIK3CA, NF1, MSH2, MET, KRAS, EGFR, APC, ALK, MET, BRAF) detected in AlmoR1 cells.

Figure 4 is a diagram showing the drug resistance verification results of the AlmoR1 cell line; wherein, Part A is a growth curve diagram drawn by continuously monitoring the proliferation of the AlmoR1 cell line using the CCK8 experiment; Part B shows a sensitivity test diagram of the EGFR-mut cell PC9 to ametinib and osimertinib detected by the MTT experiment; Part C shows a sensitivity test diagram of AlmoR1 to ametinib and osimertinib detected by the MTT experiment; Part D shows a schematic diagram of the activation of EGFR and downstream survival signals AKT and ERK of AlmoR1 cells in the control group, osimertinib treatment group and ametinib treatment group verified by Western blot.

Figure 5 is a diagram showing the verification results of the intracranial tumorigenicity of the AlmoR1 cell line; wherein, Part A is the in vivo imaging of the nude mouse brain AlmoR1 cell orthotopic implanted tumor on days 1, 4, 8, 12, 16, 20, 24, 28, and 32 3 weeks after implantation; Part B is a growth curve of the orthotopic implanted AlmoR1 cells in the brain; Part C shows the fresh brain image of the AlmoR1 tumor-bearing nude mouse, the brain of the tumor-bearing nude mouse was removed at the terminal stage of the disease, and the tumor is the part indicated by the blue arrow; Part D shows the HE and pan-CK staining images of the orthotopic implanted tumor in the mouse brain.

The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with embodiments and with reference to the accompanying drawings.

DETAILED DESCRIPTION

The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

The present application relates to an EGFR inhibitor-resistant human non-small cell lung cancer (NSCLC) brain metastasis cell line, named AlmoR1. The cell line is isolated and cultured from brain metastasis-resistant tissue of a patient with non-small cell lung cancer carrying EGFR mutations. The AlmoR1 cell line has an EGFR exon 19 deletion mutation, exhibits extensive resistance to EGFR-TKIs, and has good intracranial tumorigenicity.

The establishment of this cell line is helpful for studying the molecular mechanism of targeted resistance to EGFR inhibitors and provides valuable experimental materials for the development of new treatment methods. The AlmoR1 cell line not only retains the main molecular characteristics of the original tumor EGFR 19 exon deletion mutation, but also shows growth advantages in both in vivo and in vitro environments, making it an ideal model for studying the mechanism of resistance to EGFR inhibitors and drug screening. In addition, the AlmoR1 cell line can be used for in vivo and in vitro drug sensitivity testing, new drug development and targeted therapy research, providing a basis for the formulation of personalized treatment strategies.

The cell line AlmoR1 of the present application has been preserved in the cell bank with the deposit number CGMCC No. 45858. This cell line is the first humanized lung cancer brain metastasis cell line with driver gene mutations and acquired resistance to the third-generation EGFR-TKIs; therefore, this cell line can be used as a tool cell for targeted drug resistance research, especially in the field of targeted drug resistance research of lung cancer brain metastasis.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. The use of NSCLC cell lines in research helps to understand its biological characteristics and develop new treatments. The following are cell lines with mutations used in existing non-small cell lung cancer research: ZX2021H, NCI-H2030, NCI-H1975, NCI-H2228, NCI-H2122, NCI-H23, Calu-3, PC9, NCI-H3255, NCI-H3122, NCI-H1792, NCI-H1435. These NSCLC cell lines with mutations are sensitive to TKIs and cannot truly simulate clinical drug resistance. At present, there are no cell lines derived from brain metastases. The cell line AlmoR1 of the present invention is a NSCLC brain metastasis cell line with mutations that is resistant to TKIs, which can provide a good research platform for intracranial targeted resistance of non-small cell lung cancer.

In addition, one of the key issues in the current research on brain metastases is the modeling of brain metastases. Modeling methods for brain metastases include in situ brain metastasis models (for example, brain metastases formed by subcutaneous injection of melanoma, brain metastases formed by breast cancer cells inoculated in breast pads), ectopic brain metastasis models (tail vein injection, left ventricular injection, and carotid artery injection), and intracranial transplant tumor models. However, the currently available brain metastasis models cannot fully simulate the full steps of brain metastasis, resulting in various defects in the modeling methods of brain metastases. One of the more important defects is that lung cancer cell lines are used in the modeling process of brain metastases, which cannot simulate the intracranial tumor environment of real brain metastases, and there is currently a lack of research on targeted drug resistance of brain metastases. The AlmoR1 cell line of the present invention is directly extracted from the intracranial tumor tissue of NSCLC patients, so the use of this cell line for modeling is more in line with the actual drug resistance of brain metastases and better persuasiveness.

In summary, the purpose of this application is to provide a TKIs-resistant EGFR mutant non-small cell lung cancer brain metastasis cell line, which provides a good research platform for basic research on intracranial targeted resistance of non-small cell lung cancer.

Example 1: Obtaining a human non-small cell lung cancer brain metastasis specimen resistant to third-generation EGFR-TKIs: In order to establish a human non-small cell lung cancer brain metastasis cell line resistant to EGFR inhibitors, the present application first obtains a brain metastasis resistant tissue from a patient with non-small cell lung cancer carrying an EGFR mutation.

As shown in part A of Figure 1, the patient was diagnosed with brain metastasis from lung cancer in June 2021 and underwent brain metastasis resection in July. A new lesion in the right cerebral hemisphere was found in a cranial MRI reexamination in May 2022, and a puncture biopsy indicated EGFR exon 19 deletion combined with MET amplification. Since May 25, 2022, the patient has successively received ametinib combined with anlotinib (6 months), ametinib combined with crizotinib (2 months), and ametinib (2 months) alone until the lesion progressed, and underwent a second brain metastasis resection (March 10, 2023), and the lesions also indicated exon 19 deletion combined with MET amplification.

Subsequently, osimertinib combined with bevacizumab was given for treatment (3 months). On July 23, 2023, the lesion progressed again, and the patient underwent a third brain metastases resection ( Figure 1 , part B).

HE and immunohistochemical staining of the last surgical specimen confirmed that the lesion was a brain metastasis tissue of non-small cell lung cancer with proliferative activity (part C in Figure 1 ).

Example 2: Cultivation and identification of AlmoR1 cell line: Primary cell lines were extracted from fresh brain metastasis tissues of the last surgery and purified by differential adhesion method. After 50 generations of continuous in vitro culture, a cell line was successfully constructed and named AlmoR1.

As shown in part A of FIG2 , during the continuous in vitro culture of AlmoR1, the morphology of cells at the 1st, 10th, 20th, 30th, 40th and 50th generations was continuously collected by optical microscopy, and the observation results showed that the morphology of the cell line was stable.

Immunofluorescence technology detected that AlmoR1 cells widely expressed pan-CK, which preliminarily confirmed that the cells were epithelial tumor cells (Figure 2, part B).

Next, the AlmoR1 cell line was subjected to STR identification. By testing the information of eight STR loci (CSF1PO, D13S317, D16S539, D5S818, D7S820, THO1, TPOX, and vWA), it was found that the AlmoR1 cell line had a unique STR profile and the possibility of cross-contamination with other cells was ruled out ( Figure 2 C-Figure 2D).

The above data indicate that the present application has established a new and stable lung cancer brain metastasis cell line.

Example 3: AlmoR1 cell line has EGFR exon 19 deletion mutation: In order to clarify the mutation site information of the AlmoR1 cell line, whole exon sequencing was performed on it.

Among them, the mutation types of well-known lung cancer-related genes (TP53, PTEN, PIK3CA, NF1, MSH2, MET, KRAS, EGFR, APC, ALK, MET, BRAF) are mainly single-site mutations (SNVs) and insertion-deletion mutations (indels) (Figure 3, part A and Figure 3, part B).

Furthermore, the present application searched the sites of EGFR, ALK, and MET and found that only the EGFR E746_A750del site mutation was a sense mutation, which is the most common type of exon 19 deletion (Table 1).

The mutation sites of ALK and MET are nonsense mutations as shown in Table 2-Table 3. The above results suggest that the lung cancer brain metastasis cell line AlmoR1 constructed in the present application has an EGFR 19 exon deletion mutation.

Table 1:

Table 2:

Table 3:

Example 4: Drug resistance verification of AlmoR1 cell line: First, the present application uses CCK8 experiment to continuously monitor the proliferation of AlmoR1 cell line and draws a growth curve (Part A in Figure 4). The curve is S-shaped, indicating that AlmoR1 cells are growing exponentially and in good condition. In order to confirm that AlmoR1 cells are resistant to the third-generation EGFR-TKIs, the present application uses MTT experiment to detect the sensitivity of EGFR-mut cells PC9 and AlmoR1 to ametinib and osimertinib, and it is concluded that the IC50 of PC9 to the third-generation EGFR-TKIs is 0.9μM, and the IC50 of AlmoR1 to the third-generation EGFR-TKIs is about 7μM (Part B in Figure 4 and Part C in Figure 4). Further, the present application confirms in the Western blot experiment that ametinib and osimertinib can inhibit the phosphorylation of EGFR, but cannot inhibit the activation of downstream survival signals AKT and ERK (Part D in Figure 4). These results indicate that the primary lung cancer brain metastasis cell line AlmoR1 constructed in this application is generally resistant to EGFR-TKIs.

Example 5: Verification of intracranial tumorigenicity of AlmoR1 cell line: In order to evaluate whether AlmoR1 cells have intracranial tumorigenicity, the present application successfully constructed AlmoR1/luc stably expressing green fluorescence using a lentiviral transfection experiment, and used this cell line to construct a brain orthotopic tumor model. After 3 weeks, the results of in vivo imaging of mice showed that (3/4, 75%) brain orthotopic tumors could be formed in the nude mice, and the tumor growth was good as can be seen from the proliferation curve (Part A in Figure 5 and Part B in Figure 5). Subsequently, the present application extracted the orthotopic brain tumors of mice and performed HE and pan-CK staining. The results showed that the pathological type was highly consistent with the tissue characteristics of brain metastases surgically removed by patients (Part C in Figure 5 and Part D in Figure 5). In general, AlmoR1 cells exhibit good intracranial tumorigenicity.

In summary, the primary non-small cell lung cancer brain metastasis cell line AlmoR1 extracted in this application has an EGFR 19 exon deletion mutation, and exhibits extensive resistance to EGFR-TKIs and good intracranial tumorigenicity. This cell line is the first humanized lung cancer brain metastasis cell line, with driver gene mutations and acquired resistance to third-generation EGFR-TKIs; therefore, this cell line can be used as a tool cell for targeted drug resistance research, especially in the field of targeted drug resistance research of lung cancer brain metastasis.

The above are only preferred embodiments of the present application, and are not intended to limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the present application specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims (10)

1. The cell strain AlmoR1 is characterized in that the cell strain AlmoR1 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation address of being No. 3 of Sei No. 1, no. 3 of the Korean area North Star of Beijing, a preservation date of being 2024, a date of being 09 months of year 05, and a preservation number of being CGMCC No. 45858.

2. The cell line AlmoR1 of claim 1, wherein the cell line AlmoR has an EGFR e746_a750del (ex 19 del) mutation.

3. The cell line AlmoR1 of claim 1, wherein the cell line AlmoR has 15 snp mutation sites and 2 indel mutation sites on the EGFR gene.

4. The cell line AlmoR of claim 3, wherein the numbering of the snp mutation sites is rs712830、rs2072454、rs2270247、rs2075109、rs2075110、rs11506105、rs3735059、rs4947987、rs1558544、rs759162、rs17336919、rs6970262、rs1140475、rs139068680 and rs2692456, respectively;

The index mutation sites are numbered rs121913421 and rs34723095 respectively.

5. The cell line AlmoR of claim 1, wherein the cell line AlmoR1 has 30 snp mutation sites and 5 indel mutation sites on the ALK gene.

6. The cell line AlmoR of claim 5, wherein the numbering of the snp mutation sites is rs1728828、rs1881421、rs1881420、rs1670283、rs12619135、rs1728826、rs1670284、rs3738870、rs4666178、rs3820712、rs6748797、rs1625283、rs1534545、rs1569156、rs3795850、rs4666182、rs2276550、rs12714270、rs2256740、rs4666183、rs4589708、rs2272409、rs4666194、rs11127213、rs11127214、rs11127215、rs2293564、rs13428230、rs2256376 and rs4358080, respectively;

The index mutation sites are numbered rs1391843363, rs397706189, rs10623528, rs3832036 and rs540486879 respectively.

7. The cell line AlmoR1 of claim 1, wherein the cell line AlmoR1 has 4 snp mutation sites and 1 indel mutation site on MET gene;

Numbering of the snp mutation sites is rs62469056, rs38860, rs13223756 and rs6947629 respectively; the index mutation site is numbered rs35212357.

8. The use of the cell line AlmoR of claim 1 for testing the pharmacodynamic properties of tyrosine kinase inhibitors and for studying the molecular mechanisms of EGFR inhibitor-targeted drug resistance.

9. The use of cell line AlmoR1 according to claim 8, wherein the tyrosine kinase inhibitor comprises: the IC50 was 7.142. Mu.M of oxatinib and the IC50 was 7.289. Mu.M of almitinib.

10. The use of the cell line AlmoR according to claim 1 for the establishment of an animal model of brain metastasis from non-small cell lung cancer.