Lung cancer cell line of mouse-derived EML4-ALK V1 or V3a fusion protein, construction method and application

Abstract

This invention belongs to the field of genetic engineering cell line technology, specifically providing a lung cancer cell line, construction method, and application of mouse-derived EML4-ALK V1 or V3a fusion protein. The method includes the following steps: synthesizing the full-length coding sequence of mouse-derived EML4-ALK V1 as shown in SEQ ID NO:1 or the full-length coding sequence of mouse-derived EML4-ALK V3a as shown in SEQ ID NO:2; cloning into a lentiviral vector to obtain a recombinant lentiviral transfer plasmid of mouse-derived EML4-ALK V1 or V3a; transfecting cells to obtain lentiviral particles of mouse-derived EML4-ALK V1 or V3a; using mouse lung cancer cells as parental cells, infecting with the lentiviral particles of mouse-derived EML4-ALK V1 or V3a, and screening to obtain a lung cancer cell line stably expressing the mouse-derived EML4-ALK V1 or V3a fusion protein. This invention obtains a mouse-derived V1 / V3a fusion construct through non-mechanical homologous exon substitution. The lung cancer cell lines constructed stably expressing mouse-derived EML4-ALK V1 or V3a fusion proteins can form quantifiable, drug-intervention-enabled in vivo and in vitro models with subtype comparison value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering cell line technology, and particularly relates to lung cancer cell lines, construction methods and applications of mouse-derived EML4-ALK V1 or V3a fusion proteins. Background Technology

[0002] The EML4-ALK fusion gene is an important driver gene variant in non-small cell lung cancer. Its encoded fusion protein possesses constitutively activated ALK tyrosine kinase activity, which can continuously activate downstream signaling pathways such as MAPK, PI3K-AKT, and JAK-STAT. EML4-ALK has several fusion variant subtypes, among which V1 and V3a are the two most frequently studied in clinical and basic research. Different subtypes may differ in protein stability, cellular localization, drug sensitivity, drug resistance evolution, and metastatic biological behavior.

[0003] Existing studies have shown that EML4-ALK positive models largely rely on human tumor cell lines, exogenous human EML4-ALK expression systems, or genetically engineered mouse models. Human cell line transplantation typically requires immunodeficient mice, making it difficult to fully evaluate the host's immune system; expressing human fusion proteins in a human-mouse xenogeneic context may introduce additional immune interference; traditional genetically engineered mouse models are time-consuming, costly, and not conducive to rapidly comparing the functional differences between V1 and V3a subtypes in the same parental context.

[0004] Simply mapping human EML4-ALK V1 or V3a to mouse homologous genes by exon numbering does not directly yield mouse fusion constructs that are equivalent to human variants, have correct reading frames, and retain reasonable domains. Therefore, this invention provides a tool for obtaining mouse EML4-ALK V1 / V3a fusion genes, expression vectors, stable LLC (Lewis lung cancer cells, a classic lung cancer cell line from mice), and their application in immunocompetent mouse tumor models based on non-mechanical homologous exon substitution rules. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a lung cancer cell line, construction method, and application of mouse-derived EML4-ALK V1 or V3a fusion protein, with the aim of solving the problems mentioned in the background art.

[0006] In a first aspect, the present invention provides a method for constructing a lung cancer cell line based on a mouse-derived EML4-ALK V1 or V3a fusion protein, comprising the following steps: Step 1: Synthesize the full-length coding sequence of mouse EML4-ALK V1 as shown in SEQ ID NO:1 or the full-length coding sequence of mouse EML4-ALK V3a as shown in SEQ ID NO:2; Step 2: The full-length coding sequence of mouse EML4-ALK V1 or V3a is cloned into a lentiviral vector to obtain a recombinant lentiviral transfer plasmid of mouse EML4-ALK V1 or V3a. Step 3: Transfect cells with the recombinant lentiviral transfer plasmid of mouse EML4-ALK V1 or V3a to obtain mouse EML4-ALK V1 or V3a lentiviral particles. Step 4: Using mouse lung cancer cells as parental cells, infect them with mouse-derived EML4-ALK V1 or V3a lentiviral particles, and screen to obtain lung cancer cell lines that stably express mouse-derived EML4-ALK V1 or V3a fusion proteins.

[0007] Furthermore, in step one, a Kozak sequence is set at the 5′ end of the full-length coding sequence of mouse-derived EML4-ALK V1 or EML4-ALK V3a, and a 3×FLAG tag coding sequence is set at the 3′ end.

[0008] Furthermore, in step two, the lentiviral vector is the pLV3-CMV-3×FLAG-EF1a-CopGFP-Puro vector.

[0009] Furthermore, in step four, the mouse lung cancer cells are LLC.

[0010] Secondly, the present invention provides a lung cancer cell line that stably expresses mouse-derived EML4-ALK V1 or V3a fusion protein, which is constructed by a method for constructing a lung cancer cell line of mouse-derived EML4-ALK V1 or V3a fusion protein.

[0011] Thirdly, this invention provides the application of a lung cancer cell line stably expressing mouse-derived EML4-ALK V1 or V3a fusion protein in constructing an ALK-positive lung cancer model in immune-healthy mice, whereby the ALK-positive lung cancer model is used for non-disease treatment or diagnostic purposes.

[0012] Furthermore, ALK-positive lung cancer models include subcutaneous tumorigenesis, in situ lung cancer models, hematogenous dissemination models, and brain metastasis-related models.

[0013] Fourthly, the present invention provides a recombinant lentiviral transfer plasmid of mouse EML4-ALK V1 or V3a, wherein the recombinant lentiviral transfer plasmid contains the full-length coding sequence of mouse EML4-ALK V1 as shown in SEQ ID NO:1 or the full-length coding sequence of mouse EML4-ALK V3a as shown in SEQ ID NO:2.

[0014] This invention offers the following advantages: A murine V1 / V3a fusion construct is obtained through non-mechanical homologous exon substitution, and a stable expression cell line is formed in the same LLC parental background, reducing immune interference from heterologous proteins and transient expression fluctuations. Experimental results show that both V1 and V3a cells exhibit detectable EML4-ALK-FLAG and p-ALK, accompanied by phosphorylation of the STAT3, AKT, and ERK1 / 2 pathways; the migration phenotypes and alectinib dose-response of the two cell types can be distinguished. Therefore, lung cancer cell lines stably expressing murine EML4-ALK V1 or V3a fusion proteins can form quantifiable, drug-intervention-compatible in vivo and in vitro models with subtype comparison value, providing a stable and reproducible tool for studying brain metastasis mechanisms, ALK inhibitor efficacy, drug resistance mechanisms, and the tumor immune microenvironment. Attached Figure Description

[0015] The exemplary embodiments of the present invention can be more fully understood by referring to the following figures, in which ns indicates no statistically significant difference (P≥0.05), * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001. Figure 1 This is a schematic diagram of the mouse-derived EML4-ALK V1 / V3a fusion boundary and lentiviral expression vector structure according to an embodiment of the present invention, wherein: Figure 1 In the figure, A represents the correspondence between human and mouse EML4 and ALK exons, the fusion breakpoint, and the structure of mouse V1 and V3a fusion proteins and fusion genes. Figure 1 B in the text represents the structure of the pLV3-CMV-V1-3×FLAG-EF1a-CopGFP-Puro vector. Figure 1 C in the text represents the structure of the pLV3-CMV-V3a-3×FLAG-EF1a-CopGFP-Puro vector.

[0016] Figure 2 The diagram shows the results of expression and signaling pathway validation in stable cell lines, including: Figure 2 A in the image represents CopGFP fluorescence images of WT, V1, and V3a cells, with a scale bar of 100 μm. Figure 2 In this context, B represents the relative mRNA expression levels of ALK, V1, and V3a in WT, V1, and V3a cells. Figure 2 The diagram in C represents the mechanism by which the EML4-ALK fusion protein constitutively activates the MAPK, PI3K-AKT, and JAK-STAT pathways, illustrating the activation relationship of these three pathways. Figure 2 In the figure, D represents the detection results of EML4-ALK-FLAG fusion protein and p-ALK; Figure 2 E in the figure represents the detection results of STAT3, p-STAT3, AKT, p-AKT, ERK1 / 2, p-ERK1 / 2, and β-actin.

[0017] Figure 3 The figures shown are the results of cell migration, invasion, foci formation, and alectinib response verification in an embodiment of the present invention, wherein: Figure 3 In the figure, A represents the Transwell invasion and migration results of WT, V1, and V3a cells. The top row shows representative staining images and invasive cell counts from the invasion experiment, while the bottom row shows representative staining images and migrating cell counts from the migration experiment. The scale bar is 100 μm. Figure 3 B in the figure represents the focal formation results of NIH / 3T3-WT (empty vector), NIH / 3T3-V1 and NIH / 3T3-V3a cells. V1 and V3a cells showed focal cell stacking and clumping growth under confluence culture conditions. The scale bar is 100 μm. Figure 3 In the figure, C represents the dose-response curve and IC50 comparison results of cell viability after alectinib treatment; Figure 3 In the figure, D represents the EML4-ALK-FLAG fusion protein and p-ALK detection results after alectinib treatment; Figure 3 In the figure, E represents the detection results of STAT3, p-STAT3, AKT, p-AKT, ERK1 / 2, p-ERK1 / 2 and GAPDH after alectinib treatment.

[0018] Figure 4 The following is a diagram showing the validation results of the immune-healthy mouse brain transfer model according to an embodiment of the present invention: Figure 4 In the figure, A represents the in vivo brain imaging results of mice in the WT, V1, and V3a groups; Figure 4 B in the figure represents the quantitative result of the average imaging signal of the brain region; Figure 4 In this context, C represents the pathological result of brain tissue. Figure 4 D in the figure represents the Kaplan-Meier survival curves of the three groups of mice; Figure 4 E in the figure represents the incidence of brain metastasis in the three groups of mice; Figure 4F in the figure represents the observation results of the dorsal and ventral surfaces of the isolated brain specimen. Detailed Implementation

[0019] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0021] Example 1: Determination of the fusion boundary of mouse-derived EML4-ALK V1 / V3a This embodiment does not mechanically replace mouse sequences based on human exon numbering. Instead, it uses open reading frame continuity, preservation of the N-terminal structural contribution of EML4, preservation of the intracellular tyrosine kinase domain of ALK, and equivalence to the structural boundary of human V1 / V3a proteins as common constraints to determine the fusion boundary.

[0022] We obtained the coding region information of the human EML4 and human ALK ends corresponding to the human EML4-ALK V1 and V3a fusion variants, and obtained the coding transcripts and exon annotations of mouse Eml4 and mouse Alk. Using protein-coding coordinates rather than simple exon numbers as the basis for correspondence, we established the exon-amino acid coordinate correspondence between human EML4 and mouse Eml4, and between human ALK and mouse Alk, respectively.

[0023] Based on the above correspondence, the fusion boundaries of candidate mouse sources were confirmed step by step according to the following constraints: (1) Connect the ends of different exons of mouse Eml4 to the start of exon 20 of mouse Alk to obtain candidate open reading frames; (2) Translate each candidate open reading frame and exclude candidates that have undergone code shifting or premature termination; (3) Confirm the N-terminal structural contribution required for the retention of the EML4 end to promote oligomerization and aberrant activation of the fusion protein; (4) Confirm that the ALK terminus retains the intracellular tyrosine kinase domain starting from exon 20; (5) Compare the structural boundaries of the candidates that meet the aforementioned constraints with those of the human V1 and V3a fusion proteins for equivalence.

[0024] Only candidates that simultaneously meet the requirements of open reading frame continuity, preservation of the N-terminal structural contribution of EML4, preservation of the intracellular tyrosine kinase domain of ALK, and protein structural boundary equivalence are identified as the corresponding mouse-derived fusion boundaries. Therefore, mouse-derived V1 is determined to be fused with mouse Eml4 exons 1-14 and mouse Alk exons 20-29; mouse-derived V3a is determined to be fused with mouse Eml4 exons 1-7 and mouse Alk exons 20-29, as shown in Table 1.

[0025] Table 1. Correspondence between human EML4-ALK V1 / V3a reference and mouse fusion boundary

[0026] Example 2: Construction of recombinant lentiviral expression plasmid The mouse EML4-ALK V1 fusion gene ontology coding sequence (SEQ ID NO:1) and the mouse EML4-ALK V3a fusion gene ontology coding sequence (SEQ ID NO:2) were synthesized. These two fusion gene ontology coding sequences were inserted into the multiple cloning site of the pLV3-CMV-3×FLAG-EF1a-CopGFP-Puro lentiviral vector, respectively, downstream of the CMV promoter. In the resulting recombinant vectors, each fusion gene ontology coding sequence has a Kozak sequence at the 5′ end, and a 66 bp 3×FLAG tag coding sequence and a TAG stop codon are sequentially linked at the 3′ end. The fusion gene ontology coding sequence and the 3×FLAG tag coding sequence are kept within the same open reading frame, thus obtaining the pLV3-CMV-V1-3×FLAG-EF1a-CopGFP-Puro and pLV3-CMV-V3a-3×FLAG-EF1a-CopGFP-Puro recombinant lentiviral transfer plasmids. SEQ ID NO:1 and SEQ ID NO:2 do not contain the Kozak sequence, the 3×FLAG tag coding sequence, or the TAG stop codon. The vector and cloning operations described in this embodiment are one implementation method to achieve stable expression of the aforementioned specific fusion gene.

[0027] Based on the recombinant vector design sequence, the coding sequence of the mouse EML4-ALK V1 fusion gene is 3201 bp long, encoding 1067 amino acids, and its nucleotide and amino acid sequences are shown in SEQ ID NO:1 and SEQ ID NO:3, respectively; the coding sequence of the mouse EML4-ALK V3a fusion gene is 2379 bp long, encoding 793 amino acids, and its nucleotide and amino acid sequences are shown in SEQ ID NO:2 and SEQ ID NO:4, respectively. Neither of the coding sequences of the above fusion gene genes contains a 3×FLAG tag coding sequence. In the constructed expression vector, the 3′ end of the fusion gene gene is kept within the same open reading frame as the 66 bp 3×FLAG tag coding sequence, followed by a TAG stop codon. The complete expression open reading frames of V1 and V3a, containing the 3×FLAG tag coding sequence and the TAG stop codon, are 3270 bp and 2448 bp long, respectively.

[0028] The constructed recombinant lentiviral transfer plasmids pLV3-CMV-V1-3×FLAG-EF1a-CopGFP-Puro and pLV3-CMV-V3a-3×FLAG-EF1a-CopGFP-Puro were identified using Sanger sequencing. The effective sequencing range of the Sanger sequencing mainly covered the coding sequences of the V1 and V3a fusion genes. The effective sequencing regions were compared with the sequences shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0029] A schematic diagram of the mouse-derived EML4-ALK V1 / V3a fusion boundary and lentiviral expression vector structure is shown below. Figure 1 As shown.

[0030] Sequencing results showed that, within the effective coverage area of ​​the Sanger sequencing, the fusion gene ontology coding sequence in the V1 recombinant plasmid was consistent with the corresponding sequence shown in SEQ ID NO:1, and the fusion gene ontology coding sequence in the V3a recombinant plasmid was consistent with the corresponding sequence shown in SEQ ID NO:2. No base deletions, insertions, or substitutions were found within the effective sequencing region, and no sequence abnormalities causing changes in the open reading frame were found. These results indicate that the V1 and V3a fusion gene ontology coding sequences have been successfully introduced into the corresponding recombinant lentiviral transfer plasmids, and their effective sequencing coverage regions are consistent with the designed sequences.

[0031] Example 3: Establishment of stable LLC cell lines 1. Preparation of lentiviral particles: HEK293T cells were seeded in 10cm culture dishes using DMEM containing 10% fetal bovine serum. When cell confluence reached approximately 70%–80%, pLV3-CMV-V1-3×FLAG-EF1a-CopGFP-Puro, pLV3-CMV-V3a-3×FLAG-EF1a-CopGFP-Puro, or their corresponding empty vectors, were co-transfected into HEK293T cells with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G. The mass ratio of transfer plasmid, packaging plasmid, and envelope plasmid was 4:3:1, and the total amount of plasmid used per 10cm culture dish was 10μg, including 5μg of transfer plasmid, 3.75μg of psPAX2, and 1.25μg of pMD2.G. Linear polyethyleneimine (PEIMAX) was used as the transfection reagent, with a plasmid DNA to PEI mass ratio of 1:3.

[0032] Fresh culture medium was added 12 hours after transfection, and viral supernatant was collected at 48 and 72 hours post-transfection. The viral supernatant was centrifuged at 500×g for 10 min at 4°C to remove cell debris and then filtered through a 0.45 μm filter. The filtered viral supernatant was used directly for subsequent LLC cell infection; unused viral supernatant was aliquoted and stored at −80°C, avoiding repeated freeze-thaw cycles.

[0033] 2. LLC cell infection and drug screening: LLC mouse lung cancer cells were stored at 2 × 10⁻⁶ cells per well. 5 Cells were seeded into 6-well plates and cultured until the cell confluence reached approximately 50%. Empty vector, V1, and V3a lentiviral particles were then added for infection. 100 μL of viral supernatant was added to each well, and the culture medium was replaced with fresh medium after 48 hours of infection.

[0034] Selection was initiated 24 hours post-infection with puromycin at a final concentration of 2 μg / mL. The puromycin-containing culture medium was changed every 2 days for 4 consecutive days. LLC cells uninfected with lentivirus during the same period served as a control group. Drug selection was considered complete when all cells in this control group died. After selection, maintenance culture was performed using medium containing 2 μg / mL puromycin.

[0035] CopGFP fluorescence signals were observed or detected using an Axio Observer+Axiocam 208 inverted fluorescence microscope, and CopGFP positivity was used as the criterion for lentiviral infection and enrichment of positive cells to obtain empty vector, V1 and V3a positive cell populations.

[0036] 3. Limiting dilution and candidate clone screening: V1 and V3a positive cells selected by drug screening were digested to prepare single-cell suspensions. After cell counting, the suspensions were diluted with complete culture medium to 10 cells / mL, and seeded into 96-well plates at a rate of 1 cell per well and 100 μL per well. Eight hours after seeding, each well was observed under a microscope. Wells with only one cell observed were marked, and wells containing more than two cells or whose single-cell origin could not be confirmed were excluded.

[0037] The labeled single-cell wells were cultured for 7–10 days, with the culture medium changed every 3 days. Once the single cells formed clones and reached approximately 80% confluence, they were sequentially transferred to 24-well, 12-well, and 6-well plates, and 6cm and 10cm culture dishes for expansion culture. During expansion culture and continuous passage, the CopGFP fluorescence signal, cell growth status, and passage stability of each clone were observed. Based on the clarity of the single-cell origin, the proportion of CopGFP positivity, cell morphology, and the stability of the fluorescence signal during continuous passage, one candidate single-clone cell line was selected from groups V1 and V3a for subsequent qPCR and Western blot verification.

[0038] 4. qPCR validation of selected monoclonal cell lines: Wild-type (WT) empty vector control LLC cells, selected V1 candidate monoclonal cell lines, and selected V3a candidate monoclonal cell lines were collected. When the cells reached approximately 80% confluence, total RNA was extracted from each group using an adsorption column RNA extraction kit, and the RNA concentration and purity were determined. Equal amounts of total RNA were reverse transcribed to obtain the corresponding cDNA.

[0039] The mRNA expression levels of mouse Alk, mouse EML4-ALK V1, and mouse EML4-ALK V3a in each group of cells were detected by real-time quantitative PCR. For detecting mouse Alk, the upstream primer shown in SEQ ID NO:5 and the downstream primer shown in SEQ ID NO:6 were used; for detecting mouse EML4-ALK V1, the upstream primer shown in SEQ ID NO:7 and the downstream primer shown in SEQ ID NO:6 were used; and for detecting mouse EML4-ALK V3a, the upstream primer shown in SEQ ID NO:8 and the downstream primer shown in SEQ ID NO:6 were used.

[0040] The mouse Gapdh upstream primer shown in SEQ ID NO:9 and the mouse Gapdh downstream primer shown in SEQ ID NO:10 were used for internal reference gene detection, with an expected amplification product length of 133 bp. Real-time quantitative PCR was performed using a 10 μL reaction system, including 5 μL of 2×SYBR Green qPCR premix, 0.4 μL of 10 μmol / L upstream primer, 0.4 μL of 10 μmol / L downstream primer, 1 μL of cDNA template, and 3.2 μL of nuclease-free water.

[0041] The amplification program was as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, annealing and extension at 60℃ for 30 s, for a total of 40 cycles; melting curve analysis was performed after amplification. Each sample was prepared in triplicate, and a template-free negative control was included. The relative mRNA expression level of the target gene was calculated using the 2^-ΔΔCt method. Experiments were independently repeated in triplicate, and results are expressed as mean ± standard deviation.

[0042] The results of expression and signaling pathway validation in stable cell lines are shown in the figure below. Figure 2 As shown.

[0043] qPCR results as follows Figure 2 As shown in B, the relative mRNA expression levels of mouse EML4-ALK in WT, V1, and V3a cells were generally similar. Significantly elevated levels of mouse-derived EML4-ALK V1 transcripts were detected in the selected V1 monoclonal cell line, and significantly elevated levels of mouse-derived EML4-ALK V3a transcripts were detected in the selected V3a monoclonal cell line. These results indicate that the corresponding mouse-derived EML4-ALK fusion gene transcripts were stably expressed in the selected V1 and V3a monoclonal cell lines, respectively.

[0044] 5. Western blot validation of selected monoclonal cell lines: WT empty vector control LLC cells, selected V1 monoclonal cell lines, and selected V3a monoclonal cell lines were collected separately. After washing the cells with pre-chilled PBS, RIPA lysis buffer (1:1:1000) containing protease inhibitors and phosphatase inhibitors was added, and the cells were thoroughly lysed on ice. The cell lysates were centrifuged at 4°C and approximately 12000×g for 15 min, the supernatant was collected, and the protein concentration was determined using the BCA method.

[0045] Equal amounts of total protein were collected from each sample, separated by SDS-PAGE, and transferred to PVDF membranes. The PVDF membranes were blocked with protein-free rapid blocking buffer at room temperature for 1 hour, followed by incubation overnight at 4°C with primary antibodies against FLAG, p-ALK, STAT3, p-STAT3, AKT, p-AKT, ERK1 / 2, p-ERK1 / 2, and β-actin. After washing, the corresponding enzyme-labeled secondary antibodies were added, and the membranes were incubated at room temperature for 1 hour. Protein bands were detected using chemiluminescence immunoassay. β-actin was used as an internal control for sample loading.

[0046] Western blot results are as follows Figure 2 D and Figure 2 As shown in E in the figure. The EML4-ALK-FLAG fusion protein was not detected in the WT empty vector control LLC cells; the EML4-ALK-FLAG fusion protein was detected in both the selected V1 and V3a monoclonal cell lines, and the apparent molecular weight of the V1 fusion protein was higher than that of the V3a fusion protein, which is consistent with the theoretical length difference between the two fusion proteins.

[0047] p-ALK signaling was detected in both V1 and V3a monoclonal cell lines. Compared with WT empty vector control LLC cells, p-STAT3, p-AKT, and p-ERK1 / 2 signals were enhanced in V1 and V3a monoclonal cell lines, while the total protein levels of STAT3, AKT, and ERK1 / 2 and the β-actin internal control were generally comparable. These results indicate that the selected V1 and V3a monoclonal cell lines can stably express the corresponding mouse-derived EML4-ALK fusion protein and activate ALK and its downstream signaling pathways.

[0048] 6. Determination of stable monoclonal cell lines: Based on the combined results of single-cell origin, CopGFP fluorescence signal, continuous passage stability, qPCR results, and Western blot results, the selected V1 and V3a monoclonal cell lines were confirmed to be LLC cell lines that stably express mouse-derived EML4-ALK V1 and V3a fusion proteins, respectively.

[0049] The selected stable monoclonal cell line of V1 was named "LLC-mEML4-ALK-V1", and the selected stable monoclonal cell line of V3a was named LLC-mEML4-ALK-V3a; LLC cells introduced with the corresponding empty vector were named LLC-WT and used as empty vector control cells for subsequent experiments.

[0050] The lentiviral packaging, cell infection, drug screening, and limiting dilution steps described in this embodiment are used to obtain LLC cell lines stably expressing a specific mouse-derived EML4-ALK fusion protein. This application does not distinguish itself by general cell infection or screening procedures, but rather by introducing the V1 fusion gene ontology coding sequence shown in SEQ ID NO:1 and the V3a fusion gene ontology coding sequence shown in SEQ ID NO:2 under the same LLC parental cell background, the same lentiviral vector backbone, and the same screening system, respectively, to obtain stable cell models that can be used for parallel comparison of the two fusion subtypes.

[0051] Example 4: Verification of cell migration and focal formation phenotypes 1. Transwell invasion experiment: WT empty vector control LLC cells in logarithmic growth phase, LLC cells stably expressing mouse EML4-ALK V1 fusion protein, and LLC cells stably expressing mouse EML4-ALK V3a fusion protein were digested and prepared into single-cell suspensions. Invasion assays were performed using Transwell chambers in 24-well plates with 8 μm pore sizes. Matrigel was diluted 1:8 in serum-free high-glucose DMEM medium, and 50 μL of diluted Matrigel was added to the upper chamber of each Transwell. The cells were incubated at 37°C for 1 h to allow the Matrigel to solidify. Cells from each group were resuspended in serum-free medium and cultured at a density of 5 × 10⁶ cells per chamber. 4 200 μL of cells were seeded into the upper chamber of a Transwell cell line; 600 μL of complete culture medium containing 10% fetal bovine serum was added to the lower chamber as a chemotactic solution. Cells were cultured at 37°C and 5% CO2 for 24 h. After culture, the Transwell cells were removed, and cells that had not yet penetrated the membrane of the upper chamber were gently wiped away with a cotton swab. Cells that had penetrated the membrane and attached to the lower membrane surface were fixed with tissue fixative for 20 min, followed by staining with 0.1% crystal violet for 20 min. After washing away excess stain and air-drying, three fields of view were randomly selected from each chamber under a microscope for photographing and counting; the number of cells that penetrated the membrane represented cell invasion ability.

[0052] 2. Transwell migration experiment: Transwell migration assays were performed using 24-well plates with 8 μm pore sizes and equipped with Transwell chambers. Matrigel was not plated onto the chamber membrane. WT, V1, and V3a cells were resuspended in serum-free DMEM medium and cultured at 5 × 10⁶ cells per chamber. 4Cells were seeded at a total volume of 200 μL in the upper chamber; 600 μL of complete culture medium containing 10% fetal bovine serum was added to the lower chamber. Cells were cultured at 37°C and 5% CO2 for 24 h. After culture, unmigrated cells on the upper surface of the membrane were gently wiped away with a cotton swab. Cells that migrated to the lower surface were fixed with 4% paraformaldehyde for 20 min, followed by staining with 0.1% crystal violet for 20 min. After washing away excess stain with PBS and air-drying, three fields of view from each chamber were randomly selected under the same microscope for photographing and counting. Cell migration ability was represented by the number of cells that crossed the membrane. Results are expressed as mean ± standard deviation. Differences among the WT, V1, and V3a groups were analyzed using one-way ANOVA, and post-hoc multiple comparisons were performed using the Tukey method. P < 0.05 was considered statistically significant. Transwell invasion and migration assay results are shown below. Figure 3 As shown in Figure A, the top row contains representative staining images and invasive cell counts from the invasion assay, while the bottom row contains representative staining images and migrating cell counts from the migration assay. Compared to the WT group, both the V1 and V3a groups showed an increase in the number of invasive and migrating cells crossing the Transwell membrane, indicating that stable expression of mouse-derived EML4-ALK V1 or V3a fusion proteins can enhance the invasion and migration-related phenotypes of LLC cells.

[0053] Further focus formation experiments were conducted on lung cancer cell lines containing WT, mouse-derived EML4-ALK V1, or V3a fusion proteins. Mouse embryonic fibroblasts (NIH / 3T3) were used as parental cells and infected with lentiviruses obtained from the V1 or V3a recombinant plasmids described in this example. Equal amounts of cells were seeded at 2000 cells per well and 2 mL per well in 6-well culture plates and cultured continuously under 5% fetal bovine serum. After 2 weeks of culture, once the WT control cells reached over 90% confluence and exhibited relatively obvious contact inhibition growth characteristics, the growth morphology and focus formation of each group of cells were further observed.

[0054] The experimental results regarding the formation of cell foci in each group of cells are as follows: Figure 3 As shown in Figure B, the results indicate that the WT group cells exhibited uniform monolayer growth; while the V1 and V3a groups showed focal cluster growth areas with clear boundaries and high cell density, where cells locally stacked and formed visible cellular foci. This suggests that after the introduction and stable expression of the mouse-derived EML4-ALK V1 or V3a fusion gene, the contact inhibition growth characteristics of NIH / 3T3 cells were overcome, enabling them to continue local proliferation and form cellular foci under cell confluence conditions, thus presenting a focal formation phenotype that can be used to evaluate the oncogenic transformation activity of the fusion protein.

[0055] Example 5: Verification of alectinib sensitivity and signal inhibition 1. Alectinib dose-response experiment: WT empty vector control LLC cells in logarithmic growth phase, LLC cells stably expressing mouse EML4-ALK V1 fusion protein, and LLC cells stably expressing mouse EML4-ALK V3a fusion protein were digested and prepared into single-cell suspensions. Cells from each group were seeded at 3000 cells per well and 100 μL of complete culture medium per well in 96-well plates and cultured at 37°C and 5% CO2 for 24 h to allow cell adhesion.

[0056] Alectinib stock solution was serially diluted with complete culture medium to final concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μmol / L. Cells in each group were added to different final concentrations of alectinib, with cells containing an equal volume of solvent serving as a solvent control. Cell-free culture medium blanks were also included. The final solvent concentration remained consistent across all groups, not exceeding 0.1%. Cells were cultured for 48 hours under alectinib treatment.

[0057] After treatment, add 10 μL of CCK-8 reagent and 90 μL of serum-free DMEM medium to each well, and incubate at 37°C for 2 h. Measure the absorbance at 450 nm using a microplate reader. Calculate the relative cell viability using the following formula: Relative cell viability (%) = (Absorbance of experimental wells - Absorbance of blank wells) ÷ (Absorbance of solvent control wells - Absorbance of blank wells) × 100%.

[0058] Using the common logarithm of the final alectinib concentration as the x-axis and relative cell viability as the y-axis, a four-parameter nonlinear regression model was used to fit the dose-response curve, and the half-maximal inhibitory concentration (IC50) of cells in each group was calculated. 50 Each drug concentration was configured with 3 replicates, and the experiment was independently repeated 3 times. Results are expressed as mean ± standard deviation, and IC50 values ​​for each group are calculated. 50 Differences between the groups were compared using one-way ANOVA, with P < 0.05 indicating statistical significance.

[0059] Alectinib dose-response curve and IC50 50 The comparison results are as follows Figure 3 As shown in C. Compared with WT empty control LLC cells, the dose-response curves of V1 and V3a cells shifted to the left, IC50... 50 The decreased IC50 indicates that LLC cells stably expressing mouse-derived EML4-ALKV1 or V3a fusion proteins have higher drug sensitivity to alectinib. The IC50 values ​​between the V1 and V3a groups were significantly lower. 50 The existence of certain differences indicates that the two stable cell lines constructed in this embodiment can be used to compare the drug response characteristics of different mouse-derived EML4-ALK fusion subtypes in parallel.

[0060] 2. Detection of signaling pathways after alectinib treatment: WT, V1, and V3a cells were seeded into 6-well plates and cultured until the cell confluence reached approximately 70%. Then, alectinib at a final concentration of 10 μmol / L was added for 4 hours. Cells treated with an equal volume of DMSO served as a control.

[0061] After treatment, cells were washed with pre-cooled PBS, and total cellular protein was extracted using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined using the BCA method. Equal amounts of protein from each sample were subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking, the cells were incubated with primary antibodies against EML4-ALK-FLAG fusion protein, p-ALK, STAT3, p-STAT3, AKT, p-AKT, ERK1 / 2, p-ERK1 / 2, and GAPDH, respectively, followed by incubation with corresponding enzyme-labeled secondary antibodies. Protein bands were detected using chemiluminescence immunoassay. GAPDH was used as an internal control for sample loading.

[0062] Protein test results as follows Figure 3 D and Figure 3 As shown in E in the figure. Compared with the corresponding solvent control, alectinib treatment reduced p-ALK signaling in V1 and V3a cells, and also reduced downstream phosphorylated protein signals such as p-STAT3, p-AKT, and p-ERK1 / 2, while the total protein levels of STAT3, AKT, and ERK1 / 2 did not change significantly. These results indicate that alectinib can inhibit ALK kinase activity mediated by mouse-derived EML4-ALK V1 or V3a fusion protein and its downstream JAK-STAT, PI3K-AKT, and MAPK signaling pathways.

[0063] The results of alectinib dose-response experiments and signaling pathway detection jointly demonstrate that the LLC cell line stably expressing mouse-derived EML4-ALK V1 or V3a fusion protein constructed in this embodiment has clear ALK dependence and pharmacologically modifiable characteristics, and can be used for drug screening, efficacy evaluation, comparison of fusion subtype differences, and research on drug resistance mechanisms of ALK inhibitors.

[0064] Example 6: Validation of a brain transfer model in immune-intelligent mice Eight-week-old male C57BL / 6 mice weighing 22±2g were selected as experimental animals. Mice were randomly divided into three groups: WT, V1, and V3a, with eight mice in each group. The WT group used control LLC cells that did not express the EML4-ALK fusion protein, while the V1 and V3a groups used LLC cells that stably expressed mouse-derived EML4-ALK V1 and V3a fusion proteins, respectively. All cells used in the animal experiments were labeled with a luciferase reporter tag for subsequent in vivo bioluminescence imaging.

[0065] Cells in the logarithmic growth phase were collected from each group, digested, and single-cell suspensions were prepared. After washing with sterile PBS, the cell concentration was adjusted to 2 × 10⁻⁶ cells / mL. 6 Cells / mL. Each mouse was injected with 100 μL of cell suspension, resulting in a cell count of 2 × 10⁻⁶ cells / mL. 5 Mice were anesthetized with isoflurane inhalation and fixed in a supine position. The surgical area of ​​the neck was disinfected, and a 1 cm incision was made in the midline of the neck. The subcutaneous tissue and submandibular gland were dissected layer by layer to expose the trachea. The neck muscles and carotid sheath were bluntly dissected along the left side of the trachea, and the left common carotid artery, external carotid artery, and internal carotid artery were fully exposed while protecting the vagus nerve. The proximal end of the common carotid artery and the external carotid artery were ligated, and blood flow to the internal carotid artery was temporarily blocked.

[0066] After establishing an injection route in the common carotid artery, the temporary occlusion of the internal carotid artery was released, and 100 μL of tumor cell suspension was slowly injected via the internal carotid artery over approximately 5 minutes. After injection, the injection site was sealed to ensure no active bleeding or leakage of the cell suspension. The subcutaneous tissue and skin were then sutured layer by layer. Post-operatively, the mice were kept warm on a heating pad until they recovered from anesthesia, after which they were returned to their IVC cages for continued rearing. The cell inoculation volume, injection route, injection time, and post-operative rearing conditions were kept consistent across all three groups of mice.

[0067] On day 14 after cell inoculation, in vivo bioluminescence imaging was performed on mice. Before imaging, mice were intraperitoneally injected with 150 μL of D-fluorescein potassium working solution at a dose of 150 mg / kg body weight. Ten minutes after administration, bioluminescence images were acquired using an IVIS in vivo imaging system under isoflurane anesthesia. Image acquisition parameters were set as follows: exposure time 60 s, medium pixel binning (8×8), f / stop 1, field of view 12.5 cm, and open emission filter. The same parameters were used for image acquisition in all groups. The brain tumor region was selected as the region of interest (ROI), and the average signal within the ROI was measured. ), and quantitative analysis was performed using a consistent ROI selection and background subtraction method.

[0068] Intact brain tissue was harvested at the predetermined time or after the mice reached their survival endpoint. The ex vivo brain specimens were observed and photographed from both the dorsal and ventral sides, and the location, number, and distribution of visible lesions were recorded. After fixation with tissue fixative for 24 hours, the brain tissue was embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin. The brain tissue structure and tumor metastases were observed under a microscope. The presence of brain metastases was determined by combining in vivo bioluminescence signals, observation of ex vivo brain specimens, and pathological results of the brain tissue, and the incidence of brain metastases in each group was calculated.

[0069] Validation results of immune-infected mouse brain transfer model: Figure 4 As shown, the results indicated that the average signal intensity in the brain regions of the V1 and V3a groups was higher than that of the WT group; lesions corresponding to the imaging sites were visible in brain tissue sections, and ex vivo brain specimens also showed that the V1 and V3a groups had more extensive visible lesions. Survival analysis showed that the survival time of the V1 and V3a groups was shorter than that of the WT group, and the Kaplan-Meier curves of the three groups showed a difference of P<0.0001 according to the log-rank test. In the cohort of 8 mice in each group, the incidence of brain metastasis was 25% (2 / 8) in the WT group, 100% (8 / 8) in the V1 group, and 100% (8 / 8) in the V3a group. These results demonstrate that lung cancer cell lines derived from mouse-derived EML4-ALK V1 or V3a fusion proteins can form brain metastasis-related models in immunocompetent hosts that can be quantified by imaging, pathology, brain metastasis incidence, and survival time, and can be used for subtype differences, drug intervention, and tumor immune microenvironment studies.

[0070] In the aforementioned animal experiments, the WT, V1, and V3a groups used the same LLC parental cell background, cell seeding quantity, seeding volume, internal carotid artery seeding route, animal strain, and brain metastasis evaluation methods. The main difference between the experimental groups was whether and which mouse-derived EML4-ALK fusion protein was expressed. Therefore, the differences in brain metastasis incidence, brain bioluminescent signal, brain lesion extent, and survival time among the groups can reflect the influence of a specific mouse-derived EML4-ALK fusion construct on brain metastasis-related phenotypes.

[0071] This application does not merely mechanically replace the human EML4-ALK sequence with a mouse homologous sequence. Instead, based on protein-coding coordinates, open reading frame continuity, and functional structure preservation requirements, it determines the specific fusion boundaries of mouse EML4-ALK V1 and V3a, and obtains LLC cell lines capable of stably expressing the corresponding fusion proteins. Animal experiments further showed that, under the same modeling conditions, the brain metastasis rate in both the V1 and V3a groups was 100%, higher than the 25% in the WT group, accompanied by enhanced brain bioluminescence signals, increased brain tissue metastatic lesions, and shortened survival. These results provide functional validation that specific mouse-derived V1 and V3a fusion sequences can form immune-intact mouse models with clearly defined brain metastasis-related phenotypes.

[0072] Recombinant plasmid construction, lentiviral infection, and cell screening are methods for obtaining target cell lines. The main technical contribution of this application is reflected in the specific mouse-derived EML4-ALK V1 / V3a fusion sequence, the syngeneic mouse lung cancer cell line that stably expresses the corresponding fusion protein, and the use of this cell line in immune-healthy mice to form a model that can quantify brain metastasis-related phenotypes.

[0073] SEQ ID NO:1:

[0074] SEQ ID NO:2:

[0075] SEQ ID NO:3:

[0076] SEQ ID NO:4: ; SEQ ID NO:5:5′-TTCTCCTGCATTGTGTCACCC-3′; SEQ ID NO:6:5′-GGGCTCTGCAGTTCCATCT-3′; SEQ ID NO:7: 5′-TCAGTGTTTTGGCATTCTTGGGG-3′; SEQ ID NO: 8: 5′-AGCTGAAAAGTCACATAACTCATGGG-3′; SEQ ID NO:9: 5′-CCTCGTCCCGTAGACAAAATG-3′; SEQ ID NO: 10: 5'-TGAGGTCAATGAAGGGGTCGT-3'.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a lung cancer cell line of mouse-derived EML4-ALK V1 or V3a fusion protein, characterized by, Includes the following steps: Step 1: Synthesize the full-length coding sequence of mouse EML4-ALK V1 as shown in SEQ ID NO:1 or the full-length coding sequence of mouse EML4-ALK V3a as shown in SEQ ID NO:2; Step 2: The full-length coding sequence of mouse EML4-ALK V1 or V3a is cloned into a lentiviral vector to obtain a recombinant lentiviral transfer plasmid of mouse EML4-ALK V1 or V3a. Step 3: Transfect cells with the recombinant lentiviral transfer plasmid of mouse EML4-ALK V1 or V3a to obtain mouse EML4-ALK V1 or V3a lentiviral particles. Step 4: Using mouse lung cancer cells as parental cells, infect them with mouse-derived EML4-ALK V1 or V3a lentiviral particles, and screen to obtain lung cancer cell lines that stably express mouse-derived EML4-ALK V1 or V3a fusion proteins.

2. The construction method of claim 1, wherein, In step one, a Kozak sequence is set at the 5′ end of the full-length coding sequence of mouse-derived EML4-ALK V1 or EML4-ALK V3a, and a 3×FLAG tag coding sequence is set at the 3′ end.

3. The construction method as described in claim 1, characterized in that, In step two, the lentiviral vector is the pLV3-CMV-3×FLAG-EF1a-CopGFP-Puro vector.

4. The construction method as described in claim 1, characterized in that, In step four, the mouse lung cancer cells are LLC.

5. A lung cancer cell line stably expressing mouse-derived EML4-ALK V1 or V3a fusion protein, characterized in that, The lung cancer cell line was constructed using the method described in any one of claims 1-4 for constructing the mouse-derived EML4-ALK V1 or V3a fusion protein.

6. The application of a lung cancer cell line stably expressing mouse-derived EML4-ALK V1 or V3a fusion protein in constructing an ALK-positive lung cancer model in immunocompetent mice, characterized in that, ALK-positive lung cancer models are used for non-disease treatment or diagnostic purposes.

7. The application as described in claim 6, characterized in that, ALK-positive lung cancer models include subcutaneous tumorigenesis, in situ lung cancer models, hematogenous dissemination models, and brain metastasis-related models.

8. A recombinant lentiviral transfer plasmid of mouse-derived EML4-ALK V1 or V3a, characterized in that, The recombinant lentiviral transfer plasmid contains the full-length coding sequence of mouse EML4-ALK V1 as shown in SEQ ID NO:1 or the full-length coding sequence of mouse EML4-ALK V3a as shown in SEQ ID NO:2.

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