A human renal clear cell carcinoma high-metastatic cell line and application thereof

Abstract

The application relates to the field of biological medicine, in particular to a human renal clear cell carcinoma high-metastasis cell line, which is classified and named as human renal clear cell carcinoma high-metastasis cell 786O-M2-Luc Homo sapiens, and the preservation number is CCTCC No: C2025339. The renal clear cell carcinoma high-metastasis cell line can be applied to constructing a renal clear cell carcinoma high-metastasis cell model or an animal model. The cell line of the application can form a stable orthotopic tumor in an immunodeficient mouse, can cause distant organ metastasis such as lung metastasis, can express a GFP and Luciferase double-reporting system, and is convenient for in-vitro sorting and in-vivo IVIS imaging tracking. The cell line of the application retains the typical renal cell carcinoma characteristics of 786-O cells in vitro, but has stronger proliferation, migration and invasion capacity, and can be used as a tool model for renal cancer metastasis mechanism research and drug screening.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a highly metastatic human clear cell renal cell carcinoma cell line and its applications. Background Technology

[0002] Clear cell renal carcinoma (ccRCC) is the most common histological subtype of renal cell carcinoma, with a rising global incidence. ccRCC is highly occult, with approximately 30% of patients already having metastases at diagnosis; the lungs, bones, and brain are common sites of metastasis. About 60-70% of patients who die from advanced renal cell carcinoma have lung metastases. Although treatment for RCC has significantly improved over the past decade, shifting from high-dose cytokine therapy combined with surgical tumor resection to targeted therapy, immunotherapy, and combination therapies, only about 10% of patients show a strong treatment response, and most patients experience disease progression 8.2-23.9 months after first-line immunotherapy. Therefore, there is an urgent need to establish metastatic cell lines and animal models to study metastasis mechanisms and treatment.

[0003] Currently available experimental models for simulating the metastasis process of ccRCC (cytotoxic renal cell carcinoma) still have significant limitations. Commonly used human renal cell carcinoma lines (such as 786-O and Caki-1) possess certain proliferation and migration capabilities in vitro, but they struggle to form stable, efficient, and reproducible spontaneous metastases in mice. Existing metastasis models primarily utilize tail vein or intracardiac injection models to simulate hematogenous dissemination and distant colonization, or spontaneous tumor models formed using genetically engineered mouse models (such as combined knockout of genes like Vhl, Pbrm1, and Setd2). However, these models either bypass the complete metastasis cascade or are based on specific mouse cell lines, exhibiting low metastasis efficiency, organ-specific instability, and difficulty in obtaining stable passages of highly metastatic subline cells, thus limiting systematic research on metastasis mechanisms and anti-metastatic treatment strategies.

[0004] Chinese patent document CN112239750A discloses a circulating tumor cell line for renal cell carcinoma and a method for obtaining, isolating, and culturing it. The cell line, named 786-O-CTC, is established by isolating and culturing renal cell carcinoma circulating tumor cells from peripheral blood. Compared to the parental 786-O cells, this cell line exhibits stronger proliferation and migration capabilities and possesses the characteristics of circulating tumor cells. However, this model cannot simulate the in situ metastasis of clear cell renal cell carcinoma and cannot enhance its lung colonization ability. Therefore, constructing a human renal cell carcinoma model cell line with a clearly derived origin, lung metastasis capability, and ease of labeling and monitoring has significant scientific research and application value. Summary of the Invention

[0005] The purpose of this invention is to provide a highly metastatic human clear cell renal cell carcinoma cell line, and the application of this cell line in constructing highly metastatic renal cell carcinoma cell models or animal models. The cell line is constructed based on 786-O. After constructing a dual reporter system of GFP and luciferase via lentiviral transfection, in vivo screening was performed using in situ subcapsular injection into the kidney. Following two rounds of in vivo transfection and enrichment in mice, a 786O-derived subclonal cell line with stable lung metastasis ability was obtained, named "786O-M2-Luc".

[0006] In a first aspect, the present invention provides a human clear cell carcinoma highly metastatic cell line, which is classified and named human clear adenocarcinoma highly metastatic cell line 786O-M2-Luc Homo sapiens, deposited at the China Center for Type Culture Collection (CCTCC) on November 19, 2025, with accession number CCTCC No: C2025339.

[0007] A second aspect of the present invention provides a method for constructing a highly metastatic human clear cell renal cell carcinoma cell line as described above, comprising the following steps:

[0008] After constructing a dual reporter system of GFP and luciferase through lentiviral transfection, the 786-O cell line was injected under the renal capsule of BALB / cNude mice to establish an orthotopic renal cell carcinoma model. Primary cells were collected and cultured. After lung metastasis occurred due to re-in situ implantation, tumor cells were isolated from the lungs for culture and passage to obtain the highly metastatic human clear cell renal carcinoma cell line 786O-M2-Luc.

[0009] A third aspect of the present invention provides the application of the human clear cell carcinoma highly metastatic cell line as described above in the construction of a clear cell carcinoma highly metastatic cell model or animal model.

[0010] In a fourth aspect, the present invention provides an application of the human clear cell renal carcinoma highly metastatic cell line as described above in the study of the metastasis mechanism of clear cell renal carcinoma.

[0011] Furthermore, the aforementioned clear cell renal cell carcinoma metastasis is a lung metastasis.

[0012] In a fifth aspect, the present invention provides the application of a highly metastatic human renal clear cell carcinoma cell line as described above in drug screening.

[0013] The cell lines of the present invention have the following advantages and technical strengths:

[0014] 1. It can form stable in situ tumors in immunodeficient mice;

[0015] 2. Metastasis to distant organs such as the lungs may occur;

[0016] 3. It expresses both GFP and Luciferase dual reporter systems, which facilitates in vitro sorting and in vivo IVIS imaging tracking;

[0017] 4. It retains the typical renal cell carcinoma characteristics of 786-O cells in vitro, but has stronger proliferation, migration and invasion capabilities;

[0018] 5. It can serve as a tool model for studying the metastasis mechanism of renal cell carcinoma and for drug screening.

[0019] Preservation information for biological material samples:

[0020] Preservation Institution: China Center for Type Culture Collection (CCTCC)

[0021] Address: Wuhan University, Wuhan, China

[0022] Deposit date: November 19, 2025

[0023] Accession number: CCTCC No: C2025339

[0024] Classification and nomenclature: Highly metastatic human renal clear adenocarcinoma cells 786O-M2-Luc Homo sapiens Attached Figure Description

[0025] Figure 1 The flowchart shows the procedure for selecting highly metastatic renal cell lines from the 786-O renal cell carcinoma cell line.

[0026] Figure 2 HE slices of in situ tumors and lung metastases from multiple rounds of screening experiments; left: first in situ tumor, middle: second in situ tumor, right: second lung metastases (20× magnification, scale bar 50um).

[0027] Figure 3 The image shows in vivo imaging of M-NSG mice after tumor bearing; the left image shows 786O-Luc cells, and the right image shows 786O-M2-Luc cells.

[0028] Figure 4 Edu showed the difference in proliferation capacity of renal cell carcinoma cells 786-O, 786O-T1-Luc, and 786O-M2-Luc before and after screening.

[0029] Figure 5 To screen for differences in the Transwell migration ability of renal cell carcinoma cells 786-O, 786O-T1-Luc, and 786O-M2-Luc before and after screening.

[0030] Figure 6The results of principal component analysis (PCA) of renal cell carcinoma RNA sequencing before and after screening are shown. PCA revealed that 786O-M2-Luc was significantly different from other populations.

[0031] Figure 7 To screen for differentially expressed genes in renal cell carcinoma RNA after sequencing.

[0032] Figure 8 The results are from the differential gene enrichment analysis. Detailed Implementation

[0033] The specific embodiments of the present invention will be described in detail below with reference to examples. Unless otherwise specified, all methods described in the following examples are conventional. Unless otherwise specified, all experimental materials or reagents used in the following examples are commercially available conventional materials or reagents.

[0034] In the following specific examples, 786-O human clear cell renal cancer cells were purchased from the Chinese Academy of Sciences Type Culture Collection Committee Cell Bank (SCSP-5059), and BALB / c Nude mice and M-NSG mice were purchased from Shanghai Southern Model Biotechnology Co., Ltd. All cell culture media were 1640 medium + 10% fetal bovine serum.

[0035] Example 1: Construction of the cell line “786O-M2-Luc”

[0036] A renal cell carcinoma orthotopic model was constructed using the 786-O cell line and BALB / c Nude mice. Primary cells were collected and cultured. After re-implantation into the lungs and subsequent metastasis, tumor cells were isolated from the lungs for culture and passage to obtain highly metastatic clear cell renal cell carcinoma cells, "786O-M2-Luc". The procedure is as follows: Figure 1 As shown, the specific steps are as follows:

[0037] 1. Lentiviral virus was constructed using a dual-labeled vector (GFP-T2A-Luciferase) with a CMV promoter and packaged into 293T cells. 786-O cells were infected, and GFP-positive cells were selected and named "786O-Luc".

[0038] 2. Using 6-8 week old BALB / c Nude mice, 5×10 6 An in situ model was established by injecting a 1:1 mixture of 786O-Luc cell suspension and Matrigel under the kidney capsule. In vivo imaging was performed weekly for 6 weeks.

[0039] 3. Six weeks later, mice were euthanized by cervical dislocation, immersed in alcohol for 5 minutes, and then transferred to a clean bench for further processing. Kidney tissue was isolated, and tumor tissue attached to the kidney surface was peeled off. A portion of the tissue was fixed in fixative, embedded, sectioned, and stained with hematoxylin and eosin (HE). Another portion of the tissue was minced and digested in a digestive solution (containing collagenase IV and DNase I) at 37°C and 100 rpm for 30 minutes on a shaker. After terminating the digestion with twice the volume of culture medium, the cells were centrifuged, resuspended, filtered through a 70 μm tissue filter, washed twice with PBS, and cultured in complete medium containing antibiotics. After cell expansion, tumor cells were obtained by GFP sorting and named "786O-T1-Luc".

[0040] 4. After scaling up the culture, repeat step 2, adding 5×10 6 786O-T1-Luc cell suspension was re-injected subcapsularly into the kidney. After 4 weeks of continuous observation, in situ radical resection was performed to reduce the primary tumor burden.

[0041] 5. After 4 weeks of observation, the same procedure as in step 3 was performed. Mice were euthanized by cervical dislocation, and multiple organs (lung, liver, intestine, bone, brain, etc.) were harvested. Tissues with fluorescent signals were screened using IVIS imaging. The primary lesion and lung metastases were separated. One part was fixed, and the other part was digested and sorted by GFP to obtain tumor cells, which were named "786O-T2-Luc" and "786O-M2-Luc", respectively.

[0042] The aforementioned 786O-M2-Luc has been deposited in CCTCC (accession number: CCTCC No: C2025339). Pathological examinations of the first and second in situ tumors and the second lung metastasis are as follows: Figure 2 As shown.

[0043] Example 2: Detection of the transferability of the cell line “786O-M2-Luc”

[0044] Six- to eight-week-old male M-NSG cells were randomly divided into two groups of three each. 786O-Luc and 786O-M2-Luc cells were expanded using standard passage culture methods. After reaching the logarithmic growth phase, the cells were resuspended in sterile PBS buffer and the cell concentration was adjusted to 2 × 10⁻⁶ cells / mL. 6 Cells / 100 μL. Subsequently, the corresponding cell suspensions were injected into the two groups of mice via tail vein injection to construct a tumor lung metastasis model.

[0045] In vivo bioluminescence imaging was performed on mice on day 7 post-injection (e.g. Figure 3(As shown in the figure). The results showed that no obvious bioluminescent signal was observed in the lungs of mice injected with 786O-Luc cells, suggesting that its transfer efficiency was low; while significant bioluminescent signals were observed in the lungs of mice injected with 786O-M2-Luc cells, indicating that the cells have a stable and strong lung transfer ability.

[0046] Example 3: Detection of the proliferation capacity of cell line “786O-M2-Luc”

[0047] Using BeyoClick TM The EdU-555 kit (catalog number: C0075L, Beyotime) was used in conjunction with flow cytometry for cell proliferation analysis. An appropriate amount of cells were seeded in 6-well plates and cultured overnight to restore normal proliferation. EdU working solution was then added to the culture system to a final concentration of 10 μM, and incubation continued for 3 hours to complete EdU incorporation. After incubation, the culture medium was discarded, and cells were digested with trypsin and gently pipetted to obtain a single-cell suspension. After washing with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, and washed 1-2 times with PBS again. Cell membranes were then lysed with 0.5% Triton X-100 and incubated at room temperature for 20 minutes. After washing with PBS, a click reaction was performed. The click reaction solution was prepared according to the reagent instructions, added to the cells, and incubated in the dark for 30 minutes. After the reaction, the cells were washed twice with PBS, and finally resuspended for analysis.

[0048] Detection was performed using an Attune flow cytometer (Thermo Fisher Scientific), with fluorescence signals acquired through the PE channel. Gating was set using unstained control samples, and the percentage of EdU-positive cells was calculated. Results are as follows: Figure 4 As shown, 786O-M2-Luc cells exhibited a significantly higher EdU positivity rate compared to 786O-Luc cells, suggesting that they have stronger proliferative capacity, and the difference was statistically significant.

[0049] Example 4: Migration ability detection of cell line “786O-M2-Luc”

[0050] Cell migration ability was analyzed using Transwell chambers (8 μm pore size, Corning). 786O-Luc, 786O-M1-Luc, and 786O-M2-Luc cells were cultured using standard methods, and then starved for 12–24 hours with serum-free medium the day before the experiment. Cells were collected by trypsin digestion and centrifugation, washed 1–2 times with PBS, and resuspended in serum-free medium containing 1% BSA, adjusting the cell density to 1 × 10⁶ cells / mL. 5 per mL.

[0051] 2 × 10⁻⁶ doses per upper chamber4 Cells were placed in a transwell chamber with complete medium containing 20% ​​FBS. The transwell system was incubated at 37°C with 5% CO2 for 24 hours. The chamber was then removed, fixed with methanol for 10 minutes, and stained with 1% crystal violet for 15 minutes. Non-migrating cells were then gently wiped from the upper surface of the filter membrane, leaving only migrating cells on the lower surface. The cells were then observed and photographed under an inverted microscope (see [link to image]). Figure 5 Each group of cells was set up with 3 technical replicates.

[0052] At 40× magnification, the number of migrating cells was counted in three randomly selected fields of view, and the average value was calculated. A bar chart was plotted using GraphPad Prism 10.1.2 software, with the horizontal axis representing cell type and the vertical axis representing the number of migrating cells. The results are as follows: Figure 5 As shown, the 786O-M2-Luc cells had the highest number of migrating cells, and the difference was statistically significant, indicating that these cells have a stronger migration ability.

[0053] Example 5: Transcriptome characteristics of the cell line “786O-M2-Luc”

[0054] To verify whether the constructed 786O-M2-Luc cells have significantly enhanced migration ability, the applicant performed transcriptome sequencing analysis on cell populations at different stages of cell construction to systematically evaluate changes in their molecular characteristics.

[0055] 1. Sample Preparation: Original cells (786O-Luc), first-generation cells (786O-T1-Luc), and second-generation cells (786O-T2-Luc from the primary tumor site and 786O-M2-Luc from lung metastases) were collected, with three biological replicates for each group. Total RNA was extracted and a transcriptome sequencing library was constructed.

[0056] 2. Sequencing Platform and Data Analysis Methods: Bulk RNA sequencing was performed using the BGISEQ-T7 high-throughput sequencing platform. Raw data underwent quality control using FastQC software, alignment to the human reference genome was performed using Hisat2, transcript assembly and expression quantification were conducted using StringTie software, and gene expression levels were assessed using FPKM and TPM normalization methods. Subsequently, differential expression analysis, GO / KEGG functional enrichment analysis, and GSEA enrichment analysis were performed.

[0057] Principal component analysis showed good reproducibility among the groups. The 786O-M2-Luc (lung metastases) samples showed significant cluster separation from other groups, indicating a significant difference in their gene expression profiles (see [link to analysis]). Figure 6Differential gene analysis showed that GFRA1, FOXN3, MYO1D, TMEM98, and ITGA4 were significantly upregulated in 786O-M2-Luc cells. Related literature reports that these genes are closely related to tumor invasion and metastasis. Further analysis revealed that several key genes associated with tumor migration and epithelial-mesenchymal transition (EMT), including SNAI2, VIM, VEGFA, CXCR4, MMP2, and MMP9, were significantly more expressed in 786O-M2-Luc cells compared to other groups (see...). Figure 7 ).

[0058] GO functional enrichment analysis revealed that the 786O-M2-Luc cell population exhibited significantly high expression in pathways such as cell migration and epithelial-to-mesenchymal transition. KEGG pathway enrichment results also pointed to multiple migration and invasion-related pathways, including extracellular matrix remodeling, angiogenesis, and cell adhesion. Furthermore, GSEA analysis further confirmed that signaling pathways such as TGF-β, JAK-STAT, ECM-receptor interaction, and Focal adhesion were significantly enriched in 786O-M2-Luc (see [link to GO functional enrichment analysis]). Figure 8 ).

[0059] In summary, transcriptome sequencing confirmed that 786O-M2-Luc cells exhibit significantly enhanced migration and metastasis-related characteristics at the molecular level, further demonstrating the reliability and practicality of the cell model provided in this invention for studying the mechanism of lung metastasis in renal cell carcinoma.

[0060] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A highly metastatic human clear cell renal cell carcinoma cell line, characterized in that, Its classification name is human renal clear adenocarcinoma highly metastatic cell 786O-M2-Luc Homo sapiens, preservation number CCTCC No: C2025339.

2. A method for constructing a highly metastatic human renal clear cell carcinoma cell line as described in claim 1, characterized in that, Includes the following steps: After constructing a dual reporter system of GFP and luciferase through lentiviral transfection, the 786-O cell line was injected under the renal capsule of BALB / c Nude mice to establish an orthotopic renal cell carcinoma model. Primary cells were collected and cultured. After lung metastasis occurred due to re-in situ implantation, tumor cells were isolated from the lungs for culture and passage to obtain the highly metastatic human clear cell renal carcinoma cell line 786O-M2-Luc.

3. The application of the human clear cell carcinoma highly metastatic cell line as described in claim 1 in constructing a clear cell carcinoma highly metastatic cell model or animal model.

4. The application of the human clear cell renal carcinoma highly metastatic cell line according to claim 3 in constructing a highly metastatic cell model or animal model of clear cell renal carcinoma, characterized in that, The aforementioned high metastasis of clear cell renal cell carcinoma refers to lung metastasis.

5. The application of a highly metastatic human clear cell renal cell carcinoma cell line as described in claim 1 in the study of the metastasis mechanism of clear cell renal cell carcinoma.

6. The application of the highly metastatic human clear cell renal carcinoma cell line according to claim 5 in the study of the metastatic mechanism of clear cell renal carcinoma, characterized in that, Clear cell renal cell carcinoma metastasized to the lungs.

7. The application of a highly metastatic human renal clear cell carcinoma cell line as described in claim 1 in drug screening.

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