An immortalized cell line of human renal chromophobe cell carcinoma, its culture method and application

Abstract

The present invention belongs to the field of biomedical technology and discloses an immortalized cell line of human renal chromophobe carcinoma, a culture method and an application thereof. The immortalized cell line of human renal chromophobe carcinoma of the present invention is named human renal chromophobe carcinoma cell line Loya-710 (Homo sapiens), and its Latin name is Chromophobe renal cell carcinoma:Loya‑ 710 , deposited with CCTCC NO: C2025123. The human chromophobe renal cell carcinoma cell line Loya-710, as an in vitro model, grows rapidly in tissue culture and retains the mitochondrial mutations, vesicle structure, and classic immunohistochemical markers characteristic of ChRCC. This invention provides a valuable tool for further studying the genetic, molecular, and biological characteristics of ChRCC and offers a powerful new model for mitochondrial disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an immortalized cell line of human renal chromophobe cell carcinoma, a culture method and application thereof. Background Art

[0002] Chromophobe renal cell carcinoma (ChRCC) is the second most common non-clear cell renal cancer, accounting for approximately 5% of all renal cancers. Existing technologies lack ChRCC-derived cell line models that retain well-characterized ChRCC. This lack of cell lines, crucial tools for studying tumor biology and drug efficacy, is one of the reasons for the slow progress of ChRCC research.

[0003] Currently, there are three types of immortalized cell lines for ChRCC abroad. Gabbert’s team reported the immortalized ChRCC cell lines chrompho-A and chrompho-B, which are derived from the same chromophobe renal cell carcinoma tissue. Among them, the average population doubling time of Chrompho-B cells is t D = 43 hours, chrompho-A cells are t D = 51 hours, which is longer. Both cell lines have longer doubling times, and neither cell line produced tumors in nude mouse subrenal capsule experiments. Chrompho-A and Chromopho-B have lower growth potential. Plating efficiency for both cell lines is quite low, requiring a minimum seeding density of 100 cells per microwell. However, this model has rarely been used in subsequent studies, due to its low utilization rate and the low growth status of the cell lines.

[0004] Linehan's group developed a ChRCC-derived cell line model, UOK276, with sarcomatoid differentiation. UOK276 represents a novel in vitro and in vivo cell line model of aggressive, sarcomatoid-differentiated, TP53-mutant ChRCC. This preclinical model system can be used to investigate novel biological characteristics of aggressive, sarcomatoid ChRCC and evaluate novel therapeutic options. Currently, there is a clear lack of cell line models for non-sarcomatoid-differentiated ChRCC. Studies have shown that metastatic ChRCC with non-sarcomatoid differentiation exists, but there is a lack of corresponding cell line models for this subset of metastatic ChRCC. Although OUK276 is a cell line model for highly invasive ChRCC with sarcomatoid features, this cell line currently represents only a subset of metastatic ChRCC. In metastatic ChRCC research, more comprehensive cell line models are needed for ChRCC without sarcomatoid features but with a poor prognosis. Furthermore, current ChRCC research primarily uses 786-O and HeLa cells as models, which lack specificity. In addition, in studying the mitochondrial-related pathogenesis of ChRCC, the UOK276 cell line does not have similar mitochondrial mutations, so it is particularly important to prepare a non-sarcoma-like ChRCC in vitro cell line model with mitochondrial mutations.

[0005] Several viruses and viral proteins can immortalize (confer unlimited replication capacity) cultured mammalian cells, including Epstein Barr virus, (Simian virus 40) SV40-T antigen, adenoviruses E1A and E1b, and human papilloma virus 16 (E6 and E7 proteins). These methods are relatively reliable, but each virus type has specific cell type preferences. Exogenous expression of the telomerase reverse transcriptase component (TERT) alone has been reported to immortalize many human cell types (e.g., fibroblasts, retinal pigment epithelial cells, vascular endothelial cells, and mesothelial cells) without additional genetic alterations. However, there are currently no reports of using this approach with primary ChRCC cells. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide an immortalized cell line of human renal chromophobe carcinoma, a culture method and application thereof.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides an immortalized cell line of human renal chromophobe cell carcinoma, the deposit number of the cell line is CCTCC NO: C2025123.

[0009] The immortalized cell line of the present invention is named as human renal chromophobe carcinoma cell line Loya-710 (Homo sapiens). The information in the brackets indicates that the cell line belongs to Homo sapiens, i.e., human. Its Latin name is named Chromophore renal cell carcinoma:Loya-710 , and was deposited in the China Center for Type Culture Collection on April 16, 2025. The deposit address is the China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0010] In a second aspect, the present invention provides a method for constructing an immortalized cell line of human chromophobe renal cell carcinoma, comprising transfecting human ChRCC tumor tissue culture with lentivirus SV40-T; and passaged for no less than 30 times to obtain the immortalized cell line of human chromophobe renal cell carcinoma.

[0011] As a preferred embodiment of the construction method of the present invention, the human ChRCC tumor tissue culture is tumor cells that have been passaged 3-5 times from the tumor tissue.

[0012] As a preferred embodiment of the construction method of the present invention, the multiplicity of infection of the lentivirus SV40-T is 50-200.

[0013] As a preferred embodiment of the construction method of the present invention, the transfection time is 12h-48h.

[0014] As a preferred embodiment of the construction method of the present invention, the lentivirus SV40-T includes at least one of resistance, fluorescence, and label.

[0015] In a third aspect, the present invention provides an immortalized cell line of human renal chromophobe carcinoma, which is prepared by transfecting human ChRCC tumor tissue culture with lentivirus SV40-T comprising at least one of resistance, fluorescence, and labeling; and passaged for no less than 30 times.

[0016] In a fourth aspect, the present invention uses the immortalized human renal chromophobe cell carcinoma cell line in the preparation, screening or evaluation of therapeutic drugs for tumors or mitochondrial diseases.

[0017] In a fifth aspect, the present invention uses the immortalized human renal chromophobe cell carcinoma cell line as a tumor cell model or an animal model.

[0018] In a sixth aspect, the present invention uses the immortalized cell line of human renal chromophobe carcinoma as experimental material in basic or clinical research on tumors.

[0019] As a preferred embodiment of the application of the present invention, the tumor is human chromophobe renal cell carcinoma; preferably, the tumor is non-sarcomatous differentiated ChRCC.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention provides a new ChRCC cell line model Loya-710, named human renal chromophobe carcinoma cell line Loya-710 (Homo sapiens), which is derived from a common ChRCC tumor area. The present invention immortalizes non-sarcoma-like ChRCC cells by lentiviral-mediated viral expression of hTERT, mCdk4 and SV40-T, and ultimately successfully immortalizes ChRCC cells by exogenous expression of SV40-T antigen. Compared with lentiviral delivery of hTERT and mCDK4 or the use of exogenous hTERT expression alone, the strategy of using the SV40-T pathogen successfully immortalizes the ChRCC cell line. Experimental verification shows that the cell line belongs to the ChRCC cell line, and its phenotype is consistent with the primary culture. It will be one of the useful tools for studying the pathogenesis and treatment response of ChRCC. The results clearly showed that even in long-term culture, Loya-710 can retain the basic cytoskeleton, ultrastructure, classic immunohistochemical markers and cytogenetic characteristics of ChRCC, mainly mitochondrial mutations. Although the intermediate filament protein spectrum of Loya-710 highly retains the phenotype of ChRCC after long-term in vitro culture, in addition to still expressing cytokeratins such as CK7, CK18, and CK9, Loya-710 additionally expresses vimentin. In addition, this cell line has a higher growth potential (t D = 28 h), Loya-710 grows rapidly in tissue culture as an in vitro model.

[0022] That is, Loya-710 grows rapidly in tissue culture as an in vitro model and retains the mitochondrial mutation characteristics, vesicle structure and classic immunohistochemical markers of ChRCC.

[0023] (2) Given that ChRCC is a rare and understudied subtype of RCC, the establishment of cell line models of specific cancers is essential for tumor research. Currently, the pathogenesis of ChRCC is unclear and there is a lack of in vitro models. Loya-710 will become a valuable tool for further studying the genetic, molecular, and biological characteristics of ChRCC, and is also a powerful model for mitochondrial disease. The pathogenesis of ChRCC is unclear and there is a lack of in vitro models. The ChRCC cell line model of the present invention can be used as a model for studying the pathogenesis and metastasis mechanisms of non-sarcomatoid differentiated ChRCC and for evaluating new treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1: A. HE staining of patient tumor tissue, scale bar: 100 μm; B. IHC staining showed negative expression of vimentin in tumor tissue, scale bar: 100 μm; C. IHC staining showed positive expression of CK7 in tumor tissue, scale bar: 100 μm; D. IHC staining showed positive expression of CD117 in tumor tissue, scale bar: 100 μm; E. Microscopic cell density of each group on the third day after transfection with different lentiviruses (from left to right, Blank group, SV40-T group, hTERT group, hTERT combined with mCDK4 group), scale bar: 100 μm; F. Microscopic cell density after 4 days of culture (from left to right, Blank group, SV40-T group, hTERT group, hTERT combined with mCDK4 group), scale bar: 100 μm.

[0025] Figure 2 : A. (Top) Primary cell morphology under a 10x microscope, (bottom) primary cell morphology under a 20x microscope, scale bar: 100 μm; B. (Top) Loya-710 cell morphology under a 10x microscope, (bottom) primary cell morphology under a 20x microscope, scale bar: 100 μm; C. Loya-710 cell growth curve; D. CK7 and CD117 immunofluorescence analysis of primary cells, Loya-710 cells, and Caki-1 cells, scale bar: 200 μm; E. Western blot analysis of CK7, CD117, CK18, CK19, and Vimentin protein expressions in 7860 and Loya-710 cells.

[0026] Figure 3 : A. Electron microscopy shows cytoplasmic microspikes in Loya-710 cells (indicated by red arrows); B. Electron microscopy shows cytoplasmic microvesicles (indicated by red arrows) and coated vesicles (indicated by blue arrows) in Loya-710 cells; C-D. Electron microscopy shows mitochondria (indicated by red arrows); E. Mitochondrial gene mutations in tumor tissue and Loya-710 cells.

[0027] Figure 4 Chromosomal CNV display of Loya-710 cells (left) and chromosomal CNV display of tumor tissue samples (right);

[0028] Figure 5 The chromosome karyotype and metaphase diagram of Loya-710 cells include Cell-001: 85 chromosomes (left), Cell-002: 85 chromosomes (center), and Cell-003: 70 chromosomes (right).

[0029] Figure 6 A histogram of the somatic mutation (SNV) spectrum of Loya-710 cells and tumor tissues (left), and a comparison of the similarity of each tumor sample at the point mutation level (right);

[0030] Figure 7 The somatic mutant allele frequency distribution of Loya-710 cell line (left) and the somatic mutant allele frequency distribution of tumor tissue (right). DETAILED DESCRIPTION

[0031] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0033] Example 1: Tissue origin of Loya-710 cell line

[0034] Human renal tumor tissue was obtained from specimens collected from six patients (three men and three women) undergoing nephrectomy for RCC at Renji Hospital. The average patient age was 62-65 years. Immediately after specimen extraction, tissue samples (approximately 10 g each) were collected from areas identified as macroscopically tumorous by a urological pathologist. The nature of these areas was subsequently confirmed by histopathological evaluation of mirrored specimens. The tissues were placed in separate 50 ml centrifuge tubes, culture medium was added, and the tubes were then transferred on ice to the cell culture laboratory.

[0035] Tumor tissue was obtained from RCC surgical specimens and cultured. Renal cell isolation was performed within 30 minutes after renal tumor tissue collection. The processing of tumor tissue was based on mechanical disintegration of the tissue, followed by enzymatic digestion and screening of purified tumor cells. Specifically: (1) The tissue was transferred to a 60 mm culture dish, and any fat, blood clots, and connective tissue were peeled off from the whole tumor tissue with forceps and scissors, and repeatedly washed with PBS; (2) The tissue was cut into small pieces of approximately 1 mm with a scalpel; (3) The tissue pieces were resuspended in 5 mL of RPMI 1640 containing 10% FBS with a final concentration of 2 mg / ml collagenase I and 150 μg / ml DNase I for digestion; 37 45 minutes at 170 °C in a water bath, inverting every 5 minutes; (4) After terminating the digestion, filter the cell suspension using a 70 μm filter and centrifuge at 300 g for 4 minutes to remove the supernatant; (5) Add lysate to lyse the red blood cells for 4 minutes, stop the lysis with PBS, and remove the supernatant after centrifugation; (6) Resuspend the cells in PBS and remove the supernatant after centrifugation; (7) Resuspend the cells in complete culture medium (Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 1% amino acids, 1% penicillin and streptomycin) and inoculate them in a 6 cm culture dish. Maintain the culture at 37 °C and 5% CO2; (8) After culturing for 3-5 days and observing and photographing the culture, subculture the cells by exposing them to 0.25% EDTA (Biochrom, Berlin, Germany) to disaggregate the cells. Subsequent subcultures were performed in the same manner every 2-4 days and cryopreserved until cell growth significantly slowed.

[0036] All specimens subsequently underwent routine tissue processing (formalin fixation and paraffin embedding). Sections were histopathologically analyzed at the Renji Pathology Department to assess the type, grade, and stage of renal cancer. Of the six renal cancer cases, four were pathologically identified as non-clear cell renal cell carcinomas of other types. Of the remaining two ChRCCs, only one generated short-term culturable cells. This cell line, derived from a chromophobe renal cell carcinoma (T1bN0M0) from a 65-year-old male, measured 5 cm × 3.4 cm × 2.7 cm and was CK7+, CK8+, CD117+, VIM-, and Ki67 (3%+). Cell growth cycle arrest occurred after six passages, and subsequent studies were performed on cells from passages 3–5.

[0037] In hematoxylin-eosin (HE)-stained sections, half of the tumor cells are chromophobe cells (showing translucent reticular cytoplasm and obvious cell walls) and the other half are eosinophilic cells (cytoplasm is eosinophilic and cell walls are light-colored). Figure 1 Middle A). Immunohistochemistry showed that vimentin was not expressed ( Figure 1 B), while CK7 was observed ( Figure 1 Middle C) and CK8 ( Figure 1 Middle D) Positive staining reaction of the antibody.

[0038] Example 2: Lentiviral transfection of primary cells

[0039] Based on the improved transduction efficiency, all ChRCC cultures were transduced simultaneously with several lentiviruses: 50,000 cells were infected and transduced with 100-fold MOI of virus (lenti-SV40-T group, hTERT group, hTERT and mCDK4 group, and Blank group) in culture medium for 24 hours. Cells were then photographed to record their ability to survive and proliferate in each group. Cells were considered immortal if they could be passaged more than 30 times, as this far exceeds the capacity of primary cultures and suggests that the cell division capacity may be unlimited, achieving the goal of significant expansion to meet the needs of many downstream experiments.

[0040] After obtaining ChRCC culture and culturing primary tumor cells to P6, the cell proliferation rate decreased significantly and cell growth stagnated. Lentivirus transfection was performed on previously frozen P3-5 generations. Lentivirus was transfected in six-well plates and divided into blank group, SV40-T group, hTERT group, and hTERT / mCDK4 group. The cell density after 3 days of infection was SV40-T group>hTERT group>BLANK group>hTERT / mCDK4 group ( Figure 1 Middle E), after 4 days of culture, the Blank group, hTERT group, and hTERT / mCDK4 group still did not grow to full size ( Figure 1 (F) Except for cells that proliferate using SV40-T alone, most primary cultured cells show stagnant growth after transfection. Therefore, the lentivirus SV40-T was used to immortalize primary ChRCC tumor cultures. SV40-T without resistance, fluorescence, or labeling was then used for transfection to avoid conflicts in subsequent cell experiments. The resulting immortalized cell line was named Loya-710 (Homo sapiens), a human chromophobe renal cell carcinoma cell line (abbreviated as Loya-710). This cell line was passaged over 30 times and successfully cryopreserved and subsequently revived.

[0041] The immortalized cell line of the present invention is a human renal chromophobe carcinoma cell line Loya-710 (Homo sapiens), the Latin name of which is named

[0042] Chromophore renal cell carcinoma: Loya-710, deposited in the China Center for Type Culture Collection on April 16, 2025, with the deposit number CCTCC NO: C2025123. The deposit address is China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The contact numbers are 027-68752056; 68752319, and the postal code is 430072.

[0043] Example 3: Identification of Loya-710 cell line

[0044] DNA from tumor tissue and Loya-710 cells was extracted using the QIAamp DNA Mini Kit (51304, Qiagen) in strict accordance with the manufacturer's instructions. Blood DNA was extracted using the QIAamp DNA Blood Maxi Kit (51194, Qiagen). DNA concentrations in all samples were quantified using the Qubit dsDNA BR Assay Kit (Q32853, Thermo Fisher), and DNA integrity was assessed by 1% agarose gel electrophoresis.

[0045] Sequencing libraries were constructed using a modified version of the KAPA library preparation kit. The steps are summarized as follows: First, 1 μg of genomic DNA was sheared using a Biorupter (Diagenode, Belgium) to an average fragment size of approximately 200 bp. Subsequently, the fragments were purified using AMPureXP magnetic beads (Beckman Coulter Inc, Brae, CA, USA) to remove small fragments. Next, the DNA underwent a three-step enzymatic reaction according to the KAPA library preparation kit (Kapa, ​​Inc.) protocol: end repair, A-tailing, and ligation with Illumina paired-end index adapters. After minimal PCR amplification cycles, the libraries were quantified using a Qubit 4.0 Fluorometer (Thermo Fisher, Germany). The libraries were pooled and captured by liquid-phase hybridization using a Twist 36.6M / 56.6M (TWIST Inc.). The captured libraries were analyzed for quality on an Agilent 2100 Bioanalyzer, and DNA concentration was determined using a Qubit 4.0 Fluorometer (Thermo Fisher, Germany). Finally, the library was sequenced on the NovaSeq 6000 platform (Illumina, Inc.) to generate 2 × 150 bp paired-end read data.

[0046] The STR genotypes of immortalized cells and corresponding primary cultures were analyzed using the cell STR similarity search tool CLASTR (https: / / web.expasy.org / cellosaurus-str-search / ) and compared with the results of multiple cell banks such as ATCC, DSMZ, and JCRB.

[0047] Cell authentication genotyping: Loya-710 cells were compared with all cell line STR data included in the ATCC, DSMZ, JCRB, and RIKEN databases (STR genotype and cell bank comparison results are shown in Table 1).

[0048] Table 1 Genotyping results of STR loci and Amelogenin loci of Loya-710 cells

[0049]

[0050] However, there are no immortalized cell lines of ChRCC included in the above databases, and the EV value was found to be lower than 0.8, which means that the STR loci of the tested cells are quite different from those of the cells matched in the cell bank, and there is no correlation between the two, indicating that Loya-710 does not originate from any cell line in the cell bank.

[0051] Example 4: Characterization of Loya-710 cell line

[0052] (1) Immunofluorescence staining

[0053] Primary ChRCC cells and Loya-710 cell slides were prepared and fixed with 4% paraformaldehyde (PFA). The slides were permeabilized with 0.1% Triton X-100 solution, washed with PBS, and blocked with 5% bovine serum albumin (BSA). The slides were incubated with primary antibodies (rabbit, Cytokeratin 7-specific polyclonal antibody, 1:500, Proteintech; rabbit, CD117 / c-Kit polyclonal antibody, 1:500, Proteintech) at 4°C overnight. After washing, the slides were incubated with secondary antibodies (goat anti-rabbit IgG, 1:200) at room temperature in the dark for 1 hour and then washed. The cell nuclei were then stained with 3-5 μg mL-1 4′,6-diamidino-2-phenylindole (DAPI) staining solution (Biosharp, Hefei), washed with PBS, and mounted with anti-fluorescence quenching mounting solution. Images were collected and observed under a fluorescence microscope.

[0054] (2) Contact inhibition

[0055] Normal renal cancer cells are contact-inhibited in culture, meaning they form a monolayer and cease dividing upon reaching confluence. In this experiment, cells were routinely seeded so that the cell density reached 30-50% by the second day (Day 1). Images were taken daily for five consecutive days to document the increase in cell density, morphological changes, and whether cells demonstrated the ability to grow on top of each other (cell bodies rather than just processes). Immortalized cell cultures were evaluated for continued division and accumulation on top of each other at confluence, a characteristic of transformed cells. This latter phenotype was defined as a loss of contact inhibition.

[0056] (3) Western blot (WB)

[0057] Loya-710 cells were lysed using RIPA buffer (Cell Signaling) supplemented with 1 mM PMSF (Beyotime) and 1× protease / phosphatase inhibitor cocktail (Roche). Nuclear proteins were isolated using the Beyotime nuclear and cytoplasmic protein extraction kit, strictly following the provided protocol. Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime). Subsequently, 30 μg of each sample was loaded onto a 4–20% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) for electrophoresis. For protein identification, gel-separated proteins were transferred to a polyvinylidene difluoride membrane (Roche, 0.22 μm pore size) for immunoblotting using a Trans-Blot Turbo transfer apparatus (Bio-Rad). The membrane was then blocked in Tris-buffered saline (TBS-T) (0.1% Tween-20) containing 5% nonfat dry milk and incubated at 37°C for 3 hours. The membrane was then incubated with diluted primary antibody (prepared in Beyotime's primary antibody dilution buffer) at 4°C overnight. The membrane was then washed three times with TBS-T and incubated with a horseradish peroxidase-conjugated secondary antibody (prepared in TBS-T supplemented with 1% nonfat dry milk) for 60 minutes at room temperature. Finally, the chemiluminescent signal was visualized using an enhanced chemiluminescence system (Millipore, Billerica, MA, USA). Antibodies used included a rabbit Cytokeratin 7-specific polyclonal antibody (1:500, Proteintech) and a rabbit CD117 / c-Kit polyclonal antibody (1:500, Proteintech).

[0058] (4) Determination of cell doubling time and proliferation rate

[0059] 1.5×10 5 Cells were seeded into 6-well tissue culture plates. Viable cells were counted in duplicate wells at three time points (0, 24, and 48 hours) using trypan blue staining and a hemocytometer. Cell doubling time was calculated based on the cell count results.

[0060] (5) Transmission electron microscopy

[0061] Terminate the culture when the cell density in a 10 cm dish reaches approximately 90%. Discard the cell culture supernatant and immediately add 4°C pre-cooled electron microscopy fixative for 1-2 hours. Then, gently scrape the cells with a cell scraper at a 45-degree angle in the same direction and transfer them to a 10 mL centrifuge tube. Centrifuge at no more than 2000 rpm for 5 minutes to pellet the cells. Discard the supernatant, add 1 mL of fresh electron microscopy fixative, resuspend the cells in a 1.5 mL EP tube, and store in a refrigerator at 4°C until ready to use. Rinse three times with 0.1 M phosphate buffer (PBS) (pH 7.4) for 15 minutes each. Fix the cells with 1% osmium hydroxide in 0.1 M phosphate buffer (PBS) (pH 7.4) at room temperature (20°C) for 2 hours. Rinse three times with 0.1 M phosphate buffer (PBS) (pH 7.4) for 15 minutes each. Dehydrate the tissue in ascending order of 50%-70%-80%-90%-95%-100%-100% alcohol for 15 minutes each. Infiltrate overnight with a 1:1 mixture of acetone and 812 embedding medium, or with pure 812 embedding medium. Polymerize at 60°C for 48 hours. Section ultrathin sections (60-80 nm) using a microtome. Double stain with uranium-lead (2% saturated aqueous uranyl acetate (phosphotungstic acid) and lead citrate, each for 15 minutes). Dry sections at room temperature overnight. Observe under a transmission electron microscope, and collect images for analysis.

[0062] The Loya-710 cell line was observed to be strictly dependent on monolayer anchorage for growth, and contact inhibition occurred after growth space saturation. Microscopic morphology of Loya-710 cells showed that their shape and cell size were consistent with those of primary cells, but the chromatin of Loya-710 cells was more abundant and denser than that of primary cells ( Figure 2 (A and B). In vitro growth characteristics of Loya-710 showing the average population doubling time (t D ) is 27.97 hours ( Figure 2 (C) The saturation density was 3.82e4 ± 0.64e4 cells / cm 2 .

[0063] Compared with the CK7 immunofluorescence negative and CD117 immunofluorescence positive ccRCC cell line Caki-1, Loya-710 showed double immunofluorescence positivity for CK7 and CD117 ( Figure 2Western blot analysis showed that Loya-710 cells expressed high levels of CK7, CK18, and CK19 compared to 786O cells. It is noteworthy that Loya-710 cells, like 786O cells, expressed vimentin and CD117 ( Figure 2 Middle E).

[0064] Transmission electron microscopy revealed that tumor cells of the Loya-710 cell line were loosely attached to the wall and showed extended cytoplasmic microspikes ( Figure 3 A prominent ultrastructural feature of Loya-710 cells is the presence of cytoplasmic microvesicles (100 to 300 nm in diameter). Coated vesicles near the cell surface also indicate processes of membrane fusion or endocytosis ( Figure 3 Middle B). Electron microscopy also captured the mitochondrial characteristics of Loya-710 ( Figure 3 C and D).

[0065] Example 5: Mitochondrial genome mutations in Loya-710 cell line

[0066] Whole-exome sequencing (WES) BAM files were provided by the BCM Sequencing Center. These BAM files served as input for the MToolBox pipeline, which integrates tools such as GSNAP, MUSCLE, and SAMtools. This pipeline was used to align sequencing reads to the revised Cambridge Reference Sequence (rCRS) of human mitochondrial DNA, extract variant alleles, quantify heteroplasmy levels and associated confidence intervals, and functionally annotate identified variants. Samples with greater than 75% mitochondrial DNA sequence coverage and a variant load greater than 5% in both tumor and normal DNA were further analyzed. Somatic mutations were identified by comparing variant tables between tumor samples and corresponding normal DNA, and these mutations were classified according to standard criteria.

[0067] The TCGA study of ChRCC identified several nonsynonymous mutations in electron transport genes encoded within the mitochondrial genome that are associated with eosinophilic ChRCC. Mutations in the upstream regulatory regions of mitochondrial tRNA genes (chrM-TF, chrM-TV, chrM-TL1, chrM-TQ, and chrM-TS1) were found in primary cultured patient tumor tissue samples. These tRNA genes encode mitochondrial tRNAs that play a role in protein synthesis. They are responsible for translating mitochondrial mRNA and helping to synthesize mitochondrial proteins; in another mitochondrial tRNA, chrM-TL2, splicing region mutations and non-coding transcript exon mutations occurred; in mitochondrial protein-coding genes, chrM-ND1 underwent missense mutations, frameshift mutations, stop codon acquisition, synonymous mutations, etc., and missense mutations also occurred in chrM-ND2, and synonymous mutations in chrM-CO2. chrM-ATP8, chrM-ATP6, chrM-CO3, chrM-ND3, and chrM-ND5 all had missense mutations. In addition, chrM-CYB underwent synonymous mutations, and the upstream regulatory region of chrM-ND6 mutated ( Figure 3 Middle E).

[0068] Evaluation of primary tumors and the Loya-710 cell line revealed similar alterations in the mitochondrial genome. Mitochondrial tRNAs, including chrM-TF, chrM-TV, chrM-TL1, and chrM-TQ, retained the same upstream regulatory region mutations. Among mitochondrial protein-coding genes, chrM-ND1, chrM-ATP6, and chrM-ND5 inherited missense mutations, while the upstream regulatory region of chrM-ND6 was mutated. Additional missense mutations were found in chrM-CO1 and chrM-CYB in the cell line. Figure 3 This indicates that Loya-710 basically retains the classic mitochondrial gene mutations in ChRCC and is a good model for studying mitochondrial genome mutations.

[0069] Example 6: Chromosome copy number analysis of Loya-710 cell line

[0070] When Loya-710 cells reached 70–80% confluence, colcemid was added to the culture to a final concentration of 0.1 µg / mL and incubated for 1.5 hours before harvesting with collagenase. After centrifugation, the supernatant was removed, and the cell pellet was hypotonicized with 0.075% MHCl at 37°C for 30 minutes. The pellet was then fixed repeatedly with Carnoy's fixative (3:1 methanol / glacial acetic acid). Chromosome preparation and banding were performed according to conventional G-banding karyotyping methods. Chromosome image data acquisition and karyotype analysis were performed using the Metafer Microscopic Image Automated Scanning and Analysis System (Zeiss).

[0071] Most ChRCCs exhibit a typical pattern of chromosome loss, including single losses of chromosomes 1, 2, 6, 10, 13, 17, and 21. In somatic CNV detection, the chromophobe cell carcinoma tumor sample of the 65-year-old male patient in Example 1 showed CNV losses of whole chromosomes 1, 2, 6, 10, 11, and 17. However, these CNV losses were not observed in the Loya-710 cell line ( Figure 4 ). In the Loya-710 cell line, three cells were randomly selected as representatives to display their chromosome karyotypes ( Figure 5 (Table 2). Chromosome karyotype analysis showed that most cells were tri-tetraploid, with a mode of 85 chromosomes per cell and a range of 34-158 chromosomes. Various structural alterations were observed. This suggests that after chromosome loss, SV40-T transfection led to chromosome duplication. The particular chromosomes 1, 2, 6, 10, 13, and 17, which have received attention, likely compensated for the original chromosome loss under the influence of SV40-T, resulting in nearly all of them becoming aneuploid, with variations ranging from mosaicism to near-tetraploidy.

[0072] Table 2 Chromosome karyotypes of different Loya-710 cells

[0073]

[0074] Example 7: Loya-710 somatic mutation

[0075] Raw data were processed with skewer (version 0.23.2) to trim adapters and low-quality bases, and data quality was checked with fastqc (v0.11.9). Clean reads were aligned to the Human genome hg19 using Sentieon (Version 202112.01) bwa mem, sorted using Sentieon sort, and duplicates removed using Sentieon Dedup. Single nucleotide variants (SNVs) and indices were called using Sentieon tnplotyper (sentieon QualCal) after Indel Realigner (SentieonRealigner) and Base; variants were further annotated using ANNOVAR. Somatic signatures were recorded using MutationalPatterns (v3.4.0) using the COSMIC database. Finally, copy number variation was analyzed using CNVkit (v0.9.9).

[0076] Mutation analysis was performed on the typical chromophobe cell tumor area of ​​the primary tumor of the 65-year-old male patient in Example 1, the patient's blood, and the Loya-710 cell line. It was found that among the SNV mutations in Loya-710 cells, the C>T / G>A ratio was the highest, which was consistent with that in the tumor sample ( Figure 6 ).

[0077] Through WGS analysis, a subset of ChRCC showed kataegis, involving highly localized substitution mutations (C>T or C>G). The cell line Loya-710 and the corresponding tumor tissue showed similar kataegis. The test found that in the distribution of somatic mutation allele frequencies detected in Loya-710 and patient tumor samples, the allele mutation frequency (AF) was 0-0.1, accounting for the majority ( Figure 7 ), consistent with the perception of low mutation rates in ChRCC. Missense mutations in the TP53 gene are the most commonly mutated gene in sporadic ChRCC. 5 However, no TP53 mutations were observed in the tumor sample from this patient or in the cultured Loya-710 cell line.

[0078] In summary, this study describes a novel ChRCC-derived cell line model. ChRCC cells were immortalized by lentiviral transfection of SV40-T in cultured cells derived from chromophobe renal cell carcinoma tissue, resulting in a cell line named Loya-710. Experimental validation confirmed that this cell line is a ChRCC cell line, with a phenotype highly similar to the primary culture, particularly retaining the mitochondrial mutations characteristic of ChRCC. This will be a useful tool for studying the pathogenesis and treatment response of ChRCC, and a powerful model for mitochondrial disease.

[0079] Loya-710 is an important model for studying the pathogenicity of ChRCC. While most ChRCC cases lack classic pathogenic mutations, mitochondrial DNA (mtDNA) mutations are present in many ChRCC cases. Approximately 18% of ChRCC cases harbor loss-of-function mutations in electron transport chain complex 1 genes, with ND5 being the most commonly mutated gene. Furthermore, mtDNA mutations in the electron transport chain are more common in the acidophilic variant of ChRCC (71% vs. 22%). Expression of nearly all genes encoding enzymes in the tricarboxylic acid (TCA) cycle is elevated in ChRCC compared to normal kidneys. The mitochondrial biogenesis regulator PGC1a (PPARGC1A) and mitochondrial genome copy number are also elevated (approximately four times higher in ChRCC than in normal kidneys), indicating elevated mitochondrial biogenesis in ChRCC. However, ChRCC patients exhibit low levels of mitochondrial metabolites involved in the TCA cycle, suggesting that increased mitochondrial biogenesis is associated with and potentially driven by dysfunctional mitochondria.

[0080] Given that ChRCC samples already harbor numerous TP53 mutations, in the absence of TP53 mutations in primary Loya-710 tissue, SV40-T transfection inactivates TP53 and induces cell growth, leading to chromosomal instability and multiple chromosome duplications. Future studies will investigate p53 inhibition in Loya-710 and the downstream pathways activated by p53 inactivation. Loya-710 may reflect pathway changes associated with TP53 mutations and serve as a viable alternative model for ChRCC with TP53 mutations.

[0081] Numerous microvesicles and abnormal mitochondria were observed in the Loya-710 cell line. Microvesicles are a characteristic ultrastructural feature of ChRCC and have also been observed in intercalated cells. Cytoplasmic microvesicles are also abundant in Loya-710 cells. Coated vesicles are thought to contain the main protein component of the ATP-driven proton pump H+-ATPase and have been shown to fuse with the luminal plasma membrane of intercalated cells. The observation of coated vesicles near the plasma membrane of Loya-710 cells suggests that the process of membrane fusion or endocytosis is preserved in these cells. Intercalated cells (ICs) are mitochondria-rich, ion-regulating cells in the distal nephron. They express V-ATPase on their apical membrane and regulate acid-base homeostasis by secreting hydrogen ions into the urine. It is estimated that approximately 25% of the volume of intercalated cells is occupied by mitochondria. Consistent with their intercalated cell origin, ChRCCs highly express C-KIT (a hallmark marker of intercalated cells), subunits of V-ATPase, and the transcription factor Foxi1.

[0082] The pathological hallmarks of ChRCC include the accumulation of morphologically abnormal mitochondria, which are diffusely distributed and closely intermingled with other organelles in the cytoplasm. In all chromophobe carcinomas, varying numbers of mitochondria exhibit outer membrane protrusions, some of which also contain portions of the inner membrane. These protrusions closely resemble nearby cytoplasmic microvesicles, some of which contain a homogeneous, electron-dense, finely granular matrix similar to that observed in mitochondria. The number and shape of mitochondria in ChRCC are influenced by the number of microvesicles within the cell. Cells with relatively few microvesicles tend to have numerous mitochondria that are uniform in size and round or oval in shape. In contrast, cells with a high number of microvesicles have fewer mitochondria with greater variability in shape and size. This suggests a close relationship between microvesicles and mitochondria. The mitochondria and microvesicles observed in the Loya-710 cell line provide a powerful model for demonstrating the pathological hallmarks of ChRCC and provide valuable support for studying the pathogenic mechanisms of ChRCC.

[0083] CK7 is an important marker in ChRCC, showing diffuse positive expression in 94% of ChRCCs. However, CK7 is mostly negative in oncocytic adenomas (ROs), which are difficult to distinguish from ChRCCs, with only occasional single positive cells. CD117 is also diffusely positive in approximately 88% of ChRCCs. Therefore, CK7 and CD117 are frequently used as immunohistochemical markers to differentiate ChRCC from ROs or ccRCCs. Coexpression of vimentin and cytokeratins is a characteristic of many epithelial cell lines derived from various carcinoma types, which do not express vimentin in their primary tumors. The intermediate filament protein profile of Loya-710 cells does not highly retain the ChRCC phenotype after long-term in vitro culture. In addition to expressing cytokeratins such as CK7, CK18, and CK9, Loya-710 cells additionally express vimentin, suggesting that vimentin activation is a cellular adaptation associated with SV-40T transfection.

[0084] This study demonstrates the practicality and effectiveness of immortalizing primary human ChRCC cells. The results clearly demonstrate that even in long-term culture, Loya-710 retains the basic cytoskeleton, ultrastructure, classical immunohistochemical markers, and cytogenetic characteristics of ChRCC, primarily mitochondrial mutations, and that the cell line possesses a higher growth potential (t D = 28 hours), Loya-710 rapidly grows in tissue culture as an in vitro model. The intermediate filament protein profile of Loya-710 highly retains the ChRCC phenotype even after long-term in vitro culture. Therefore, Loya-710 will be a valuable tool for further investigation of the genetic, molecular, and biological characteristics of ChRCC, and a powerful model for mitochondrial disease. Given the current lack of clear in vitro models for ChRCC pathogenesis, this model can also be used to study the pathogenesis and metastasis mechanisms of non-sarcomatoid differentiated ChRCC and to evaluate new treatments.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An immortalized cell line of human renal chromophobe cell carcinoma, characterized in that: The deposit number of the cell line is CCTCCNO: C2025123.

2. Use of the immortalized human chromophobe renal cell carcinoma cell line according to claim 1 in screening or evaluating ChRCC therapeutic drugs.

3. Use of the immortalized human chromophobe renal cell carcinoma cell line according to claim 1 as a ChRCC cell model.

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