HUMAN BLADDER CANCER CELL LINE 847 BlCan LEB

Abstract

FIELD: biotechnology.SUBSTANCE: human bladder cancer cell line 847 BlCan LEB is proposed for use in the preparation of biomedical cell products, the creation of experimental models, and the testing of antitumor drugs. The invention makes it possible to expand the arsenal of tumor cell lines that can be used for experimental and clinical purposes, namely for the creation of antitumor biomedical cell products.EFFECT: use of the resulting cell line for testing the activity of various pharmaceutical drugs, creating diagnostic kits, test systems, and experimental models for developing new therapeutic approaches and drugs.1 cl, 2 ex

Description

[0001] The invention relates to the field of biotechnology. The resulting new bladder cancer cell line, 847 B1Can LEB, possesses stable morphological and cultural characteristics. This cell line can be used in active specific immunotherapy of solid tumors to create biomedical cell products and is suitable for laboratory research to develop diagnostic kits and test systems for determining the activity of pharmaceuticals. The cell line is deposited in the Vertebrate Cell Culture Collection of the Irkutsk Scientific Center of the Russian Academy of Sciences under registration number KKKP 823D.

[0002] Bladder cancer (BC) is one of the most frequently diagnosed malignant neoplasms of the urinary system, developing from the epithelial cells lining the wall of the bladder, which plays a key role in storing urine. In developed countries, urothelial cancer is the predominant form of BC, accounting for more than 90% of all cases [Bladder cancer biomarkers / Godlewski D., Bartusik-Aebisher D., Czech S., Szpara J., Aebisher D. / / Explor Target Antitumor Ther. - 2025. - Mar 25; 6:1002301. - doi: 10.37349 / etat.2025.1002301].

[0003] Despite recent key advances in the diagnosis and treatment of urothelial carcinoma, approximately 11% of patients with urothelial carcinoma are diagnosed at an advanced stage and are not amenable to surgical treatment. For decades, platinum-based chemotherapy has been the standard first-line treatment for metastatic urothelial carcinoma, but the median survival is only 14-15 months [Changing landscape of first-line treatment for locally advanced or metastatic urothelial carcinoma: the progression from platinum-based chemotherapy to platinum-free therapy / Liu Z., Chen C., Yin J., Cong X., Liu Z. / / Front Immunol. - 2025. - Jun 25;16:1604395. - doi: 10.3389 / fimmu.2025.1604395].

[0004] Currently, the main focus of researchers is on finding bladder cancer biomarkers, targets that could be used in the development of new approaches to targeted therapy [Mihai IM, Biomarkers for predicting bladder cancer therapy response / Mihai IM, Wang G. / / Oncol Res. - 2025. - Feb 28; 33(3):533-547. - doi: 10.32604 / or.2024.055155].

[0005] The development of new treatment methods for this type of malignant neoplasm requires the involvement of cellular technologies based on the production and use of well-characterized bladder cancer cell lines.

[0006] The technical result of the invention is an expansion of the arsenal of tumor cell lines that can be used for experimental and clinical purposes, namely, for the creation of antitumor biomedical cell products, the use of which can improve treatment efficacy and prolong survival in the treatment of malignant neoplasms. Furthermore, the resulting new cell line can be used to test the activity of various pharmaceuticals, create diagnostic kits, test systems, and experimental models for the development of new therapeutic approaches and drugs.

[0007] The specified technical result is achieved by obtaining a new human bladder cancer cell line 847 B1Can LEB, expressing specific tumor-associated antigens, used for the preparation of biomedical cell products, the creation of experimental models and testing of antitumor drugs, stored in the Vertebrate Cell Culture Collection of the IRC RAS ​​under registration number KKKP 823D.

[0008] The obtained cell line 847 B1Can LEB has stable cultural and morphological characteristics.

[0009] The pedigree of 847 B1Can LEB cell line is shown as follows.

[0010] Tumor cell culture was isolated from the primary tumor - muscle-invasive low-differentiated G3 bladder cancer of patient L.E.B., 65 years old, obtained as a result of cystectomy performed in a specialized oncology medical institution.

[0011] The 847 B1Can LEB cell line was obtained as follows.

[0012] Tumor tissue was obtained surgically. Tumor samples (1-3 mm) were crushed with a sterile scalpel. 2 ) were mechanically disaggregated automatically by placing them on sterile knives for 30 seconds to 1 minute. The resulting cell suspension was passed successively through sterile nylon filters (100 µm, 70 µm, and 50 µm), suspended in complete medium, and transferred to a culture vessel, where it was cultured for an extended period. To improve cell attachment to the substrate, a 2% gelatin solution was used, pre-coating the culture vessel. A stably growing cell line was obtained at passage 40.

[0013] The morphological features of the 847 B1Can LEB cell line are characterized as follows.

[0014] The culture consists of small polygonal cells with large, round, light nuclei containing 1-2 nucleoli. The cytoplasm has a pronounced granularity, concentrated around the nucleus. Giant multinucleated cells are present. At passage 40, the proliferative marker Ki-67 is detected in 90% of the cells.

[0015] The marker features of 847 B1Can LEB cell line are as follows.

[0016] The PCR method revealed hyperexpression of testicular cancer genes NY-ESO-1, MAGEA1, PRAME.

[0017] The cultural properties of 847 B1Can LEB cell line are presented as follows.

[0018] The cell line is cultured in DMEM / F12 (80%) nutrient medium supplemented with 20% fetal bovine serum, antibiotics (penicillin and streptomycin at a concentration of 100 U / ml and 100 μg / ml, respectively); 37°C, 5% CO2, 100% humidity. For better cell attachment to the substrate, a 2% gelatin solution is used, which is pre-coated with the culture dishes. The culture has an adhesive monolayer growth type. In 25 cm flasks 2 1×10 is inoculated into 5 ml of medium 6 cells. Subculture once a week in a 1:1 ratio using equal volumes of 0.25% trypsin solution and 0.02% versene solution.

[0019] The conditions for cryopreservation are as follows.

[0020] For long-term storage, cells are preserved by freezing in liquid nitrogen. Cryo-medium: DMEM / F12 50%, fetal bovine serum 40%, DMSO 10%; 3×10 6cells / ml per ampoule. Cells of the cell line are resuspended in freezing medium. Freezing mode: liquid nitrogen, temperature decrease by 1°C per minute to -25°C, then rapid freezing to -70°C. Storage in liquid nitrogen at -196°C. Rapid defrosting at 37°C. Cells are diluted in 10 ml of serum-free medium and pelleted by centrifugation, resuspended in 5 ml of the same medium containing 20% ​​fetal calf serum, and transferred to a 25 cm culture flask. 2 Cell viability is assessed by trepan blue staining. Cell viability after cryopreservation is 88%.

[0021] Contamination was investigated as follows.

[0022] During long-term observation, bacteria and fungi were not detected in the culture. PCR tests were performed for the presence of Mycoplasma orale, M. arginini, M. fermentans, M. hominis, M. hyorhinis, M. gallisepticum, M. genitalium, M. penetrans, M. pneumonia, M. salivarium, and Acholeplasma laidlawii - the mycoplasma test was negative.

[0023] The karyological characteristics of the 847 B1Can LEB cell line are as follows.

[0024] 53,X(?)i(1q),+del(1)(p22),del(1)(q12),del(1)q11,del(1)q23),del(3)p11),+d er(6),del(7)(q12),+t(9;?),-10,del(11)(p13),der(11),-13,-15,+16,-17,-18,+20,-21 - 55.6% of cells

[0025] 44,X(?),del(1)(p22),del(1)(q11), der(6),+t(9;?),-10,del (11)(p13),der(11),-13 -33% of cells

[0026] 38,X(?),del(1)(p22),del(1)(q11), t(9;?),-10,del (11)(p13),der(11),-13,-15,-17,-18,-21 - 11.4% of cells

[0027] Species specificity: human tumor cells.

[0028] The use of 847 B1Can LEB cell line is represented by the following examples.

[0029] Example 1. Use of the 847 B1Can LEB cell line for the preparation of antitumor vaccines based on activated dendritic cells

[0030] 1. The 847 B1Can LEB cell line was cultured in plastic flasks in a complete DMEM / F12 nutrient medium supplemented with 20% fetal bovine serum, penicillin (100 U / ml), and streptomycin (100 μg / ml). Cultivation was carried out at 37°C, 5% CO2, and 100% humidity in a Heracel CO2 incubator (Termo Electron LTD GmbH, Germany).

[0031] 2. Upon reaching a confluent monolayer, the cells were reseeded and the culture was further passaged with a 1:1 dispersal.

[0032] 3. The culture medium was removed from the flask, the surface of the flask was washed with a small amount of enzyme solution (0.25% trypsin and 0.02% versene solution in equal proportions), and then 1-3 ml of this solution was added to the cells. The flasks were placed in a CO2 incubator and visually inspected for cell status.

[0033] 4. Cells were collected with a serological pipette and washed by double centrifugation in 10 ml of 0.9% saline at 1000 rpm for 10 min.

[0034] 5. Cell counting and viability assessment were performed.

[0035] 6. To prepare tumor lysate from 847 B1Can LEB cells, 6 consecutive cycles of instant freezing to -196°C and thawing to room temperature were performed in phosphate-buffered saline without cryoprotectant; the quality of lysis was monitored using 0.1% trypan blue and a light microscope.

[0036] 7. Cell debris was precipitated by centrifugation (10 min, 3000 rpm), the supernatant fraction was filtered through a 0.2 μm filter, and the tumor lysate was packaged in cryovials for storage at -20°C until use.

[0037] 8. The lysate with the 847 B1Can LEB cell line was added to the cultures of immature dendritic cells from patients with bladder cancer on the 5th day of cultivation at a ratio of 3 lysed tumor cells: 1 DC, growth factors were added: granulocyte-macrophage growth factor (72 ng / ml), interleukin 4 (20 ng / ml) and tumor necrosis factor alpha (20 ng / ml). Incubation was carried out at 37°C, 5% CO2, 100% humidity in a Heracel CO2 incubator (Termo Electron LTD GmbH, Germany) for 48 hours.

[0038] 9. As a result, activated DCs with the immunophenotype CD14- / CD1a- / CD83+ / CD80+ / CD86+ / HLADR+ / CD209+ / CCR7+ were obtained.

[0039] Example 2. Study of chemoresistance of RMP cells when cultured in the presence of cisplatin-based drugs

[0040] 1. The 847 B1Can LEB cell line was cultured and passaged as described above.

[0041] 2. The 847 B1Can LEB cell line suspension was seeded into the wells of 24-well Lumox culture plates designed for confocal studies (Sarstedt, Germany) at a rate of 1×10 4 cells per well.

[0042] 3. After adherence of 847 B1Can LEB cells to the substrate, complete nutrient medium was added, supplemented with specific concentrations of cisplatin, corresponding to 10, 50, and 100% of its peak concentration in the patient's blood plasma. The incubation time with cisplatin corresponded to the half-life of the drug in the patient's bloodstream (3 hours). Untreated 847 B1Can LEB cells served as a control.

[0043] 4. Next, cisplatin was removed from the system and the culture medium was changed. A solution of 5-ethynyl-2'-deoxyuridine, which is a component of the Click-iT EdU Cell Proliferation Kit for Imaging, and Alexa Fluor™ 647 dye (Thermo Fisher Scientific, USA) were added to the system. The system was incubated overnight at 37°C in a Heracel CO2 incubator (Thermo Electron LTD GmbH, Germany).

[0044] 5. The preparations were processed according to the Click-iT EdU Cell Proliferation Kit for Imaging instructions, and the nuclei of 847 B1Can LEB cell line were stained with DAPI.

[0045] 6. Proliferating cells were detected by nuclear fluorescence using an Olympus FV 3000 confocal microscope (Japan) and the number of luminous nuclei was counted when excited by a 640 nm laser relative to the total number of cells.

[0046] 7. Results were obtained showing no inhibition of proliferation of the RMP 847 B1Can LEB cell line when using a cisplatin concentration corresponding to the therapeutic dose of the drug (10% of the peak plasma concentration).

[0047] The new cell line obtained expands the arsenal of tumor cell lines that can be used for experimental and clinical purposes, namely, for the creation of antitumor biomedical cell products, the use of which makes it possible to increase the effectiveness of treatment and increase life expectancy in the treatment of malignant neoplasms.

[0048] Can also be used to test the activity of various pharmaceutical drugs, create diagnostic kits, test systems and experimental models for the development of new therapeutic approaches and drugs.