Human pleomorphic liposarcoma cell line 7702 LS den

Abstract

FIELD: biotechnology.SUBSTANCE: human pleomorphic liposarcoma cell line 7702 LS DEN is proposed and is used to create biomedical cell products, cell models, and test antitumor drugs. The invention makes it possible to expand the range of cell lines used to create biomedical antitumor cell products.EFFECT: use of the resulting cell line for testing the activity of various pharmaceuticals, creating diagnostic kits and test systems for the development of new therapeutic approaches and drugs.1 cl, 2 ex

Description

[0001] The invention pertains to the field of biotechnology. The resulting new human pleomorphic liposarcoma cell line, 7702 LS DEN, possesses stable morphological and cultural characteristics. This cell line can be used in cellular 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 824D.

[0002] Liposarcomas (LPS) are malignant tumors of adipocytic differentiation. They are the most common subtypes of soft tissue sarcomas (STS), accounting for approximately 15% to 20% of all STS [Clinical and Molecular Spectrum of Liposarcoma / Lee ATJ, Thway K., Huang PH, Jones RL / / J. Clin. Oncol. - 2018. - Jan 10;36(2):151-159. doi: 10.1200 / JCO.2017.74.9598].

[0003] The causes of liposarcoma remain unknown. Risk factors for the development of liposarcoma include radiation (especially radiation therapy used to treat other malignancies), certain familial cancer syndromes, injury / trauma to the lymphatic system, and exposure to toxic chemicals. However, there are many cases in which patients have no obvious risk factors for this disease [Zafar R., Liposarcoma / Zafar R., Wheeler Y. / / In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing, Jan 2025].

[0004] Pleomorphic liposarcoma (PLS) is the rarest high-grade liposarcoma subtype, accounting for only 5% of LPS, and genetically carrying complex genomic aberrations with numerous chromosomal imbalances and frequent mutations in the TP53 and RB1 genes. PLS has the worst prognosis among LPS, distant metastasis develops in 30-50% of patients after radical surgery, tumor-related mortality is observed in 50% of patients, and effective chemotherapy regimens for this type of LPS have not yet been found [Clinical and Molecular Spectrum of Liposarcoma / Lee ATJ, Thway K., HuangP.H., Jones RL / / J. Clin. Oncol. - 2018. - Jan 10; 36 (2): 151-159. doi: 10.1200 / JCO.2017.74.9598; Establishment and characterization of NCC-DDLPS5-C1: a novel patient-derived cell line of dedifferentiated liposarcoma / Sin Y., Yoshimatsu Y., Noguchi R., Tsuchiya R et al. / / Hum Cell. 2022 May;35(3):936-943. doi:10.1007 / s 13577-022-00689-2].

[0005] At the current stage of research, several different molecular genetic subtypes of LPS with different biology and clinical behavior have been identified. Additional research is needed to better understand the biology specific to each subtype and to identify new targets for targeted therapy [Liposarcoma: Novel Approaches to Systemic Therapy and Multidisciplinary Care / Jonczak E., Trent J., Roland C, Haddad EN / / Hematol. Oncol. Clin. North. Am. -2025. - May 24:S0889-8588(25)00048-6. doi: 10.1016 / j.hoc.2025.04.010].

[0006] Patient-derived tumor cell lines are important tools for understanding the biology of malignancies and the molecular mechanisms underlying their development. The use of malignant cell lines also facilitates drug screening and the investigation of numerous anticancer agents and their modes of action, since in most cases, the properties of cultured tumor cells correlate with the histological characteristics of the tumor and its vulnerability to anticancer agents [Establishment and characterization of NCC-PLPS2-C1: a novel cell line of pleomorphic liposarcoma / Akiyama T., Yoshimatsu Y., Noguchi R., Sin Y. et al. / / Hum Cell. - 2023. - Jan; 36 (l): 468-475. doi: 10.1007 / s 13 577-022-00828-9].

[0007] Nowadays, the work on preparing cell lines has become systematic, but each newly obtained cell line has individual, unique properties and characteristics, since it is a product obtained from the biological material of a specific individual.

[0008] The objective of the present invention is to obtain a new tumor cell line of human pleomorphic liposarcoma, which can potentially be used as a cell model for studying the molecular basis of carcinogenesis and developing new innovative methods for treating this type of malignant neoplasm.

[0009] The technical result of the invention is an expansion of the arsenal of cell lines used to create biomedical antitumor cell products, which makes it possible to improve treatment efficacy and extend survival in the treatment of liposarcoma. Furthermore, the resulting new cell line can be used to test the activity of various pharmaceuticals, create diagnostic kits and test systems for the development of new therapeutic approaches and drugs.

[0010] This technical result is achieved by obtaining a new cell line, 7702 LS DEN, from a human pleomorphic liposarcoma tumor sample. This cell line expresses specific antigens and produces specific molecules. The resulting 7702 LS DEN cell line possesses stable cultural and morphological characteristics and is stored in the Specialized Collection of Vertebrate Cell Cultures of the Russian Collection of Cell Cultures under registration number KKKP 824D.

[0011] The cell line was isolated from a tumor metastasis in the right hemithorax of patient D.E.N., 48, who was undergoing treatment at the N.N. Petrov Cancer Center. The material was obtained during surgical removal of the metastatic lesion.

[0012] The 7702 LS DEN cell line was obtained as follows.

[0013] 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 sec to 1 min. The resulting cell suspension was passed successively through sterile nylon filters of 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.

[0014] A stably growing cell line was obtained at passage 70.

[0015] The morphological features of the 7702 LS DEN cell line are characterized as follows.

[0016] The culture is represented by a population of polygonal cells containing large nuclei with one or two nucleoli. The cytoplasm contains numerous mitochondria, localized primarily near the nucleus, and lysosomes. The actin cytoskeleton is well defined (according to fluorescence confocal microscopy). The proliferation marker Ki-67 is immunocytochemically detected in 69% of the cells.

[0017] The marker features of 7702 LS DEN cell line are as follows.

[0018] The PCR method revealed hyperexpression of testicular cancer genes GAGE, PRAME.

[0019] The PCR method revealed the expression of the genes beta-tubulin III, PDGFRa / b, C-kit. The enzyme-linked immunosorbent assay method revealed high production of the ligand of the activation receptor of cytotoxic T-lymphocytes MICA (1.12 ng / ml / 10 6 cl), vascular endothelial growth factor VEGF (2.5 nt / ml / 10 6 cl), interleukins IL-6 (467.31 pg / ml / 10 6 cl), IL-8 (1.21 ng / ml / 10 6 cl).

[0020] The cultural properties of 7702 LS DEN cell line are presented as follows.

[0021] The 7702 LS DEN cell line is characterized by an adhesive monolayer growth pattern. The cell line is cultured in DMEM / F12 (80%) nutrient medium supplemented with 20% fetal bovine serum and antibiotics (penicillin and streptomycin at a concentration of 100 U / ml and 100 μg / ml, respectively). Cultivation is carried out at 37°C, 5% CO2, and 100% humidity. The culture has an adhesive monolayer growth pattern. In 25 cm flasks 2 1x10 are inoculated into 5 ml of medium 6 cells. Subculture is performed twice a week in a 1:3 ratio using equal volumes of 0.25% trypsin solution and 0.02% versene solution.

[0022] The cryopreservation conditions are as follows.

[0023] For long-term storage, cells are preserved by freezing in liquid nitrogen. Cryomedium: DMEM / F12 50%, fetal bovine serum 40%, DMSO 10%; 3×106 cells / ml per ampoule. The cells of the cell line are resuspended in the freezing medium. Freezing mode: liquid nitrogen, temperature decrease by 1°C per minute to -25°C, then quick freezing to -70°C. Storage in liquid nitrogen at -196°C. Quick defrosting at 37°C. The 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 bovine serum, and transferred to a 25 cm2 culture flask. Cell viability is assessed by trepan blue staining. Cell viability after cryopreservation is 85%.

[0024] Contamination was investigated as follows.

[0025] During long-term observation, bacteria and fungi were not detected in the culture. PCR testing with the Myco Real-Time test system (Eurogen, Russia) was performed to detect the presence of DNA from microorganisms of the Mollicutes class, including the most common contaminants of cell cultures in laboratory conditions: 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.

[0026] The karyological characteristics of the 7702 LS DEN cell line are as follows.

[0027]

[0028] Species specificity: human tumor cells.

[0029] The use of 7702 LS DEN cell line is represented by the following examples.

[0030] Example 1. Using the 7702 LS DEN cell line to create a personalized model system to assess the sensitivity of tumor cells to cytostatics.

[0031] 1. The 7702 LS DEN cell line was cultured as described above. Upon reaching a confluent monolayer, the cells were detached from the substrate using an equilibrium mixture of 0.25% trypsin and 0.02% versene.

[0032] 2. The cells were washed twice in complete culture medium, cell counting and viability assessment were performed.

[0033] 3. To assess chemosensitivity, 7702 LS DEN tumor cells were pre-seeded in E-Plate 16 PET plates (Acea Bioscience Inc., USA) in an amount of 1×10 4 cells per well in 100 µl of complete nutrient medium. Incubated for 24 hours in an xCelligence automated real-time cell analyzer (Acea Bioscience Inc., USA), 100 µl of the test drug solution were then added.

[0034] 4. Ifosfamide, doxorubicin, gemcitabine, and docetaxel were used as chemotherapeutic agents. The chemotherapeutic agents were added at a final concentration corresponding to 10% of the peak plasma concentration in the patient. This concentration corresponds to the therapeutic concentration (Salmon SE, 1981). The duration of incubation with the chemotherapeutic agents corresponded to the half-life of the drug in circulation.

[0035] 5. 7702 LS DEN cells without treatment were used as a positive control.

[0036] 6. After adding cytostatics to the system, observation was carried out for 72 hours. The total observation time was 96 hours.

[0037] 7. The effect of cytostatics was determined by changes in the cellular index—the ratio of electrical impedance at the "zero" point to the impedance at a given time point. The device measures the electrical resistance of sensor electrodes, which is proportional to the number of cells attached to the sensors, allowing for cell growth measurements at various time intervals.

[0038] 8. The 7702 LS DEN cell line was found to be chemoresistant to the individual drugs studied. When exposed to the combination drugs doxorubicin / ifosfamide and gemcitabine / docetaxel, the chemoresensitivity of the 7702 LS DEN cell line was 54% and 32%, respectively.

[0039] Example 2. Use of the 7702 LS DEN cell line for the preparation of antitumor vaccines based on activated dendritic cells.

[0040] 1. The 7702 LS DEN cell line was cultured as described above. Upon reaching a confluent monolayer, the cells were detached from the substrate using an equilibrium mixture of 0.25% trypsin and 0.02% versene.

[0041] 2. The culture medium was removed from the flask, the surface of the flask was rinsed with a small amount of enzyme solution, and then 1-3 ml of this solution was added to the cells. The flasks were placed in a CO2 incubator and the cell condition was visually inspected.

[0042] 3. 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.

[0043] 4. Cell counting and viability assessment were performed.

[0044] 5. To prepare the tumor lysate, 7702 LS DEN cell line was subjected to 6 consecutive cycles of instant freezing to -196°C and thawing to room temperature in phosphate-buffered saline without cryoprotectant. The quality of lysis was monitored using 0.1% trypan blue and a light microscope.

[0045] 6. 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.

[0046] 7. The lysate of the 7702 LS DEN cell line was added to the culture of autologous immature dendritic cells from a patient with liposarcoma 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.

[0047] 8. As a result, activated DCs with the immunophenotype CD14- / CD1a- / CD83+ / CD80+ / CD86+ / HLADR+ / CD209+ / CCR7+ were obtained.

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