RU2796357C1 - HUMAN SKIN MELANOMA CELL LINE mel-Alx-LP - Google Patents

Abstract

FIELD: biotechnology; obtaining a cell line.

SUBSTANCE: obtained cell line of human skin melanoma mel-Alx-LP can be used as a model system for studying the processes of carcinogenesis and metastasis in vitro, searching for new potential targets in the therapy of skin melanoma, as well as testing antitumor drugs and new therapeutic approaches, it is stored in a specialized collection of cultures cells of the Center for Collective Use “Collection of Vertebrate Cell Cultures” of the Institute of Cytology of the Russian Academy of Sciences under the number “РККК(П) 812Д”.

EFFECT: mel-Alx-LP cell line has stable cultural, morphological, and molecular characteristics and can be used as a positive control in immunohistochemical and immunocytochemical analyzes for the detection of skin melanoma markers; it is characterized by stable expression of the following melanocytic markers: SOX10, MITF.

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Description

The invention relates to the field of medicine, biotechnology, and in particular to the production of a cell line. The resulting cell line can be used as a model system for studying the processes of carcinogenesis and metastasis in vitro, searching for new potential targets in the treatment of skin melanoma, as well as testing anticancer drugs and new therapeutic approaches. The mel-Alx-LP cell line has stable cultural, morphological, and molecular characteristics.

The cell line can also be used as a positive control for immunohistochemical and immunocytochemical analyzes in detecting skin melanoma markers, since it is characterized by stable expression of melanocytic markers: SOX10, MITF.

Skin melanoma accounts for ~5% of all skin cancer cases and accounts for >75% of skin cancer deaths [1]. The 5-year relative survival rates for patients with localized or regional disease are 98% and 64%, respectively. In contrast, in patients with metastatic melanoma, the 5-year survival rate is reduced to 23%.

Metastatic melanoma was historically considered a untreatable disease until the FDA approved therapeutic strategies based on targeted therapy and immunotherapy in 2011 and 2014.

For patients with BRAF ^V600E/K mutant melanoma, significant efficacy has been observed with the combination of a BRAF inhibitor and a MEK inhibitor, with a response rate of ~76% [2]. At the same time, >80% of patients subsequently relapse on the BRAF/MEK inhibitor cocktail, leaving them suitable only for anti-PD-1 and anti-CTLA4 immunotherapy [3].

The activity of combination therapy with checkpoint inhibitors using anti-PD-1 and anti-CTLA-4 antibodies demonstrated a long-term response, which is a therapeutic strategy suitable for all genotypes. However, 60-70% of patients with melanoma do not respond to checkpoint inhibitor therapy due to toxicity, intrinsic resistance, and other reasons that are not fully understood. At the same time, persistent melanoma cells that do not respond to current standard treatment strategies represent a problem that researchers and clinicians are trying to overcome [4].

Treatment of metastatic melanoma is challenging. Although targeted and immune therapies have improved patient survival, in most cases, tumor cells develop resistance to therapy. Dynamic interactions of melanoma cells with other cellular and acellular components of the tumor microenvironment provide additional mechanisms of homeostatic regulation that are critical for the effectiveness of therapy [5]. Despite recent technological advances in the study of melanoma of the skin, the role of the microenvironment in the resistance of melanoma to therapy remains a question.

To develop new strategies for the treatment of melanoma of the skin that can overcome the current clinical plateau observed in the treatment of patients with targeted and immune therapeutic strategies, it is necessary that melanoma model systems be as close as possible to the natural tumor system, reproducing the diverse complex mechanisms of resistance in vivo in patients.

With the help of traditional cell cultures, it is possible to study the phenotypic portrait of a tumor, cytotoxicity, proliferation, mobility, invasiveness, adaptation to hypoxic conditions, leading to its resistance and dissemination, to study drug sensitivity, protein expression and plasticity of molecular pathways, genomic and genetic characteristics (sequencing RNA, whole exome sequencing).

In total, there are >2000 melanoma cell lines worldwide. For most of these cell lines, systematic genetic and molecular tests, as well as high-throughput drug screenings, have provided comprehensive information on the main genes and molecular pathways responsible for the progression of melanoma and its resistance to therapy. And the creation of three-dimensional models from two-dimensional cultures with the introduction of stromal cell cultures makes it possible to complicate the interactions of tumor cells and the microenvironment, which significantly brings such a model closer to natural conditions.

Therefore, an important and urgent task today is to expand the arsenal of characterized cell lines with the aim of using them as model systems for studying the processes of carcinogenesis, testing anticancer drugs and testing new therapeutic approaches in the treatment of malignant neoplasms, as well as to search for new potential targets.

Closest to the claimed cell line - the prototype is the line NCI-H838 [H838] Lung Adenocarcinoma Human from the ATCC collection with collection number CRL-5844 (https://www.atcc.org/products/crl-5844). According to the conducted STR-analysis with the study of markers present in the ATCC database, the percentage of coincidence is 82%. The NCI-H838 cell line was derived from lung adenocarcinoma metastases to a lymph node. The culture is characterized by epithelial morphology and adhesive type of growth.

The technical result of the invention is the expansion of the arsenal of cell lines with metastatic potential and tumorigenicity, which can be used to test the activity of new antitumor drugs, as well as to search for new potential targets in the treatment of skin melanoma and the development of new therapeutic approaches in the treatment of malignant neoplasms. The cell line can also be used as a positive control for immunohistochemistry.

and immunocytochemical analyzes in the detection of skin melanoma markers, since it is characterized by a stable expression of melanocytic markers: SOX10, MITF.

The specified technical result of the invention is achieved by a new human skin melanoma cell line mel-Alx-LP, which retains the key characteristics of the tumor sample: expression of skin melanoma markers, the ability to invade and migrate, high proliferative potential, from which the culture was obtained, is stored in a specialized collection of cell cultures Center for Collective Use "Collection of Vertebrate Cell Cultures" of the Institute of Cytology of the Russian Academy of Sciences under the number RKKK(P) 812D.

Obtaining cell line mel-Alx-LP was carried out in the following way:

The mel-Alx-LP cell line was isolated from human malignant melanoma metastasis in the inguinal lymph node. The sample was characterized by high mitotic activity (21 mitoses per 1 sq. mm of tumor).

Intraoperatively obtained tumor tissue fragments were placed in a sterile tube containing complete nutrient medium (DMEM/F-12, 10% FBS, penicillin 25000 U/500 ml of medium + streptomycin 25 mg/500 ml of medium) and transported to the laboratory. The samples were transferred to a laminar and crushed on a sterile Petri dish with a sterile scalpel with the addition of 5-7 ml of DMEM/F-12 into pieces no larger than 2 mm ³ in size. Next, the tumor pieces were placed in a sterile medicon (Medicon, Sterile, BD™) and subjected to mechanical disaggregation inside the medimachine (Medimachine, BD™) for 1 min. The resulting cell suspension is passed through sterile filters with a diameter of 50-70 μm using a sterile syringe 1 cm ³ . The resulting filtered cell suspension was centrifuged at 1000 rpm for 5 min, the supernatant was carefully removed and planted in a 25 cm ² vial with the addition of 5 ml of DMEM/F-12 nutrient medium containing 10% fetal bovine serum (FBS), antibiotics ( penicillin with streptomycin at a concentration of 100 units / ml and 100 μg / ml, respectively). Cells were incubated at 37°C, 5% CO ₂ , 100% humidity.

A stably proliferating culture was obtained at passage 10.

The culturing conditions for the mel-Alx-LP cell line are as follows.

Growth type - adhesive.

In vials with a volume of 25 cm ² in 5 ml of medium, 1×10 ⁶ cells are seeded.

The cells are cultured in a DMEM/F-12 nutrient medium containing 10% fetal bovine serum (FBS), antibiotics (penicillin with streptomycin at a concentration of 100 U/ml and 100 μg/ml, respectively) in a 25 cm ² culture flask and incubated at 37°C, 5% CO ₂ , 100% humidity.

Reseeding 1 time in 3-4 days in a ratio of 1:1 using equal volumes of 0.25% Trypsin solution and 0.02% Versen solution.

Cell viability is assessed by the inclusion of trypan blue using automatic counters or a Goryaev camera. Cell viability after reseeding ranges from 90-99%.

Morphological features of the mel-Alx-LP cell line are characterized as follows:

Cells are predominantly epithelioid cell morphology (75%) and spindle cell features (25%). The nuclei are large, rounded. Single cells are binuclear and multinuclear.

Multiplex analysis of 19 STR markers and the human amelogenin locus is presented in Table 1.

The identified genotype was analyzed using the ATCC reference database.

Reference database search results: The maximum degree of genotype match was established for the cell line - NCI-H838Lung AdenocarcinomaHuman (82%). According to the ATCC recommendations: cell lines demonstrating 100% match for the studied markers present in the ATCC database can be considered identical. Cell lines demonstrating at least 80% genotype match for the studied markers present in the ATCC database can be considered related (derived from a common precursor line). Cell lines showing less than 80% genotype match for the studied markers present in the ATCC database cannot be reliably considered related lines based on the data obtained (Capes-Davis et al., Match criteria for human cell line authentication: Where do we draw the line (Int. J. Cancer. 2012 Nov 8. doi:0.1002/ijc.27931).

Marker features of the mel-Alx-LP cell line:

SOX10 - 40-45% express cells of epithelioid-cell morphology, 5% - spindle-shaped cell morphology, the remaining cells have lost the expression of this marker. MITF - 40-45%, of which 20% stain most brightly.

Proliferation marker Ki-67 was detected in 92% of cells.

The conditions for cryopreservation of the mel-Alx-LP cell line are as follows:

Cryomedia: fetal calf serum 90%, DMSO 10%.

Volume of cells per cryovial: 1.5-3×10 ⁶ cells/ml.

Cells are resuspended in a cryogenic medium until a homogeneous suspension is formed, and transferred to cryovials. Cryotubes are placed in a refrigerator container to maintain a constant cooling rate of 1°C/min., the container is placed in a refrigerator at -80°C for 24 hours. For long-term storage, the tubes are transferred to a cryobank at -196°C.

The defrosting of the line is carried out at 37°C.

Cells are diluted in 10 ml of serum-free medium and precipitated by centrifugation, resuspended in 5 ml of the same medium containing 10% fetal calf serum, antibiotics (penicillin with streptomycin at a concentration of 100 U/ml and 100 μg/ml, respectively), transferred to a culture flask volume of 25 cm ² and incubated at 37°C, 5% CO ₂ , 100% humidity.

Cell viability is assessed by the inclusion of trypan blue using automatic counters or a Goryaev camera. Cell viability after cryopreservation is 85-99%.

The contamination of the skin melanoma cell line mel-Alx-LP was investigated as follows.

The PCR method was used to study the presence of mycoplasma - mycoplasma was not detected. During long-term cultivation in the medium, contamination by bacteria and fungi was not detected.

The use of the skin melanoma cell line mel-Alx-LP is represented by the following examples.

Example 1 Use of the mel-Alx-LP skin melanoma cell line as a positive control for the SOX10 transcription factor (SRY-Box Transcription Factor 10)

The cells are cultured in a DMEM/F-12 nutrient medium containing 10% fetal bovine serum (FBS), antibiotics (penicillin with streptomycin at a concentration of 100 U/ml and 100 μg/ml, respectively) in a 25 cm ² culture flask and incubated at 37°C, 5% CO ₂ , 100% humidity. After 3 days, cells are reseeded in a ratio of 1:1 using equal volumes of 0.25% Trypsin solution and 0.02% Versen solution.

When a confluent monolayer is reached, the supernatant is removed, 1 ml of a solution of 0.25% Trypsin solution and 0.02% Versene solution (in a ratio of 1:1) is added and placed in a CO ₂ incubator at 37 ° C, 5% CO ₂ , 100% humidity for 2 minutes. After the specified time has elapsed, the vial is removed from the incubator, cells are removed from the bottom of the vial using a 1 ml automatic dispenser, placed in a sterile tube (15 ml) with the addition of DMEM/F-12 containing 10% fetal calf serum (FBS) and centrifuged 5 minutes at 1000 rpm.

At the end of centrifugation, the supernatant is removed and the cells are washed in a single phosphate buffer solution (PBS).

Next, cell blocks are prepared on the basis of 2% agar and the block is processed in the same way as biopsy material: fixation in 10% buffered formalin (up to 12 hours), wiring, embedding in paraffin and microtomy. The control section with the cell line is mounted on the same slide with the test sample.

For immunohistochemical studies, deparaffinization, rehydration, unmasking and peroxide treatment of the obtained sections are performed.

Primary monoclonal antibodies against SOX10 (EP268, Rabbit Monoclonal Antibody, Cell Marque) are incubated for 30 minutes at 25°C. After incubation, they were washed with TBS buffer three times. The EnVision FLEX system, High pH (Link) (Agilent) is used as an imaging system, incubation with secondary antibodies is carried out for 30 minutes at 25°C. After incubation, they were washed with TBS buffer and visualized with diaminobenzidine at room temperature for 10 minutes. The cells were washed with TBS buffer and the cell nuclei were stained with Mayer's hematoxylin. Samples are dehydrated by a series of immersions in isopropyl alcohol, xylene and sealed.

As a result, the expression of SOX10 by the mel-Alx-LP cell line was observed using a light microscope as nuclear staining.

Example 2 Use of the mel-Alx-LP skin melanoma cell line as a model system for evaluating the cytotoxic effect of anticancer drugs.

Cell lines are planted in flat-bottomed 96-well plates and placed in CO ₂ -incubator (+37°C, 5-6.5% CO ₂ ) for 3 days. The entire tablet is divided into the following groups: control group - 3 wells; experimental groups: addition of the studied antitumor drug at various concentrations - 3 wells.

After 3 days, the investigated antitumor drug is added to the medium. The incubation time in the conditions of a CO ₂ incubator (+37°C, 5-6.5% CO ₂ ) is determined in advance. At the end of cultivation with the preparation, images of monolayer cultures are obtained using a phase contrast microscope Zeiss Primo Vert (4×0.10) (Zeiss, Germany).

Then, 10 μl of MTT (Roche, Basel, Switzerland) at a final concentration of 0.5 mg/ml are added to the wells where monolayer cell lines were cultivated and incubated for 4 hours in a CO ₂ -incubator (+37°C, 5-6.5 % CO ₂ ).

After 4 hours, the supernatant was removed and 100 μ1 DMSO was added for 1 hour. The optical density (OD) of the obtained solutions was measured on an iMark microplate spectrophotometer (Bio-Rad Laboratories, USA) at a wavelength of 595 nm. To convert units of optical density to percent viability, use the formula: (OD of the test sample - OD of the control group) * 100%.

As a result, a dose-response curve is obtained, which characterizes the change in the effect of the anticancer drug depending on the concentration on the cell line.

The new human skin melanoma cell line mel-Alx-LP allows expanding the arsenal of cell lines with metastatic potential and tumorigenicity, which can be used to test the activity of new anticancer drugs, as well as to search for new potential targets in the treatment of skin melanoma and develop new therapeutic approaches. in the treatment of malignant neoplasms. The cell line can also be used as a positive control for immunohistochemical and immunocytochemical analyzes in detecting skin melanoma markers, since it is characterized by stable expression of melanocytic markers: SOX10, MITF.

Information sources:

1. Matthews NH, Li WQ, Qureshi AA, et al. epidemiology of melanoma. In: Ward WH, Farma JM, editors. Cutaneous Melanoma: Etiology and Therapy. Brisbane (AU): Codon Publications; 2017 Dec 21. Chapter 1. doi: 10.15586/codon.cutaneousmelanoma.2017.chl.

2 Dummer, R. et al. Overall survival in patients with BRAF-mutant melanoma receiving encorafenib plus binimetinib versus vemurafenib or encorafenib(COLUMBUS): a multicentre, open-label, randomised, phase 3 trial. Lancet Oncol. 19, 1315-1327 (2018).

3. Schreuer, M. et al. Combination of dabrafenib plus trametinib for BRAF andMEK inhibitor pretreated patients with advanced BRAF(V600)-mutant melanoma: an open-label, single arm, dual-centre, phase 2 clinical trial. Lancet Oncol. 18, 464-472 (2017).

4. Rogiers, A. et al. Long-term survival, quality of life, and psychosocial outcomes in advanced melanoma patients are treated with immune checkpoint inhibitors. J. Oncol. 2019, 5269062 (2019).

5. Falcone I, Conciatori F, Bazzichetto C, Ferretti G, Cognetti F, Ciuffreda L, Milella M. Tumor Microenvironment: Implications in Melanoma Resistance to Targeted Therapy and Immunotherapy. Cancers (Basel). 2020 Oct 6;12(10):2870. doi: 10.3390/cancersl2102870.

Claims (1)

Human skin melanoma cell line mel-Alx-LP, intended for testing the activity of new anticancer drugs and studying tumor biology, to search for new potential targets in the treatment of skin melanoma and develop new therapeutic approaches in the treatment of malignant neoplasms, as well as for positive control during immunohistochemical and immunocytochemical analyzes in the detection of skin melanoma markers, characterized by stable expression of melanocytic markers: SOX10, MITF; is stored in the specialized collection of cell cultures of the Central Collective Use Center "Collection of Vertebrate Cell Cultures" of the Institute of Cytology of the Russian Academy of Sciences under the number RKKK(P) 812D.