RU2572569C1 - METHOD OF PRODUCTION OF SUBDERMAL XENOGRAFTS OF CELL LINE OF PEOPLE SKIN MELANOMA mel Cher WITH MUTATION V600E BRAF FOR PRECLINICAL STUDY OF ANTINEOPLASTIC TARGETED AGENTS - Google Patents

Abstract

FIELD: bioengineering.

SUBSTANCE: method of production of subdermal xenografts of cell line of people skin melanoma mel Cher with mutation V600E BRAF for preclinical study of antineoplastic targeted agents includes acclimation of the cell line of people skin melanoma mel Cher to rise of Balb/c nude at immunodeficient mice by means of multiple passaging of up to nine subcultures of suspensions of tumour cells (50-60 mg per mouse) with stable rise kinetics, and testing for presence of the mutation V600E BRAF. The subdermal xenografts of cell line of people skin melanoma mel Cher with mutation V600E BRAF for preclinical study of antineoplastic targeted agents are produced.

EFFECT: higher compositions use efficiency.

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Description

The invention relates to medicine, namely to experimental oncology, and for the creation of subcutaneous xenografts of human skin melanoma for preclinical study of antitumor agents.

Disseminated human skin melanoma is characterized by extremely low sensitivity to various types of therapy [Demidov L.V., Orlova K.V. Individualization of drug treatment of skin melanoma // Practical Oncology, 2013, T.14, No. 4, S. 239-246].

Mutation of V600E in exon 15 of the BRAF gene leads to hyperactivation of serine threonine BRAF kinase followed by abnormal cell proliferation and rapid progression of melanoma [Graeme J. Walker, et al. Modeling melanoma in mice // Pigment Cell Melanoma Res., 2011, 24, P. 1158-1176; Davies H., et al. Mutations of the BRAF gene in human cancer // Nature, 2002, 417, P. 949-954; Tsai J. et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity // Proc. Natl. Acad. Sci. USA, 2008, 10, P. 3041-3046].

Selective blocking of BRAF kinase determined the creation of anticancer targeted agents, among which the most effective in the treatment of disseminated melanoma is vemurafenib [Ribas A., et al. BRIM-2: an open label, multicenter phase II study of vemurafenib in previously treated patients with BRAFV600E mutation positive melanoma // J. Clin. Oncol., 2011, 29, P. 8509].

The objective of the invention is to obtain subcutaneous xenografts of the cell line melanoma of human skin mel Cher with a mutation V600E BRAF for preclinical studies of anticancer targeted agents.

The problem is solved by the fact that to obtain subcutaneous xenografts, the cell line of human skin melanoma mel Cher was used, adapted for growth in immunodeficient Balb / c nude mice with stable growth kinetics, tested for the presence of the V600E BRAF mutation and used for preclinical studies of anticancer targeted drugs.

For preclinical studies of antitumor target agents, the mel melanoma cell line of human skin mel Cher was used, deposited in the Cell Culture Collection of the Institute of Cytology of the Russian Academy of Sciences RKKK (P) 704D. The cell line of human skin melanoma mel Cher has stable cultural, morphological and individual phenotypic characteristics and high mitotic potential [RF patent No. 2364624].

In the mel Cher cell line of human skin mel Cher, a V600E mutation was determined in exon 15 of the BRAF gene.

The technical result of the invention.

Subcutaneous xenografts of the mel Cher human skin melanoma cell line with the V600E BRAF mutation were obtained for preclinical studies of anticancer targeted agents.

The method was carried out as follows.

The adaptation of the mel Cher human skin melanoma cell line to growth in immunodeficient Balb / c nude mice was carried out by repeated passage of mice with different vaccination doses of cells. In stable passages of subcutaneous xenografts, the V600E BRAF mutation was determined, followed by the use of vemurafenib.

The following transplant characteristics were used to control the adaptation of the mel Cher human skin melanoma cell line to in vivo growth: graft dose of cells for 100% obtaining subcutaneous xenografts, cytologically identical to the mel Cher human skin melanoma cell line after implantation in mice (first passage); stable multiple transplantation until the ninth passage with a suspension of tumor tissue; steady kinetics of growth of melanoma of human skin mel Cher in mice in late passages; histological verification of melanoma.

The criterion for the use of subcutaneous xenografts of mel Cher human skin melanoma for preclinical studies of antitumor target agents was the presence of the V600E BRAF mutation and high sensitivity to vemurafenib.

The transplant characteristics of the mel Cher human melanoma cell line mel Cher were obtained as follows.

An inoculum for implantation was the mel Cher cell line of human skin melanoma cultured in CO ₂ . The cells washed with physiological saline from the culture medium were placed in a nutrient medium 199 and implanted subcutaneously in doses of 0.1 × 10 ⁷ ; 0.5 × 10 ⁷ ; 1.0 × 10 ⁷ cells per mouse in 0.2 ml of nutrient medium 199. In the study, 12 immunodeficient Balb / c nude mice, 4 mice per cell dose, were used. Cell graft control was carried out by the presence of palpable tumors in mice with registration of the latency for 21 days. At the end of the study, we determined the optimal vaccination dose of 1.0 × 10 ⁷ cells per mouse, providing 100% vaccination.

Obtaining subcutaneous xenografts of the mel Cheroma human melanoma cell line with the V600E BRAF mutation.

Subcutaneous xenografts of mel Cher human skin melanoma were obtained on 10 Balb / c nude mice. Repeated passages were performed by suspension of tumor tissue 50-60 mg per mouse diluted 1:20 with nutrient medium 199. The presence of transplanted tumors in mice at the same time with repeated passage indicated the achievement of stable kinetic characteristics of subcutaneous xenografts in vivo.

The growth kinetics of subcutaneous xenografts was determined by the average tumor volume, starting from the appearance of all palpable tumors, taking into account the latent growth phase. A period of an increase in the average tumor volume (V _cp = a × b × c) two or more times determined the duration of the exponential growth phase, and stabilization of tumor growth at a level of less than a double increase determined the duration of the stationary growth phase. The tumor growth rate was judged by the ratio of the subsequent average volume to the previous one (V _t / V _t-1 ) when measuring the tumor every 3-4 days for 2-4 weeks.

Statistical analysis of the growth parameters of the fourth passage was performed using the standard Fisher method. The absence of significant differences in the growth curves of the tumor of the two passages in the semilogarithmic coordinate system confirmed the stability of the kinetic characteristics of the growth of subcutaneous xenografts of mel Cher human skin melanoma in Balb / c nude mice.

Histological verification of subcutaneous xenografts of skin melanoma to compare the cell composition with the original cell line of human melanoma mel Cher was performed in the ninth passage with stable growth kinetics using light microscopy of tumor sections stained with hematoxylin and eosin.

The invention is illustrated in tables (1-4) and figures (1-3).

Tab. 1. The choice of the optimal vaccination dose of mel Cher human skin melanoma cells for obtaining subcutaneous xenografts in Balb / c nude mice.

Tab. 2. Tumor growth dynamics after implantation of 1.0 × 10 ⁷ mel Cher human skin melanoma cells in Balb / c nude mice (first passage).

Tab. 3. The average volume of a mel Cher human skin melanoma tumor in Balb / c nude mice (fourth passage).

Tab. 4. The average tumor volume of mel Cher human skin mel mel in Balb / c nude mice (ninth passage).

FIG. 1. The histological picture of the subcutaneous xenograft of melanoma of human skin mel Cher on the 14th day of tumor growth (ninth passage).

FIG. 2. Detection of the V600E BRAF mutation in subcutaneous xenographs of mel Cher human skin melanoma in Balb / c nude mice (passage 9).

FIG. 3. The antitumor activity of vemurafenib in mice with subcutaneous xenografts of melanoma of human skin mel Cher.

In the table. Figure 1 shows the dependence of tumor growth after subcutaneous implantation of mel Cher human skin melanoma cells on a graft dose of cells. The optimal vaccination dose was 1.0 × 10 ⁷ cells per mouse. When applying the optimal inoculation dose of 1.0 × 10 ⁷ cells per mouse on day 10 (latent phase), palpable tumors were determined in 100% of the mice.

In the table. Figure 2 shows that the growth kinetics of the first passage tumors were relatively slow (21 days), the tumor volume did not reach 1.0 cm, and the phase of exponential growth was practically absent. The multiplicity of tumor growth during this period was less than 2.0. The average tumor volume was: on the 10th day 42.2 ± 10.5 mm ³ , on the 15th day 112.0 ± 35.1 mm ³ . Then by 21 days the tumor volume reached 133.0 ± 27.8 mm ³ , exceeding the initial volume by 3.2 times.

In the table. Figure 3 shows that the kinetics of tumor growth progressively changed at the fourth passage. The latent phase was reduced to 8 days, the average tumor volume was 68.0 ± 25.0 mm ³ , almost 1.6 times the tumor volume of the first passage. On day 11, the average tumor volume was 200 ± 67.0 mm ³ with a tumor growth rate of 2.9. On days 14, 17 and 21, corresponding to the stationary phase, the tumor growth rate varied between 1.9; 2.1 and 1.8. The average tumor volume at 21 days reached 1526.3 ± 496.0 mm ³ , 10 times the average tumor volume of the first passage.

The steady kinetics of tumor growth achieved by the fourth passage was maintained until the ninth passage with the same temporal characteristics: latent phase up to 8 days, exponential phase up to 14 days and stationary phase up to 21 days after transplantation (Table 4).

A histological examination of subcutaneous xenografts of mel Cher human skin melanoma of the ninth passage revealed the presence of large polymorphic cells identical to the cytological characteristic of mel Cher human skin melanoma cells (Fig. 1).

The V600E BRAF mutation was detected in mel Cheroma human skin tumor cells of the ninth passage at the peak of the exponential growth phase.

In FIG. Figure 2 shows the V600E BRAF mutation in subcutaneous xenographs of Mel Cher human skin melanoma in mice (a - wild type, b - mutation 1799T> A, leading to the replacement of valine with glutamic acid at position 600 (V600E), triplet GTG (GAG is highlighted in gray) .

To assess the sensitivity to a BRAF kinase inhibitor, vemurafenib in a single dose of 75 mg / kg was injected into the stomach of 10 mice daily from 4 to 19 days after tumor transplantation. The control group of mice did not receive vemurafenib. The dynamics of tumor growth in both groups was evaluated during and after the use of vemurafenib every 4-5 days for 20 days.

In FIG. Figure 3 shows a continuous progressive increase in tumor volume in mice of the control group, while vemurafenib completely inhibited tumor growth for 7 days followed by tumor growth (gray curve is the control group, black curve is the experimental group).

Claims (1)

A method for producing subcutaneous xenografts of the mel Cher human skin melanoma cell line with the V600E BRAF mutation for preclinical study of antitumor targeted agents, including adaptation of the mel Cher human skin melanoma cell line to growth by repeatedly passaging up to nine passages of a suspension of tumor cells of 50-60 mg per mouse stable growth kinetics and testing for the presence of the V600E BRAF mutation.

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