Application of human skin melanoma cell line mel cher as positive model of vasculogenic mimicry

FIELD: medicine.

SUBSTANCE: cell line of human skin melanoma mel Cher possesses stable culture and morphological characteristics, is stored in Specialised collection of cell cultures of Institute of CYTOLOGY RAS under number PKKK ("П") 704"Д". Ctll line of human skin melanoma mel Cher expresses growth factors, such as VEGF, of VEGF receptors of I-st and II-nd types, markers of endothelial cells, such as VIII as.ag., Laminin 5 gamma 2.

EFFECT: obtained cell line is able in stable manner to reproduce process of vasculogenic mimicry in vitro and in vivo.

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The invention relates to the field of medical biotechnology, namely the creation of tumor models to study mechanisms of carcinogenesis and screening of anticancer drugs unique mechanism of action.

Malignant disease of the skin is about 25% of all malignant tumors. The proportion of melanoma is up to 3%. Traditionally, the skin melanoma is a malignant tumor with variable and often unpredictable clinical course. So, making structurally not more than 10% of all skin cancers, it is responsible for 80% of deaths attributable to the group of malignant tumors of the skin. The search for new methods of treatment of malignant melanoma of the skin is important.

Currently, there are active research in the development of new anticancer agents with directional mechanism of action, the so-called "targeted" drugs. A distinctive feature of these drugs is the impact on a specific mechanism for the progression of malignant tumors through blocking a protein target inside the tumor. However, there is the problem of finding tumor models in vitro and in vivo for effective evaluation of the action of drugs with directional mechanism of action.

Conducted research on finding new antiangiogenic anticancer drugs. This class is the courthouse square blocks the formation of new blood vessels in tumors and "normalizes" the already formed a vascular network within the tumor tissue. Currently used in clinical practice Avastin (monoclonal antibody against VEGF). Antiangiogenic therapy in combination with chemotherapy improves treatment efficiency by 15-25% [1].

Resistance to antiangiogenic therapy may be explained by the emergence of a tumor alternative routes of blood supply. The most likely mechanism of resistance to antiangiogenic therapy is the appearance of the component of vasculogenic mimicry (VM) and mosaic vessels. VM is the ability of highly aggressive tumor cells, such as cells of melanoma, to form in the absence of endothelial cells (EC) and fibroblasts highly structured vascular channels restricted to the basal membrane [2]. This new type of tumor vascularization was opened relatively recently in 1999 [3]. In vivo it was shown that the long introduction of angiostatin, a natural inhibitor of angiogenesis, some mice were resistant to treatment. Histological analysis of samples of tumors insensitive to the action of the drug, showed that in these tumors, in contrast to the sensitive component is present VM. Was also revealed high correlation between the ability to VM and frequency of metastasis of primary malignant melanoma of the skin and eyes [4]. In this regard, the mu is the need for studying the mechanisms of this process and the creation of new areas of treatment, influencing the process of vasculogenic mimicry in malignant tumors, can be considered as relevant.

For VM study and evaluate the possibility of using antitumor substances as potential inhibitors is necessary to create models of cell lines of human tumors, are capable of reproducing process VM in experimental conditions. This tumor model VM should have the following characteristics: stable play sosudistogo patterns (SPS) in vitro and in vivo implantation in immunodeficient mice, to give reproducible results testing of new anticancer agents in short-term tests in vitro (up to 24 hours), to give reproducible results when testing anticancer substances in vivo. It is assumed that different types of tumors should be developed separate models, because the mechanisms of formation of the VM may vary in different types of tumors.

A large number of studies devoted to the search for an adequate model VM for human cancers in experimental conditions. It is shown that the available cell lines of human tumors either do not form ATP in vitro, or form ATP in vitro in long-term tests (up to 3 weeks). To date, the main cell line for studying the VM is CL the exact line uveal melanoma MUM2B (prototype). The prototype has the following disadvantages: derived from tumor cells uveal melanoma (a rare form of melanoma), generates ATP in vitro in long-term tests (up to 3 weeks), the formation of the BM in vivo occurs under certain conditions (implantation spheroids cells) [5, 6]. Taken as a prototype cell line uveal melanoma MUM2B does not meet all the requirements for a positive model of vasculogenic mimicry metastatic melanoma.

The present invention is to develop a positive model VM cell lines of metastatic melanoma human skin, stably reproducing sosudistogo patterns (SPS) in vitro and in vivo implantation in immunodeficient mice, giving reproducible results testing of new anticancer agents in short-term tests in vitro (up to 24 hours), suitable for screening antitumor substances and the subsequent study of the mechanisms of formation of the VM.

A study of more than 10 cell lines of metastatic melanoma human skin to create the model VM in vitro and in vivo. Assessed the ability of cell lines to form and maintain at least 24 hours PCA on the Matrigel to form tubular structures in long-term tests (up to 3 weeks), to form tumors when implanted in immunodeficient mice, MPW is ideological study formed tumors should be determined by the characteristics of the VM. Studies have shown that only cell line Mel Cher can be used as a positive model VM.

This cell line has a stable cultural and morphological characteristics. Is stored in the cell culture collection of the Institute of Cytology RAS number RCCC(P) D.

Cell line metastatic melanoma Mel Cher consistently reproduces the PCA in vitro by incubation in the matrix of the basal membrane (Matrigel) in short-term tests. ATP are formed within 8 hours after application of the cells and stored up to 24 hours. Educated patterns have the form of an ordered network, where the cells form an interconnected tubes, repeating the behavior of endothelial cells (Example 2, Figa).

Incubation of cell line Mel Cher with antitumor substances developed in the short-term test in vitro allows you to identify substances that block the formation of VM. In this case, the cells planted on Matrigel, form or short, open tubes, unlimited contact with other tubes or collected in a separate, unrelated to each other, the clusters of cells (Example 3, Figb).

Also cell line melanoma Mel Cher is capable of forming tubular structures in long-term tests (up to 3 weeks). Cells are incubated in the basal matrix of memb is an (Matrigel) or proteins of the extracellular matrix (collagen, fibronectin) in high density within 3 weeks. The formation of tubular structures begins three days after the landing of the cells and ends by the end of 3 weeks (Example 4, Figure 2).

The analysis of the expression of some proteins on the cell line Mel Cher immunocytochemical method. It is shown that this line expresses markers of endothelial cells, which allows its use for studying mechanisms VM (example 5).

It is shown that implantation of mice 1-5 million cells Mel Cher observed the formation of tumors within 20 days (Example 6, Fig 3). Histological examination of sections from paraffin blocks of the removed tumor is shown that tumors formed PAS-positive structures (parallel, parallel, crossing, arches etc.) (Figure 4), characteristic of human cancers [6]. Staining with Schiff's reagent (PAS staining) allows you to select the basal membrane in tumor tissue, characteristic of blood vessels and channels, limited tumor cells (VM). Additional double staining with antibodies against CD34+ showed that PAS-positive structures are not limited to CD34 - positive blood vessels, which also indicates the presence of a VM inside of implanted tumors. Characteristics of tumors and histological features of the tumor was maintained in 3 independent is independent experiments.

Lineage cell line mel Cher

Cell line derived from a tumor sample of the patient CNO, and/b 98/10437, were treated in 2003 with a diagnosis of disseminated melanoma skin back. The material derived from a metastatic lymph node. Previous treatment: surgery, chemotherapy, immunotherapy.

Obtaining cell lines mel Cher

Tumor tissue obtained by surgery when removed melanoma metastases. The obtained cell suspension was seeded into flasks and cultured in a long time. Steadily growing cell line was obtained at passage 15.

Morphological features mel Cher

Culture of melanoma mel Cher has a polymorphic cellular structure and represented by cells of different sizes and shapes: elongated, elongated, atrocity, veretenoobraznoj with long, thin spikes of different length, round, oval and irregular shapes.

The cytoplasm is relatively abundant, heterogeneous, painted in basophilic tones of varying intensity, sometimes with a pinkish tinge and often contains a significant amount of fine-grained pigment melanin.

The nucleus of tumor cells polymorphic, labor and hyperchromic with 1-3 nucleoli. The chromatin structure is fine-grained or mercogliano. In part of the cells are budding and fragmentaciyata.

A lot of ugly giant polynuclear cells rounded, elongated elongated, atrocity and irregular in shape with a Central or peripheral location of the nuclei. There are also giant single-core and 2-core cells. Commonly identified mitoses.

Karyological characteristics mel Cher

The culture is characterized by different diameters of the nuclei at the same density of color and the state of chromatin, characteristic of interphase nuclei. Analyzed 54 metaphases. The number of chromosomes in 39 cells ranges from 40 to 47 chromosomes, reflecting the diploid set (2n). There are multiple complex transformations; identify the point of chromosome impossible. 15 metaphases contain from 64 to 80 chromosomes. The modal number of chromosomes in these metaphases corresponds triploid set of (3n). In 13 cells observed chromosomal aberrations, mainly dicentrics chromosomes, chromatide exchanges, multiple aberrations. Therefore, this culture consists of at least two cell lines (diploid and triploid) and is characterized by high chromosomal variability.

Examples of the use of cell lines melanoma human skin mel Cher

Example 1. Cultivation of cell lines mel Cher

Tumor tissue obtained by surgery with removal of melanoma metastases were divided mechanically into fragments the size of 2-3 mm 3in the medium RPMI-1640, then, using Cell dissociation sieve-tissue kit" (Sigma), was obtained cell suspension. The number of viable cells was determined by a standard method in the camera Goryaeva, using 0.5% solution Trypanosoma blue in PBS. In the culture flasks of 25 cm2in 5 ml of medium were seeded at 1×106cells. The cultivation temperature of 37°C. Cells were cultured in medium RPMI 1640 containing 20% fetal calf serum, 2 mm L-glutamine, 1% HEPES, penicillin (100 u/ml), streptomycin (100 μg/ml) and a complex of amino acids and vitamins (Flow Lab.) in the culture flasks (Costar). After 15 passages received steadily growing cell line.

Example 2. The formation of the PCA cell line mel Cher in short-term tests in vitro

Cells were removed with a solution of Versene was loose for 5 minutes in a clean environment RPMI, counted the number of cells in 1 ml Goryayev camera. Matrigel (+4°C) was applied onto the bottom of wells of a 24-hole flat-bottomed plates in a quantity of 100 μl on ice (to prevent crystallization of the gel)in the next 24-hole plate was placed in an incubator with 37°C for 20 minutes. Cell was added in the amount of 200 thousand per well in 10% RPMI medium. The result was evaluated after 16 and 24 hours. Cell line mel Cher has formed a network of elongated in the form of tubes of cells occupying the entire surface of the Matrigel in the LUN is E. The network has survived more than 24 hours (Figa).

Example 3. The use of cell lines mel Cher for screening of drugs aimed at inhibiting the VM in vitro and mechanisms of the process VM

Cells were removed with a solution of Versene was loose for 5 minutes in a clean environment RPMI, counted the number of cells in 1 ml Goryayev camera. Cell line Mel Cher dissipated by 12-hole die in the amount of 100 thousand to 1 hole in 1 ml of 10% RPMI medium, the plate was placed in an incubator for 24 hours. After 24 hours, the original medium was carefully removed, and the environment with the test substances in most recitations concentration was added to all wells, except the control. The plate was again placed in the incubator for 1 hour or 24 hours. Next Wednesday with the tested substances was carefully removed by pipetting, and the cells in each well was added a solution of Versene for a few minutes to unpin from plastic, the cells were loose. Matrigel (+4°C) was applied onto the bottom of wells of a 24-hole flat-bottomed plates in a quantity of 100 μl on ice (to prevent crystallization of the gel)in the next 24-hole plate was placed in an incubator with 37°C for 20 minutes. The cells with medium containing the test substance was applied to 24-hole die with ready depolimerization the Matrigel. The die was placed in the incubator. Rez is ltat compared with the positive control was assessed after 16 and 24 hours.

As a test substance can be used anticancer medicinal drugs or blockers of proteins and genes in tumor cells that may participate in the molecular mechanisms of the formation of the VM.

The substance was evaluated as blocking VM, if after 24 hours was not observed the formation of ATP. Cells can form either a short, open tubes, unlimited contact with other tubes or collected in a separate, unrelated to each other, the clusters of cells (Figb).

Example 4. The formation of the PCA cell line mel Cher in long-term tests in vitro

Incubation of cells on Matrigel. Cells were removed with a solution of Versene was loose for 5 minutes in a clean environment RPMI, counted the number of cells in 1 ml Goryayev camera. Matrigel (+4°C) was applied on the cover glass size 24×24 mm in the amount of 150 μl on ice (to prevent crystallization of the gel), in the subsequent cover glass Petri dish was placed in an incubator with 37°C for 20 minutes. Cell cultures were prepared and counted in a known manner, was applied in the amount of 500 thousand on top of the glass. After adding cells waited 20 minutes before their attachment to the matrix, then the Petri dish was added 10% RPMI medium in the amount of 12 ml, and it was placed in the incubator. Outcome was assessed at 3, 7, 1, and 21 days. Cell line melCher formed tubular structure representing the ordered structure of the cells of the three-dimensional configuration (Figure 2).

Incubation of cells on fibronectin. Fibronectin was diluted in 10 ml of sterile PBS. To prepare a working concentration of original original solution of fibronectin was diluted another 10 times and was applied at 2 ml per cover glass Petri dish with a cover glass was placed in the incubator for 30-40 minutes. Then after this time nezapominayuschiesya fibronectin was carefully removed by pipette. Cells of melanoma in the amount of 500 thousand was placed on the prepared matrix, waited 20 minutes to attach and added 12 ml of 10% RPMI medium. Outcome was assessed at 3, 7, 14 and 21 days. Cell line melCher formed tubular structure representing the ordered structure of the cells of the three-dimensional configuration.

Example 5. Molecular biological characterization of cell lines Mel Cher.

Cell line mel Cher using the methods of immunocytochemistry was investigated on different antigens.

Differencirovanie melanoma markers that define this line to melanoma, was investigated using monoclonal antibodies against CD63, NPS, MelanA, Tyrosinase, HMW (table 1).

The table is a 1
Expression differencirovannyh antigens on the cell line mel Cher.
Differencirovanie antigens
CD63Expressed
NMWExpressed
MelanANot expressed
TyrosExpressed
HMWExpressed

The study of the expression of molecular biological markers involved in angiogenesis, using monoclonal antibodies to VEGF, VEGFR-1, VEGFR-2, VIII as ag and Laminin 5 gamma 2 (Table 2 and 3).

Table 2
Determination of expression of the primary vascular VEGF and receptors to him on the cell line mel Cher.
VEGF and its receptors
VEGF (factor vascular endothelial growth)Expressed
VEGFR1 (receptor type I)Expressed
VEGFR2 (receptor type II)Expressed

Table 3
The expression of markers of endothelial cells on the cell line mel Cher.
Markers of endothelial cell
VIII as.agExpressed
Laminin 5 gamma 2Expressed
CD34Not expressed
CD31Not expressed

Thus, the cell line melanoma human skin mel Cher has its own individual phenotype of tumor markers, implying the presence of differencirovannyh antigens (CD63, HMW, HMB45, Tyrosinase). This cell line expresses growth factors such as VEGF, VEGF receptors I, II types, markers of EC, such as VIII as.ag., Laminin 5 gamma 2.

Example 6. The formation of VMS in vivo implantation of cell lines mel Cher immunodeficient mice

For the experiment, cells of metastatic melanoma mel Cher was perebivalis immunodeficient mice in the amount of 1-5 million cells subcutaneously. The growth dynamics of previwe who's tumors were measured every 3 days, starting from the 7th day after inoculation. The size of the tumor, namely tumor volume was determined as the ratio of the width to the length and half the width. The experiment was ended on day 21, all the tumors were enclosed in paraffin blocks for further immunohistochemical study.

PAS-staining at the basal membrane revealed the presence of parallel, parallel with the intersection of the channels and arches in the tumor tissue (Figure 4). In the implanted tumors when combined staining for vascular CD34+ (dilution 1:50) followed by PAS-staining at the basal membrane were identified patterns VM.

The technical result consists in the fact that the cell line melanoma human skin mel Cher can be used as a positive model of vasculogenic mimicry.

References

1. Miller KD, Sweeney SJ, Sledge GW. The Snark is a Boojum: the continuing problem of drug resistance in the antiangiogenic era. // Annals of Oncology. - 2003. V.14 - P.20-28.

2. Stepanova E.V., Vartanyan A.A., Lichinitser MR // Vascular mimicry in malignant formations. // Molecular medicine. - 2006. No. 1.

3. Maniotis, A., Folberg R, Hess A. et al. Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. // Am. J.Pathol. - 1999. - Vol.55 - P.739-752.

4. Warso M.A., Maniotis AJ, Chen X. et al. Prognostic significance of periodic acid-schiff-positive patterns in primary cutaneous melanoma. // Clin. Cancer Res. - 2001. - Vol.7 - P.473-477.

5. Folberg, R., Hendrix, .J., and Maniotis, A.J. Vasculogenic mimicry and tumor angiogenesi. // Am. J. Pathol. - 2000. - Vol.156. - P.361-381.

6. Donovan D., Brown N.J., Bishop E.T., et al. Comparison of three in vitro human angiogenesis assays with capillaries formed in vivo. // Angiogenenesis. - 2001. - Vol.4. - P.113-121.

The use of cell lines melanoma human skin mel Cher as a positive model of vasculogenic mimicry.



 

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77 cl, 9 dwg, 3 tbl

FIELD: biotechnology, molecular biology.

SUBSTANCE: method involves transfection of cells HKB with vector pCIS25DTR comprising a selective marker and a sequence encoding protein eliciting procoagulating activity of factor VIII. Cells are selected using the selecting agent and clones with high level for expressing protein eliciting procoagulating activity of factor VIII are isolated. Invention provides preparing the protein eliciting activity of factor VIII with high yield, and strain of cells HKB with improved production under protein-free conditions also. Invention can be used for preparing the protein eliciting activity of factor VIII in industrial scale.

EFFECT: improved preparing and isolating methods.

8 cl,, 6 dwg, 1 tbl, 5 ex

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