RU2722268C1 - Biotherapy method of laboratory rats with grafted glioblastoma - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and oncology, and concerns a biotherapy method of lab rats with grafted glioblastoma. That is ensured by introducing once day for 7 days of granulocyte colony-stimulating factor 4 mg per 140–160 g of body weight and bacterial lipopolysaccharide in amount of 5 mg per kg of body weight of rat, and 4 hours after administering the last dose of bacterial lipopolysaccharide, 9 mg of interferon-γ is administered once.

EFFECT: invention allows suppressing production of TGF-β1 in a grafted glioblastome of laboratory rats and localizing a tumor center.

3 cl, 9 dwg

Description

The invention relates to medicine, more specifically to oncology, and can be used to suppress the production of transforming growth factor - betta (TGF-β1) in transplanted laboratory rat rat glioblastoma.

Known methods of therapy in oncology, when laboratory rats are administered granulocyte colony stimulating factor (hereinafter - G-CSF).

Linsenmann T, Jawork A, Westermaier T et al. Tumor growth under rhGM-CSF application in an orthotopic rodent glioma model. Oncol Lett. 2019 Jun; 17 (6): 4843-4850.

The disadvantage of this model is the emphasis on the immobilization of CD8 lymphocytes, while the introduction of G-CSF is carried out for 9 days without monitoring the effectiveness of immobilization of hematopoietic stem cells and their homing into the tumor focus.

Clavreul A, Delhaye M, Jadaud E, Menei P. Effects of syngeneic cellular vaccinations alone or in combination with GM-CSF on the weakly immunogenic F98 glioma model. J Neurooncol. 2006 Aug; 79 (1): 9-17.

The disadvantage of this model is the synchronous use of G-CSF with cancer vaccine, while the control is performed in relation to CD11b / c and their presence in the tumor is ignored.

Obi R, Tohda M, Zhao Q et al. Chotosan enhances macrophage colony-stimulating factor mRNA expression in the ischemic rat brain and C6Bu-1 glioma cells. Biol Pharm Bull. 2007 Dec; 30 (12): 2250-6.

The disadvantage of this work is the lack of emphasis on the immobilization of hematopoietic stem cells.

Tseng SH, Chen Y, Chang CJ et al. Induction of T-cell apoptosis in rats by genetically engineered glioma cells expressing granulocyte-macrophage colony-stimulating factor and B7.1. Clin Cancer Res. 2005 Feb 15; 11 (4): 1639-49.

In this work, there is no emphasis on the immobilization of bone marrow stem cells; moreover, tumors modified by genetic engineering are used.

The disadvantages of the known analogues is the fact that stimulation of G-CSF does not provide the formation of a vector for the transformation of hematopoietic stem cells into microglia cells, however, under the influence of anti-inflammatory cytokines produced by tumor cells (for example, transforming growth factor beta (TGF-β1) and interleukin- 10 (IL-10)), they will acquire the M2 phenotype and will synthesize wnt proteins that activate tumor stem cells.

The problem to which the invention is directed is the development of effective biotherapy for laboratory rats with transplanted glioblastoma.

The technical result achieved by solving the problem is expressed in the suppression of TGF-β1 production in transplanted laboratory rat rat glioblastoma and the localization of the tumor focus.

The problem is solved in that the method of biotherapy of laboratory rats with transplanted glioblastoma, in which a granulocyte colony stimulating factor is introduced, is characterized in that once a day for 7 days a granulocyte colony stimulating factor is introduced in an amount of 4 mg per 140-160 g of body weight and bacterial lipopolysaccharide in the amount of 5 mg per kg of body weight, and 4 hours after the last dose of the bacterial lipopolysaccharide is administered, 9 mg of interferon-γ is administered once.

In addition, granulocyte colony stimulating factor and bacterial lipopolysaccharide are administered simultaneously.

In addition, granulocyte colony stimulating factor and bacterial lipopolysaccharide are administered sequentially.

A comparative analysis of the set of essential features of the proposed technical solution and the set of essential features of the prototype and analogues indicates its compliance with the criterion of "novelty".

The distinctive features of the claims solve the following functional tasks.

The sign “enter granulocyte colony stimulating factor” ensures the recruitment of cells, including mononuclear CD45 + red bone marrow cells, into the systemic circulation.

The signs “enter granulocyte colony stimulating factor once a day for 7 days in the amount of 4 mg per 140-160 g of weight” describe the dose and mode of application of G-CSF.

The signs “administer bacterial lipopolysaccharide” and “interferon-γ are administered” trigger a systemic inflammatory response in which microglial cells polarize into M1-activated macrophages and produce tumor necrosis factor alpha (TNFα), interleukin 1 (IL-1) and others pro-inflammatory factors.

The signs “administer bacterial lipopolysaccharide once a day for 7 days in the amount of 5 mg per kg of body weight” describe the dose and mode of application of the bacterial lipopolysaccharide.

Signs “4 hours after the last dose of bacterial lipopolysaccharide is administered, 9 mg of interferon-γ are given once” describe the dose and mode of application of interferon-γ.

The features of the dependent claims describe the sequence of application of granulocyte colony stimulating factor and bacterial lipopolysaccharide.

In FIG. 1 shows glioma tissue on day 14 for the control group (group I):

a - neoplastic cells;

b - blood vessels;

in - dense congolomerates;

g - PCNA + cells;

d, e - reaction of astrocytic glia,

W, h - the reaction of microglia.

In FIG. 2 shows glioma tissue on day 28 for the control group (group I):

a, b - zones of necrosis;

c, d - PCNA + cells;

d - GFAP + cells;

e - Iba + microglia cells;

g, h - the invasive edge of the tumor.

In FIG. 3 shows the morphochemical characteristic of glioma for the control group (group I) on the 14th and 28th day:

a - the ratio of the total area of necrosis to the total area of the drug;

b - the number of PCNA + cells per unit area of the drug;

in - the number of microglia cells;

g is the content of TGF-β1.

In FIG. 4 shows the number of HSCs (SC) in the structure of the pool of leukocytes (Lei):

a - for group II;

b - for group III;

in - for group IV;

g - the ratio of HSC and white blood cells before and after exposure to G-CSF;

In FIG. 5 shows the morphometric characteristic of glioma on the 28th day for different groups:

a is the number of PCNA + cells;

b - the number of microglia cells;

c - pro-inflammatory population of microglia (CD86);

g - anti-inflammatory population of microglia (CD206).

In FIG. 6 shows glioma tissue on day 28:

a - for group I in the center;

b - for group I on the periphery;

in - for group IV in the center;

g - for group IV on the periphery.

In FIG. 7 shows the immunohistochemical characterization of glioma for group IV:

a - pro-inflammatory CD86 microglia cells in the center;

b - pro-inflammatory CD86 microglia cells in the periphery;

c - anti-inflammatory CD206 microglia cells in the center;

g - anti-inflammatory CD206 microglia cells in the periphery.

In FIG. 8 shows the content of cytokines for different groups:

a - TGF-β1;

b - IL-10;

c - TNFα;

g - IL-1.

In FIG. 9 shows the Kaplan-Meier rat survival curve.

The inventive method is based on the following principles.

The introduction of granulocyte colony stimulating factor leads to the recruitment of mononuclear CD45 + red bone marrow cells, a significant part of which are hematopoietic stem cells (HSCs), into the systemic circulation.

HSCs, when introduced into the bloodstream of animals with transplanted glioma, migrate to the tumor site, penetrate into the neoplastic tissue, accumulate in the zones of invasion and necrosis and transform into cells identical in a number of leading immunohistochemical markers to their own brain microglia, which is accompanied by an increase in the processes of microglial proliferation and infiltration in tumor tissue by microglia cells.

In addition, in addition to HSCs, when G-CSF is introduced into the bloodstream of animals, CD45 + (CD8 +) T-lymphocytes / killers are recruited, which recognize and attack cells that do not have antigens of the main histocompatibility complex of the first type.

Migrating into a tumor, T-lymphocytes / killers trigger the “transplant against tumor” reaction and, on the other hand, they themselves are attacked as foreign agents by the patient’s own T-lymphocytes. As a result, a permanent pro-inflammatory modification of the tumor site occurs, without the introduction of bacterial lipopolysaccharide and interferon-γ.

Carrying out pro-inflammatory therapy, in which bacterial lipopolysaccharide and interferon-γ are introduced, sets the vector of HSC transformation into microglia cells and provides a local increase in the inflammatory response.

A systemic inflammatory reaction is triggered, under which microglia cells are polarized into M1-activated macrophages and produce tumor necrosis factor alpha (TNFα), interleukin 1 (IL-1) and other pro-inflammatory factors. As you know, pro-inflammatory factors reduce the content of wnt proteins in the tumor (a key pathway for the proliferation of cancer cells) and inhibit the synthesis of a transforming growth factor - betta (TGF-β1), which triggers invasion processes.

Pro-inflammatory therapy with G-CSF stimulation causes polarization of macroglia / macrophage populations, which is accompanied by an increase in antigen presentation processes, a decrease in the production of TGF-β1 and IL-10, and an increase in the synthesis of pro-inflammatory cytokines TNFα and IL-1 in the tumor focus and the adjacent brain substance , remodeling of the tumor matrix and increased survival of experimental animals.

Thus, the controlled introduction of G-CSF into the systemic circulation and the recruitment of own mononuclear CD45 + cells from the laboratory rat into the blood provides constant “pumping” of M1-activated macrophages producing pro-inflammatory cytokines that regulate cancer cells (including cancer stem cells) into the tumor.

It should be noted that by the 28th day of observation, the tumor becomes denser and more visible with the formation of clear boundaries between the tumor tissue and the brain substance, which allows you to effectively remove it surgically.

To implement the method used the following starting materials.

1. Experimental animals . This work on animals has been agreed and approved by the Ethics Committee of the FEFU School of Biomedicine (Minutes No. 1 and 2 of 03/05/2019). The maintenance and care of animals was carried out in accordance with the requirements of GLP standards and the Helsinki Declaration on the Humane Treatment of Animals.

We used 150 male Wistar rats with a body weight of 150 ± 10 g and an age of 12-14 weeks. Rats were obtained from the nursery of the research and production unit of the Institute of Bioorganic Chemistry branch. Academicians M.M. Shemyakina and Yu.A. Ovchinnikova (Pushchino, Moscow Region, Russia).

2. Tumor cells .We used low-grade C6 glioma cells (cat.no ATCC® CCL-107 ™). Cells were cultured to 80% confluency and used in the experiment after the third passage from receipt from the manufacturer. The cell line was tested for mycoplasma contamination using the Universal Mycoplasma Detection Kit (ATCC® 30-1012K ™).

3. Reagents .

GKS-F (Filgrastim, cat.no.Y0001173 Sigma-Aldrich, Saint Louis, MO, US);

bacterial lipopolysaccharide E. coli serotype O111: B4 (cat. no L2630 Sigma-Aldrich, Saint Louis, MO, US);

recombinant rat IFNγ (cat. no. I3275 Sigma-Aldrich, Saint Louis, MO, US).

The inventive method is carried out according to known technology in several stages.

1. Animals were divided into 5 groups :

I) a control group (N = 60);

II) a group of rats (N = 30) that received granulocyte colony stimulating factor (G-CSF) for the recruitment of mononuclear CD45 + bone marrow into the systemic circulation (group - G-CSF);

III) a group of rats (N = 30) who received pro-inflammatory therapy to trigger a systemic inflammatory reaction by introducing bacterial lipopolysaccharide (LPS) and interferon-γ (IFNγ) (proinflamation therapy, PT group);

IV) rats (N = 30) that received G-CSF stimulation followed by pro-inflammatory therapy (G-CSF + PT group).

2. Modeling a glial brain tumor.

Animals were anesthetized by intraperitoneal administration of 2 mg of a mixture containing zoletil 100 (Virbac, France) + rometar (Bioveta a.s. Czech) in a 1: 4 ratio. The soft tissues of the head were dissected, a trepanation hole was applied using an Ideal Micro Drill Surgical Drill (Harvard apparatus, US).

0.5 × 10 ⁶ tumor cells in 5 μl of sterile 0.14M NaCl solution were injected using a Hamilton syringe (600 Series, Hamilton, US) at a speed of 1 μl / min into the caudoputamen region using the SR-5R stereotactic apparatus for rats -HT (Narishige, Japan) at the coordinates of the rat brain atlas (Paxinos & Watson, 2007): Ap-1; L-3.0; V-4.5, TBS-2.4 mm.

Tumor verification was performed 10 days later by magnetic resonance imaging using a Bruker’s PharmaScan® unit (Massachusetts, US).

3. Recruitment of mononuclear CD45 + cells.

Animals once a day for 7 days were injected subcutaneously with granulocyte colony stimulating factor in the amount of 4 mg per 140-160 g of body weight.

The immobilization of mononuclear CD45 + cells and HSCs was confirmed by testing blood samples of experimental animals on a Navios ™ flow cytometer (Beckman Coulter, US).

Antibodies against surface CD45 + rat antigen (clone OX-1 conjugated to APC / Cy7, cat.no. 202216, Biolegend Inc., US) were used in the work. Processing of cytofluorimetric data was carried out using the programs Navios Software v.1.2 and Kaluza ™ v.1.2 (Beckman Coulter, US).

4. Pro-inflammatory therapy.

To initiate a systemic inflammatory reaction once a day for 7 days at the same time or after administration of a granulocyte colony-stimulating factor, bacterial lipopolysaccharide was injected intraperitoneally in an amount of 5 mg per kg of body weight (dissolved in 0.5 ml of sterile physiological saline).

And 4 hours after the last dose of bacterial lipopolysaccharide was administered, animals were intraperitoneally injected with 9 mg of recombinant rat interferon-γ (dissolved in 0.5 ml of sterile physiological saline).

5. Morphological study.

Rat brain tissue was obtained after decapitation of animals. The material was divided into two parts: one part of the material was used for morphological and immunohistochemical studies, the second part of the material was used to assess the content of cytokines in the brain tissue according to the procedure described below.

Material for morphological and immunohistochemical studies was fixed (4% PFA) for 12 hours, followed by washing with PBS. For morphological analysis, paraffin sections of the brain with a thickness of 7 μm were dewaxed and stained with hematoxylin-eosin according to standard methods. All reagents used are manufactured by Sigma-Aldrich (Saint Louis, MO, US).

6. Immunohistochemical study.

The study was performed according to standard methods. Primary antibodies against microglia / macrophage-specific iba-1 protein (ab107159), proliferating cells of the nuclear antigen PCNA (ab 29) and membrane protein antigen-presenting cells CD86 (ab213044) and mannose receptor CD206 (ab64693) were used in accordance with the manufacturer's instructions - Abcam company (USA). Secondary antibodies labeled with horseradish peroxidase PI-1000 (anti-rabbit); PI-2000 (anti-mouse) was used in accordance with the manufacturer's instructions - Vector Laboratories. For the immunoperoxidase reaction, the NovaREDsubstratekit chromogen (Vector Laboratories; SK-4800) was used. Slices were thoroughly washed with 0.1 M PBS, dehydrated and baled, studied using an AxioScope A1 light microscope (Carl Zeiss, Germany).

7. Enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assay (ELISA) of brain tissue for the content of key cytokines IL-1 (ab100768), IL-10 (ab100765), TNF-α (ab100747) and TGF-β1 (ab119558) was performed using commercial kits from Abcam (USA). The rat brain was removed, the site was separated for morphological and immunohistochemical studies, and the rest of the material was immediately frozen in liquid nitrogen at -70 ° C. For analysis, glioma tissue was taken with the capture of brain matter within 5 mm from the edge. Tissue homogenates were prepared using a buffer: sucrose 0.25 M, Tris-HCl 0.01 M, EDTA 0.001 M, HEPES 0.01. All reagents manufactured by Sigma-Aldrich (Saint Louis, MO, USA). Lysis buffer was added to the tissue at the rate of 5 ml per 1 mg of tissue, tissue homogenization, centrifugation, and selection of the supernatant for analysis were performed. Protein content was determined using a commercial Pierce BCA Protein Assay Kit (Thermo Scientific). The optical density was determined at a wavelength of 450 nm on a tablet spectrophotometer (BioRad xMark).

8. Statistical processing of results.

The data was processed using Graph Pad Prism 4.00 software (GraphPad Software Inc., US). According to the obtained data, the arithmetic mean and its standard deviation were calculated. The results are presented as M ± SEM. For paired samples, Student t-test was used. Differences between groups were considered significant at P <0.05. Survival analysis was performed according to Kaplan-Mayer.

9. Analysis of the results.

Patterns of TGF-β1 production in brain tissue at various stages of the tumor process.

Implantation of glioma cells led to the rapid formation of bulky tumors towering above the convexital surface of the brain in the form of a pale pink node, contrasting with the color of the brain tissue. The development of the tumor was accompanied by edema and deformation of the brain structures, smoothness of the main grooves and convolutions, compression of the ventricles of the brain and a gross deformation of the brain structures.

On the 14th day of the experiment during morphological examination, glioma tissue was represented by an accumulation of neoplastic cells (Fig. 1a) forming numerous blood vessels (Fig. 1b). In some areas of the tumor node, the glioma cell group became denser and, concentrating around the blood vessels, they formed dense conglomerates in the form of balls or "rosettes", limited to the periphery by foci of necrosis (Fig. 1c). Along the periphery of the tumor node, glioma cells without clear boundaries invaded the brain substance containing numerous solitary tumor elements and secondary conglomerates.

The formed tumor was characterized by high proliferation rates. PCNA + cells (Fig. 1d) were evenly distributed in the center of the glioma node and the brain substance adjacent to the tumor, where they were localized in the blood vessels and formed secondary, satellite foci. The development of a tumor in the brain of rats was accompanied by an intense reaction of astrocytic glia (Fig. 1e, 1e). GFAP + cells with a large body and short processes were localized in brain tissue adjacent to the tumor adjacent to the tumor node, as well as at a certain distance from it, and were absent in the tumor tissue. In turn, dense clusters of cell elements immunoreactive with respect to microglia / macrophage-specific iba-1 protein were detected both in the center of the neoplastic node (Fig.1g) and on the periphery of the tumor (Fig.1h), as well as in brain tissue, blood vessels surrounding the tumor.

On the 28th day of the experiment, the main place in the morphological picture of glioma began to occupy large areas of necrosis (figa, 2b), containing desolate blood vessels, surrounded by clusters of a few cellular elements. The intensity of proliferation processes by this time sharply decreased, while PCNA + cells tightly grouped along the boundaries of the tumor and were detected in areas of invasion of dystrophicly altered brain matter (Figs. 2c, 2d). The localization of elements of astrocytic glia did not reveal significant differences in comparison with the 14th day of observations. GFAP + cells were localized along the edge of the tumor site (fig.2d) had a star shape and numerous processes that are closely in contact with each other.

The number of microglia cells in the tumor tissue sharply decreased by the 28th day of observation. Iba + cells in the center of the glioma node were distributed in the form of focal clusters or surrounded zones of necrosis forming a kind of “pseudo-palisade” structure (Fig. 2e). In the region of the invasive edge of the tumor, the group of microglia cells significantly compacted, forming a rim between the tumor tissue and the brain substance (Figs. 2g, 2h), which is dotted with tumor cells. The decrease in the proliferation rate and the number of microglia cells against the background of intensive necrosis processes in the center of the tumor node by the 28th day of the experiment was accompanied by an increase in the content of TGF-β1 in the brain tissue (Fig. 3d). In this regard, the 28th day was chosen as a control point to continue the experiment.

Recruitment of autologous mononuclear CD45 + bone marrow cells into the bloodstream of animals with inoculated C6 glioma.

Administration of G-CSF to animals with C6 glioma was accompanied by an increase in the proportion of CD45 + cells in the total number of lymphocytes recruited into the bloodstream. Moreover, among mononuclear bone marrow cells immunopositive for the membrane antigen CD45, the number of HSCs by 7 days of the experiment increased by more than 19 times (Fig. 4).

The effect of a systemic inflammatory response on the production of TGF-β1 in a tumor site during the immobilization of mononuclear CD45 + bone marrow cells into the bloodstream of rats with C6 glioma.

On the 28th day of the experiment, in animals of groups II (G-CSF) and IV (G-CSF + PT), there was a significant (Fig. 5a) increase in the number of PCNA + cells in the tumor tissue compared with the control (group I) and rat group receiving only PT (group III). However, in contrast to the control (Figs. 2c, 2d), numerous PCNA + cells in group IV rats (G-CSF + PT) were scattered throughout the tissue of the tumor node (Fig. 6c) and tightly grouped in the zone of invasion of neoplastic cells into the brain substance (Fig.6g).

Enhanced proliferation in tumor tissue in animals of groups II (G-CSF) and IV (G-CSF + PT) was combined with an increase in the area of staining of the glioma site with antibodies against the microglia / macrophage protein iba-1 (Fig.5b). In contrast to the control (Fig. 2f), cells of a round and amoebiform shape were localized both in the center of the tumor node (Fig. 6c) and in the region of the invasive edge of the tumor (Fig. 6d), as well as visualized in the substance of the brain adjacent to the tumor. . In group III rats that received only PT, the general architecture of the microglia reaction did not differ fundamentally from the control.

On the 28th day of the experiment, an increase in the number of Iba1 + microglia elements in the tumor tissue in rats of groups II and IV was accompanied by polarization of the microglia populations (Figs. 5c, 5d). In rats of the control group, single microglia expressing the CD86 marker were detected in glioma tissue and brain substance adjacent directly to the tumor site, indicating a low level of antigen presentation in brain tissue (Fig. 7a). In animals of groups II and III, the number of these cells increased markedly (Fig. 5c), and reached a maximum in rats of Group IV, where numerous CD86 + cellular elements of a round, star or polygonal shape were tightly grouped along the boundaries of the tumor and actively invaded neoplastic tissue (Fig. .7b).

In contrast, cells immunopositive for the mannose receptor CD206 were detected in large numbers in the tumor (Fig. 7c) and in the brain tissue adjacent to the tumor focus (Fig. 7g). Their number increased slightly in group II rats treated with G-CSF, but in animals of group III and IV their number was minimal (Fig. 5g).

Enzyme-linked immunosorbent assay revealed significant differences in the level of key pro- and anti-inflammatory cytokines in rats of experimental groups. By the 28th day of the experiment in rats of the control group, the content of anti-inflammatory cytokines TGF-β1 (figa) and IL-10 (fig.8b) in the tumor and adjacent brain tissue was maximum. In groups II (G-CSF) and III (PT), the content of these cytokines was significantly reduced and reached a minimum in rats of group IV (G-CSF + PT). A similar effect was accompanied by a sharp increase in the content of proinflammatory cytokines TNFα (Fig. 8c) and IL-1 (Fig. 8g) in animals of this group.

Comparative study of the survival of animals with glioma C6.

Animals of all experimental groups showed an increase in survival rates compared with the control group, but the best rates were recorded in rats of group IV (G-G-CSF + PT) (Fig. 9).

Summarizing.

As follows from the results of the experiment, the stereotactic implantation of C6 glioma cells into the brain of immunocompetent Wistar rats allows the formation of a glial tumor, which is characterized by invasive growth, high proliferation of cancer cells, intense angiogenesis, and early appearance of foci of necrosis, which shows it to be classified as high-grade gliomas malignancy according to the classification of Ste. Anne-Mayo System (SAMS), or Grade IV, in accordance with the criteria of the World Health Organization.

It is extremely important that this tumor in its development reproduces the basic pathophysiological mechanisms characteristic of MGB. So, despite intensive angiogenesis, already on the 14th day foci of necrosis are formed in the tumor, on the 28th day there is a progressive increase in the area of these zones of necrosis (Fig. 2a, 2b and 3a), which begin to occupy the main place in the morphological picture, and a decrease in the rate of proliferation of tumor cells, which indirectly indicates the key role of the hypoxia factor as the main regulator of morphological changes in tumor tissue.

In this case, the tumor does not die, and at the border of the tumor with the substance of the brain (Fig. 2c), there is an active invasion of neoplastic cells and the formation of satellite foci.

It is noteworthy that proliferation is maintained at the borders with unchanged brain matter (Figs. 2c, 2d), where the degree of oxygenation is higher than in the center of the tumor node. In this case, it is precisely the high level of energy-consuming proliferation of many tumor cells that, on the one hand, causes hypoxia and the formation of foci of necrosis, and on the other hand, induces the synthesis of TGF-β1 and VEGF in glioma cells. The first, simultaneously with anti-inflammatory effects, stimulates the formation of structures of the intercellular matrix, while the second factor triggers angiogenesis associated with the structures of the matrix, and thereby provokes invasion, which is confirmed by the results of morphological analysis.

Already these results of the morphological study are sufficient grounds for an in-depth analysis of the role of TGF-β1 in the course of the tumor process and the pathophysiological significance of its enhanced production in the region of the invasive edge of the tumor and the brain substance.

Claims (3)

1. The method of biotherapy of laboratory rats with transplanted glioblastoma, in which enter granulocyte colony stimulating factor, characterized in that once a day for 7 days enter granulocyte colony stimulating factor in the amount of 4 mg per 140-160 g of body weight and bacterial lipopolysaccharide in the amount of 5 mg per kg of body weight, and 4 hours after the last dose of bacterial lipopolysaccharide was administered, 9 mg of interferon-γ was administered once. 2. The method according to claim 1, characterized in that the granulocyte colony stimulating factor and bacterial lipopolysaccharide are administered simultaneously. 3. The method according to claim 1, characterized in that the granulocyte colony stimulating factor and bacterial lipopolysaccharide are administered sequentially.

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