RU2538243C1 - Method for simulating liver metastases experimentally - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves surgical subcutaneous separation of the spleen in white outbread rats. After the incision wound is healed, a tumour cell suspension of sarcoma 45 in normal saline is implanted into the spleen tissue in an amount of 0.1 ml at a dilution of 1×10⁶. After 2.5-3 weeks, a morphological examination of the liver follows.

EFFECT: while avoiding the effect of stress factors of the metastasis process and approaching the natural process, the method enables following the growth rates of a primary tumour node and provides simulating the liver metastasising by the most prominent haematogenic spread.

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Description

The invention relates to medicine, namely to experimental oncology, and can be used to reproduce the metastatic process in the liver in rats and in the future to develop new methods for their experimental therapy.

One of the most important tasks in the treatment of malignant tumors is the fight against metastases. Complicating the situation is that the primary tumor and metastases may have different sensitivity to certain drugs, as a result of which drugs that cope well with the primary tumor may be powerless against metastases. Metastasis is common to many types of cancer. For example, colorectal cancer, which most often metastases to the liver. Therefore, the development of experimental models of metastatic liver damage is an urgent issue.

Known methods for creating an intraorgan liver tumor in rats by puncture injection of 0.1 ml of a 20% suspension of cells of a subcutaneously transplanted hepatocarcinoma (G-27) and a model of a multicentric tumor of a liver lesion using intraportal injection of a suspension of cells of a transcutaneous subcutaneous alveolar mucous cancer (RS-1) Catalytic therapy of experimental liver tumors. / Mogirev S.V., 2005, Moscow, abstract of dissertation, candidate of medical sciences, p. 18).

The disadvantage of these methods is the need for surgical intervention at the time of introducing tumor suspension of cells either into the liver of rats or into the portal vein on the one hand, and on the other hand, the resulting tumor process in the liver cannot be considered metastasis, since there is no primary focus giving metastatic screenings.

It is known that during metastasis, the process is not limited to simple transfer and multiplication of cells in places of a new dislocation: tumor cells and metastases continue to actively change. In particular, Wu et al. (2012) concluded that only some tumor cells at the early stage of its development are capable of forming metastases. Then, in the primary tumor and in metastases, new mutations accumulate. Mutations of certain genes identified by the authors can disrupt metabolic and signaling pathways, and these pathways can serve as targets for effective therapy (Xiaochong Wu, Paul A. Northcott, Adrian Dubuc et al. Clonal selection drives genetic divergence of metastatic medulloblastoma // Nature. 2012 V.482, P.5629-5630).

Another group of researchers found that metastases branched off from the primary tumor quite early, and then the tumors and metastases evolved independently of each other, accumulating different mutations. Only 30-37% of mutations were common for tumor cells from the primary tumor and its metastases (Marco Gerlinger, Andrew J. Rowan, Stuart Horswell et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing // The New England Journal of Medicine, 2012 . V.366. P.883-892).

It is known that experimental rat colon adenocarcinoma obtained by intraperitoneal injection of the chemical carcinogen 1,2-dimethylhydrazine often metastasizes to the liver (Morphofunctional state of the thymus under conditions of colon tumor development. / Struchko G.Yu., Merkulova L.M., Moskichev E .V. Et al. // Health of Chuvashia No. 3, 2009. P.47-52; Morphology of colon cancer after application of 1,2-dimethylhydrazine dehydrochloride / Moskvichev EV, Struchko G.Yu., Merkulova L.M ., Kostrova O.Yu., Bull. Experimental biol. And honey., 2009, No. 1, p.76).

The disadvantage of this method is that reproduction of adenocarcinoma in rats is 40-65%, and liver metastases are recorded even less frequently. It is also necessary to note the toxic effect of carcinogen on the animal’s body, the long experiment time is 150 days.

The closest in technical essence to the proposed are methods for modeling liver metastases by injection into the spleen under general anesthesia: in mice - suspensions of species-specific tumor cells: Ehrlich ascites tumor (Method for modeling liver metastases. / Udintsev S.N., Fomina T.I. ., Razina TG / patent No. 4906903, 1995) or colon adenocarcinoma (AKATOL) (Study of the effectiveness of the heheoprotector Lohein in the complex treatment of experimental malignant tumors. / Malinovskaya EA, Siberian oncologists eskij zhurnal, 2003, No. 4, pp. 17-21), in rats of the BDJX strain, suspensions of tumor cells without indicating the type of tumor (Means for local use and a method for the treatment and prevention of tumor damage to the liver and peritoneum with its use. / Alexandrov V. B., Razbirin V.N., Tsaranov K, N., patent No. 223365, 2004).

These methods also have a number of significant disadvantages:

- ACATOL and Ehrlich ascites tumor are species-specific for mice;

- to recreate the process of metastasis in rats, it is necessary to use linear animals;

- not specified% reproduction of metastatic liver damage in mice and rats;

- the introduction of a suspension of tumor cells into the spleen is carried out during surgery, under anesthesia, which in themselves are stressful factors that affect the process of tumor metastasis;

- it is impossible to control the growth dynamics of the primary tumor node.

Together, these shortcomings do not allow standardizing the model, which is necessary for evaluating new methods of treatment.

The aim of the present invention is to develop a method for metastatic liver damage in white outbred rats with visual control of the growth dynamics of the primary tumor node and a large percentage of metastases in a short period of time (from the moment of transplantation of sarcoma 45 into the spleen to the appearance of a tumor process in the liver).

This goal is achieved by the fact that previously on outbred rats an operation is carried out to remove the spleen under the skin. After 2 weeks, after healing of the surgical wound, 0.1 ml of suspension of sarcoma tumor cells 45 in physiological saline in a dilution of 1 * 10 ⁶ is administered to rats intralienally. Metastatic liver damage is recorded after 17-21 days.

The invention "A method for producing liver metastases in an experiment" is new, since it is not known in the field of experimental studies in oncology about the reproduction of a metastatic process in the liver in rats.

The novelty of the invention lies in the fact that, prior to the intraliennal administration, a suspension of tumor cells of the spleen is excreted under the skin, which makes it possible to visually control the growth of the primary tumor, eliminates the influence of stress factors (surgery, anesthesia) at the time of tumor inoculation, which brings the experimental metastasis closer to natural. Also, this method involves the use of nonlinear animals, since a tumor transplanted into the spleen leads to a gradual destruction of the organ, and therefore to a violation of the immune antitumor defense. In addition, sarcoma 45 is a transplantable non-invasive tumor, which does not metastasize with standard subcutaneous transplantation; therefore, received liver metastases during intralienal administration of tumor suspensions are truly hematogenous metastases (tumor cells entering through the inferior vena cava is the most significant metastasis pathway), in contrast , for example, from adenocarcinoma, in which both hematogenous and lymphogenous metastasis are possible.

The invention "A method for producing liver metastases in an experiment" is industrially applicable, since it can be used in cancer research institutions to reproduce an experimental model of liver metastasis of a malignant tumor in order to develop experimental methods for the treatment of this pathology.

To explain the invention, drawings are provided.

Figure 1 shows the expansion of blood vessels in the microvasculature of the liver, hemo- and plasmastasis, and a violation of the beam-radar structure of the liver lobules. Hematoxylin-eosin stain. SW one hundred.

Figure 2 shows numerous mitoses of tumor cells, including atypical ones (arrows). Hematoxylin-eosin stain. SW 1000.

Figure 3 shows a tumor growing in the liver tissue (arrows), destroying it and disrupting its structure. Hematoxylin-eosin stain. SW 400.

Figure 4 shows the hematogenous dissemination of the tumor. Hematoxylin-eosin stain. SW 400.

Figure 5 shows a micropreparation of altered liver tissue. Blood vessels with plasmastasis and the bile duct are visualized - collapsed, irregular in shape, with a multilayer altered endothelium (arrows). Hematoxylin-eosin stain. SW 400.

The method of obtaining liver metastases in the experiment is as follows.

All manipulations with animals are made in boxing. Tools, dishes, hands are sanitized in the usual way. The assistant fixes the rat in a supine position, removes hair from the front surface of the body, lubricates the skin of the abdomen with 70% alcohol and gives ether anesthesia. Then the experimenter under sterile conditions performs the upper median laparotomy, gently separates the skin from the muscles, forming a “pocket”, and makes a small vertical incision in the muscle layer of the anterior abdominal wall under the left costal arch, sufficient to allow the spleen to pass through it without damage.

After the experimenter has removed the spleen from the hole in the muscles, he sutures this incision so that the spleen is fixed under the skin in his pocket. Next, the assistant sutures the surgical wound and processes the suture with a 5% alcohol solution of iodine.

Approximately 2 weeks later (possibly at a later date) after healing of the surgical wound and restoration of the animal, 0.1 ml of suspension of tumor cells of sarcoma 45 in physiological saline is diluted 1 × 10 ⁶ . To do this, observing all the aseptic conditions described above, the assistant fixes the rat on his back. The spleen is visualized as a tubercle on the rat’s stomach. Above the place of elevation, the assistant shaves off the hair, treats the skin with a 5% alcohol solution of iodine. The experimenter with his hand in a sterile glove captures the skin fold with a protruding spleen and at the place of its maximum elevation, which approximately corresponds to the center of the organ, with a 5 cm ³ syringe needle pierces the skin and spleen superficially, approximately to the middle of the organ, and introduces the tumor suspension. Then it removes the needle, and the injection site is tightly, without pressing much, pressed with a cotton swab dipped in 70% alcohol with a small addition of iodine for 1 minute to prevent leakage of the introduced suspension.

2.5-3 weeks after intralienal administration of C-45 suspension, tumor growth in the spleen began to be recorded. 45 days after the intra-splenic inoculation of C-45, some animals showed icteric staining of the skin and mucous membranes, and bilirubin in the blood increased.

The procedure was performed on 40 animals. Liver metastases appeared in 95% of rats.

After opening the rats, a primary tumor node was found in the spleen from 9.75 cm ³ to 16.2 cm ³ in size. The spleen lost the structure of the organ, its tissue was represented only by single small islands on the surface of the tumor node. As a rule, 1-2 focal tumor growths of 18.71-34.56 cm ³ in size were visualized in the liver. On the surface of the changed organ tissue were single cysts filled with bile.

Morphologically, the primary tumor node in the spleen consisted of densely spaced polymorphic spindle-shaped cells with a moderate number of mitoses, without signs of damage, surrounded by a loose connective tissue capsule, with the exception of a few degeneratively altered elements and small areas of necrosis; as well as small single islets of spleen tissue.

Microscopic examination of the liver showed significant blood supply to the organ with foci of hemorrhage. The vessels of the microvasculature were dilated and full-blooded, hemo- and plasmostasis were noted (Fig. 1).

The beam-radar structure of the hepatic lobules in large sections of the sections is broken (Fig. 1). In part of the parenchyma cells, there are dystrophic changes: vacuolization of the cytoplasm and nuclei, some nuclei are in a state of pycnosis, in others - chromatin lysis. Only a small number of hepatocytes with signs of their regeneration in the form of fission of nuclei and the cells themselves. Small leukocyte infiltrates were recorded.

The picture of destructive changes in the liver was supplemented by an abundance of homogeneous cells, most often rounded with hyperchromic large nuclei, with numerous figures of mitosis (10 or more in sight), including atypical ones (Fig. 2).

This kind of undifferentiated cells can be attributed to the immature form of sarcoma, namely squamous cell sarcoma. There was little stroma in such a tumor. The tumor grew into the liver tissue, destroying it and disrupting its structure (Fig. 3).

On some micropreparations, vessels are visible with the spread of tumor cells along their walls and with the presence of tumor conglomerates in the lumens (hematogenous dissemination of the tumor) (Fig. 4).

In addition, analyzing the micrograph of the bile ducts and adjacent liver tissue, we came to the conclusion about the morphological signs of cholestasis (swelling of the sinusoid lining, their degenerative changes, and the presence of vacuoles were observed).

The described changes appeared, most likely, due to a violation of the outflow of bile due to the germination of tumor cells, not only blood vessels, but also bile ducts.

Some bile ducts did not have a clear lumen, they were formed by several rows of oval large cells with a large elongated nucleus and basophilic cytoplasm (Fig. 5), which indicated their proliferation, apparently had an adaptive-compensatory value and was aimed at correcting bile secretion.

Thus, liver metastases, in our opinion, had histological signs of greater “tumor aggression” than the primary tumor.

The technical and economic efficiency of the “Method for obtaining liver metastases in the experiment” lies in the fact that the received metastases of a malignant tumor of the liver are morphologically confirmed squamous cell sarcoma, reproduce in 95% of cases, grow and lead to the death of animals. The method is carried out on white outbred rats, eliminates the influence of surgical intervention and general anesthesia on the reconstruction of the metastasis process, allows you to visually control the growth of the primary tumor node, suggests the spread of metastases to the liver only in the most significant way - the hematogenous route. The method is economical, affordable, provides the reproduction of tumors in a predicted amount.

Claims (1)

A method of reproducing the metastatic process in the liver of rats, including the intra-splenic injection of tumor cells, characterized in that the white outbred rats first expose the spleen under the skin, after healing of the surgical wound, a suspension of sarcoma 45 tumor cells in physiological solution in a volume of 0.1 ml is implanted in a dilution of 1 × 10 ⁶ and after 2.5-3 weeks, a morphological study of the liver is performed.

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