RU2753144C1 - Method of transplantation of a fragment of a human colon tumor into the descending colon of immunodeficient mice - Google Patents

Abstract

FIELD: medicine, experimental oncology.

SUBSTANCE: invention relates to medicine, namely experimental oncology. Skin, muscles and tissues of the abdominal wall of the lumbosacral area of the back are scarified along the axis of body 3 mm away from the backbone. The 10-mm insection starts 7 mm away from the tailhead. As the operating wound is expanded using surgical forceps, the colon is being visualized. At 5 mm from the transition of the descending colon into the back gut, a 3-mm lengthwise section of the seromuscular layer of the colon is made. Using a suture, a 27-mm³ fragment of a human colon tumor is fixed above the section made earlier, the abdominal space wall and the mouse’s skin are sutured.

EFFECT: the method makes it possible to create an orthotopic patient-like model of descending colon cancer most accurately reproducing the characteristics of human descending colon tumor and necessary to carry out pre-clinical studies of the mechanisms of oncogenesis and new therapeutic agents and methods.

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Description

The invention relates to medicine, namely to experimental oncology, and concerns a method for transplanting a fragment of a human colon tumor into the descending colon of immunodeficient mice to obtain an orthotopic patient-like model that most accurately reflects the molecular-biochemical, cytological and histological features of carcinogenesis occurring in the human body ...

Colon cancer is currently one of the most common forms of malignant neoplasms in all countries. Every year, about 1 million new cases of colon cancer are detected in the world (Imyanitov E.N. Clinical and molecular aspects of colorectal cancer: etiopathogenesis, prevention, individualization of treatment // Practical Oncology. - 2005. - V. 6, No. 2. . 65-70). Moreover, in recent decades, there has been a tendency to increase the number of patients with this diagnosis (Helm JF, Sandler RS Colorectal cancer screening // Med. Clin. North. Am. - 1999. - Vol. 83. - P. 1403-1422; Jean Faivre Screening for colorectal cancer: who, when, haw // 3 International conference, June 7-9, 2001, Dublin, Ireland. - P. 13.). According to the WHO, colon cancer takes 3-4 place in the structure of cancer incidence. This pathology accounts for 15% of all initially diagnosed tumors of all localizations. Moreover, about 6% of cases are associated with the hereditary nature of the disease. (Bazin I.S. Colon cancer - the state of the problem // Russian medical journal. - 2003. - T. 11. - No. 11. - Access mode: http://www.rmj.ru/main.htm/rmj /t11/n11/674.htm.).

Every year about 100 thousand people die from colorectal cancer (Axel E.M., Barulina N.M. Pp. 40-46; Khanevich MD Colorectal cancer. The choice of surgical treatment for colonic obstruction. - SPb .: Agraf +, 2008. - 136 p.). The maximum mortality is noted in the Czech Republic, Hungary and New Zealand (Hanevich MD Colorectal cancer. Choice of surgical treatment for colonic obstruction. - SPb .: Agraf +, 2008. - 136 p.). Five-year survival in Europe ranges from 50% (in Switzerland, Finland, the Netherlands, Spain) to 30-39% (in Italy, Denmark, Great Britain, Germany and France) (Myrhoy T. Hereditary nonpolyposis colorectal cancer: clinical features and survival // Progr. Colorect. Cane. - 1998. - Vol. 2. - P. 1-12; Longo WE Risk factors for morbidity and mortality after colectomy for colon cancer // Dis. Colon. Rectum. - 2000. - Vol. 43. - P. 83-91). It is generally accepted that colon tumors are a more typical disease for industrialized countries, which is associated with high consumption of animal products, as well as excessive nutrition in combination with low physical activity (Boyle P., Leon ME Epidemiology of colorectal cancer // Brit. Med Bull. - 2002. - Vol. 64. - P. 1-25; Imyanitov EH Clinical and molecular aspects of colorectal cancer: etiopathogenesis, prevention, individualization of treatment // Practical Oncology. - 2005. - V. 6, No. 2. - S. 65-70).

A big problem is the high mortality rate: for 100 newly diagnosed patients, there are more than 70 deaths, of which in the 1st year from the moment of diagnosis - about 40%. This is due to the high percentage of diagnostics (71.4%) of this disease in the late stages (III-IV stages) (Davydov M.I., Axel E.M. Malignant neoplasms in Russia and the CIS countries in 2000. - M., 2002 .-- 281 s.).

Recently, there has been significant progress in the treatment of colon cancer, due to which the average life expectancy has been increased from 4-6 months during symptomatic therapy to 20-24 months in the case of joining chemotherapy (XT) targeted drugs (Chau I., Cunningham D . Treatment in advanced colorectal cancer: what, when and how? // Br J Cancer. - 2009. - 100. - P. 1704-1719). At the same time, there is a transition from monotherapy with 5-fluorouracil to combined treatment regimens with the inclusion of a surgical component, which significantly increases life expectancy (Nordlinger V., van Cutsem E., Rougier P. et al. Does chemotherapy prior to liver resection increase the potential for cure in patients with metastatic colorectal cancer? A report from the European Colorectal Metastases Treatment Group // Eur J Cancer. - 2007. - 43 (14). - P. 2037-2045). Hence it follows that there is a justified need to develop new therapeutic agents for the treatment of this disease, an indispensable stage of which are preclinical studies. Animal models, including tumor xenografts in immunodeficient mice, are an important tool in studies of the effectiveness of new drugs.

Numerous studies have established a difference between the proximal (right) colon, which includes the cecum, the ascending and transverse colon, and the distal (left) colon, which includes the descending colon, sigmoid colon, and rectum. The proximal and distal colon show significant differences in embryonic origin, morphological and biochemical characteristics, and gene expression patterns. (Bufill J.A. Colorectal cancer: evidence for distinct genetic categories based on proximal or distal tumor location // Ann. Int. Med. - 1990. - Vol. 113. - P. 779-788). An important fact is that the proximal colon, in contrast to the distal, is relatively sensitive to therapy with fluoropyrimidines and its analogues (Iacopetta B. Are there two sides to colorectal cancer // Int. J. Cancer. - 2002. - Vol. 101. - P. 403-408; Lindblom A. Different mechanisms in the tumorigenesis of proximal and distal colon cancers // Curr. Opin. Oncol. - 2001. - Vol. 13. - P. 63-69). Therefore, in studies of new methods and means of treatment of the descending colon, it is necessary to create an adequate model of cancer of this localization.

A known method of subcutaneous transplantation of fragments of a colon tumor into the flank of immunodeficient mice (see Mitsunobu Takeda, Jun Koseki, Hidekazu Takahashi et al. Disruption of Endolysosomal RAB5 / 7 Efficiently Eliminates Colorectal Cancer Stem Cells // Cancer Res. - 2019. - 79 (7 ). - P. 1426-1437; Magdalena Cybulska, Tomasz Olesinski, Krzysztof Goryca et al. Challenges in Stratifying the Molecular Variability of Patient-Derived Colon Tumor Xenografts // BioMed Research International. - 2018. - P. 1-9; Ruidi Teng, Jingjing Zhao, Yiding Zhao et al. Chimeric Antigen Receptor-modified T Cells Repressed Solid Tumors and Their Relapse in an Established Patient-derived Colon Carcinoma Xenograft Model // J Immunother. - 2019. No. 2. - P. 33-42 ). The advantage of this model is its convenience in monitoring its development. The disadvantage of this model is the implantation of tumor material into a site with a foreign microenvironment, the influence of which contributes to the appearance of characteristics that are not characteristic of a real human tumor.

There is a known method of creating an orthotopic model of intestinal cancer, in which in the first passage from a fragment of a patient's tumor, a suspension is made in Matrigel, which is injected subcutaneously into the side of a mouse. When the tumor reaches the size required for transplantation, it is divided into fragments that are sutured to the intestine (Mukohyama J., Shimono Y., Yamashita K., Sumi Y., Mukohara T., Minami H., Kakeji Y. Effect of Xenotransplantation Site on MicroRNA Expression of Human Colon Cancer Stem Cells // Anticancer Research. - 2016. - 36. - P. 3679-3686). The disadvantage of this method is the implantation in the first passage of the tumor material under the skin of the mouse, where the processes of carcinogenesis will be influenced by a foreign microenvironment, which can lead to a significant change in the molecular-biochemical and histological characteristics of the tumor. Ultimately, the orthotopic model obtained in the second and subsequent passages cannot be considered adequate.

The technical result of the present invention is the development of a method for transplanting a fragment of a human colon tumor into the descending part of the colon of immunodeficient mice, the advantage of which is optimal access to the desired organ of the mouse from the dorsal surface of the the section of the large intestine of the mouse has to be removed into the surgical wound practically the entire intestine due to the fact that this section of the large intestine is adjacent to the dorsal surface of the abdominal cavity. This method makes it possible to obtain an adequate model that most accurately reflects the molecular-biochemical characteristics of human colon tumors.

The technical result of the invention is achieved by transplanting a fragment of a human colon tumor into the descending colon of immunodeficient mice, including anesthesia of mice, layer-by-layer dissection of the skin, muscles and tissues of the abdominal wall, characterized in that they dissect the skin, muscles and tissues of the abdominal wall lumbar - the sacral region of the back along the axis of the trunk at a distance of 3 mm from the spine, a 10 mm incision begins at a distance of 7 mm from the base of the tail, expanding the surgical wound with the help of anatomical tweezers, visualize the rectum, at a distance of 5 mm from the transition point of the descending colon into the rectum, a longitudinal dissection of the serous-muscular layer of the colon is performed over a length of 3 mm, a fragment of a human colon tumor with a volume of 27 mm ^{3 is} fixed with a ligature over the previously made incision, the abdominal wall and mouse skin are sutured in layers.

The method is carried out as follows.

A fragment of the patient's colon tumor immediately after excision is placed in a 5% solution of gentamicin in Hanks medium. Immediately before transplantation, pieces of 27 mm ³ are cut out. For successful engraftment of tumor material, it is necessary that the time from the moment of resection of tumor material in humans to implantation into the cecum of an immunodeficient mouse should not exceed 20 minutes.

Immunodeficient Balb / c Nude mice are the recipient of the tumor material.

Anesthesia of animals is carried out using xylazine with a concentration of 20 mg / ml and zoletil with a concentration of 22.57 mg / ml.

The lumbosacral region of the back of the mouse is treated with 10% povidone-iodine solution.

With the help of scissors, the skin, muscles, and also the tissues of the abdominal wall in the lumbosacral region of the back are dissected along the axis of the body at a distance of 3 mm from the spine. A 10 mm long incision begins at a distance of 7 mm from the base of the tail. With the help of anatomical tweezers, the surgical wound is expanded and the rectum is visualized. At a distance of 5 mm from the transition zone of the descending colon to the rectum, a longitudinal dissection of the serous-muscular layer of the colon is performed over a length of 3 mm. A fragment of a human colon tumor with a volume of 27 mm ^{3 is} sutured using a ligature to the mouse colon over the previously made incision. The wall of the abdominal cavity and the skin of the mouse are sutured in layers.

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

Figure 1 shows the implantation of a human tumor fragment into the descending colon of an immunodeficient Balb / c Nude mouse.

Figure 2. depicts a fixed fragment of a human tumor on the descending colon of an immunodeficient Balb / c Nude mouse.

FIG. 3. Shows the final view after transplantation of a human tumor fragment into the descending colon of an immunodeficient Balb / c Nude mouse.

In this way, a fragment of a human colon tumor was transplanted into the descending colon of 20 Balb / c Nude immunodeficient mice.

The technical and economic efficiency of this method consists in creating an orthotopic patient-like model of cancer of the descending colon, which most accurately repeats the characteristics of tumors of the descending colon in humans and is necessary for preclinical studies of the mechanisms of oncogenesis and new therapeutic agents and methods.

Example

This method was used to operate a female immunodeficient Balb / c Nude mouse, 7 weeks old, weighing 24 g. A fragment of a tumor of the descending part of the human colon was used as the transplanted material, which immediately after isolation from the human body was placed in Hanks medium. The interval between the isolation of tumor material from the donor organism before its implantation into the animal's body was 12 minutes.

Premedication was carried out with xylazine at a concentration of 20 mg / ml. Zoletila with a concentration of 22.57 mg / ml was used as the main anesthetic substance.

The sacro-lumbar region of the back of the mouse was treated with a 10% povidone-iodine solution. Next, the skin, muscles and tissues of the abdominal wall were dissected in layers in the lumbosacral region of the back along the axis of the body at a distance of 3 mm from the spine. The beginning of the cut was located 7 mm from the base of the tail. The incision length was 10 mm. Further, pushing apart the edges of the surgical wound, the rectum was visualized. After that, the serous-muscular layer of the descending section of the mouse colon was dissected with a length of 3 mm at a distance of 5 mm from the place of the transition of the descending section of the large intestine into the rectum. After that, a fragment of a human colon tumor with a volume of 27 mm ^{3 was} isolated, which was then sutured with a ligature to the mouse colon over the incision made earlier. The transplantation procedure was completed by layer-by-layer suturing of the wall of the abdominal cavity and the skin of the mouse.

The implantation procedure was successfully and without complications transferred by the recipient mouse.

Claims (1)

A method of transplanting a fragment of a human colon tumor into the descending colon of immunodeficient mice, including anesthesia of mice, layered dissection of the skin, muscles and tissues of the abdominal wall, characterized in that the skin, muscles and tissues of the abdominal wall of the lumbosacral region of the back are dissected along the trunk axis at a distance of 3 mm from the spine, an incision with a length of 10 mm begins at a distance of 7 mm from the base of the tail, expanding the surgical wound with the help of anatomical tweezers, visualize the rectum, at a distance of 5 mm from the transition of the descending colon to the rectum, perform a longitudinal dissection serous -muscular layer of the large intestine for 3 mm, with the help of a ligature, a fragment of a human colon tumor with a volume of 27 mm ^{3 is} fixed over the previously made incision, the wall of the abdominal cavity and the skin of the mouse are sutured in layers.

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