RU2725273C1 - Method of human pancreatic neuroendocrine tumor fragment transplantation into immunodeficiency mice pancreatic gland - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely to experimental oncology. Anesthesia is introduced in immunodeficient mice. Skin and abdominal wall tissues are incised perpendicularly to the middle line of the body in the left hypochondrium for 22 mm. Beginning of the incision is located 5 mm from the left costal arch, and its end extends 3 mm beyond the middle line into the right half of the animal's body. Gastrolienal lobe of pancreas is dissected in cranial-caudal direction at distance of 3 mm from caudal edge and 5 mm from left edge of pancreas with depth of 2 mm, length 2 mm. Fragment of human pancreatic neuroendocrine tumor 15 mmby means of ligature is fixed to wall of pancreas above dissection. Abdominal cavity is closed.EFFECT: method enables to obtain an orthotopic patient-like model of neuroendocrine pancreatic cancer, most adequately reflecting features of oncogenesis occurring in the human body, and is a valuable test system for preclinical studies of various methods of treating neuroendocrine pancreatic cancer.1 cl, 3 dwg, 1 ex

Description

The invention relates to medicine, namely to experimental oncology, and relates to a method for transplanting a fragment of a neuroendocrine tumor of a human pancreas into the pancreas of immunodeficient mice to obtain an orthotopic patient-like model having structural and physiological features that most adequately reflect the picture of the development of neuroendocrine tumors in humans.

Pancreatic neuroendocrine tumors are rare diseases. Together with neuroendocrine tumors of the gastrointestinal tract, they make up less than 2% of all neoplasms of these localizations (see Maistrenko N.A., Romashchenko P.N., Lysanyuk M.V. Diagnosis and surgical treatment of neuroendocrine tumors of the pancreas and gastrointestinal tract // Annals of surgical hepatology. - 2016.Volume 21. - No. 1. - S. 13-20).

At the same time, there are a number of difficulties in the treatment of this disease. The difficulty in making a diagnosis at the prehospital stage is increased by the period of detection of neuroendocrine pancreatic tumors (see Pinsky S.B., Beloborodoe V.A., Batoroev Yu.K., Dvornichenko V.V. Neuroendocrine tumors of the pancreas // Siberian Medical Journal. - 2013. - No. 5. - S. 12-17). A feature of this tumor is its high metastasis potential.

So gastrinomas, vipomas and glucagonomas in 80% of cases metastasize to the liver. The main problem is the high percentage of cases (65-70%) of patients who had metastases to the liver and other organs at the time of initial treatment (see Yankin A.V. Neuroendocrine tumors of the gastrointestinal tract // Practical Oncology. - 2005. - T 6. - No. 4. - S. 227-233).

Currently, in clinical practice there is no unified approach to the treatment of this disease (see Chernousov A.F., Egorov A.V., Musaev G.Kh. et al. Neuroendocrine pancreatic tumors: 30 years of experience at the Faculty of Surgery Clinic named after NN Burdenko // Surgery. - 2013. - No. 7. - S. 13-19). The key treatment is surgical. Today, the question remains of the development of more effective enzyme and cytostatic drugs, as well as the study of the etiology of this disease.

One of the key tools in studying the features of neuroendocrine pancreatic cancer and developing new therapeutic drugs is the creation of models of this disease that most fully reflect the real picture of the pathological process. An orthotopic patient-like model of neuroendocrine pancreatic cancer can meet this requirement, since its cellular composition is heterogeneous like real tumors, and the orthotopic microenvironment has an effect on oncogenesis, similar to that in which tumors develop in humans.

In addition, with orthotopic implantation of tumor material, the probability of developing metastases becomes high (see Kholodenko I.V., Doronin I.I., Kholodenko R.V. Tumor models in the study of oncological diseases // Immunology. - 2013. - No. 5. - S. 282-286 .; Yu Wang, Jiujie Cui, Liwei Wang. Patient-derived xenografts: a valuable platform for clinical and preclinical research in pancreatic cancer // Chin Clin Oncol. - 2019 .-- No. 8 (2): 17 P. 1-11; Wanglong Qiu, Gloria H. Su. Challenges and advances in mouse modeling for human pancreatic tumorigenesis and metastasis // Cancer Metastasis Rev. - 2013. - No. 32. - P. 83-107).

A known method for creating a patient-like model of pancreatic cancer is when a tumor fragment from a patient of 1-2 mm ^{3 is} implanted subcutaneously (see Linshi Zhang, Jiarong Zhou, Yingcai Yan et al. Excipient-free nanodispersion of 7-ethyl-10-hydroxycamptothecin exerts potent therapeutic effects against pancreatic cancer cell lines and patient-derived xenografts // Cancer Letters. - 2019 .-- No. 465 - P. 36-44 .; Guoyun Zhu, Davide Foletti, Xiaohui Liu et al. Targeting CLDN18.2 by CD3 Bispecific and ADC Modalities for the Treatments of Gastric and pancreatic Cancer // Scientific RepoRts. - 2019 .-- No. 9: 8420. - P. 1-11). The disadvantage of this method is the unnatural site of implantation of tumor material, suggesting the effect of a foreign microenvironment on the tumor.

A known method of orthotopic implantation of pancreatic cancer, including dissection of the skin and abdominal wall of a mouse, injection of a tumor suspension obtained from a fragment of a human pancreatic tumor under a connective tissue capsule of the pancreas (see Wanglong Qiu, Gloria N. Su. Challenges and advances in mouse modeling for human pancreatic tumorigenesis and metastasis // Cancer Metastasis Rev. - 2013. - No. 32. - P. 83-107). The advantage of this method is the orthotopic site of implantation.

However, the tumor material in the form of a suspension develops more slowly, is less capable of metastasis compared to that transplanted in the form of a fragment (see Treschalina E.M. Immunodeficient Balb / c Nude mice and modeling of various tumor growth options for preclinical studies // Russian Biotherapeutic Journal . - 2017. - T. 16. - No. 16. - S. 6-13). To improve implant survival, other authors suggest introducing the tumor suspension in a mixture with Matrigel, which prevents the elimination of cancer cells (see Matthew C. Hernandez, John R. Bergquist, Jennifer L. Leiting et al. Patient-Derived Xenografts Can Be Reliably Generated from Patient Clinical Biopsy Specimens // Journal of Gastrointestinal Surgery. - 2019. - No. 23. - P. 818-824). The disadvantage of this method is the complexity of the preparatory phase, and accordingly its duration, which may have a negative impact on the success of the engraftment of the tumor material.

There is a method of transplanting a pancreatic carcinoma cell culture COLO 357 and L3.3 first by subcutaneous injection, and then extracting the grown tumor from the body of the immunodeficient mouse and transplanting it as a fragment into the outer part of the pancreas (see Treschalina E.M. Immunodeficient mice Balb / c Nude and modeling of various tumor growth options for preclinical studies // Russian Biotherapeutic Journal. -2017. - T. 16. - No. 16. - P. 6-13). The disadvantage of this method is the implantation of a cell culture, rather than heterogeneous material, taken directly from the patient. As is known, the heterogeneous composition of the cell population of tumors most adequately reflects the real picture of oncogenesis.

A method is known for transplanting a human pancreatic tumor fragment 1-2 mm3 in size orthotically into the pancreas of an immunodeficient mouse (see Eunsung Jun, Seung-Mo Hong, Hyun Ju Yoo et al. Genetic and metabolic comparison of orthotopic and heterotopic patient-derived pancreatic-cancer xenografts to the original patient tumors // Oncotarget, Advance Publications. - 2017 .-- P. 1-15). The disadvantage of this method is the small size of the implant, which can have a significant impact on its engraftment.

The technical result of the present invention is the development of a method for transplanting a fragment of a neuroendocrine tumor of a human pancreas into the pancreas of immunodeficient mice, which allows to obtain an orthotopic patient-like model of neuroendocrine pancreatic cancer that most accurately reflects the characteristics of this disease in humans.

The technical result of the invention is achieved by the fact that the incision of the skin and tissues of the abdominal wall is perpendicular to the midline of the trunk in the region of the left hypochondrium for 22 mm, the beginning of the incision is 5 mm from the left costal arch, and its end extends 3 mm beyond the middle line to the right half of the animal’s body, cut the gastro-splenic pancreatic lobe in the cranial-caudal direction at a distance of 3 mm from the caudal edge and 5 mm from the left edge of the pancreas 2 mm deep, 2 mm long, a fragment of a neuroendocrine tumor of the human pancreas with a volume of 15 mm3 help ligatures are fixed to the wall of the pancreas over the dissection, the abdominal cavity is sutured. The method is as follows.

A fragment of neuroendocrine pancreatic cancer after excision is immediately placed in Hanks's environment. Just before transplantation, 15 mm3 pieces are cut out. The time from the moment of resection of the tumor material in humans to implantation of an immunodeficient mouse in the pancreas should not exceed 20 minutes. The recipient of the tumor material is immunodeficient Balb / c Nude mice. Xylazine at a concentration of 20 mg / ml is used as premedication. Animals were anesthetized with zoetil at a concentration of 22.57 mg / ml.

To provide access to the gastro-splenic lobe of the pancreas, the skin and tissues of the abdominal wall are cut perpendicular to the midline of the trunk in the region of the left hypochondrium for 22 mm. The incision begins 5 mm from the left costal arch, and its end extends 3 mm beyond the midline into the right half of the body. Next, dissect the gastro-splenic lobe of the pancreas in the cranial-caudal direction at a distance of 3 mm from the caudal edge and 5 mm from the left edge of the pancreas 2 mm deep, 2 mm long. A fragment of a human pancreatic neuroendocrine tumor with a volume of 15 mm3 is fixed using a ligature to the pancreas wall above a dissection. After transplantation of the tumor material, the abdominal cavity is sutured.

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

Figure 1. shows the fixation of a fragment of a neuroendocrine tumor of a pancreas of a person to the wall of the gastro-splenic lobe of the pancreas of an immunodeficient mouse Balb / c Nude.

Figure 2. depicts a fragment of a neuroendocrine tumor of a human pancreas, fixed on the pancreas wall of an immunodeficient mouse Balb / c Nude.

Figure 3. depicts an immunodeficient Balb / c Nude mouse after orthotopic transplantation of a fragment of a human pancreatic neuroendocrine tumor.

This method transplanted a fragment of a neuroendocrine tumor of a human pancreas into the pancreas of 20 immunodeficient Balb / c Nude mice.

Example.

Using this method, a female Balb / c Nude immunodeficient mouse, 6 weeks old weighing 21.5 g, was operated on. The transplant was a 15 mm3 neuroendocrine pancreatic cancer fragment, which, after isolation from the human body, was placed in Hanks medium. The time from resection of the tumor material in humans to implantation of an immunodeficient mouse in the pancreas was 13 minutes. Premediation was performed using xylazine at a concentration of 20 mg / ml. Anesthesia of animals was carried out using zoletil concentration of 22.57 mg / ml.

To provide access to the gastro-splenic pancreatic lobe of an immunodeficient mouse, skin and abdominal wall tissues were cut perpendicular to the midline of the trunk in the left hypochondrium for 22 mm. The section began 5 mm from the left costal arch, and its end went 3 mm beyond the midline into the right half of the body. After this, the gastro-splenic lobe of the pancreas was cut in the cranial-caudal direction at a distance of 3 mm from the caudal edge and 5 mm from the left edge of the pancreas with a depth of 2 mm and a length of 2 mm. A fragment of a human pancreatic neuroendocrine tumor with a volume of 15 mm3 was fixed using a ligature to the pancreas wall above a dissection. The abdominal cavity was sutured. The mouse successfully underwent surgery, without complications.

The technical and economic efficiency of this method lies in the fact that it allows you to get an orthotopic patient-like model of neuroendocrine pancreatic cancer, which most adequately reflects the features of oncogenesis that occurs in the human body, and is a valuable test system for preclinical studies of various methods of treating pancreatic neuroendocrine cancer.

Claims (1)

A method for transplanting a fragment of a neuroendocrine tumor of a human pancreas into the pancreas of immunodeficient mice, including anesthesia of immunodeficient mice, layerwise dissection of the skin and tissues of the abdominal wall, stitching the tumor fragment to the wall of the pancreas, characterized in that the incision of the skin and tissue of the peritoneal wall of the abdominal wall in the region of the left hypochondrium for 22 mm, the beginning of the incision is located 5 mm from the left costal arch, and its end extends 3 mm beyond the midline into the right half of the animal’s body, the gastro-splenic lobe of the pancreas is cut in the cranial-caudal direction on a distance of 3 mm from the caudal edge and 5 mm from the left edge of the pancreas, 2 mm deep, 2 mm long, a fragment of a human pancreatic neuroendocrine tumor with a volume of 15 mm ^{3 is} fixed using a ligature to the pancreas wall above the dissection, and the abdominal cavity is sutured.

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