RU2818462C1 - Method for creating orthotopic model of human soft tissue sarcoma growing into bone in immunodeficient mice of balb/c nude line - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to experimental oncology, and can be used to create an orthotopic model of human soft tissue sarcoma growing into a bone in immunodeficient Balb/c Nude mice. Skin and thigh muscles are incised in layers parallel to the hip bone for 15 mm; the incision is started 20 mm below the sacral region of the back, and the incision is continued so that the incision is made on the entire femoral region of the mouse leg. Muscle straining the fascia lata is incised at the same distance by moving the subcutaneous muscle of the tibia with the surgical forceps, and with the help of a surgical ball-shaped bur with diameter of 2 mm below 1 mm from the third trochanter and 5 mm above from the popliteal surface, a periosteum and an upper layer of bone tissue are injured at length of 1.5 mm and depth of 1 mm on a femoral body. 4 mm × 1 mm × 1 mm human soft tissue sarcoma tumour is placed over the defect. One ligature knot is fixed in the centre, the biceps muscle of thigh is sutured, the postoperative wound is closed with a continuous blanket suture.

EFFECT: method provides the most accurate display of oncogenesis features and is a necessary test system for preclinical studies of various methods of treating soft tissue sarcoma.

1 cl, 5 dwg

Description

The invention relates to medicine, namely to experimental oncology, and can be used to create an orthotopic model of human soft tissue sarcoma growing into bone in immunodeficient mice of the Balb/c Nude line.

Sarcoma is a malignant neoplasm that is formed from various types of connective tissue. They are divided into 2 large groups - soft tissue sarcomas and bone sarcomas. Soft tissue sarcomas (STS) are relatively rare malignancies, accounting for 1% of tumors in adults and 7–15% in children. This is a heterogeneous group of tumors of mesenchymal origin that can occur anywhere in the body, with the extremities of the body being the most common primary location, accounting for 60% (see Fedenko A.A., Tararykova A.A. The role of trabectedin in the treatment of sarcomas soft tissue: a review of current evidence. Sarcomas of bone, soft tissue and skin tumors 2021;13(3):16-26. ). According to the current WHO classification, there are currently more than 50 different subtypes of STS, and the most common aggressive tumors are liposarcoma, leiomyosarcoma and undifferentiated pleomorphic sarcoma (see Poon E., Quek R. Soft tissue sarcoma in Asia //Chin Clin Oncol. – 2018. – T. 7. – No. 4. – P. 46). The most common bone sarcomas are Ewing sarcomas or osteosarcomas (OS). A common feature of these cancers is the extremely poor survival of patients with metastatic disease or relapse (see Cillo AR, Mukherjee E, Bailey NG, Onkar S, Daley J, Salgado C, Li X, Liu D, Ranganathan S, Burgess M, Sembrat J , Weiss K, Watters R, Bruno TC, Vignali DAA, Bailey KM. Ewing Sarcoma and Osteosarcoma Have Distinct Immune Signatures and Intercellular Communication Networks. Clin Cancer Res. 2022 Nov 14;28(22):4968-4982. 1078-0432.CCR-22-1471. PMID: 36074145; PMCID: PMC9669190).

Thus, there is a need to study the characteristics of sarcomas to better understand the course of the disease. Animal models are used to study the biology of this disease, which is an important part of fundamental and translational research in oncology. The main task of such models is to reproduce the course of human diseases. At present, sarcomas have not been fully studied, so there remains a need to create experimental models that reflect a more accurate picture of the development of the disease.

There is a known method for creating a model of SMT in BALB/c mice by subcutaneous injection of mouse soft tissue sarcoma cells CCRF S-180II into the back of mice in the amount of 2-4×105 cells in a volume of 0.2 ml of nutrient medium (see Kasama F, Tsuchie H , Nagasawa H, Hongo M, Kasukawa Y, Nozaka K, Kudo D, Shoji R, Igarashi S, Harata S, Okamoto K, Oya K, Miyakoshi N. Effects of Soft Tissue Sarcoma and Doxorubicin on Bone Metabolism in Vivo. 2023 Jul-Aug;37(4):1532-1539. doi: 10.21873/invivo.13238. PMID: 37369484; The advantage of this method is the production of a large number of tumor-carrying animals. The disadvantage of the method is the transplantation site and tumor cells, which are murine and not human, which may not provide an informative characteristic of the tumor for further clinical studies.

There is a known method for subcutaneous transplantation of a human osteosarcoma tumor fragment into Balb/c Nude mice (see Wu N. F. et al. The first mouse model of primary osteosarcoma of the breast //in vivo. – 2021. – T. 35. – No. 4. – S. 1979-1983). A surgical specimen measuring approximately 2 mm3 was transplanted using the Hozumi method, namely, a 1 cm long skin incision was made on the dorsal side of the mouse body and a pocket was created by separating the skin from the dermis, then a fragment of the primary tumor was placed along with surrounding normal tissue, after which the wound was sutured . The advantage of this method is the high percentage of engrafted tumor, which was 90.2%, that is, out of 51 mice, growth of the tumor node was observed in 46 mice. However, the disadvantage of the method is the unnatural site of implantation of the tumor material, suggesting the influence of a foreign microenvironment on the tumor.

There is a known method for creating orthotopic transplantation of human osteosarcoma into the tibia of Balb/c Nude mice (see Inoue M. et al. Trabectedin suppresses osteosarcoma pulmonary metastasis in a mouse tumor xenograft model //Journal of Orthopedic Research®. - 2022. - Vol. 40. – No. 4. – P. 945-953). To achieve this goal, an 8 mm skin incision was made on either the right or left thigh of the mice, then the paw was flexed at the knee joint in order to expose the quadriceps and gastrocnemius muscles through the incision. The muscles were also cut. A 5 mm blade was then used to puncture the proximal tibia. After the puncture was made, the blade was inserted 2.5 mm into the bone and rotated several times to create a hole in the bone. Thus, a hole with a diameter of 1 mm was formed and then a tumor fragment of the size corresponding to the formed hole was placed. The last stage was layer-by-layer suturing of the surgical wound. The advantage of the method is that it reflects a more accurate clinical picture of the tumor model, simulating the disease of a person with OS. However, the disadvantages are the labor-intensive transplantation process, the small volume of the tumor fragment, which can reduce the percentage of its engraftment and, as a result, the growth of the tumor node, and the difficulty of determining the volume of tumor growth.

There is a known method for creating orthotopic transplantation of human osteosarcoma into Balb/c Nude mice (see Blattmann C. et al. Establishment of a patient-derived orthotopic osteosarcoma mouse model //Journal of translational medicine. – 2015. – T. 13. – P. 1-10.). The operation was performed under general anesthesia using isoflurane inhalation. Tumor samples taken from the patient were divided into pieces measuring 1×1×1 mm ³ . Then an incision was made in the right tibia of the mouse in the medial part, then by drilling with a drill with a diameter of 0.5 mm, a bone tissue defect was created and a tumor fragment was inserted, which was in contact with the bone marrow. After this, the surgical wound was sutured. The advantage is the success rate, which was 92%, and the implantation site is natural for the tumor material. But the method has a significant drawback. The tumor fragment was not fixed, which may change the location of the tumor fragment during suturing of the surgical wound; also, the size of the tumor pieces is small and, thus, can have a significant impact on the engraftment of the material.

The technical result of the present invention is the development of a method that makes it possible to create an orthotopic model of human soft tissue sarcoma growing into bone in Balb/c Nude mice.

The technical result is achieved by making an incision in the skin and muscles of the thigh layer by layer parallel to the hip bone over a length of 15 mm, the beginning of the incision is made 20 mm below the sacral area of the back, ending on the stationary area of the paw , so that the incision is on the entire femoral area of the mouse's paw, then an incision is made in the tensor fascia lata muscle at the same distance, moving the subcutaneous muscle of the tibia with surgical tweezers, and using a surgical spherical bur with a diameter of 2 mm below 1 mm from the third trochanter and 5 mm above from the popliteal surface, the periosteum and the upper layer of the bone are damaged tissue 1.5 mm long and 1 mm deep on the body of the femur, a fragment of a human soft tissue sarcoma tumor measuring 4 × 1 × 1 mm ³ is placed on top of the defect, secured with a single ligature in the center, the biceps femoris muscle is sutured, the postoperative wound is sutured with a continuous twist seam

The method is carried out as follows.

The experiment was carried out on Balb/c Nude mice. After excision of the tumor, its fragment is placed in Hanks' medium. Before transplantation, a fragment measuring 25 mm ³ is cut out and divided into equal pieces for transplantation into each animal. The time from the moment of resection of tumor material in humans to implantation into the body of the femur of a mouse should not exceed 15 minutes. To perform the surgical procedure, animals are put under anesthesia. For premedication, the drug xylazine is used at a concentration of 20 mg/kg. Mice are anesthetized with zoletil at a concentration of 50 mg/kg (see Patent for invention RU No. 2712916, published 02/03/2020, Bulletin No. 4).

To ensure transplantation of a fragment of a human SMT tumor into the diaphyseal region of the bone, an incision is made in the skin and muscles of the thigh in layers parallel to the hip bone over a length of 15 mm. The incision begins 20 mm below the sacral region of the back, and continues so that the incision is across the entire femoral region of the mouse's paw. Next, an incision is made into the muscle that strains the fascia lata at the same distance. By moving the subcutaneous muscle of the tibia with surgical tweezers and using a surgical spherical bur with a diameter of 2 mm below 1 mm from the third trochanter and 5 mm above from the popliteal surface, damage is carried out to the periosteum and the upper layer of bone tissue 5 mm long and 1 mm deep on the body of the femur , a fragment of a human SMT tumor measuring 4 mm by 1 mm by 1 mm is placed on top of the defect, secured with one ligature in the center, the biceps femoris muscle is sutured, and the postoperative wound is sutured with a continuous encircling suture.

The invention is illustrated by figures (1-5).

Figure 1 shows access to the femur of a Balb/c Nude mouse.

In FIG. Figure 2 shows damage to the upper layer of bone tissue on the body of the femur of a Balb/c Nude mouse using a surgical drill.

In FIG. Figure 3 shows the transplantation of a human SMT tumor fragment onto the damaged femur of a Balb/c Nude mouse.

In FIG. Figure 4 shows the fixation of a human SMT tumor fragment on the damaged femur of a Balb/c Nude mouse.

In FIG. Figure 5 shows the final view of a fixed human SMT fragment on the damaged femur of a Balb/c Nude mouse.

Using this method, a fragment of a human SMT tumor was transplanted onto the femur into 18 Balb/c Nude mice.

Example of application of the method.

Transplantation was performed into a male Balb/c Nude mouse weighing 26 g, 10 weeks old.

After excision of the tumor, its fragment is placed in Hanks' medium. Before transplantation, a tumor fragment measuring 25 mm ³ is cut out and divided into equal pieces for transplantation into each animal. The time from the moment of resection of tumor material in humans to implantation into the body of the femur of a mouse should not exceed 15 minutes.

To perform the surgical procedure, animals are put under anesthesia. For premedication, the drug xylazine is used at a concentration of 20 mg/ml. Mice are anesthetized with zoletil at a concentration of 22.57 mg/kg.

To ensure transplantation of a human tumor fragment, a human SMT was carried out into the diaphyseal region of the bone, an incision was made in the skin and the thigh muscles were carried out in layers parallel to the hip bone for 15 mm. The beginning of the incision is made 20 mm below the sacral region of the back, and continues so that the incision is on the entire femoral region of the mouse's paw. Next, an incision is made in the muscle that strains the fascia lata at the same distance. Using surgical tweezers, moving the subcutaneous muscle of the tibia and using a surgical spherical bur with a diameter of 2 mm below 1 mm from the third trochanter and 5 mm above from the popliteal surface, the periosteum and the upper layer of bone tissue 1.5 mm long and 1 mm deep are damaged on the body femur, a fragment of a human SMT tumor measuring 4 mm by 1 mm by 1 mm is placed on top of the defect, secured with one ligature in the center, the biceps femoris muscle is sutured, and the postoperative wound is sutured with a continuous enveloping suture.

The technical and economic efficiency of this method lies in the possibility of creating an orthotopic patient-like model of human STS growing into the bone in Balb/c Nude mice for further study of the pathogenesis of the disease. This model most accurately reflects the features of oncogenesis, and is a necessary test system for preclinical studies of various methods of treating STS.

Claims (1)

A method for creating an orthotopic model of human soft tissue sarcoma growing into the bone in immunodeficient mice of the Balb/c Nude line, which consists in making an incision in the skin and muscles of the thigh in layers parallel to the hip bone for 15 mm, the beginning of the incision is made 20 mm below the sacrum area of the back, and continue so that the incision is on the entire femoral region of the mouse's paw, then make an incision in the tensor fascia lata muscle at the same distance, moving the subcutaneous muscle of the tibia with surgical tweezers, and using a surgical spherical bur with a diameter of 2 mm lower 1 mm from the third trochanter and 5 mm above the popliteal surface, the periosteum and the upper layer of bone tissue 1.5 mm long and 1 mm deep are damaged on the body of the femur; a fragment of a human soft tissue sarcoma tumor measuring 4 mm × 1 mm × is placed on top of the defect. 1 mm, secured with one ligature knot in the center, sutured the biceps femoris muscle, and sutured the postoperative wound with a continuous encircling suture.