Construction method of tumor-associated meningeal metastasis model

Abstract

The invention discloses a construction method of a tumor-associated meningeal metastasis model, and particularly relates to the technical field of biology. The method comprises the following steps: treating tumor cells according to a cell passage mode and centrifugal parameters, adding a serum-free culture medium, centrifuging again, adjusting the cell density by using a serum-free fresh culture medium, and sub-packaging to obtain a tumor cell suspension; the tumor cell suspension is injected into the L4-L5 intervertebral space of the mouse. After injection, the mouse resuscitates and normally diets; the growth of the tumor is monitored through a small animal living body imaging technology, and the dissected organ is subjected to in-vitro imaging to judge the invasion range of the tumor. The mouse tumor meningeal metastasis model is constructed in a simple, repeatable and efficient manner.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a tumor-related meningeal metastasis model. Background Art

[0002] Cancer is a major disease that threatens human life. According to data published in the Journal of Clinical Oncology in 2021, there were 19.3 million new cancer cases worldwide in 2020. Melanoma usually refers to malignant melanoma, which is a highly malignant tumor derived from melanocytes. It often occurs in the skin and can also be seen in mucous membranes and internal organs. Its incidence and mortality rates are increasing worldwide. Surgical resection is the main treatment for melanoma at all stages. The treatment of advanced or metastatic melanoma is usually combined with adjuvant treatments such as immunotherapy, radiotherapy or chemotherapy. The death of melanoma patients is closely related to tumor metastasis. The most common distant metastasis site of melanoma is the brain. Among all patients with brain metastases, about 6-11% of brain metastases are derived from melanoma. Once brain metastasis occurs, the patient's prognosis is extremely poor.

[0003] Chinese patent CN202210183048.5, a method for constructing a tumor dura mater metastasis model. The method of the present invention includes: the cultivation of breast cancer cell lines (4T1), lung cancer cell lines (LLC) and human embryonic kidney cell lines (293T); lentiviral packaging; lentiviral infection; obtaining breast cancer cell lines and lung cancer cell lines that co-express luciferase and neomycin genes; and the construction of mouse models of breast cancer and lung cancer dura mater metastasis. Dura mater metastasis is a type of intracranial metastasis of malignant tumors, which can cause a variety of neurological symptoms such as brain edema and cerebrospinal fluid circulation disorders. It is to construct B16 luc+ cells, repeatedly screen the pro-meningeal metastasis cells through the cerebrospinal fluid after injection into the cerebellomedullary cistern, and construct a meningeal metastasis model by left ventricular injection after the screening is completed.

[0004] Chinese patent CN202110968953.7, a method for constructing a melanoma pia mater metastasis model. The method of the invention includes: culturing melanoma cell lines and 293T cell lines; lentiviral packaging; lentiviral infection; obtaining melanoma cell lines co-expressing luciferase and fluorescent protein; and constructing a melanoma pia mater metastasis mouse model. It is to construct 4T1 luc+ cells, inject tumor cells directly into the dura mater through craniotomy, repeatedly screen for pro-meningeal metastasis cell lines, and finally construct a meningeal metastasis model through carotid artery injection.

[0005] There are two patented technologies for the construction of animal models of mouse meningeal metastasis, both of which are registered by Fudan University. Both technologies require complex surgical operations to be performed. In particular, injection into the cerebellar medullary cistern requires not only high surgical skills but also the support of more complex and precise instruments. Both methods of constructing brain metastasis models involve screening brain-friendly cell lines after surgery, and then injecting tumor cells through the carotid artery or intracardiac injection to construct a meningeal metastasis model.

[0006] The two existing patented technologies not only further increase the difficulty of model construction and the requirements for experimental platforms and instruments (intracardiac injection requires ultrasound-guided injection, and carotid artery injection requires a stereo microscope) and experimental techniques. At the same time, because injection through the ventricle or carotid artery directly injects tumor cells into the arterial system, although cells have been screened in the early stages, the objective laws of blood circulation are still unavoidable and bone metastasis or metastasis to other internal organs is very likely to occur. Summary of the invention

[0007] To this end, the present invention provides a method for constructing a tumor-related meningeal metastasis model, so as to obtain a simple, repeatable and efficient way to construct a mouse meningeal metastasis model.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] According to the present invention, a method for constructing a tumor-related meningeal metastasis model is provided, the method comprising:

[0010] Step 1: Cell preparation

[0011] Treat the tumor cells according to the cell passage method and centrifugation parameters, add serum-free medium, centrifuge again, adjust the cell density with serum-free fresh medium, and aliquot to obtain a tumor cell suspension;

[0012] Step 2: Modeling

[0013] Tumor cell suspension was injected into the L6 lumbar vertebra of mice, and the mice were fed a normal diet after recovery. The growth of the tumor was monitored using small animal in vivo imaging technology, and the dissected organs were imaged in vitro to determine the extent of tumor invasion.

[0014] Furthermore, in step 1, the cell density is adjusted to 5×107 / ml.

[0015] Furthermore, in step 2, the method for injecting the tumor cell suspension is to use a 31G BD300 μl microsyringe to extract 10 μl of the mixed tumor cell suspension, with the needle tip bevel facing the mouse head, and slowly puncture into the dural cavity of the mouse.

[0016] Furthermore, in the step 2, after the injection is completed, a clean cotton swab is used to press the puncture point for 1 minute, and after there is no bleeding or exudation, the mouse is placed on a heating pad to wait for recovery.

[0017] Furthermore, the method also includes preoperative preparation.

[0018] Furthermore, the preoperative preparation is that the mice are fasted and deprived of water one day before the operation, and the dorsal side of the mice near the base of the tail is shaved and the skin is prepared.

[0019] Furthermore, the method also includes the disinfection of all instruments and operating tables before surgery.

[0020] The present invention has the following advantages:

[0021] The modeling method of the present invention constructs a meningeal metastasis model through intrathecal injection, which is not only technically easy to perform, but also greatly shortens the process of preliminary cell screening through a one-step operation, and can specifically construct a meningeal metastasis model with extremely strong specificity.

[0022] The present invention constructs a mouse meningeal metastasis model in a simple, repeatable and efficient manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0024] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0025] Figure 1 This is a live imaging diagram of a mouse model of meningeal metastasis of tumor cells injected intrathecally in the present invention 1; wherein A-C57 mice were injected intrathecally with 5×10 4 After B16 luc tumor cells were injected, the tumor growth showed local growth and continued along the spinal cavity into the skull; B-shows intrathecal injection of 5×10 4 After B16 luc tumor cells were injected, the tumor growth showed a jump along the spinal cord cavity into the skull, and no obvious tumor growth was observed at the injection site. C-BalB / C mice were injected intrathecally with 5×104 4T1 luc mouse breast cancer tumor cells showed local tumor growth after injection, and the first intracranial signal (arrow) appeared 17 days after injection; D- Direct view of melanoma meningeal metastasis, black tumor tissue can be seen attached to the ventral side of the brain and deep into the brain sulci; E- In vitro imaging of brain tissue with breast cancer meningeal metastasis;

[0026] Figure 2 The BalB / C mice provided by the present invention were intrathecally injected with 5×10 4 Coronal MRI images 28 days after B16 luc tumor cells;

[0027] Figure 3 The C57 mice provided by the present invention were intrathecally injected with 5×10 4 Pathological images of brain tissue 20 days after B16 luc tumor cells were injected; A-coronal plane shows melanoma cells attached to the meninges on the ventral side of the hypothalamus, and tumor invasion of brain parenchyma and melanoma cells invading upward along the cerebral cortex and brainstem; B-sagittal plane shows tumor invasion of the dorsal and ventral meninges and invasion of normal brain parenchyma. DETAILED DESCRIPTION

[0028] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example

[0030] 1. Cell Preparation

[0031] Treat tumor cells (breast cancer or malignant melanoma; the cells used in 57 mice were mouse melanoma cells B16; the tumor cells used in BALB / C mice were 4T1 mouse breast cancer cells) according to the cell passage method and centrifugation parameters, add 10 mL of serum-free medium and centrifuge again for 2 times. Finally, use fresh serum-free medium to adjust the cell density to 5×10 7 / ml. Aliquot the cells into 1.5mL EP tubes and place them on ice for inoculation. Note that due to limited epidural space and to avoid injection reflux, the injection volume is 10 μl per mouse.

[0032] 2. Preoperative Preparation

[0033] The day before the operation, the mice were fasted and deprived of water, and 2 cm2 of hair was shaved off the dorsal area of ​​the animals near the base of the tail to prepare the skin for better observation during puncture.

[0034] 3. Surgical Procedures

[0035] 1. Check the instruments needed for the operation and place them in a clean bench for UV disinfection for 25 minutes;

[0036] 2. Weigh the mouse and mark it. Since the operation is short and trauma-free, you can choose to puncture the mouse while it is awake and fix it;

[0037] 3. Find the L6 lumbar vertebra, which is the most prominent dorsal vertebra, and fix the vertebra around this area by gently pressing, and disinfect with alcohol;

[0038] 4. Use a 31G BD300 μl microsyringe to extract 10 μl of the mixed tumor cell suspension, with the needle tip facing the mouse head, and slowly puncture into the mouse's dural cavity. The appearance of a tail-flick reflex indicates that the needle has punctured in place;

[0039] 5. Immediately after observing the tail-flick reflex, carefully hold the needle in place with one hand and slowly and steadily push the needle with the other hand;

[0040] 6. After the injection is completed, use a clean cotton swab to press the puncture point for 1 minute. After there is no bleeding or exudation, place the mouse on a heating pad and wait for recovery;

[0041] 7. Transfer the awakened mice to the cage and feed them normally;

[0042] 8. Monitor tumor growth through small animal in vivo imaging technology and determine the extent of tumor invasion through ex vivo imaging of dissected organs.

[0043] 4. Model building effect

[0044] 1. In vivo imaging of the mouse model of intrathecal injection of tumor cells into the meningeal metastasis Figure 1 shown.

[0045] Depend on Figure 1 visible: Figure 1The present invention provides a mouse model of meningeal metastasis established by intrathecal injection of tumor cells in vivo; wherein, A: after C57 mice were intrathecally injected with 5×104B16luc melanoma tumor cells, tumor growth was manifested as local growth in the injection area and continuous growth along the spinal cavity into the skull; B: intrathecal injection of melanoma tumor growth was manifested as jumping into the skull along the spinal cavity, and no obvious tumor growth was observed in the injection area; C: after BalB / C mice were intrathecally injected with 5×1044T1luc mouse breast cancer tumor cells, local tumor growth was observed, and intracranial signals (arrows) appeared for the first time on the 17th day after injection; D: gross anatomical diagram of melanoma meningeal metastasis, with black tumor tissue attached to the ventral side of the brain and deep into the cerebral sulci; E- in vitro imaging diagram of brain tissue with breast cancer meningeal metastasis;

[0046] 2. Coronal MRI images of mouse meningeal metastasis Figure 2 shown.

[0047] Depend on Figure 2 It can be seen that BalB / C mice were injected intrathecally with 5×10 4 28 days after B16 luc breast cancer cells were implanted, 7.0T small animal MRI coronal image. T2-weighted images clearly show thickened meninges near the nasal side (white arrows) and dilated and high-pressure ventricular system (red arrows).

[0048] 3. Pathological picture of meningeal metastasis after intrathecal injection Figure 3 shown.

[0049] Depend on Figure 3 It can be seen that C57 mice were injected intrathecally with 5×10 4 HE staining of brain tissue 20 days after B16 luc melanoma tumor cells were inoculated. Figure 3 The coronal view of A shows that melanoma cells can be seen attached to the meninges on the ventral side of the hypothalamus, the tumor invades the brain parenchyma, and the melanoma cells invade upward along the cerebral cortex and brainstem. Figure 3 B Sagittal view shows invasion of the dorsal and ventral meninges by the tumor and invasion of the adjacent normal brain parenchyma.

[0050] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for constructing a tumor-related meningeal metastasis model, characterized in that: The method comprises: Step 1: Cell preparation After the tumor cells are processed according to the cell passage method and centrifugation parameters, a serum-free medium is added and centrifuged again, the cell density is adjusted using a serum-free fresh medium, and the tumor cell suspension is obtained by aliquoting; Step 2: Modeling Tumor cell suspension was injected into the L4-L5 intervertebral space of mice. After the injection, the mice recovered and ate normally. The growth of the tumor was monitored by small animal live imaging technology, and the dissected organs were imaged in vitro to determine the invasion range of the tumor.

2. The method for constructing a tumor-related meningeal metastasis model according to claim 1, characterized in that: In step 1, the cell density was adjusted to 5×10 7 / ml.

3. The method for constructing a tumor-related meningeal metastasis model according to claim 1, characterized in that: In the step 2, the method for injecting the tumor cell suspension is to use a 31G BD 300 μl microsyringe to extract 10 μl of the mixed tumor cell suspension, with the needle tip bevel facing the mouse head, and slowly puncture into the mouse's dural cavity.

4. The method for constructing a tumor-related meningeal metastasis model according to claim 1, characterized in that: The step 2 also includes pressing the puncture point with a clean cotton swab for 1 minute after the injection is completed, and placing the mouse on a heating pad to wait for recovery after there is no bleeding or exudation.

5. The method for constructing a tumor-related meningeal metastasis model according to claim 1, characterized in that: The method also includes preoperative preparation.

6. The method for constructing a tumor-related meningeal metastasis model according to claim 5, characterized in that: The preoperative preparation is that the mice are fasted and deprived of water one day before the operation, and the surgical area near the tail of the mouse's dorsal side is shaved and skin prepared.

7. The method for constructing a tumor-related meningeal metastasis model according to claim 1, characterized in that: The method also includes the disinfection of all instruments and the operating table before the operation.

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