Mouse model for advanced high metastatic renal clear cell carcinoma, mouse model for radical operation of renal clear cell carcinoma and construction method of mouse model

Abstract

The invention provides a construction method of an advanced high metastatic renal clear cell carcinoma mouse model, which comprises the following steps: implanting Renca cancer cells into a renal capsule of a mouse, suturing a wound, and detecting colonization of tumor cells. The invention also provides a construction method of the mouse model for radical operation of renal clear cell carcinoma, and the method comprises the following steps: implanting Renca cells into a renal capsule of a mouse, suturing a wound, and detecting colonization of tumor cells; and performing a radical renal resection operation on the mouse, and continuing to detect the colonization of the tumor cells in the mouse. The method is high in success rate and easy to operate. The constructed advanced high metastatic clear cell renal carcinoma mouse model and the constructed clear cell renal carcinoma radical operation mouse model can be used for screening drugs for treating clear cell renal carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to mouse models of advanced highly metastatic clear cell renal cell carcinoma and mouse models of radical resection of clear cell renal cell carcinoma, and their construction methods. Background Technology

[0002] Renal cell carcinoma (RCC), also known as kidney cancer, is one of the ten most common malignant tumors, accounting for 4% of all adult malignant tumors. Worldwide, more than 250,000 new cases of kidney cancer are diagnosed each year. 1 More than 140,000 people die from kidney cancer each year. The incidence of renal cell carcinoma varies globally, with higher rates in developed countries than in developing countries. It is predominantly male, with a male-to-female ratio of 1.5:1, and peaks in age between 60 and 70 years. 2 .

[0003] Clear cell RCC (CCRCC) is the most common type of RCC (accounting for approximately 75% of all cases). 3 In addition, there are papillary RCC (pRCC) and chromophilic RCC (chRCC). Because clear cell renal cell carcinoma is often only discovered at an advanced stage, ccRCC has the worst disease-specific survival rate.

[0004] Renal cell carcinoma is clinically classified into four stages based on its development within the kidney, local spread, lymph node involvement, and metastatic spread. Intrarenal tumors are classified as stage I; stage II is when the intrarenal tumor extends to Gerota's fascia; stage III is when the tumor spreads beyond Gerota's fascia or extends to the inferior vena cava; and stage IV is when the tumor has metastasized to distant sites. 4 Among these, patients diagnosed at stage I or II have a 5-year survival rate of 70%-80% after treatment. 5 However, the 5-year survival rate for patients diagnosed at stage III and IV is extremely low.

[0005] It has been reported that approximately 20-30% of patients with renal cell carcinoma (RCC) have metastases at initial diagnosis, with the lungs being the most common site of distant metastasis. Nearly one-third of patients with localized renal cell carcinoma who undergo radical nephrectomy develop distant metastases. Among patients with metastatic RCC, 45-76% have lung metastases. 6 Furthermore, the prognosis for patients with metastasis is uncertain, with a 2-year survival rate of only 10%-30%. 7 Therefore, advanced and metastatic renal cell carcinoma is a major cause of death in patients with renal cell carcinoma. 8 Meanwhile, the treatment of highly metastatic advanced renal cell carcinoma has become extremely urgent.

[0006] However, there are no reports of successfully preparing metastatic animal models using renal cancer cells, nor are there reports of successfully preparing mouse models of radical resection of clear cell renal carcinoma.

[0007] (1)Shenoy, N.; Pagliaro, L. Sequential pathogenesis of metastatic VHLmutant clear cell renal cell carcinoma: putting it together with a translational perspective. Annals of Oncology 2016, 27(9), 1685-1695. DOI: 10.1093 / annonc / mdw241.

[0008] (2) Levi, F.; Ferlay, J.; Galeone, C.; Lucchini, F.; Negri, E.; Boyle, P.; LaVecchia, C. The changing pattern of kidney cancer incidence and mortality in Europe. BJU International 2008,101(8),949-958.DOI:10.1111 / j.1464-410X.2008.07451.x.

[0009] (3) Chadwick, BP; Ricketts, CJ; Linehan, WMGender Specific MutationIncidence and Survival Associations in Clear Cell Renal Cell Carcinoma (CCRCC). Plos One 2015, 10 (10). DOI: 10.1371 / journal.pone.0140257.

[0010] (4) Delahunt, B.; Eble, JN; Samaratunga, H.; Thunders, M.; Yaxley, JW; Egevad, L. Staging of renal cell carcinoma: current progress and potential advances. Pathology 2021, 53(1), 120-128. DOI: 10.1016 / j.pathol.2020.08.007.

[0011] (5)Richard E Gray 1,GTHRenal Cell Carcinoma:Diagnosis andManagement.2019Feb 1,99(3),179-184.

[0012] (6) Price, M.; Wu, CC; Genshaft, S.; Sadow, PM; 2018,210(6),1181-1191.DOI:10.2214 / ajr.18.19645.

[0013] (7)1,RCFDebulking nephrectomy in metastatic renal cancer.10(18Pt2):6335S-41S2004Sep 15.DOI:doi:10.1158 / 1078-0432.CCR-sup-040026. Summary of the Invention

[0014] To address the shortcomings of existing technologies, this invention provides a method for constructing a mouse model of advanced highly metastatic clear cell renal carcinoma. The method includes: implanting Renca cells under the renal capsule of a mouse, suturing the wound, feeding the mouse, and detecting tumor cell colonization.

[0015] Renca (mouse renal cell carcinoma) is an epithelial cell line isolated from the kidneys of male mice with renal cortical carcinoma. Its growth pattern is remarkably similar to that of adult renal cell carcinoma, particularly in spontaneous lung and liver metastases. It does not express the TGF-β-II receptor and is frequently used in experimental studies to establish nude mouse Renca tumor models.

[0016] In some embodiments of the present invention, the Renca cells are a cell suspension, wherein the density of the Renca cells in the cell suspension is 1 to 9 × 10⁻⁶. 7 The injection volume is 10–40 μl per ml.

[0017] The density of Renca cells in the cell suspension was selected from 1 × 10⁻⁶. 7 cells / ml, 2×10 7 cells / ml, 3×10 7 cells / ml, 4×10 7 cells / ml, 5×10 7 cells / ml, 6×10 7 7 x 10 cells / ml 7 8 x 10 cells / ml 7 9×10 cells / ml 7 per ml.

[0018] The injection volume was selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 μl.

[0019] In some embodiments of the present invention, the mouse is a Balb / c mouse.

[0020] BALB / c is a white variant of the laboratory mouse, originating from the house mouse (Mus musculus), and is widely used in animal experiments in immunology and physiology. Like many commonly used substrains, BALB / c mice are derived from the house mouse (Mus musculus). BALB / c mice are inbred, bred from siblings, resulting in less individual variation, purer genetic material, and better overall health.

[0021] The mice of this invention may also be selected from: C57BL / 6 mice, DBA / 2 mice, NIH Swiss mice, SCID mice, NOD mice, CD-1 mice, KK-Ay mice, China No. 1 (CI) mice, Jinbai No. 1 (TA1), Jinbai No. 2 (TA2), 615 mice, AMMS / 1, A and A / He mice, BAL / c mice, AKR-Furth mice, C3H mice, DBA mice, C57BL mice, C57BR mice, C58 mice, 129 / ter Sv subspecies mice, KK and KR strain mice, or SWR strain mice.

[0022] In some embodiments of the present invention, the Renca cells are Renca cells carrying luciferase, and the detection of tumor cell colonization is performed by in vivo imaging.

[0023] As in some embodiments of the present invention, the Renca cells are Renca cells cultured to 80% cell coverage.

[0024] In some embodiments of the present invention, the cell suspension is obtained by resuspending Renca cells and matrix gel in a 2:1 ratio.

[0025] In vivo imaging is a technique that uses imaging methods and a set of highly sensitive optical instruments to conduct qualitative and quantitative studies of biological processes at the cellular and molecular levels in living animals without causing harm to the experimental animals. This technique allows for non-invasive and direct observation of biological processes such as tumor growth, metastasis, disease development, and gene expression changes in living animals, enabling the tracking and observation of various biological behaviors of the same experimental subject at different time points. Due to its extremely simple operation, intuitive results, and high sensitivity, it is now widely used in life sciences, medical research, and drug development.

[0026] As some embodiments of the present invention, the tumor cell colonization detection is the colonization of the tumor cells in the lungs.

[0027] The present invention also provides a mouse model of advanced highly metastatic clear cell renal cell carcinoma constructed according to the described construction method.

[0028] The present invention also provides a method for constructing a mouse model of radical renal cell carcinoma, the method comprising the following steps: (1) implanting Renca cells under the renal capsule of a mouse, suturing the wound, feeding the mouse, and detecting the colonization of tumor cells; and (2) performing radical nephrectomy on the mouse in step (1), feeding the mouse, and continuing to detect the colonization of tumor cells in the Balb / c mouse.

[0029] In some embodiments of the present invention, the Renca cells are a cell suspension, wherein the density of the Renca cells in the cell suspension is 1 to 9 × 10⁻⁶. 7 The injection volume is 10–40 μl per ml.

[0030] The density of Renca cells in the cell suspension was selected from 1 × 10⁻⁶. 7 cells / ml, 2×10 7 cells / ml, 3×10 7 cells / ml, 4×10 7 cells / ml, 5×10 7 cells / ml, 6×10 7 7 x 10 cells / ml 7 8 x 10 cells / ml 7 9×10 cells / ml 7 per ml.

[0031] The injection volume was selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 μl.

[0032] The mice of this invention may also be selected from: C57BL / 6 mice, DBA / 2 mice, NIH Swiss mice, SCID mice, NOD mice, CD-1 mice, KK-Ay mice, China No. 1 (CI) mice, Jinbai No. 1 (TA1), Jinbai No. 2 (TA2), 615 mice, AMMS / 1, A and A / He mice, BAL / c mice, AKR-Furth mice, C3H mice, DBA mice, C57BL mice, C57BR mice, C58 mice, 129 / ter Sv subspecies mice, KK and KR strain mice, or SWR strain mice. In some embodiments of this invention, the mice are Balb / c mice.

[0033] In some embodiments of the present invention, the Renca cells are Renca cells carrying luciferase, and the detection of tumor cell colonization is performed by in vivo imaging.

[0034] As in some embodiments of the present invention, the Renca cells are Renca cells cultured to 80% cell coverage.

[0035] In some embodiments of the present invention, the cell suspension is obtained by resuspending Renca cells and matrix gel in a 2:1 ratio.

[0036] As some embodiments of the present invention, the tumor cell colonization detection is the colonization of the tumor cells in the lungs.

[0037] As some embodiments of the present invention, the detection of tumor cell colonization in step (1) is the colonization of the tumor cells in the kidney.

[0038] As some embodiments of the present invention, the tumor cell colonization detection in step (2) is the colonization of the tumor cells in the lungs.

[0039] As some embodiments of the present invention, the radical nephrectomy in step (2) is performed on the 10th day after the Renca cells in step (1) are implanted under the renal capsule of the mouse.

[0040] The present invention also provides a mouse model of radical resection of clear cell renal cell carcinoma constructed according to the described construction method.

[0041] The present invention also provides the application of the aforementioned advanced highly metastatic clear cell renal cell carcinoma mouse model or the aforementioned clear cell renal cell carcinoma radical resection mouse model, the application being for screening drugs for the treatment of clear cell renal cell carcinoma and for use in medical research.

[0042] The advanced highly metastatic clear cell renal cell carcinoma mouse model and the clear cell renal cell carcinoma radical resection mouse model of the present invention can be used to study the effects and safety of drugs in humans. Through rich data, the efficacy, toxicity and pharmacokinetic characteristics of drugs can be evaluated. In the process of drug screening, animal models can help researchers select potential drug candidates and evaluate their safety and efficacy.

[0043] As described above, the advanced highly metastatic clear cell renal cell carcinoma mouse model and the clear cell renal cell carcinoma radical resection mouse model of the present invention, and their construction methods, have the following beneficial effects:

[0044] The methods for constructing advanced highly metastatic clear cell renal cell carcinoma mouse models and mouse models of clear cell renal cell carcinoma radical resection according to the present invention are simple, easy to operate, and have a high success rate.

[0045] The mouse models of advanced highly metastatic clear cell renal cell carcinoma and radical resection of clear cell renal cell carcinoma of the present invention successfully simulate the development stages of advanced highly metastatic clear cell renal cell carcinoma and the metastatic development stage after radical resection of clear cell renal cell carcinoma. They have high value in clinical and scientific research for patients in the middle and late stages of renal cancer and patients with metastasis after radical resection. They are also helpful in screening clinical drugs for the middle and late stages of cancer and metastasis after radical resection of renal cancer. Attached Figure Description

[0046] Figure 1 This is a schematic diagram illustrating the construction of a mouse model of advanced highly metastatic clear cell renal cell carcinoma according to the present invention.

[0047] Figure 2 In vivo imaging can be used to detect the growth of tumors in situ, as well as the organ sites and time of distant metastasis in advanced renal cell carcinoma. Figure 2 a represents the result of in vivo imaging. Figure 2 Figure b shows the quantitative statistics of the in vivo imaging results;

[0048] Figure 3 The generation of metastatic lesions in a model of advanced, highly metastatic clear cell renal cell carcinoma was determined by hematoxylin and eosin staining of tissue layers. Figure 3 a represents the HE result of the in situ tumor. Figure 3 b represents the HE result of the metastatic foci;

[0049] Figure 4 The diagram shows the construction of a mouse model of radical resection of clear cell renal cell carcinoma according to the present invention;

[0050] Figure 5 In vivo imaging is used to detect the growth of the in situ tumor before surgery and to investigate the occurrence of distant metastases after radical surgery. Figure 5 a represents the result of in vivo imaging. Figure 5 b represents the quantitative statistics of the in vivo imaging results;

[0051] Figure 6 The occurrence of metastatic lesions after radical surgery was determined by hematoxylin and eosin staining of tissue layers. Figure 6 a represents the HE result of the in situ tumor. Figure 6 b represents the HE result of the metastatic foci. Detailed Implementation

[0052] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0053] [Experimental Materials]

[0054] Renca cells were purchased from Pronosei, catalog number CL-0568.

[0055] Balb / c mice were purchased from the Model Animal Research Center of Nanjing University.

[0056] The Matrigel Matrix was purchased from Corning, item number: 354248.

[0057] Sumu was purchased from YEASEN, 60502ES60, Shanghai, China.

[0058] Erythromycin was purchased from Seville Biotechnology, product code G1001-100ML.

[0059] The liveness detection system was purchased from IVIS Lumina XR System.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0061] Example 1: Construction of a mouse model of advanced highly metastatic clear cell renal carcinoma

[0062] Figure 1 This diagram illustrates the construction of a mouse model of advanced, highly metastatic clear cell renal cell carcinoma according to the present invention. The specific construction method is as follows:

[0063] Renca cells equipped with luciferase were cultured to a confluent level (80% cell coverage) in cell culture flasks. The luciferase-equipped Renca cells were then resuspended in a 2:1 mixture with Matrigel. Matrigel's main components are laminin, type IV collagen, heparan sulfate proteoglycan (HSPG), and nestin, and it also contains various growth factors such as TGF-β, EGF, IGF, and FGF. At room temperature, Matrigel polymerizes to form a biologically active three-dimensional matrix that mimics the structure, composition, physical properties, and function of the in vivo cell basement membrane, thus facilitating cell culture and differentiation in vitro.

[0064] Renca cells carrying luciferase, mixed with a matrix gel suspension, were implanted subcapsularly into the kidneys of Balb / c mice. Six-week-old male Balb / c mice were intraperitoneally injected with 10% sodium pentobarbital solution during surgery, and their body temperature was maintained using a heating block. On a sterile operating table, the hair on the left subcostal region was shaved and fixed laterally. After routine disinfection with povidone-iodine, an incision was made approximately 1-2 cm along the left midline to separate the left kidney from the left costal margin, exposing the left kidney. 25 μL of a suspension of Renca cells carrying luciferase was implanted into the subcapsular capsule of the left kidney. After the surgery, the incision was sutured, and the mice were allowed to recover. Tumor cell colonization was detected by in vivo imaging on day 3.

[0065] Subsequently, the growth of the orthotopic tumor in mice was examined on days 10, 13, and 16 post-tumor transplantation. Simultaneously, the heart, liver, spleen, lungs, and non-tumor-transplanted kidneys of the mice were removed. In vivo imaging detected fluorescent signals in the lungs on day 13 (see [link to original text]). Figure 2 -a live imaging results and Figure 2(Statistics on the time of metastasis shown in Figure b) indicate that distant lung metastases occurred on day 13 at the time of initial diagnosis. Furthermore, the histopathological HE sections also revealed the morphology of the in situ tumor and adjacent tissues, with the same tumor morphology appearing on the lung foci (see...). Figure 3 ,in Figure 3 a represents the HE result of the in situ tumor. Figure 3 (b represents the HE results of metastatic lesions), confirming the occurrence of distant lung metastases from clear cell renal cell carcinoma. These results demonstrate the successful invention and construction of a model for advanced, highly metastatic clear cell renal cell carcinoma.

[0066] This application successfully constructed a mouse model of advanced, highly metastatic clear cell renal cell carcinoma by implanting Renca cell suspension into the renal capsule of Balb / c mice. This model can simulate the distant metastasis that occurs in clinical renal cancer patients at the initial diagnosis. The construction method of this application has high reproducibility, is simple to operate, and is easy to apply in industry.

[0067] Example 2: Construction of a mouse model of radical resection for clear cell renal cell carcinoma

[0068] Figure 4 The diagram shown illustrates the construction of a mouse model for radical resection of clear cell renal cell carcinoma according to the present invention. The specific construction method is as follows:

[0069] Renca cells with luciferase were cultured to a confluent level (80% cell coverage) in cell culture flasks. The luciferase-containing Renca cells were then resuspended in a 2:1 mixture with Matrigel. This suspension was then implanted subcapsularly into Balb / c mice. Six-week-old male Balb / c mice were intraperitoneally injected with 10% sodium pentobarbital solution during surgery, and their body temperature was maintained using a heating block. On a sterile operating table, the hair on the left subcostal region was shaved and fixed laterally. After routine disinfection with povidone-iodine, an incision was made approximately 1-2 cm along the left midline to separate the left kidney from the left costal margin, exposing the left kidney. 25 μL of a luciferase-containing Renca cell suspension was implanted into the subcapsular capsule of the left kidney. After the surgery, the incision was sutured, and the mice were allowed to recover. Tumor cell colonization was detected by in vivo imaging on day 3.

[0070] A radical nephrectomy was performed on day 10: the surgical area was strictly disinfected, mice were anesthetized, and the tumor-bearing left kidney was exposed through a lateral ventral incision. The renal artery, vein, and ureter were bound with 4-0 silk sutures, and the kidney was removed. After blood clotted, the surgical incision was sutured. The mice maintained normal body temperature throughout the procedure. After recovery from anesthesia, the mice were returned to the rearing room, and postoperative lung metastasis was detected by in vivo imaging on day 28 after tumor grafting (see...). Figure 5 ,in, Figure 5 a represents the result of in vivo imaging. Figure 5b represents the quantitative statistics of the in vivo imaging results.

[0071] Meanwhile, the histopathological HE sections also showed that the lesions in the lung tissue had the same tumor morphology as the in situ tumor (see...). Figure 6 , Figure 6 a represents the HE result of the in situ tumor. Figure 6 b represents the HE result for the metastatic foci, from... Figure 6 The results confirmed the occurrence of metastatic lesions after radical resection surgery, and determined that distant lung metastases occurred after radical resection. In summary, these results demonstrate the successful invention and construction of a lung metastasis model after radical resection of clear cell renal cell carcinoma.

[0072] This application successfully constructed a mouse model of clear cell renal cell carcinoma radical resection by implanting Renca cell suspension into the renal capsule of Balb / c mice. This model simulates the metastatic situation that occurs in clinical patients with renal cancer after curative surgical resection. The construction method of this application has high reproducibility, is simple to operate, and is easy to apply in industry.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for constructing a mouse model of advanced highly metastatic clear cell renal cell carcinoma, characterized in that, The construction method includes: implanting Renca cells under the renal capsule of mice, suturing the wound, feeding the mice, and detecting the colonization of tumor cells.

2. The construction method according to claim 1, characterized in that, Renca cells are in the form of a cell suspension, and the density of Renca cells in the cell suspension is 1–9 × 10⁻⁶. 7 The injection volume is 10–40 μl per ml. Preferably, the Renca cells are Renca cells cultured to 80% cell coverage; Preferably, the cell suspension is obtained by resuspending Renca cells and matrix gel in a 2:1 ratio; The mice in question were Balb / c mice.

3. The construction method according to claim 1, characterized in that, The Renca cells are Renca cells equipped with luciferase, and the detection of tumor cell colonization is performed by in vivo imaging. Or the tumor cell colonization detection indicates that the tumor cells are colonized in the lungs.

4. A mouse model of advanced, highly metastatic clear cell renal cell carcinoma, characterized in that, Constructed according to the construction method of any one of claims 1 to 3.

5. A method for constructing a mouse model of radical resection for clear cell renal cell carcinoma, characterized in that, The construction method includes the following steps: (1) Renca cells were implanted under the renal capsule of mice, the wound was sutured, the mice were fed, and tumor cell colonization was detected; and (2) The mice in step (1) underwent radical nephrectomy, were fed, and the colonization of tumor cells in the mice was continued to be detected.

6. The construction method according to claim 5, characterized in that, Renca cells are in the form of a cell suspension, and the density of Renca cells in the cell suspension is 1–9 × 10⁻⁶. 7 The injection volume is 10–40 μl per ml. Preferably, the Renca cells are Renca cells cultured to 80% cell coverage; Preferably, the cell suspension is obtained by resuspending Renca cells and matrix gel in a 2:1 ratio; The mice in question were Balb / c mice.

7. The construction method according to claim 5, characterized in that, The Renca cells are Renca cells equipped with luciferase, and the detection of tumor cell colonization is performed by in vivo imaging. Or the tumor cell colonization detection indicates that the tumor cells are colonized in the lungs.

8. The construction method according to claim 5, characterized in that, The detection of tumor cell colonization in step (1) refers to the colonization of the tumor cells in the kidney; Or, the tumor cell colonization detection in step (2) is the colonization of the tumor cells in the lungs. The radical nephrectomy in step (2) was performed on day 10 after Renca cells were implanted under the renal capsule of mice in step (1).

9. A mouse model of radical resection of clear cell renal cell carcinoma, constructed according to the construction method of any one of claims 6 to 8.

10. The application of the advanced highly metastatic clear cell renal cell carcinoma mouse model of claim 4, or the radical renal cell carcinoma mouse model of claim 9, characterized in that, The applications mentioned include screening drugs for the treatment of clear cell renal cell carcinoma and applications in medical research.

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