Improved pdx model construction method and reagent combination thereof

Abstract

The application provides an improved PDX model construction method and a reagent combination thereof, comprising a treatment method of patient tumor tissue after being separated from a body, an activation reagent during transplantation and mouse pretreatment, and the like. The method of the application can significantly shorten tumor formation days and improve tumor formation rate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cancer treatment, and particularly relates to a construction method of a modified patient-derived tumor xenograft (PDX) and a reagent combination thereof. BACKGROUND

[0002] Globally, cancer (malignant tumor) has become the first factor threatening human health, and the mortality rate caused by cancer is much higher than that of other diseases (cardiovascular and cerebrovascular diseases, etc.). Malignant tumors are divided into two categories: primary and secondary. Primary malignant tumors generally originate from tissue epithelium or mesenchymal tissue. Secondary or metastatic cancer refers to the migration and invasion of cancer cells originating from multiple organs to target organs. Clinically, common organs that are prone to cause cancer and target organs include brain, lung, breast, liver, stomach, esophagus, biliary tract, pancreas, colorectum, ovary, uterus, prostate, etc.

[0003] Currently, surgery is a common method for treating middle and early stage cancer, but it has the problems of great trauma, easy residual and recurrence of cancer cells, so drug therapy is more common. Drug therapy mainly includes chemotherapy and interventional drugs. Common clinical chemotherapy drugs include fluorouracil, paclitaxel, cisplatin, doxorubicin, mitomycin, gemcitabine, oxaliplatin and bevacizumab, etc. These drugs are mostly given by intravenous infusion, which can inhibit the development and metastasis of tumors to a certain extent (Ming G, et al. Chinese New Drug Journal, 2007; Hua B, et al. Chinese Pharmaceutical Industry, 2011). However, chemotherapy drugs have some drawbacks. First, due to the side effects of chemotherapy drugs, patients may experience nausea, vomiting, loss of appetite, and other gastrointestinal reactions, as well as allergic reactions such as hair loss and skin rash. In severe cases, rare and serious conditions such as ketonuria, heart disease, liver disease, and kidney failure may occur, and even death (Ding S, et al. Chinese Journal of General Surgery, 2016; Song X, et al. Practical Medical Journal, 2006). Therefore, chemotherapy has certain requirements for the physical fitness and immunity of cancer patients, and patients with particularly weak bodies and low resistance are often not recommended for radiotherapy. Second, most chemotherapy drugs lack selectivity for normal cells and tumor cells, with the disadvantage of “friend or foe”. While tumor cells are killed, normal cells are also greatly damaged, causing serious side effects in patients. The white blood cell (especially neutrophils and lymphocytes) and platelet content in most patients decreases significantly during chemotherapy, leading to infection and bleeding, and even death (Wang W, et al. Chinese Journal of Cancer Prevention and Treatment, 1999; Wang Z, et al. Seek Medicine and Ask Medicine, 2012; Zhong J, et al. Journal of Clinical Rational Drug Use, 2018). For patients with recurrent tumors, the recurrent tumors have lost their initial sensitivity to many chemotherapy drugs, and can only be treated with other sensitive chemotherapy drugs. However, due to the low physical fitness of most cancer patients, they cannot withstand multiple drug trials, so the application of patient-derived tumor xenograft (PDX) animal models is necessary and important. PDX models are human xenograft models established by transplanting tumor tissue from patients subcutaneously or intracorporeally into immunodeficient mice (mostly nude mice). Through the construction of this model, the microenvironment and genetic characteristics of the primary tumor of the cancer patient are preserved, and most of the characteristics of the primary tumor are preserved at the histopathological, molecular biological and genetic levels, with good predictive ability for clinical efficacy. It is commonly used in new drug development, especially in clinical trials of target drugs, patient screening and predictive biomarker research, and is also often used to help sensitive drug screening for patients with recurrent or advanced cancer, to help predict patient efficacy, toxicity and absorption.In February 2016, the National Cancer Institute (NCI) of the United States put PDX into guidelines and recommended that PDX models be widely used for preclinical anti-tumor drug research and clinical sensitive anti-tumor drug screening. The construction of PDX models has become an indispensable technical means in precision medicine.

[0004] Human primary tumors are implanted subcutaneously or in vivo in immunodeficient mice, mainly depending on the number and viability of tumor cells surviving in vivo. It is reported that the success rate of PDX models in colon cancer, liver cancer and breast cancer is about 36.4%-47%, 29.5%-33.8% and 17%-20%, respectively. From the day of transplantation, the average time for tumor tissue to grow to 200mm 3 The average time is about 55 days, 67 days and 80 days, respectively (Hidalgo et al. Cancer Discovery, 2014.; Owonikoko et al. Journal of Translational Medicine, 2016. Izumchenko et al. Annals of Oncology, 2017.; Hidalgo et al. Cancer Discovery, 2014.; Owonikoko et al. Journal of Translational Medicine, 2016.). Considering the poor quality of life of patients with tumor recurrence and advanced cancer, it is necessary to "fight for every second" with tumor growth and metastasis to prolong the survival period of patients as much as possible and improve the quality of life, therefore, how to improve the success rate of PDX models and shorten the tumor formation time has become a difficult problem to be solved in the clinical treatment of tumors. SUMMARY

[0005] To improve the above technical problems, the present application provides an improved method for establishing a PDX model and a reagent combination thereof.

[0006] In the first aspect of the application, the improvement of the method over the prior art includes a pretreatment step of the mouse.

[0007] Further, the pretreatment is injecting heparin into the mouse before inoculation.

[0008] Further, the pretreatment is injecting heparin and matrigel matrix glue into the mouse before inoculation.

[0009] Further, the pretreatment is injecting heparin into the mouse subcutaneously in the abdominal cavity and injecting matrigel matrix glue subcutaneously at the inoculation site before inoculation.

[0010] According to an embodiment of the present application, the specific operation of the pretreatment is as follows: 2-3 hours before inoculation, the inoculation point on the back of the mouse is gently wiped with a cotton ball containing 75% medical alcohol, 1 mg / kg of body weight of heparin injection is injected subcutaneously in the abdominal cavity with a 1 mL sterile syringe (total volume is 200 μL), and 100 μL of matrigel matrix glue (pre-stored on ice) is slowly injected subcutaneously at the inoculation point on the back with a 1 mL sterile syringe.

[0011] In the second aspect of the application, the improvement of the method over the prior art comprises a post-treatment method of tumor tissue ex vivo.

[0012] Further, the treatment method is to treat the tumor tissue ex vivo with a collagen combination.

[0013] Further, the collagen combination is a combination of types II, IV and V, preferably in a mass ratio of 1.5-2.5:2.5-3.5:5.5-6.5.

[0014] Further, the mass ratio of the collagen combination of types II, IV and V is 2:3:6.

[0015] According to an embodiment of the present application, the post-treatment method of tumor tissue ex vivo is as follows:

[0016] In a sterile operating table, remove the mucosa, blood scab or neoplastic tissue around the tumor tissue on ice, rinse the tissue with ice PBS solution containing 1% double antibody + 2.5% collagen combination (types II, IV and V, mass ratio 2:3:6) for 15 seconds, soak the tissue for 30 seconds, repeat 3 times. Then cut the tissue block into small pieces of 0.5 cm x 0.5 cm x 0.5 cm in size with a sterile scalpel and place it in the tissue preservation solution.

[0017] In the third aspect of the application, the improvement of the method over the prior art comprises activating the tumor mass at the time of transplantation.

[0018] Further, the activation is matrigel matrix glue treatment.

[0019] Further, the activation is 25% matrigel matrix glue treatment.

[0020] According to an embodiment of the present application, the activation reagent formula at the time of transplantation is as follows:

[0021] 75% sterile PBS + 25% matrix glue component, prepared on ice, prepared and used in a sterile operating table, the tumor mass is soaked in the reagent for 30 seconds before transplantation, and then the animal is inoculated.

[0022] According to an embodiment of the present application, the activation reagent formula at the time of transplantation is as follows:

[0023] 73% sterile PBS + 25% matrigel + 2% anti-apoptotic components, prepared on ice, extemporaneously in a sterile bench, used extemporaneously, the tumor pieces are immersed in this reagent for 30 seconds before being removed and inoculated in the animals.

[0024] The anti-apoptotic components are optionally components known in the prior art having anti-apoptotic activity, such as components of the following groups or combinations thereof: aurothioglucose (ATA), zinc ions, antioxidants, peptides and cytokines having anti-apoptotic activity.

[0025] According to a preferred embodiment of the application, the above-mentioned second and third aspects are used in combination.

[0026] According to a preferred embodiment of the application, the anti-apoptotic components are added during ex vivo tissue preservation.

[0027] According to one embodiment of the application, the tissue preservation solution has the following formulation:

[0028] 9.9% DMSO + 0.1% anti-apoptotic components + 30% fetal bovine serum + 60% DMEM medium. Prepared extemporaneously in a sterile bench, the small pieces of tumor tissue can be preserved in this tissue preservation solution for 4-8 hours, 4 hours at 25°C and 8 hours at 4°C.

[0029] The anti-apoptotic components are optionally components known in the prior art having anti-apoptotic activity, such as components of the following groups or combinations thereof: aurothioglucose (ATA), zinc ions, antioxidants, peptides and cytokines having anti-apoptotic activity.

[0030] According to one embodiment of the application, the cancer is colon cancer, breast cancer and liver cancer.

[0031] According to one embodiment of the application, the genetically deficient mice used to construct the PDX model are BALb / c nude mice.

[0032] The application is not limited to these three types of cancer, but also includes various types of tumor tissue; it is not limited to the use of genetically deficient mice (BALb / c nude mice or SCID mice), but also includes other types of genetically deficient mice and various small mammals.

[0033] The application also provides a reagent combination for constructing a PDX model, comprising heparin, matrigel and a collagen combination.

[0034] Further, the collagen combination is a combination of type II, type IV and type V collagen.

[0035] Further, the collagen combination is a combination of type II, type IV and type V collagen, in a mass ratio of 1.5-2.5:2.5-3.5:5.5-6.5.

[0036] Further, wherein the collagen combination is a combination of type II, type IV and type V in a mass ratio of 2:3:6.

[0037] The reagent combination for constructing the PDX model further contains an anti-apoptosis component.

[0038] The anti-apoptosis component is optionally a component of the following group or a combination thereof: aurothioglycolic acid (ATA), zinc ions, an antioxidant, a peptide with anti-apoptotic activity, and a cytokine.

[0039] Beneficial effects

[0040] The present application provides an improved PDX construction method, which can significantly shorten the tumor formation time of PDX, improve the probability of subcutaneous tumor formation of genetically deficient mice, and facilitate the screening of sensitive drugs for cancer patients in a clinical range. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The results of the detection of the influence of the construction of BALb / c nude mouse PDX models, intraperitoneal subcutaneous injection of heparin and injection of matrigel matrix glue at the back of the inoculation site on the tumor formation time of colon cancer, breast cancer and liver cancer tissues in Example 1 are shown.

[0042] Figure 2 The results of the detection of the influence of the construction of BALb / c nude mouse PDX models, ex vivo treatment of tissues + activation reagent treatment on the tumor formation time of colon cancer, breast cancer and liver cancer tissues in Example 2 are shown.

[0043] Figure 3 The results of the detection of the influence of the construction of BALb / c nude mouse PDX models, ex vivo treatment of tissues + activation reagent treatment on the tumor formation rate of colon cancer, breast cancer and liver cancer tissues in Example 3 are shown.

[0044] Figure 4 After the ex vivo treatment of tissues + activation reagent treatment of colon cancer, breast cancer and liver cancer tissues, pathological detection and analysis showed that the tumor tissues were rich in blood vessels, the cancer cells were uniformly distributed, and the vitality was normal, and there was no obvious abnormality compared with the control group. DETAILED DESCRIPTION

[0045] The technical solutions of the present application will be further described in detail below in combination with specific embodiments. It should be understood that the following examples are only illustrative and explanatory of the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of protection intended by the present application.

[0046] The starting materials and reagents used in the following examples are commercially available or can be prepared by known methods unless otherwise stated.

[0047] Effect of mouse pretreatment on tumorigenicity in Example 1

[0048] To evaluate the effect of intraperitoneal injection of heparin and injection of matrigel at the inoculation site on the tumorigenicity of colon cancer, breast cancer and liver cancer tissues, 72 male BALb / c nude mice weighing 20-22 g were selected to construct PDX models, which were divided into two parts. One part was treated according to the conventional method reported in the literature and served as a control group. The other part was treated with 75% medical alcohol to wipe the inoculation site of the mouse back, and 1 mg / kg body weight of heparin injection solution (total volume 200 μL) was injected intraperitoneally with a 1 mL sterile syringe, and at the same time, 100 μL of matrigel (pre-stored on ice) was slowly injected subcutaneously at the inoculation site with a 1 mL sterile syringe, which served as an improved treatment group. Each part was divided into three groups (colon cancer group, breast cancer group and liver cancer group), with 12 nude mice in each group. The human tumor tissue was removed into a sterile operating table as soon as possible, and the mucosa, blood scab or neoplastic tissue around the tumor tissue was removed on ice. The tissue was rinsed with ice PBS solution for 15 seconds and soaked for 30 seconds, repeated 3 times. Then the tissue block was cut into small pieces of 0.5 cm x 0.5 cm x 0.5 cm in size with a sterile scalpel, and each piece was loaded into a 0.75 cm diameter trocar and injected into the back of the nude mouse. One nude mouse was injected once, i.e. one nude mouse with one tumor piece. From the first day of inoculation, the tumorigenicity was observed every day until the 60th day. The number of mice with tumors was counted, and the tumorigenicity rate (%) of each group was calculated as follows: number of mice with tumors / 12 x 100%.

[0049] The experimental results are shown in Table 1. Figure 1 As shown in Table 1, compared with the control group (50.0%), the tumorigenicity rate of the improved treatment group was increased to 75.0% in the colon cancer model; compared with the control group (25.0%), the tumorigenicity rate of the improved treatment group was increased to 50.0% in the breast cancer model; and compared with the control group (41.7%), the tumorigenicity rate of the improved treatment group was increased to 66.7% in the liver cancer model.

[0050] Effect of tissue ex vivo treatment + activation reagent treatment on shortening the tumorigenicity time of colon cancer, breast cancer and liver cancer in Example 2

[0051] To evaluate the effect of tissue ex vivo treatment + activation reagent treatment on the tumor formation time of colon cancer, breast cancer and liver cancer tissues, 72 male BALb / c nude mice weighing 20-22 g were selected to construct PDX models, which were divided into two parts. One part was treated with conventional treatment reported in the literature as a control group; the other part was treated with tissue ex vivo treatment + activation reagent treatment as an improved treatment group. Each part was divided into three groups (colon cancer group, breast cancer group and liver cancer group), and each group contained 12 nude mice. The tumor tissue was placed in the tissue preservation solution (10% DMSO + 30% fetal bovine serum + 60% DMEM medium) for the first time after ex vivo. As soon as possible, it was moved to a sterile operating table and the mucosa, blood scabs or neoplastic tissue around the tumor tissue was removed on ice. The control group was washed with ice PBS solution, and the improved treatment group was washed with ice PBS solution containing 1% double antibody + 2.5% collagen combination (types II, IV and V, mass ratio 2:3:6). The tissue was washed for 15 seconds and soaked for 30 seconds, repeated 3 times. Then the tissue block was cut into small pieces of 0.5 cm x 0.5 cm x 0.5 cm in size with a sterile surgical knife and placed in the tissue preservation solution (10% DMSO + 30% fetal bovine serum + 60% DMEM medium). Then the epidermis of the nude mice was disinfected with 75% ethanol, and the tumor tissue pieces were treated with activation reagent on ice. The activation reagent for the control group was sterile PBS solution, and the activation reagent for the improved treatment group was 75% sterile PBS + 25% matrigel glue (soaked for 30 seconds before transplantation). Then the tumor tissue pieces were loaded into a 0.75 cm diameter trocar one by one and injected into the back of the nude mice. One nude mouse was injected once, that is, one nude mouse was injected with one tumor piece. From the first day of inoculation, the tumor formation was observed every day, and the length and width of the tumor were detected with a vernier caliper. The tumor volume (mm 3 ) = 0.5 x length (mm) x width (mm) 2 The tumor volume was calculated, and the number of days when the tumor volume reached 200 mm 3 was the tumor formation time (days).

[0052] The experimental results are shown in Figure 2 . In the colon cancer model, compared with the control group (24.5 ± 5.6 days), the tumor formation time of the improved treatment group was shortened to 11.9 ± 2.5 days; in the breast cancer model, compared with the control group (36.8 ± 7.5 days), the tumor formation time of the improved treatment group was shortened to 14.3 ± 3.3 days; in the liver cancer model, compared with the control group (28.8 ± 5.1 days), the tumor formation time of the improved treatment group was shortened to 15.7 ± 3.4 days. In the three cancer tissue models, the improved treatment significantly shortened the subcutaneous tumor formation time of the nude mice. The symbol * indicates that there is a significant statistical difference compared with the control group without any treatment, P < 0.05.

[0053] Example 3 Effect of tissue ex vivo treatment + activation reagent treatment on improving the tumor formation rate of colon cancer, breast cancer and liver cancer

[0054] To evaluate the effect of tissue ex vivo treatment + activation reagent treatment on the tumorigenicity of colon cancer, breast cancer and liver cancer tissues, 72 male BALb / c nude mice weighing 20-22 g were selected to construct PDX models, which were divided into two parts. One part was treated with conventional treatment reported in the literature as a control group; the other part was treated with tissue ex vivo treatment + activation reagent treatment as an improved treatment group. Each part was divided into 3 groups (colon cancer group, breast cancer group and liver cancer group), and each group contained 12 nude mice. The tumor tissues were placed into the tissue preservation solution at the first time after ex vivo. The control group used 10% DMSO + 30% fetal bovine serum + 60% DMEM medium, and the improved treatment group used 10% DMSO + 0.1% anti-apoptotic component + 30% fetal bovine serum + 60% DMEM medium. As soon as possible, the tumor tissues were removed from the sterile operating table and the mucosa, blood scabs or neoplastic tissues around the tumor tissues were removed on ice. The control group used ice PBS solution, and the improved treatment group used ice PBS solution containing 1% double antibody + 2.5% collagen. The tissues were washed for 15 seconds and soaked for 30 seconds, respectively, and the process was repeated 3 times. Then the tissue blocks were cut into small pieces with a size of 0.5 cm x 0.5 cm x 0.5 cm using a sterile surgical knife, and were placed into the tissue preservation solution. The control group used 10% DMSO + 30% fetal bovine serum + 60% DMEM medium, and the improved treatment group used 10% DMSO + 0.1% anti-apoptotic component + 30% fetal bovine serum + 60% DMEM medium. The small pieces of tumor tissues could be stored in the tissue preservation solution for 4-8 hours, 4 hours at 25°C and 8 hours at 4°C. Then the epidermis of the nude mice was disinfected with 75% concentration of ethanol, and the small pieces of tumor tissues were placed into the activation reagent on ice. The control group used sterile PBS solution, and the improved treatment group used 75% sterile PBS + 25% matrigel glue (soaked for 30 seconds before transplantation). Then the small pieces of tumor tissues were loaded into the sleeve needle with a diameter of 0.75 cm one by one, and were injected into the back of the nude mice. One nude mouse was injected once, i.e. one nude mouse with one tumor piece. From the day of inoculation of the tumor pieces, the subcutaneous tumor formation on the back of the mice was observed until the 20th day. The tumorigenicity rate (%) of each group was calculated as follows: the number of mice with tumor formation / 12 mice x 100%.

[0055] The experimental results are shown in Table 1. Figure 3 As shown in Table 1, in the colon cancer model, the tumorigenicity rate of the improved treatment group was 91.7%, compared with the control group (58.3%); in the breast cancer model, the tumorigenicity rate of the improved treatment group was 83.3%, compared with the control group (25.0%); and in the liver cancer model, the tumorigenicity rate of the improved treatment group was 83.3%, compared with the control group (41.7%). In the three cancer tissue models, the improved treatment significantly improved the subcutaneous tumorigenicity rate of the nude mice.

[0056] In addition, the improved treatment significantly improved the survival rate of the nude mice, and the survival rate of the improved treatment group was 91.7%, compared with the control group (58.3%). Figure 4As shown, after the improved treatment of colon cancer, breast cancer and liver cancer tissue tumorigenesis, the pathological detection and analysis show that the tumor tissue is rich in blood vessels, the cancer cell distribution is uniform, the vitality is normal, and there is no obvious abnormality compared with the control group, which further confirms that the improved treatment can improve the tumorigenicity of tumor tissue and shorten the tumorigenicity time, and at the same time will not affect the tumor quality, which is helpful for further clinical drug trials.

[0057] The above describes the embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing a PDX model, comprising the following three steps: 1) a pretreatment step for mice; 2) a post-treatment step for tumor tissues ex vivo; 3) activation of tumor mass at the time of transplantation; The pretreatment is subcutaneous injection of heparin in the abdominal cavity of mice and subcutaneous injection of matrigel at the inoculation site before inoculation; wherein the dose of heparin injection is 1 mg / kg of body weight, and the dose of matrigel injection is 100 μL; The post-treatment method for tumor tissues ex vivo is to treat tumor tissues ex vivo with a collagen combination, wherein the collagen combination is a combination of types II, IV and V, and the mass ratio is 1.5-2.5:2.5-3.5:5.5-6.5; The activation of tumor mass at the time of transplantation is matrigel treatment.

2. The method for constructing a PDX model according to claim 1, wherein the specific operation of the pretreatment is as follows: 2-3 hours before inoculation, the inoculation site on the back of the mouse is gently wiped with a cotton ball containing 75% medical alcohol, 1 mg / kg of body weight of heparin injection is subcutaneously injected into the abdominal cavity of the mouse with a 1 mL sterile syringe, the total volume is 200 μL, and 100 μL of matrigel is slowly injected subcutaneously into the inoculation site on the back of the mouse with a 1 mL sterile syringe.

3. The method for constructing a PDX model according to claim 1, wherein the collagen combination is a combination of types II, IV and V, and the mass ratio is 2:3:

6.

4. The method for constructing a PDX model according to claim 3, wherein the post-treatment method for tumor tissues ex vivo is as follows: In a sterile operating table, remove the mucosa, blood scab or neoplastic tissue around the tumor tissue on ice, rinse the tissue with ice PBS solution containing 1% double antibody + 2.5% collagen combination for 15 seconds, soak the tissue for 30 seconds, repeat 3 times, then cut the tissue block into small pieces of 0.5 cm x 0.5 cm x 0.5 cm in size with a sterile surgical knife, and place them in tissue preservation solution.

5. The method for constructing a PDX model according to claim 1, wherein the activation is 25% matrigel treatment.

6. The method for constructing a PDX model according to claim 5, wherein the activation is as follows: 75% sterile PBS + 25% matrigel components, prepared on ice, prepared and used immediately in a sterile operating table, the tumor mass is soaked in the reagent for 30 seconds at the time of transplantation, and then the animal is inoculated.

7. The method for constructing a PDX model according to claim 5, wherein the activation is as follows: 73% sterile PBS + 25% matrigel + 2% anti-apoptotic components, prepared on ice, prepared and used immediately in a sterile operating table, the tumor mass is soaked in the reagent for 30 seconds at the time of transplantation, and then the animal is inoculated.

8. The method for constructing a PDX model according to claim 7, wherein the anti-apoptotic components are optionally the components of the following group or a combination thereof: aurine tricarboxylic acid, zinc ions, antioxidants, peptides with anti-apoptotic activity, and cytokines.

9. The method for constructing a PDX model according to any one of claims 1-8, further comprising a step of ex vivo tissue preservation, and adding an anti-apoptotic component during ex vivo tissue preservation.

10. The method of claim 9, wherein 0.1% of an anti-apoptotic component is added to the tissue preservation solution.

11. The method of claim 9, wherein the tissue preservation solution is 9.9% DMSO + 0.1% anti-apoptotic component + 30% fetal bovine serum + 60% DMEM medium.

12. The method of claim 11, wherein the anti-apoptotic component is optionally a component or combination of components from the group consisting of aurothioglucose, zinc ions, antioxidants, peptides with anti-apoptotic activity, and cytokines.

13. The method of any one of claims 1-8, wherein the method is applicable to cancers or tumors selected from the group consisting of colon cancer, breast cancer, and liver cancer.

14. The method of any one of claims 1-8, wherein the method is applicable to genetically deficient mice.

15. The method of any one of claims 1-8, wherein the method is applicable to fresh tumor tissue.

16. A reagent combination for constructing a PDX model, comprising heparin, matrigel, and a collagen combination, wherein the collagen combination is a combination of type II, type IV, and type V collagen in a mass ratio of 1.5-2.5:2.5-3.5:5.5-6.

5.

17. The reagent combination of claim 16, wherein the collagen combination is a combination of type II, type IV, and type V collagen in a mass ratio of 2:3:

6.

18. The reagent combination of claim 16, further comprising an anti-apoptotic component.

19. The reagent combination of claim 18, wherein the anti-apoptotic component is optionally a component or combination of components from the group consisting of aurothioglucose, zinc ions, antioxidants, peptides with anti-apoptotic activity, and cytokines.

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