Rapid PDX model efficacy evaluation method

Abstract

The invention discloses a rapid PDX model efficacy evaluation method, which comprises the following steps: 1, collecting tumor tissues, treating, and washing with a culture medium; step 2, taking a PVDF material as a biomembrane tube implanted in vivo, injecting the cut tumor tissue into the biomembrane tube, and blocking two ends of the biomembrane tube with matrigel; step 3, implanting the biological membrane tube filled with the tumor tissue under the renal capsule of the severe combined immunodeficiency mouse, and establishing a PDX model; and 4, performing drug effect test on the P0-generation mice, and evaluating the drug effect by pathological HE staining and tunel staining methods. The detection time is short, the detection cost is low, and the drug effect result is closer to the clinical actual condition of a patient.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a rapid PDX model drug efficacy evaluation method. Background Art

[0002] Predicting which treatment a patient will respond to is one of the key goals of achieving personalized medicine and improving patient survival. Individual differences in treatment outcomes for cancer patients remain a fundamental challenge. Research in the field of predictive biomarkers has mainly focused on genetic biomarkers, but clinical methods for predicting treatment efficacy are still limited. In addition, genetic biomarkers such as RAS and BRAF mutation status cannot reliably predict treatment responses to targeted therapies. As a result, many patients receive ineffective and expensive drug treatments and endure unnecessary toxic reactions.

[0003] Using the patient's fresh tumor tissue to inoculate into immunodeficient mice to reconstruct the patient's own tumor PDX (patient-derived tumor xenografts, PDX) model can better maintain the tumor microenvironment, the basic morphology of tumor cells, and the internal vascular network structure of the tumor. Its drug sensitivity test results are highly consistent with the clinical treatment effect. However, its operation process is complicated, costly, and time-consuming, which greatly limits its application in clinical practice and new drug development. Summary of the invention

[0004] Technical problems solved: In response to the above technical problems, the present invention provides a rapid PDX model efficacy evaluation method, which has a short detection time, low detection cost, and the efficacy results are closer to the actual clinical situation of patients.

[0005] Technical solution: A rapid PDX model efficacy evaluation method, comprising the following steps: Step 1: Collect tumor tissue, wash with culture medium after treatment; Step 2: Using PVDF material as a biofilm tube for in vivo implantation, injecting the cut tumor tissue into the biofilm tube, and sealing both ends of the biofilm tube with matrix glue; Step 3: Implant the biofilm tube containing tumor tissue under the renal capsule of severe combined immunodeficient mice to establish a PDX model; Step 4: Perform drug efficacy test on P0 mice and evaluate the drug efficacy by pathological HE staining and tunel staining.

[0006] Preferably, in step 1, the collected tumor tissue is kept at 4° C. to effectively maintain the freshness of the tissue sample for no less than 48 hours.

[0007] Preferably, in step 1, the culture medium is L-15 culture medium.

[0008] Preferably, the inner diameter of the biofilm tube is 1 mm, and the size of the cut tumor tissue is 3 mm*1 mm*1 mm.

[0009] Preferably, the biofilm tube allows drugs with a molecular weight not higher than 500 KD to pass through.

[0010] Preferably, in step three, the mice used are female severe combined immunodeficiency mice aged 6 to 8 weeks.

[0011] Preferably, in step three, the opening of the renal capsule is 1-2 mm.

[0012] Preferably, in step 4, pathological HE staining is first used to determine the tumor regression grade, and the drug administration group that produces drug efficacy is determined according to the ratio of tumor cells to stromal cells, and then combined with TUNEL staining, the drug efficacy difference between the drug administration groups is determined according to the cell apoptosis situation.

[0013] Furthermore, the criteria for determining the tumor regression grade are as follows: Grade 0: no fibroblasts were observed and the tumor did not shrink; Grade 1: The fibroblast content is less than 25%, and the tumor shrinks slightly; Grade 2: Fibrocyte content is 26% to 50%, and the tumor shrinks moderately; Grade 3: The fibroblast content is higher than 50%, and the tumor shrinks well; Grade 4: Only fibroblasts are seen, no cancer cells are seen, and the tumor has completely retracted.

[0014] Preferably, the tumor tissue is rectal cancer tumor tissue.

[0015] Beneficial effects: The model and method established in the present invention can maintain a high degree of consistency with the results of traditional PDX models, and the modeling time and efficacy time are significantly shortened, which greatly reduces the experimental cost and improves the accessibility of clinical application of technology and new drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the rapid PDX model efficacy evaluation method of the present invention; Figure 2 This is the tumor growth curve of each group in the traditional PDX model drug efficacy test; Figure 3 It is a comparison of tumor volumes between the blank control group and the drug-treated group after the experiment. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. Example 1

[0018] A method for rapid PDX model efficacy evaluation comprises the following steps: Step 1: Collect tumor tissue, wash with culture medium after treatment; Step 2: Using PVDF material as a biofilm tube for in vivo implantation, injecting the cut tumor tissue into the biofilm tube, and sealing both ends of the biofilm tube with matrix glue; Step 3: Implant the biofilm tube containing tumor tissue under the renal capsule of mice to establish a PDX model; Step 4: Perform drug efficacy test on P0 mice and evaluate the drug efficacy by pathological HE staining and tunel staining.

[0019] Taking rectal cancer tumor tissue as an example, the specific process of establishing the above model is as follows: Receive and prepare patient tumor specimens (<2 cm 2 ) Use Miltenyi MACS® Tissue Storage Solution and add 5-10 mL of the solution into a sterile collection tube to collect tumor specimens. Ex vivo specimens should be used immediately.

[0020] Removing a tumor sample from the tissue storage solution. Note: Rectal cancer tumor tissue is removed by the surgeon and collected for testing by the pathologist. Collect the tissue into a sterile collection tube of MACS® Tissue Storage Solution.

[0021] A biofilm tube made of PVDF was selected, sterilized under high pressure, moistened in sterile water, and cut into small sections of about 3 mm.

[0022] Tissue sample processing In a biosafety cabinet, place the collected tumor samples in a sterile plastic culture dish (90 mm in diameter and 20 mm in depth) and wash the tumor tissue 2 to 3 times with cold culture medium.

[0023] Using autoclaved scissors and forceps, under a stereomicroscope, the tumor tissue blocks were cut into small pieces of 3*1*1 mm, placed in a sterile plastic culture dish (30 mm in diameter*15 mm in depth) containing L-15 culture medium, and placed on a biological ice box.

[0024] Use autoclaved small scissors and forceps to inject a 3*1*1mm small piece of tumor tissue into the biofilm tube, seal both ends with matrix gel, put it in culture medium, and place it on a biological ice box.

[0025] Implantation of patient-derived tumor xenografts For each individual patient-derived tumor tissue, female highly immunodeficient mice aged 6 to 8 weeks were used. Mice implanted with the original human tumor were designated as the F0 generation, and then the next generation was generated by the implanted tumor of the previous generation and named F1, F2, F3, etc.

[0026] In the clean bench, mice were placed in turn in an induction anesthesia box connected to an isoflurane anesthesia machine (2%-3% isoflurane and 3%-4% oxygen were used to maintain anesthesia).

[0027] Observe the pain reflex of the mouse to ensure that the mouse is fully anesthetized. All instruments need to be sterilized under high pressure. Place the anesthetized mouse on the operating table and maintain anesthesia by oral and nasal inhalation. Wipe the surgical area of ​​the mouse with an alcohol cotton ball. Cut an incision of about 1.5 cm in the skin above the kidney area on the back of the mouse. Bluntly separate the muscle tissue. Under the dissecting microscope, remove the kidney. Cut a 1~2 mm opening in the renal capsule with fine scissors. Use forceps to clamp the tumor tissue, penetrate under the renal capsule, and close the incision to prevent the tumor from falling out. Use 5-0 sutures to close the wound. Wipe the surgical area with iodine.

[0028] Mice were injected with ceftriaxone in the abdominal region, after which each mouse was individually caged and monitored until the mouse was awake and moving.

[0029] Drug sensitivity test of human tumor tissue transplants The growth and health of the mice were monitored for 10 days. The tumors were allowed to attach and grow freely within 3 days after transplantation. Clinical drug sensitivity testing was performed on the fourth day. CPT-11 was given at 50 mg / kg, twice, and the control group was given the same volume of normal saline.

[0030] On the 7th day after drug intervention, the tumor tissue was removed and stored in 4% multidimensional polyformaldehyde for relevant pathological examination.

[0031] 4.3 Innovative use of clinical neoadjuvant therapy efficacy evaluation criteria: first use pathological HE staining to determine the tumor regression grade, and determine the drug administration group that produces drug efficacy based on the ratio of tumor cells to stromal cells. Then, combined with the tunel staining method, the drug efficacy difference between the drug administration groups is determined based on cell apoptosis.

[0032] The criteria for determining the tumor regression grade are as follows:

[0033] 4.4 Determine that the PDX tumor retains its corresponding clinical tumor histopathological characteristics and has not differentiated into other tumors.

[0034] The traditional PDX model was constructed using rectal cancer tumor tissue from the same patient. After successful modeling, drug efficacy was passed on until the tumor volume reached an average of 100 mm3 Afterwards, they were randomly divided into a control group and a CPT-11 group, with 4 mice in each group. The tumor volume was measured twice a week, and the experiment ended 21 days after administration, with a total duration of about 80 days. Figure 2 and Figure 3 The results showed that irinotecan could significantly inhibit tumor growth and had a significant therapeutic effect on the colorectal cancer model (i.e., the patient).

[0035] The efficacy of the present invention was evaluated 7 days after administration: pathological HE staining was used to determine the tumor regression grade and TUNEL staining was used to confirm the difference in efficacy. The results are as follows:

[0036] The efficacy test of the rapid PDX model of the present invention shows that the irinotecan group can significantly reduce the tumor cell content, the tumor regression grade is higher than the blank group, and the proportion of apoptotic cells is significantly higher than the blank group, and the treatment effect is significant. It can be seen that the model and method established in the present invention can maintain a high degree of consistency with the results of the traditional PDX model, and the modeling time and efficacy time are significantly shortened, which greatly reduces the experimental cost and improves the accessibility of the clinical application of the technology and the development and application of new drugs.

Claims

1. A rapid PDX model efficacy evaluation method, characterized in that: The steps include: Step 1: Collect tumor tissue, wash with culture medium after treatment; Step 2: Using PVDF material as a biofilm tube for in vivo implantation, injecting the cut tumor tissue into the biofilm tube, and sealing both ends of the biofilm tube with matrix glue; Step 3: Implant the biofilm tube containing tumor tissue under the renal capsule of severe combined immunodeficient mice to establish a PDX model; Step 4: Perform drug efficacy test on P0 mice and evaluate the drug efficacy by pathological HE staining and tunel staining.

2. A rapid PDX model efficacy evaluation method according to claim 1, characterized in that: In the step 1, the collected tumor tissue is kept at 4° C. to effectively maintain the freshness of the tissue sample for no more than 48 hours.

3. A rapid PDX model efficacy evaluation method according to claim 1, characterized in that: In the step 1, the culture medium is L-15 culture medium.

4. A rapid PDX model efficacy evaluation method according to claim 1, characterized in that: The inner diameter of the biofilm tube is 1 mm, and the size of the cut tumor tissue is 3 mm*1 mm*1 mm.

5. A rapid PDX model efficacy evaluation method according to claim 1, characterized in that: The biofilm tube allows drugs with a molecular weight not higher than 500KD to pass through.

6. A rapid PDX model efficacy evaluation method according to claim 1, characterized in that: In the step 3, the mice used are 6-8 week old female severe combined immunodeficient mice.

7. A rapid PDX model efficacy evaluation method according to claim 1, characterized in that: In step 3, the opening of the renal capsule is 1-2 mm.

8. A rapid PDX model efficacy evaluation method according to claim 1, characterized in that: In the step 4, pathological HE staining is first used to determine the tumor regression grade, and the drug administration group that produces drug efficacy is determined according to the ratio of tumor cells to stromal cells. Then, combined with the tunel staining method, the drug efficacy difference between the drug administration groups is determined according to the cell apoptosis situation.

9. A rapid PDX model efficacy evaluation method according to claim 8, characterized in that: The criteria for judging the tumor regression grade are as follows: Grade 0: no fibroblasts were observed and the tumor did not shrink; Grade 1: The fibroblast content is less than 25%, and the tumor shrinks slightly; Grade 2: Fibrocyte content is 26% to 50%, and the tumor shrinks moderately; Grade 3: The fibroblast content is higher than 50%, and the tumor shrinks well; Grade 4: Only fibroblasts are seen, no cancer cells are seen, and the tumor has completely retracted.

10. A rapid PDX model efficacy evaluation method according to claim 1, characterized in that: The tumor tissue is rectal cancer tumor tissue.

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