Method for assisting chemotherapy precise medication of gastric cancer patient by utilizing PTC (Positive Temperature Coefficient)

Abstract

The invention discloses a method for performing drug sensitivity detection by using a patient-derived micro-tumor cell cluster (PTC) and assisting a gastric cancer patient in accurately selecting a chemotherapy regimen, and particularly relates to a method for performing drug sensitivity detection by using a patient-derived micro-tumor cell cluster (PTC). The method comprises the following steps: constructing a PTC micro-tumor by using fresh surgery, biopsy or malignant effusion samples of a patient, co-culturing PTC and four common gastric cancer drug schemes of PF, XELOX, TF and DF for 168 hours, comparing the PTC area change ratio PA before and after co-culture, evaluating the drug killing efficiency of different drug schemes on PTC, and assisting the patient in accurately selecting a chemotherapy scheme in combination with clinical actual conditions. Compared with other methods, the method has the advantages that the tumor microenvironment can be simulated more truly, so that the drug sensitivity detection result based on the method is close to the clinical real situation, and individualized precise medical service is better provided.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a method for using PTC to assist in precise chemotherapy drug use for gastric cancer patients. Background Art

[0002] Gastric carcinoma is a common digestive tract tumor originating from gastric mucosa epithelium. The incidence of gastric carcinoma ranks the 5th globally and is the 3rd leading cause of death from malignant tumors. In 2019, there were over 1 million newly diagnosed cases of gastric carcinoma and 783,000 death cases. The main treatment for gastric carcinoma is surgery, supplemented by radiotherapy and chemotherapy. Early-stage gastric cancer patients can have their tumor tissues resected surgically and receive adjuvant radiotherapy and chemotherapy postoperatively, with a 5-year survival rate of up to 95%. The treatment for advanced gastric cancer mainly relies on palliative systemic chemotherapy, but the median survival time after treatment is only 4 - 8 months. The most commonly used chemotherapy regimens for gastric cancer include PF, XELOX, TF, and DF. All four regimens contain 5-fluorouracil as the basis. Among them, the PF regimen (cisplatin + 5-fluorouracil) and the XELOX regimen (oxaliplatin + 5-fluorouracil) use platinum drugs in combination, while the TF regimen (paclitaxel + 5-fluorouracil) and the DF regimen (docetaxel + 5-fluorouracil) use taxane drugs in combination. Due to the high heterogeneity of advanced gastric cancer, there are differences in the treatment efficacy and prognosis of different patients. The treatment efficacy of some patients is unstable, and it is necessary to precisely select the chemotherapy regimen for gastric cancer patients.

[0003] Tumor drug sensitivity detection technology is an important part of translational medicine and personalized medicine, and plays an important role in screening the effectiveness of traditional chemotherapy drugs or new targeted drugs. In vitro drug sensitivity screening methods at different technical levels have explored the feasibility of guiding clinical practice, including in vitro drug sensitivity screening based on patient-derived xenografts (PDXs), drug sensitivity testing using in vitro cultured tumor organoids, and drug sensitivity screening research based on in vitro tumor cell culture. Although different technical methods have shown their respective advantages and corresponding application prospects, large-scale clinical verification and effectiveness analysis have not been carried out, and there are still deficiencies in each technology. For example, the tumor formation rate of PDX modeling is low, the tumor formation cycle is long, the cost of model modeling is high, and its guiding role for clinical application is limited; the success rate and throughput of organoid culture are relatively low, which to a certain extent limits its clinical application.

[0004] Patient-derived tumor-like cell clusters (PTCs) are an emerging 3D cell model for in vitro drug sensitivity testing. PTCs contain various cell components such as tumor cells, normal epithelial cells, fibroblasts, and macrophages, and are highly consistent with the patient tumor tissue samples from which they are derived in terms of cell morphology, pathological characteristics, genetic variations, etc. Currently, PTC models derived from breast cancer patients and colorectal cancer patients have been successfully constructed, and the results of drug sensitivity tests on the PTC models are 96.6% consistent with the clinic. Therefore, the present invention constructs a PTC model using tumor samples from gastric cancer patients to perform in vitro drug screening on behalf of the patients, which is of great significance in the research of precision chemotherapy for gastric cancer. Summary of the Invention

[0005] In view of the deficiencies in the traditional chemotherapy for gastric cancer patients, the present invention develops a method for using PTC to assist in the precision medication of gastric cancer patients, aiming to improve the clinical chemotherapy benefits of gastric cancer patients by guiding the precision chemotherapy of gastric cancer patients. This method constructs a PTC model using tumor samples from gastric cancer patients, conducts drug sensitivity tests on common gastric cancer chemotherapy regimens such as PF, XELOX, TF, and DF, and evaluates the effects of different drug combinations in the in vitro model to provide support for the individualized precision medicine of patients.

[0006] The present invention adopts the following scheme:

[0007] Step 1, collect fresh tumor samples from gastric cancer patients and construct a PTC micro-tumor model;

[0008] Step 2, co-culture the constructed PTCs with different drug combinations for 168 hours to conduct drug sensitivity tests;

[0009] Step 3, after the drug sensitivity test, count and analyze the cell viability of the PTC micro-tumors and the area changes before and after the test;

[0010] Step 4, compare the sensitivity differences of the PTCs of the same patient to different drug combinations, and combine the clinical manifestations of the patient to assist in selecting a precision chemotherapy regimen.

[0011] Further, the specific method for constructing gastric cancer PTC in Step 1 includes the following steps:

[0012] 1.1 For the tumor tissue sample, remove the fat and necrotic tissue, physically shear it into a paste with a medical scissors, and then add a digestive solution and dissociate it at 37°C;

[0013] 1.2 After dissociation, filter and collect the filtrate with a filter screen, centrifuge it, and resuspend it with PTC gastric cancer medium;

[0014] 1.3 Resuspend the cells and inoculate them into the special medium for gastric cancer PTC at a density of 10 5 cells / cm 2 , and culture them to allow the tumor cells derived from the patient to self-assemble with other component cells to form a PTC micro-tumor model.

[0015] Furthermore, the specific method for co-culturing PTC with drugs in step two includes the following steps:

[0016] 2.1 After the PTC micro-tumor model is successfully cultured, filter and centrifuge to collect micro-tumor PTC with a diameter greater than 40 μm, resuspend and inoculate it with 100 μL of PTC gastric cancer medium, then add 120 μL of culture solution containing different drug combinations, and co-culture it in an incubator at 37 °C and 5% CO2. Repeat each drug combination 3 times;

[0017] Furthermore, the specific method for statistics and analysis in step three includes the following steps:

[0018] 3.1 At the beginning of the co-culture of PTC and drugs and after 168 hours of co-culture, collect the PTC area through the PTC micro-tumor image acquisition and analysis system, and compare the difference in the area between the two times P A , and evaluate the effects of different drug combinations on PTC;

[0019] 3.2 Add CellTiter-Glo reagent to the test sample and mix well. After standing at room temperature for 20 min, detect the cell viability with a microplate reader, compare the effects of different drug combinations on the PTC cell viability, and evaluate the effects of different drug combinations on PTC.

[0020] Furthermore, if the tissue sample in step 1.1 is a surgical or biopsy tissue, it needs to be more than 20 mg, and if it is a puncture tissue, it needs to be more than three pieces.

[0021] Furthermore, for the malignant effusion sample in step 1.1, choose the erythrocyte lysate for erythrocyte lysis according to the sample situation.

[0022] Furthermore, the enzyme in the digestive solution in step 1.1 is a mixed solution of collagenase I, II, and IV.

[0023] Furthermore, during the digestion process in step 1.1, pipette and mix every 10 min.

[0024] Furthermore, the drug combinations in step 2.1 include PF regimen (cisplatin + 5-fluorouracil), XELOX regimen (oxaliplatin + 5-fluorouracil), TF regimen (paclitaxel + 5-fluorouracil), and DF regimen (docetaxel + 5-fluorouracil).

[0025] Further, the effective drug concentrations of cisplatin, 5-fluorouracil, oxaliplatin, paclitaxel, and docetaxel in step 2.1 are: 9 μmol / L, 2 μmol / L, 4 μmol / L, 0.1 μmol / L, and 0.03 μmol / L, respectively.

[0026] Further, in step 3.1, when P A < 0.7, it is judged that the PTC drug sensitivity test result indicates that the patient may be sensitive to the drug regimen.

[0027] The present invention adopts the above technical solutions, and compared with the prior art, has the following technical effects:

[0028] The present invention uses the drug sensitivity test based on the PTC in vitro model as an evaluation method for the drug sensitivity of gastric cancer patients, and the evaluation result is closer to the therapeutic effect of the drug on gastric cancer in the actual clinical scenario. By simultaneously evaluating multiple commonly used chemotherapy drug regimens for gastric cancer, the sensitivity differences of different chemotherapy regimens to the PTC of specific patients can be more intuitively reflected, assisting patients to accurately select chemotherapy regimens, and having better clinical reference value. Description of the Drawings

[0029] Figure 1 It is the PTC model of gastric cancer for patient GC-01; Detailed Embodiments

[0030] The present invention will be clearly and completely described below with reference to the drawings and specific embodiments.

[0031] Example 1

[0032] Patient GC-01, male, 69 years old, postoperative tissue sample of gastric cancer

[0033] 1. A total of 1.53 g of the patient's surgical tissue sample was obtained, fat and necrotic tissue were removed and cut into minced meat, digestive juice was added, dissociation was carried out at 37 °C for 60 min to obtain a dispersed cell population, dissociation was terminated by adding a digestion termination solution, filtered through a 40-μm filter mesh, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and resuspended with a special medium for gastric cancer PTC, and inoculated into a low-attachment well plate for culture at a density of 10 5 cells / cm 2 of density.

[0034] 2. After 3 days of culture of the dispersed cells, stable PTC microtumor cell clusters were formed by self-assembly ( Figure 1) Filter and centrifuge to collect microtumors PTC with a diameter of more than 40 μm, inoculate the PTC into a new low-attachment well plate, and then add culture media containing drugs of PF, XELOX, and DF regimens into different wells respectively. Repeat each regimen in 3 wells, photograph with a microscope and calculate the PTC area of each well, and then place it in an incubator at 37 °C and 5% CO2 for 168 h.

[0035] 3. After the co-culture is completed, photograph each well again using a microscope, and calculate the ratio P of the PTC area before and after adding drugs through a microtumor image analysis system. A , and then calculate the drug killing efficiency of the corresponding drug regimen. The results of in vitro drug sensitivity testing of PTC show that the drug killing efficiencies of PF, XELOX, and DF on the PTC of patient GC-01 are 87%, 90%, and 85% respectively (Table 1).

[0036] 4. The results of the PTC drug sensitivity test of patient GC-01 suggest that the patient is sensitive to the platinum-containing PF and XELOX regimens and the taxane-containing DF regimen, and the clinical choice can be made in combination with the actual situation such as the drug resistance of the patient.

[0037] Example 2

[0038] Patient GC-02, female, 62 years old, postoperative tissue sample of gastric cancer

[0039] 1. Obtain a total of 0.62 g of the patient's surgical tissue sample, remove fat and necrotic tissue and cut it into minced meat, add digestive juice, dissociate at 37 °C for 40 min to obtain a dispersed cell population, add a digestion termination solution to terminate dissociation, filter through a 40 μm filter net, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend with a special medium for gastric cancer PTC, and inoculate into a low-attachment well plate for culture at a density of 10 5 cells / cm 2 .

[0040] 2. After 3 days of culture of the dispersed cells, stable PTC microtumor cell clusters are formed through self-assembly. Filter and centrifuge to collect microtumors PTC with a diameter of more than 40 μm, inoculate the PTC into a new low-attachment well plate, and then add culture media containing drugs of XELOX and TF regimens into different wells respectively. Repeat each regimen in 3 wells, photograph with a microscope and calculate the PTC area of each well, and then place it in an incubator at 37 °C and 5% CO2 for 168 h.

[0041] 3. After the co-culture is completed, photograph each well again using a microscope, and calculate the ratio P of the PTC area before and after adding drugs through a microtumor image analysis system. A, and then calculate the drug killing efficiency of the corresponding drug regimen. The results of in vitro drug sensitivity testing of PTC showed that the drug killing efficiencies of XELOX and TF against PTC of patient GC-02 were 44% and 2% respectively (Table 1).

[0042] 4. The results of PTC drug sensitivity test of patient GC-02 suggest that the patient is sensitive to the platinum-containing XELOX regimen, but not sensitive to the taxane-containing TF. Clinically, it is recommended to choose the platinum-containing XELOX regimen for treatment.

[0043] Example 3

[0044] Patient GC-03, male, 75 years old, gastric cancer with ascites

[0045] 1. Obtain 260 mL of ascites sample from the patient. After centrifugation, discard the supernatant. Resuspend with PBS and add lymphocyte separation medium. After centrifugation at 400 x g and 4 °C for 20 min, take the middle layer cells, centrifuge at 1500 rpm for 5 min, discard the supernatant, and resuspend with the special medium for gastric cancer PTC. Inoculate into a low-attachment well plate at a density of 10 5 cells / cm 2 and culture.

[0046] 2. After 3 days of culture of the dispersed cells, stable PTC microtumor cell clusters are formed by self-assembly. Filter and centrifuge to collect microtumor PTC with a diameter of more than 40 μm. Inoculate the PTC into a new low-attachment well plate, and then add culture solutions containing drugs of PF, XELOX, and DF regimens into different wells respectively. Repeat each regimen in 3 wells. Take pictures with a microscope and calculate the PTC area of each well, and then place it in an incubator at 37 °C and 5% CO2 for 168 h.

[0047] 3. After the co-culture is completed, take pictures of each well again with a microscope, and calculate the ratio P of the PTC area before and after adding drugs through the microtumor image analysis system A , and then calculate the drug killing efficiency of the corresponding drug regimen. The results of in vitro drug sensitivity testing of PTC showed that the drug killing efficiencies of PF, XELOX, and DF against PTC of patient GC-03 were 19%, 14%, and 42% respectively (Table 1).

[0048] 4. The results of PTC drug sensitivity test of patient GC-03 suggest that the patient is not sensitive to the platinum-containing PF and XELOX regimens, but is sensitive to the taxane-containing DF regimen. Clinically, it is recommended to choose the taxane-containing DF regimen for treatment.

[0049] Table 1 Summary of PTC drug sensitivity test results of different gastric cancer patients

[0050]

[0051] Note: Red indicates that the drug sensitivity test result suggests that the patient is sensitive to this treatment plan; " / " indicates that this treatment plan has not been tested.

[0052] The above are only the preferred embodiments of the present invention, and any equivalent substitutions or transformations made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims

1. A method for precisely guiding chemotherapy drug use in gastric cancer patients with the assistance of PTC, characterized in that, It includes the following steps: Step 1: Collect fresh tumor samples from gastric cancer patients and construct PTC micro-tumor models; Step 2: Co-culture the constructed PTCs with different drug combinations for 168 hours for drug sensitivity tests; Step 3: After the drug sensitivity tests, count and analyze the cell viability of PTC micro-tumors and the area changes before and after the tests; Step 4: Compare the sensitivity differences of PTCs from the same patient to different drug combinations, and combine with the patient's clinical manifestations to assist in accurately selecting chemotherapy regimens.

2. The method according to claim 1, characterized in that The fresh tumor samples in Step 1 include, but are not limited to, postoperative tissues, biopsy tissues such as gastroscopy and puncture, and malignant effusion samples of pleural and peritoneal fluids.

3. The method according to claim 1, wherein The construction of the PTC micro-tumor model in Step 1 includes the following steps: Step 1: Disperse fresh tumor samples from gastric cancer patients into single cells by physical shearing / enzyme digestion; Step 2: Inoculate the dispersed cells in Step 1 into a special medium for gastric cancer PTC at a density of 10 5 cells / cm 2 and culture them, enabling the patient-derived tumor cells to self-assemble with other component cells to form a PTC microtumor model.

4. The gastric cancer PTC special culture medium according to claim 3, wherein, Using AdvancedDMEM medium as the base, add components including 1 mM hydrogen ion buffer (HEPES), 1×GlutaMAX, 100 U / mL penicillin-streptomycin, 1×B27, 1×non-essential amino acids, 40 ng / mL epidermal growth factor (EGF), 20 ng / mL fibroblast growth factor (FGF-basic), 500 ng / mL R-spondin 1, 100 ng / mL Noggin, 10 mM nicotinamide, 0.5 μM A83-01, 10 μM SB202190, 10 μM ROCK inhibitor (Y-27632-07), 1 mM N-acetyl-L-cysteine, 30 ng / mL hepatocyte growth factor (HGF), 5 ng / mL MSP, 10 nM prostaglandin E-2, 100 mg / ml Primocin, 20 μM CHIR99021, 250 ng / mL Wnt3a, and 10 nM gastrin.

5. The method according to claim 1, wherein The different drug combinations used for drug sensitivity tests in Step 2 are PF, XELOX, TF, and DF regimens.

6. The pharmaceutical combination PF according to claim 5, wherein Add 9 μmol / L cisplatin and 2 μmol / L 5-fluorouracil to the co-culture system with PTC.

7. The pharmaceutical combination XELOX according to claim 5, characterized in that, Add 4 μmol / L oxaliplatin and 2 μmol / L 5-fluorouracil to the co-culture system with PTC.

8. The pharmaceutical combination TF according to claim 5, wherein Add 0.1 μmol / L paclitaxel and 2 μmol / L 5-fluorouracil to the co-culture system with PTC.

9. The pharmaceutical combination DF according to claim 5, wherein Add 0.03 μmol / L docetaxel and 2 μmol / L 5-fluorouracil to the co-culture system with PTC.

10. The method according to claim 1, characterized in that, The counting and analysis in Step 3 include the following steps: Step 1: At the start of the co-culture of PTC and the drug, and after 168 hours of co-culture, collect the area of PTC through the PTC micro-tumor image acquisition and analysis system, and compare the difference in the areas between the two times, P A , and evaluate the effects of different drug combinations on PTC; Step 2: Add CellTiter-Glo reagent to the test samples and mix well. After standing at room temperature for 20 min, detect the cell viability with a microplate reader, compare the effects of different drug combinations on the cell viability of PTC, and evaluate the effects of different drug combinations on PTC.

11. According to the method described in claim 1, wherein, The sensitivity difference in the fourth step is based on the PTC area ratio P before and after drug addition A and the comprehensive result of the cell viability value.

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