Methods for assessing anticancer drug sensitivity based on patient-derived tumor tissue
By using a three-dimensional culture system based on patient-derived tumor tissue and flow cytometry, the problems of speed and heterogeneity simulation in drug sensitivity assessment in existing technologies have been solved, enabling rapid and accurate personalized medication guidance, especially for the precision treatment of brain tumors.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
- Filing Date
- 2026-04-03
- Publication Date
- 2026-06-30
AI Technical Summary
Current technologies lack an in vitro drug sensitivity assessment platform that can achieve high throughput, speed, and mimic the tumor microenvironment and heterogeneity in vivo as closely as possible. Traditional models cannot accurately predict the responsiveness of patient tumors to chemotherapy or targeted drugs, and they also suffer from species differences and high costs.
A three-dimensional support culture system based on patient-derived tumor tissue was used. Tumor tissue fragments were prepared for in vitro culture, and after adding anticancer drugs, they were digested to obtain single-cell suspensions. The cell viability was detected by flow cytometry to assess drug sensitivity.
It preserves tumor heterogeneity and microenvironment to the greatest extent, has a short operation cycle, and can quickly and accurately guide individualized clinical medication, especially suitable for solid tumors such as brain tumors.
Smart Images
Figure CN122303367A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cancer research, and more particularly to a method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue. Background Technology
[0002] In the clinical treatment and research of malignant tumors, especially solid tumors such as gliomas, the development of personalized treatment strategies is crucial. One of the core challenges lies in how to accurately predict the responsiveness of a patient's tumor to specific chemotherapy or targeted drugs before treatment. Traditional preclinical drug evaluation models have significant limitations. Immortalized tumor cell lines undergo genetic and phenotypic drift during long-term culture, failing to represent the heterogeneity of tumors in a patient's body. While patient-derived xenograft (PDX) models can preserve tumor characteristics relatively well, they are time-consuming, costly, have unstable success rates, and exhibit species differences, making them difficult to meet the needs of rapid clinical decision-making. Two-dimensional culture models disrupt the three-dimensional structure of tumor tissue and cell-cell and cell-matrix interactions, leading to significant deviations between drug penetration and cellular response mechanisms and the actual in vivo conditions.
[0003] Therefore, current technologies lack a high-throughput, rapid, and in vitro drug sensitivity assessment platform that can simulate the tumor microenvironment and heterogeneity in vivo as closely as possible. Developing a simple, short-cycle method that can obtain results directly from fresh patient tissue is urgently needed to advance precision medicine, optimize clinical medication regimens, improve treatment efficiency, and reduce the side effects of ineffective treatments.
[0004] Therefore, this invention proposes a method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue. Summary of the Invention
[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue includes the following steps: S1: Obtain fresh tumor tissue samples from the patient; S2: Prepare tumor tissue samples into tumor tissue fragments; S3: Place tumor tissue fragments in a three-dimensional support culture system for in vitro culture; S4: Add the anticancer drug to be tested during the culture process; S5: After culturing, digest tumor tissue fragments to obtain a single-cell suspension; S6: Flow cytometry is used to detect single-cell suspensions and the sensitivity of anticancer drugs is assessed by analyzing cell viability.
[0007] Preferably, the tumor tissue in S1 originates from a brain tumor, including ependymoma, DIPG, medulloblastoma, or glioma; the size of the tumor tissue fragment is 1±5% mm³.
[0008] Preferably, it also includes the steps of cryopreserving and thawing tumor tissue fragments before culture; Frozen storage refers to storage at -80℃; The resuscitation process involved rapid resuscitation in a 37°C water bath followed by washing with pre-warmed liquid.
[0009] Preferably, in step S3, the three-dimensional support culture system is a three-dimensional gel matrix containing Matrigel and / or collagen; the culture conditions are 37°C and 5% CO2; and the culture time is 48 to 72 hours.
[0010] Preferably, in step S4, the anticancer drug to be tested includes a PARP inhibitor and / or a topoisomerase I inhibitor; specifically, it is olaparib monotherapy, topotecan monotherapy, or a combination of olaparib and topotecan.
[0011] Preferably, in step S5, the digestion step is as follows: Accutase is added to the cultured tumor tissue fragments, digestion is carried out at 37°C for 1 hour, the tissue is pipetted during digestion, and centrifuged and filtered after digestion to obtain a single-cell suspension.
[0012] Preferably, in step S6, the flow cytometry detection includes live / dead staining using DAPI or Zombie dye.
[0013] Preferably, in step S6, the flow cytometry detection further includes staining for intracellular markers; specifically, this includes staining with GFAP antibody after perforating the cells using Triton.
[0014] Preferably, in step S6, the criterion for evaluating drug sensitivity is: if the proportion of dead cells is higher and / or the proportion of live cells is lower in the drug-treated group compared with the untreated control group, then it is considered sensitive.
[0015] Application of a method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue in the preparation of products or systems for personalized drug screening, evaluation of combination therapy regimens, or preclinical drug screening.
[0016] The beneficial effects of this invention are as follows: This invention can preserve tumor heterogeneity and microenvironment to the greatest extent, has a short operation cycle, and can provide rapid and accurate guidance for individualized clinical medication, especially suitable for solid tumors such as brain tumors. Attached Figure Description
[0017] Figure 1 This is a flowchart of the anticancer drug sensitivity assessment method based on patient-derived tumor tissue proposed in this invention. Detailed Implementation
[0018] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.
[0019] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.
[0020] Example 1: 1. Sample Source and Processing Tumor tissues are obtained from fresh samples taken from patients during surgery or after biopsy in the hospital. After acquisition, the samples are immediately placed in a pre-cooled collection solution and transported to the laboratory on ice to shorten the time from ex vivo to processing and ensure tissue viability.
[0021] 2. Preparation of patient-derived tumor tissue fragments (PDTFs) Under sterile, low-temperature conditions, tumor tissue is placed in a culture dish to remove visible necrotic areas, blood clots, and non-tumor components. Subsequently, the tumor tissue is cut into small tissue pieces of approximately 1 mm³ using a sterile scalpel. During the cutting process, squeezing, tearing, and tissue drying should be avoided as much as possible. To reduce the impact of intratumoral heterogeneity on experimental results, tissue pieces from different regions can be mixed before being used in subsequent experiments.
[0022] 3. Cryopreservation and thawing of PDTFs The prepared PDTFs were added to a cryopreservation solution containing serum and dimethyl sulfoxide (such as Bambanker cryopreservation solution) and stored at -80°C using a programmed cooling method, or transferred to liquid nitrogen for long-term storage. Before the experiment, the cryopreservation tubes were rapidly thawed in a 37°C water bath (about 1 minute) and removed when only a small amount of ice crystals remained. The tissues and cryopreservation solution were transferred together into a centrifuge tube, pre-warmed culture medium was added and mixed well, and the tubes were centrifuged at 500×g for 5 minutes. The supernatant was discarded to remove any residual cryopreservation solution and obtain the thawed PDTFs.
[0023] 4. Three-dimensional in vitro culture of PDTFs A three-dimensional culture medium was constructed using Matrigel and collagen. The specific procedure was as follows: a bottom layer of the medium was added to a pre-cooled 96-well plate and incubated at 37°C to solidify it; then, one PDTF was placed in each well. Next, the top layer of medium was added, and the plate was incubated again at 37°C to completely solidify the gel; finally, an appropriate amount of tumor culture medium was added. To account for tumor heterogeneity, PDTFs of different sizes, colors, and morphologies should be evenly distributed among the different treatment groups; the culture plates were incubated at 37°C in a 5% CO2 incubator for 48 hours, which could be extended to 72 hours depending on the sample condition.
[0024] 5. Drug intervention Add the anticancer drug to be tested to the culture system; the following groups can be set up: untreated control group, Olaparib monotherapy group, Topotecan monotherapy group, and Olaparib and Topotecan combination therapy group; after adding the drug, continue to culture in the incubator until the predetermined time.
[0025] 6. Preparation of single-cell suspensions After culture, PDTFs were collected. Accutase digestion solution was added, and the cells were digested at 37°C and 5% CO2 for about 1 hour. During digestion, the cells were gently pipetted and mixed every 10 minutes, with intermittent shaking to promote complete tissue dissociation. After digestion, serum-containing culture medium was added to stop the reaction, and the cells were centrifuged at 500×g for 5 minutes. The supernatant was discarded. The cells were resuspended in culture medium and filtered through a 70μm cell filter to obtain a single-cell suspension.
[0026] 7. Flow cytometry detection 7.1 Live and Dead Staining One of the following two options can be adopted: Option 1 (DAPI staining): Centrifuge an appropriate amount of single-cell suspension, discard the supernatant, add 500 μL of PBS containing 10% heat-inactivated FBS to resuspend the cells; add 0.5 μL of DAPI dye in the dark, incubate for several minutes, and then it is ready for instrumental detection.
[0027] Option 2 (Zombie staining): Centrifuge an appropriate amount of single-cell suspension, discard the supernatant, and resuspend the cells in 100 μL PBS (containing 10% heat-inactivated FBS); add 0.35 μL of Zombie dye and incubate at room temperature in the dark for 25 minutes, gently pipetting intermittently during incubation. After incubation, add 1 mL of PBS, centrifuge at 600×g for 5 minutes, and discard the supernatant.
[0028] 7.2 Intracellular staining (using GFAP as an example) If intracellular marker staining is required, after completing Zombie staining, add 200 μL of 0.02% Triton X-100 to the cell pellet to perforate the cell membrane, and incubate at room temperature for 15 minutes. Stop the reaction by adding 1 mL of PBS, centrifuge at 600×g for 5 minutes, and discard the supernatant. Add 5 μL of GFAP antibody and incubate at 37°C in the dark for 45 minutes. After staining, centrifuge at 600×g for 5 minutes, discard the supernatant, and resuspend the cells in 200 μL of PBS (containing 10% heat-inactivated FBS) for further analysis.
[0029] 7.3 On-machine testing and analysis Flow cytometry was used for detection. First, gating was performed based on forward scattered light (FSC) and side scattered light (SSC) to exclude cell debris. Then, FSC-A / FSC-H gating was used to exclude cell aggregates and delineate single-cell populations. Next, live and dead cells were distinguished based on the fluorescence signals from DAPI or Zombie staining. If intracellular staining was performed, the expression ratio of specific markers (such as GFAP) in live cells or specific cell subpopulations could be further analyzed.
[0030] 8. Result Interpretation and Statistical Analysis Drug sensitivity is assessed by comparing cell viability (e.g., increased proportion of dead cells or decreased proportion of live cells) between each drug treatment group and the untreated control group; if a treatment group shows a higher proportion of dead cells and / or a lower proportion of live cells, the patient's tumor is considered sensitive to the drug or combination regimen.
[0031] Experiments should be biologically replicated, and data should be expressed as mean ± standard deviation. Statistical analysis should be performed using GraphPad Prism or R software. Intergroup comparisons should be performed using independent samples t-tests or one-way ANOVA, with P < 0.05 considered statistically significant.
[0032] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue, characterized in that, Includes the following steps: S1: Obtain fresh tumor tissue samples from the patient; S2: Prepare tumor tissue samples into tumor tissue fragments; S3: Place tumor tissue fragments in a three-dimensional support culture system for in vitro culture; S4: Add the anticancer drug to be tested during the culture process; S5: After culturing, digest tumor tissue fragments to obtain a single-cell suspension; S6: Flow cytometry is used to detect single-cell suspensions and the sensitivity of anticancer drugs is assessed by analyzing cell viability.
2. The method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue according to claim 1, characterized in that, The tumor tissue in S1 originates from a brain tumor, including ependymoma, DIPG, medulloblastoma, or glioma; the size of the tumor tissue fragment is 1 ± 5 mm³.
3. The method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue according to claim 1, characterized in that, It also includes the steps of cryopreserving and thawing tumor tissue fragments before culture; Frozen storage refers to storage at -80℃; The resuscitation process involved rapid resuscitation in a 37°C water bath followed by washing with pre-warmed liquid.
4. The method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue according to claim 1, characterized in that, In step S3, the three-dimensional support culture system is a three-dimensional gel matrix containing Matrigel and / or collagen; the culture conditions are 37°C and 5% CO2; and the culture time is 48 to 72 hours.
5. The method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue according to claim 1, characterized in that, In step S4, the anticancer drugs to be tested include PARP inhibitors and / or topoisomerase I inhibitors; specifically, olaparib monotherapy, topotecan monotherapy, or a combination of olaparib and topotecan.
6. The method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue according to claim 1, characterized in that, In step S5, the digestion step is as follows: Accutase is added to the cultured tumor tissue fragments, digestion is carried out at 37°C for 1 hour, the tissue is pipetted during digestion, and centrifuged and filtered after digestion to obtain a single-cell suspension.
7. The method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue according to claim 1, characterized in that, In step S6, flow cytometry detection includes live / dead staining using DAPI or Zombie dye.
8. The method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue according to claim 1, characterized in that, In step S6, flow cytometry detection also includes staining for intracellular markers; specifically, this includes staining with GFAP antibody after perforating the cells using Triton.
9. The method for assessing the sensitivity of anticancer drugs based on patient-derived tumor tissue according to claim 1, characterized in that, In step S6, the criteria for assessing drug sensitivity are: if the proportion of dead cells is higher and / or the proportion of live cells is lower in the drug-treated group compared to the untreated control group, then the group is considered sensitive.
10. The application of the anticancer drug sensitivity assessment method based on patient-derived tumor tissue as described in any one of claims 1-9 in the preparation of products or systems for personalized drug screening, evaluation of combination therapy regimens, or preclinical drug screening.