Construction method and application of in-vivo and in-vitro drug screening platform for treating breast cancer

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a construction method and application of an in-vivo and in-vitro drug screening platform for treating breast cancer, and the construction method comprises the following steps: constructing a primary tumor cell (PTC) model and carrying out in-vitro drug screening, constructing a micro-human xenotransplantation (Micro-PDX) model and carrying out in-vivo drug screening, and carrying out in-vivo drug screening. And then selecting an active small molecule drug with half tumor cell inhibition rate or more in vivo and in vitro by combining an in-vitro drug screening result of the PTC model and an in-vivo drug screening result of the Micro-PDX model as a final drug combination for performing subsequent treatment on a patient. The rapid and accurate drug screening system is constructed by integrating PTC and Micro-PDX models, in-vivo and in-vitro drug effect evaluation can be completed within 10-12 days, screening and discovery of potential anti-tumor drugs are further deepened, and the clinical reasonable drug use guiding capacity for breast cancer patients is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for constructing and applying an in vitro and in vivo drug screening platform for treating breast cancer. Background Art

[0002] Breast cancer is the most common malignant tumor in women. As the disease progresses and treatment deepens, recurrence, metastasis, and tumor resistance occur, resulting in a lack of effective treatment options, which is a major challenge currently facing clinical practice. In in vitro drug screening studies, existing traditional 2D tumor cell lines cannot simulate the patient's disease state and lack tumor heterogeneity and tumor microenvironment. In addition, the screening results are unreliable. The tumor cells do not come from the patient and cannot guide the next step of clinical treatment. In in vivo drug screening studies, traditional 2D tumor cell line animal models do not have the individualized characteristics of patients, and drug screening results cannot reflect the actual clinical situation. The more advanced human xenograft model (PDX), although its genetic characteristics are well preserved and have a high degree of clinical similarity, however, the traditional PDX construction method directly cuts up the patient's tumor tissue and uses a cannula needle to inoculate subcutaneously in mice. The success rate of breast cancer PDX is extremely low and time-consuming, making it difficult to meet the needs of clinical drug screening. Summary of the Invention

[0003] To provide a method for constructing an integrated in vitro and in vivo drug screening system, the present invention provides a method for constructing an in vitro and in vivo drug screening platform for the treatment of breast cancer. The PTC model is used instead of the traditional 2D cell line for in vitro drug screening; the Micro-PDX model is used instead of the traditional PDX model for in vivo drug screening; and the screening results of the PTC model and the Micro-PDX model are combined to determine active small molecule drugs with a tumor cell inhibition rate of more than half in vivo and in vitro as the final drug combination for subsequent treatment of patients.

[0004] The present invention provides a method for establishing a drug screening platform by combining a Micro-PDX model with a PTC model, comprising the following steps: Establish a PTC model and conduct in vitro drug screening: Resuspend tumor cells in complete culture medium to obtain a PTC suspension. Inoculate 90 μL to 100 μL of the PTC suspension into each well and culture for 24 to 48 hours before in vitro drug screening. Constructing a Micro-PDX model and conducting in vivo drug screening: Tumor tissue is digested to create a tumor cell suspension, which is then infused into a hollow fiber membrane, heat-sealed, and cut into several fiber tubes. One fiber tube was taken and implanted on both sides of the back of immunodeficient mice to obtain a Micro-PDX mouse model, namely a breast cancer Micro-PDX model, for in vivo drug screening; Combine the in vitro drug screening process of the PTC model and the in vivo drug screening process of the Micro-PDX model as a drug screening platform; The drug screening criteria of the drug screening platform are: select active small molecule drugs with a tumor cell inhibition rate of more than half in both the in vitro drug screening results of the PTC model and the in vivo drug screening results of the Micro-PDX model as the drug combination for subsequent treatment of patients.

[0005] The Micro-PDX of the present invention digests the patient's tumor tissue and seals it into a fiber tube, which is then embedded subcutaneously in mice. The success rate is increased to 90% to 95%, and the modeling time is shortened. The drug screening system that integrates PTC and Micro-PDX models can perform in vivo and in vitro efficacy evaluation in a short period of time (PTC: 3 to 5 days, Micro-PDX: 7 days), saving time, screening potential therapeutic drugs, and guiding rational clinical drug use.

[0006] Furthermore, the in vitro drug screening criteria of the PTC model are: screening for active small molecule drugs with an inhibition rate of more than half of the tumor cells.

[0007] Furthermore, the in vivo drug screening criteria of the Micro-PDX model are: screening for active small molecule drugs with an inhibition rate of more than half of the tumor cells.

[0008] Furthermore, the culture conditions were: 37 °C, 5% CO2.

[0009] Furthermore, tumor cells are obtained by mincing the tumor tissue and performing digestion and dissociation treatment.

[0010] Furthermore, the complete culture medium system is: Advanced DMEM / F12 + 5% patient autologous serum + 5% FBS + EGF (final concentration 10 ng / mL) + FGF4 (final concentration 5 ng / mL) + FGF10 (final concentration 5 ng / mL) + 1% double antibody + GlutaMAX (final concentration 2 mM) + HEPES (final concentration 10 mM).

[0011] The present invention also provides a use of a drug combination in preparing a drug for treating breast cancer, wherein the drug combination is obtained by screening using the method of claim 1; The drug combination is a combination of cyclophosphamide, docetaxel, trastuzumab and pyrotinib; or, a combination of lapatinib, apellisib, and trametinib.

[0012] Furthermore, the dosage of the drug combination is as follows: 25 mg / kg cyclophosphamide, 20 mg / kg docetaxel, 5 μg / g trastuzumab, and 10 mg / kg pyrotinib; or, 100 mg / kg lapatinib, 25 mg / kg apellisinib, and 2 mg / kg trametinib.

[0013] The present invention also provides a method for screening drugs for treating breast cancer, which uses the drug screening platform to perform screening; The screening criteria are: select active small molecule drugs with a tumor cell inhibition rate of more than half in both the in vitro drug screening results of the PTC model and the in vivo drug screening results of the Micro-PDX model as the drug combination for subsequent treatment of patients.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention effectively improves the PDX model: Traditional PDX models directly mince patient tumor tissue and inoculate it subcutaneously in mice using a trocar. The success rate of breast cancer PDX is approximately 20% to 30%. However, the present invention's Micro-PDX model digests the patient's tumor tissue, seals it into a fiber tube, and then embeds it subcutaneously in the mouse. This increases the success rate to 90% to 95%, and shortens the modeling time from 3 to 6 months for PDX to 7 days for the present invention's Micro-PDX, reducing both experimental and time costs. This demonstrates that the use of Micro-PDX in the present invention not only improves the success rate of in vivo xenograft models but also significantly shortens the drug screening cycle. This present invention is the first to utilize a drug screening system that integrates PTC and Micro-PDX models, enabling drug screening in a short period of time (PTC: 3 to 5 days, Micro-PDX: 7 days), saving valuable time for patients with advanced cancer, screening potential therapeutic drugs, and guiding rational clinical drug use. The present invention screened and obtained drug combinations for treating tumors, which are a combination of cyclophosphamide, docetaxel, trastuzumab and pyrotinib; and a combination of lapatinib, apellisinib and trametinib. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 Construct a flow chart for an integrated in vitro and in vivo drug screening platform.

[0017] Figure 2 This is a statistical chart of PTC drug screening results for patients with triple-negative breast cancer.

[0018] Figure 3 This is a statistical chart of PTC drug screening results for patients with triple-positive breast cancer.

[0019] Figure 4 This is a statistical chart of Micro-PDX drug screening results for patients with triple-negative breast cancer.

[0020] Figure 5 This is a statistical chart of the Micro-PDX drug screening results for patients with triple-positive breast cancer. DETAILED DESCRIPTION

[0021] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0022] The present invention constructs an in vitro and in vivo integrated drug screening system PTC+Mirco-PDX for screening individualized precision therapeutic drugs for patients with advanced breast cancer. This drug screening system solves the problem that traditional 2D cell lines do not have patient individualization characteristics through the constructed PTC model; and solves the problems of traditional CDX models and PDX models such as long time consumption, low success rate, and low cost through the constructed Micro-PDX model. It is an effective clinical drug guidance model with potential application value. Screening drugs for the treatment of breast cancer through the described in vitro and in vivo integrated drug screening system greatly improves the clinical individualized precision treatment capabilities, benefits a large number of cancer patients, and has extremely high clinical and social value.

[0023] Example 1: Construction and application of an in vitro and in vivo drug screening platform for treating breast cancer 1. Establishment of PTC and In Vitro Drug Screening 1. Establishment of PTC Breast tumor tissue was minced into 2 mm pieces. 3Dissociate the tumor tissue into small pieces and place them in a centrifuge tube. Add 4 mL of serum-free Advanced DMEM / F12 medium (Gibco) containing 1% double-streptomycin (final concentration of 100 U / mL penicillin and 0.1 mg / mL streptomycin) and 1 mL of 12.5 mg / mL Liberase TH digestion enzyme. Shake the tube in a 37°C waterbath for 50 minutes. Once the tumor tissue is completely dissociated, repeatedly pipette the tissue to dissociate the tumor cells. Let the tube stand for 3 minutes to allow the tumor cells to settle to the bottom of the centrifuge tube. Filter the supernatant through a 70 μm cell sieve to remove cell clumps and debris, ensuring a single-cell suspension. After filtration, collect the filtrate and add 5 mL of serum-free Advanced DMEM / F12 medium (Gibco). Filter again to collect the cell suspension. Repeat this step five times. The final filtered cell suspension was centrifuged at 1200 rpm for 5 minutes at 4°C. The cell pellet was collected and resuspended in 3 mL of PBS containing 1% anti-antibody (antibody) for washing. The cell pellet was centrifuged again at 1200 rpm for 5 minutes at 4°C. The cells were then resuspended in complete culture medium to prepare a PTC suspension. The complete culture medium system consisted of Advanced DMEM / F12 supplemented with 5% patient autologous serum, 5% FBS, 10 ng / mL EGF, 5 ng / mL FGF4, 5 ng / mL FGF10, 1% anti-antibody (antibody) supplemented with 2 mM GlutaMAX, and 10 mM HEPES. The PTC suspension was then plated at a density of 100 μL / 3,000 cells per well of a 96-well plate. The plate was then incubated at 37°C, 5% CO2, and incubated for 24 hours before drug screening.

[0024] 2. PTC in vitro drug screening Two representative patients with advanced breast cancer were selected for in vitro PTC drug screening, one of whom had triple-negative breast cancer (Basal-like). The present invention selected the patient's proposed subsequent treatment plan, the treatment plan recommended by NCCN and CSCO, and small molecule inhibitors of the corresponding signaling pathway selected based on the patient's genetic profile for in vitro screening. These drugs mainly included conventional chemotherapy drugs, molecular targeted drugs, and P53 agonists. The treatment plan used in this example is shown in Table 1.

[0025] Relevant drugs were selected for in vitro PTC drug screening. Each drug was initially explored in five replicate wells with three concentration gradients (0.1 μM, 1 μM, and 10 μM). 100 μL of drug was added to each well of a 96-well plate and incubated for 48 hours. Subsequently, 20 μL of CCK-8 was added to each well, and the cells were incubated in an incubator for 3 hours. The absorbance at 450 nm was measured using a microplate reader.

[0026] Cell inhibition rate (%) = (average absorbance of control group - average absorbance of drug group) / (average absorbance of control group - average absorbance of blank group) × 100%. Graph Pad 8.0 was used for inhibitory curve fitting and calculation of IC50: Log (inhibitor) versus response-variable slope (four parameters).

[0027] The results are as follows Figure 2 As shown, docetaxel + epirubicin + cyclophosphamide, gemcitabine + cisplatin, and paclitaxel + carboplatin demonstrated strong inhibitory activity, while olaparib (a PARP inhibitor) and apellix (a PI3K inhibitor) showed poor activity. The remaining regimens showed some activity at higher concentrations. In another case with triple-positive breast cancer (HER2+, HR+), in vitro drug screening was performed using a combination of HER2 monoclonal antibodies, HER2-targeting small molecule tyrosine kinase inhibitors, and chemotherapeutic agents. The treatment regimens are shown in Table 2.

[0028] The results are as follows Figure 3 As shown in the data, the regimens of trastuzumab + pertuzumab + docetaxel + carboplatin, cyclophosphamide + docetaxel + trastuzumab + pyrotinib, lapatinib + apellisib + trametinib showed good therapeutic effects; capecitabine + lapatinib showed inhibitory activity at a higher drug concentration, and the other regimens had poor effects.

[0029] 2. Micro-PDX Establishment and In Vivo Drug Screening 1. Establishment of Micro-PDX The present invention digests breast tumor tissue obtained from patients with advanced breast cancer after surgery, prepares a cell suspension, and injects it into a hollow fiber membrane. The two ends of the hollow fiber membrane are heat-sealed and cut into fiber tubes of 2 cm in length. The hollow fiber membrane has a diameter of 1 mm and a pore size of 500 kD, which can allow biomacromolecules and drug molecules to enter and exit normally, but restricts the entry and exit of cells. The prepared fiber tubes are buried on both sides of the mouse subcutaneously. After seven consecutive days of drug administration, the fiber tubes are removed and the activity of tumor cells in the fiber tubes is tested. The specific steps are as follows:

[0030] (1) Tumor tissue digestion and tumor cell extraction: Cut the tumor tissue into 2 mm pieces 3 Dissociate the tumor tissue into small pieces and place them in a centrifuge tube. Add 4 mL of serum-free Advanced DMEM / F12 medium (Gibco) containing 1% double-antibody-containing anti-tumor antibodies and 1 mL of Liberase TH digestive enzyme (12.5 mg / mL) and shake in a 37°C waterbath for 50 minutes. Once the tumor tissue is completely dissociated, repeatedly pipette the dissociated tumor tissue to separate the cells. Allow the tissue to settle to the bottom of the centrifuge tube for 3 minutes, collect the supernatant, and filter it through a 70 μm cell strainer. Add 4 mL of serum-free Advanced DMEM / F12 medium (Gibco) containing 1% double-antibody-containing anti-tumor antibodies to the tissue pellet again. Repeat the pipetting-collection-filtration process five times to obtain a cell suspension containing single tumor cells. The cell suspension was centrifuged at 4°C, 1200 rpm for 5 min, and the cell pellet was resuspended in 3 mL of PBS buffer containing 1% antibiotics for washing. The cell pellet was centrifuged again at 4°C, 1200 rpm for 5 min, and the cell pellet was collected and resuspended in complete culture medium to obtain a tumor cell suspension.

[0031] (2) Pretreatment of fiber membrane A KrosFlo polyvinylidene fluoride (PVDF) hollow fiber membrane (referred to as fiber membrane) with an inner diameter of 1 mm and a molecular weight cutoff of 500 kD was activated in 100% ethanol solution for 30 min. The fiber membrane was then completely immersed in PBS buffer and sterilized at 121°C for 20 min before use.

[0032] (3) Preparation of fiber tube The obtained tumor cell suspension was perfused into the hollow fiber membrane using a 1 mL syringe. The two ends of the hollow fiber membrane were heat-sealed using a surgical needle holder to form several fiber tubes with a length of 2 cm. The number of cells in each fiber tube was 1×10 4 It should be noted that there should be no air bubbles in the fiber tube, otherwise it will seriously affect the experimental results.

[0033] (4) Preparation of Micro-PDX Mouse Model The prepared fiber tubes were immersed in complete culture medium containing 10% FBS to prevent drying. After completion, two fiber tubes were taken and buried on both sides of the back of immunodeficient mice (BALB / c-Nude) (one on each side) to obtain the Micro-PDX mouse model.

[0034] 2. Micro-PDX in vivo drug screening Micro-PDX was used to validate the in vitro drug screening results in vivo. Several Micro-PDX mouse models were constructed according to the aforementioned method. Three mice were assigned to each group, including a control group and multiple experimental groups, numbered 1, 2, 3, etc. The control group received only the drug vehicle, while the experimental groups were divided according to different dosing regimens (treatment regimens for patients with triple-negative breast cancer are shown in Table 3, and treatment regimens for patients with triple-positive breast cancer are shown in Table 4). Mice were treated with different drug combinations for 7 consecutive days. After the 7-day experiment, the mice were sacrificed, the fiber tubes were removed, and the tubes were washed with PBS. The tubes were then transferred to a 24-well plate containing 200 μL of serum-free DMEM medium. The tubes were cut with sterile scissors to allow tumor cells to extravasate. 200 μL of CTG reaction solution was added to each well, and the plate was shaken at 80 rpm in the dark for 25 minutes. After the reaction, 100 μL of the reaction solution was transferred to a 96-well white plate and assayed using a GloMax DISCOVER microplate reader. Data were analyzed using GraphPad Prism 8.0. The CTG reaction solution was derived from the CellTiter-Glo® Luminescent Cell Viability Assay Kit.

[0035] Note: The letters in the table mean: ip intraperitoneal injection; ig oral gavage; qd once a day; qod once every other day; biw twice a week.

[0036] In vivo screening results of triple-negative breast cancer patients Figure 4 As shown, in vivo results for docetaxel + epirubicin + cyclophosphamide and gemcitabine + cisplatin were consistent with in vitro results, while the paclitaxel + carboplatin regimen showed no inhibitory activity. While olaparib and apellisib showed no activity in vitro, they showed good activity in vivo. Furthermore, the apellisib + everolimus + COTI-2 (P53 agonist) regimen showed high activity. Therefore, for this patient with triple-negative advanced breast cancer, this example recommends the following subsequent treatment regimens: docetaxel + epirubicin + cyclophosphamide and gemcitabine + cisplatin.

[0037] Note: ip intraperitoneal injection; ig gavage; qd once a day; qod once every other day; qw once a week; biw twice a week.

[0038] In vivo drug screening results for patients with triple positive syndrome Figure 5As shown, consistent with triple-negative patients, the present invention selects treatment strategies with higher activity both in vivo and in vitro: cyclophosphamide + docetaxel + trastuzumab + pyrotinib, lapatinib + apellisib + trametinib as follow-up treatment options for patients. The inconsistency of drug activity in vivo and in vitro is mainly due to the absorption, distribution, metabolism and excretion processes of the drug in the in vivo experiment, which enables the active metabolites of the drug to act on tumor cells and have higher consistency, but in vitro drug screening can predict some drug reactions more quickly and verify some in vivo screening conclusions from the side. The two screening methods complement each other and are indispensable. This also fully illustrates that the use of only a single drug screening method is likely to cause the misuse of the treatment plan, bringing poor treatment and prognosis to patients. Therefore, the present invention combines the PTC+Mirco-PDX screening results, selects treatment plans with more than half of the tumor cell inhibition rates both in vivo and in vitro, and performs subsequent treatment on patients, thereby ensuring a higher consistency of clinical treatment responsiveness, bringing hope of treatment to patients with advanced breast cancer.

[0039] This invention effectively improves the PDX model, using Micro-PDX to not only increase the success rate but also significantly shorten the drug screening cycle. Therefore, we are the first to use a drug screening system that integrates PTC and Micro-PDX models. This system enables in vitro and in vivo efficacy evaluation within a short time (PTC: 3-5 days, Micro-PDX: 7 days), saving valuable time for patients with advanced cancer, screening potential therapeutic drugs, and guiding rational clinical drug use.

[0040] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0041] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. A method for establishing a drug screening platform using a Micro-PDX model combined with a PTC model, characterized in that: The steps include: Establish a PTC model and conduct in vitro drug screening: Resuspend tumor cells in complete culture medium to obtain a PTC suspension. Inoculate 90 μL to 100 μL of the PTC suspension into each well and culture for 24 to 48 hours before in vitro drug screening. Constructing a Micro-PDX model and conducting in vivo drug screening: Tumor tissue is digested to create a tumor cell suspension, which is then infused into a hollow fiber membrane, heat-sealed, and cut into several fiber tubes. The fiber tubes were implanted into the backs of immunodeficient mice to create a Micro-PDX mouse model, a breast cancer Micro-PDX model, for in vivo drug screening. Combine the in vitro drug screening process of the PTC model and the in vivo drug screening process of the Micro-PDX model as a drug screening platform; The drug screening criteria of the drug screening platform are: select active small molecule drugs with a tumor cell inhibition rate of more than half in both the in vitro drug screening results of the PTC model and the in vivo drug screening results of the Micro-PDX model as the drug combination for subsequent treatment of patients.

2. The method for establishing a drug screening platform by combining the Micro-PDX model with the PTC model according to claim 1, characterized in that: The in vitro drug screening criteria of the PTC model are: screening for active small molecule drugs with an inhibition rate of more than half of the tumor cells.

3. The method for establishing a drug screening platform by combining the Micro-PDX model with the PTC model according to claim 1, characterized in that: The in vivo drug screening criteria of the Micro-PDX model are: screening for active small molecule drugs with an inhibition rate of more than half of the tumor cells.

4. The method for establishing a drug screening platform by combining a Micro-PDX model with a PTC model according to claim 1, characterized in that: Culture conditions were: 37 °C, 5% CO2.

5. The method for establishing a drug screening platform by combining a Micro-PDX model with a PTC model according to claim 1, characterized in that: Tumor cells are obtained by mincing tumor tissue and performing digestion and dissociation.

6. The method for establishing a drug screening platform by combining a Micro-PDX model with a PTC model according to claim 1, characterized in that: The complete culture medium system is: Advanced DMEM / F12 + 5% patient autologous serum + 5% FBS + EGF (final concentration 10 ng / mL) + FGF4 (final concentration 5 ng / mL) + FGF10 (final concentration 5 ng / mL) + 1% double antibody + GlutaMAX (final concentration 2 mM) + HEPES (final concentration 10 mM).

7. Use of a drug combination in preparing a drug for treating breast cancer, characterized in that: The drug combination is obtained by screening the method of claim 1; The drug combination is a combination of cyclophosphamide, docetaxel, trastuzumab and pyrotinib; or, a combination of lapatinib, apellisib, and trametinib.

8. Use of the drug combination according to claim 7 in preparing a drug for treating breast cancer, characterized in that: The dosage of the drug combination is as follows: 25 mg / kg cyclophosphamide, 20 mg / kg docetaxel, 5 μg / g trastuzumab, and 10 mg / kg pyrotinib; or, 100 mg / kg lapatinib, 25 mg / kg apellisinib, and 2 mg / kg trametinib.

9. A method for screening drugs for treating breast cancer, characterized in that: Screening using the drug screening platform of claim 1; The screening criteria are: select active small molecule drugs with a tumor cell inhibition rate of more than half in both the in vitro drug screening results of the PTC model and the in vivo drug screening results of the Micro-PDX model as the drug combination for subsequent treatment of patients.

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