Application of 3D automatic drug sensitivity screening system for esophageal cancer organs in esophageal cancer drug screening

Abstract

The invention provides application of an esophageal cancer organ 3D automatic drug sensitivity screening system in esophageal cancer drug screening, and belongs to the technical field of biomedicine. The drug sensitivity screening system constructed by the invention can better simulate individual genetics characteristics, stably replicates growth characteristics of source tumor tissues, provides a treatment scheme with good curative effect and small toxic and side effects for esophageal cancer patients, contributes potential value for improving poor prognosis and survival rate of advanced esophageal cancer patients, and has a wide application prospect. A theoretical basis is provided for individualized precise treatment of the patient with the advanced esophageal cancer, and a large number of medical resources are saved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an application of a 3D automated drug sensitivity screening system for esophageal cancer organoids in esophageal cancer drug screening. Background Art

[0002] Esophageal cancer is a common digestive tract tumor, with esophageal squamous cell carcinoma (ESCC) being the main type, accounting for more than 90% of esophageal cancer. 90% of esophageal cancer patients are in the middle and late stages when they are first treated. Even with multiple treatments such as surgery, radiotherapy and chemotherapy, their prognosis is still very poor, with an overall 5-year survival rate of less than 20%. With the advancement of medical technology, research on the pathogenesis of esophageal cancer has made great progress at the genomic level, and a variety of targeted therapeutic drugs have been used in clinical practice. This personalized precision prevention, precision diagnosis and precision treatment plan based on the specificity of individual genes, environment and lifestyle of esophageal cancer patients has brought huge benefits to the survival of esophageal cancer patients, but so far there is no good clinical precision treatment and efficacy monitoring technology.

[0003] The term organoid first appeared in the study of dermoid cysts. In 1977, Rheinwald et al. successfully reconstructed 3D tissue structure using artificially cultured human stem cells for the first time, pushing organoid research into a boom. Organoids are three-dimensional cell complexes with a certain spatial structure formed by the offspring cells differentiated from stem cells cultured in vitro. They have structural characteristics and functional properties similar to the source organs and can be stably expanded in an in vitro 3D culture system. In recent years, more tissue organoid construction has made breakthroughs, and now organoid models of normal or tumor tissues such as liver, pancreas, stomach, breast, and lung can be successfully constructed. In the past, the success rate of constructing traditional 2D cultured tumor tissue xenograft models varied greatly. After 3-5 generations, the growth characteristics of the source tumor tissue were lost. The success rate of constructing 3D cultured tumor organoid models is as high as 80%-90%. At the same time, the construction cost is low. Patient-derived tumor tissue can be fully cultivated in a suitable environment. It usually takes only 1-2 weeks to complete the construction of tumor organoid models. Organoid models can be expanded and cultured in vitro for a long time, and can maintain the structural function of the source organ and the stability of gene expression, which better simulates the human tumor microenvironment and is expected to be of greater help in the development of tumor drugs and the selection of the best treatment plan. In 2017, organoid technology was named one of the top ten advances and breakthroughs of the year by Nature Methods magazine, which shows that organoid technology has become a necessary technology for further advancing clinical precision tumor treatment.

[0004] In addition, in recent years, another cutting-edge science and technology has been developed that can simulate the complex microstructure, microenvironment and physiological functions of specific human organs or tumors. It is called "organ chip" (also called microphysiological system), which is also a unique and dynamic emerging field in biotechnology, and has important strategic significance for human health and the development of the bioindustry. The representative progress in the field was the first paper published in Science in 2010 by the Ingber research group of Harvard University on the lung chip, which can simulate the "breathing" process of the lung and is closer to the human in vivo environment. This micro cell culture system is developed based on micro-electromechanical processing technology, microfluidics technology and biomimetic principles. Through bionic design and micro-fine processing, the physiological microstructure and mechanical, chemical, electrical and other physiological microenvironments of specific functional units of human organs are simulated, and organ-related disease models are constructed inside the microfluidic chip, so as to better predict the pharmacological activity or biological toxicity of drugs and gain a deeper understanding of drug metabolic behavior.

[0005] Organoids are three-dimensional (3D) cell cultures that contain some key characteristics of the organs they represent. Such in vitro culture systems include a self-renewing stem cell population that can differentiate into multiple organ-specific cell types, have similar spatial organization to the corresponding organs, and can reproduce some of the functions of the corresponding organs, thereby providing a highly physiologically relevant system. Organ chips are multi-channel 3D microfluidic cell culture chips that simulate the activities, mechanical characteristics, and physiological responses of entire organs and organ systems. Organ chips use microfluidic chips as a platform and aim to simulate the internal microenvironment of the human body to the greatest extent in vitro through bioengineering and other means. Combining organ chip technology with organoids to form "organoid chip" technology is regarded as the next generation of organoid technology. The combination of the two technologies complements each other and makes it a more user-friendly and predictive preclinical test model. By using tissue samples from patients as organoids, organoids can create specific disease models and achieve precision medicine. At the same time, organoid chips can also realize the development of personalized precision medicine and regenerative medicine through high-throughput analysis and screening of organoids with optimized in vitro conditions. For this reason, Science journal also conducted a special issue report in 2019.

[0006] Organoid chips can be considered as organoid culture based on chip laboratories. First, the physical microenvironment such as stiffness required by cells is constructed on the chip, and micro-nano processing technology is used to form microstructures for cell growth and culture. At the same time, corresponding external stimuli are applied to build organoid models. Subsequently, nutrients, oxygen and growth factors are provided to micro-organs through microfluidic channels, which can achieve long-term cell culture (up to 2-3 weeks). Drugs can also be added to the culture medium through microfluidics for drug screening. Therefore, organoid chips have the characteristics of both chip laboratories and organoids, including: ① High-throughput screening: Organ chips use micro-nano processing methods on chips 1. Arrangement of sample arrays. Usually, a chip can contain thousands or even tens of thousands of samples, and multiple sets of detection data can be obtained at the same time, which saves time and effort compared with traditional methods; 2. Easy to form concentration gradients: By designing the microfluidics on the organ chip, different ratios of liquid components can be achieved. For example, the "Christmas tree"-shaped microfluidics can achieve gradual changes in concentration in different channels, and the formation of concentration gradients plays an important role in drug screening; 3. Simulate the physical environment in the body, including matrix stiffness adjustment, processing electrodes to electrically stimulate cells, etc.; 4. The results are easy to digitally process: the organ chip can convert the signal to be detected into an electrical signal or an optical signal, which is convenient for digital collection and subsequent processing. As a material, PDMS has the advantages of low price, easy operation, air permeability (ensuring oxygen supply to cells) and transparent properties-imaging.

[0007] At present, various solid tumor organoid models including esophageal cancer are still in their infancy in my country, facing limitations of existing conventional organoid culture technology, including: complex organoid culture operation, large initial cell sample size, large amount of matrix gel used, and difficulty in achieving high throughput. How to overcome the above technical difficulties and achieve high-throughput automated drug sensitivity screening on this basis is an issue that needs to be urgently addressed at this stage. Summary of the invention

[0008] The purpose of the present invention is to provide an application of an esophageal cancer organoid 3D automated drug sensitivity screening system in esophageal cancer drug screening, to construct a high-throughput drug sensitivity screening platform based on esophageal cancer organoid chips through tumor chemotherapy drug sensitivity tests, and to establish standards and specifications for guiding esophageal cancer chemotherapy.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides an application of an esophageal cancer organoid 3D automated drug sensitivity screening system in esophageal cancer drug screening, characterized in that the system consists of an esophageal cancer organoid microfluidic chip and an automated drug sensitivity screening auxiliary device;

[0011] The esophageal cancer organoid microfluidic chip comprises a microfluidic chip fluid layer and a microfluidic chip micropore array culture layer; the microfluidic chip fluid layer has a microchannel structure, and the microfluidic chip micropore array culture layer is used for culturing esophageal cancer organoids;

[0012] The preparation steps of the fluid layer of the microfluidic chip include the following steps:

[0013] (4) Making a mask plate;

[0014] (5) Making a SU-8 silicon wafer positive mold;

[0015] (6) preparing a PDMS fluid layer with a microchannel structure;

[0016] The steps of preparing the microfluidic chip microwell array culture layer include:

[0017] (4) Making a mask plate;

[0018] (5) Make AZ positive photoresist silicon wafer anode film;

[0019] (6) making a PDMS microwell array culture layer;

[0020] The culturing steps of the esophageal cancer organoids include:

[0021] (6) Digest fresh esophageal cancer tissue into single cell suspension;

[0022] (7) adding matrix gel to the single cell suspension to obtain a cell-gel mixture;

[0023] (8) adding the cell-gel mixture dropwise to the microwell array culture layer of the microfluidic chip for culture;

[0024] (9) After the culture is completed, a cell dissociation reagent is added to the microwell array culture layer to collect the dissociated esophageal cancer organoid clumps;

[0025] (10) Washing the esophageal cancer organoid aggregates and then subculturing them.

[0026] The esophageal cancer organoid 3D automated drug sensitivity screening system provided by the present invention is a new type of esophageal cancer organoid model. It not only has outstanding advantages in simulating the tumor microenvironment of esophageal cancer, but also can perform rapid and efficient high-throughput anti-tumor drug screening, predict and evaluate the effectiveness of clinical drug application, and provide a reference for patients to receive reasonable, safe and effective individualized treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of constructing an esophageal cancer organoid microfluidic chip using soft photolithography in Example 1;

[0028] Figure 2 Example 1 is a schematic diagram of the structure of an automated drug sensitivity screening device;

[0029] Figure 3 This is the dose-effect curve of the inhibition rate of chemotherapeutic drugs on organoid tumor cells in Experimental Example 1;

[0030] Figure 4 The corrected AUC values ​​of each dosage regimen in Experimental Example 1 are ranked. DETAILED DESCRIPTION

[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] Example 1

[0033] Construction of a 3D automated drug sensitivity screening system for esophageal cancer organoids

[0034] 1.1 Fabrication of esophageal cancer organoid microfluidic chip

[0035] 1.1.1 Construction of the fluid layer of the esophageal cancer organoid microfluidic chip ( Figure 1 )

[0036] (1) Draw the mask. Use Coreldraw 11.0 drawing software to design the chip structure (positive mold or negative mold). Use a laser imagesetter to output the film with a resolution of 3000 dpi (200 lines) as the mask plate.

[0037] (2) Preparation of SU-8 silicon wafer positive mold: The required silicon wafer is immersed in a boiling piranha solution (concentrated H2SO4:H2O2=3:1) for 20 minutes, taken out and washed with deionized water, and dried on a hot plate for 10 minutes to completely dehydrate the silicon wafer. After the silicon wafer is placed in a coating machine and coated with SU-8 photoresist at an appropriate speed for 50 seconds, it is left at room temperature for 20 minutes, and then placed on a hot plate and slowly heated to 95°C and maintained at the temperature for 20 minutes, and then slowly cooled to room temperature to complete the photoresist pre-baking process. The silicon wafer coated with photoresist after pre-baking is moved to the photolithography machine, exposed to the mask for 70 seconds, and then heated at 95°C for about 15 minutes to perform heat treatment (intermediate baking). After cooling to room temperature, it is immersed in a developer to develop and obtain a photoresist positive film with a pattern structure.

[0038] (3) Preparation of PDMS fluid layer: PDMS prepolymer and curing agent were uniformly mixed in a weight ratio of 10:1, degassed in a vacuum oven for 20 min, cast onto a SU-8 positive template, cured at 85 °C for 2 h, and then cooled and slowly peeled off the template. The fluid layer with a microchannel structure was obtained by cutting, punching and assembling a liquid storage tank.

[0039] 1.1.2 Construction of esophageal cancer organoid microfluidic chip array culture layer

[0040] (1) Chip mask production: same as the production method in 1.1.1.

[0041] (2) Preparation of AZ positive photoresist film on silicon wafer: Immerse the single crystal silicon wafer in boiling piranha solution for 20 minutes, wash with deionized water, and dry on a hot plate for 10 minutes to completely dehydrate the silicon wafer. Place the silicon wafer on a photoresist spreader at an appropriate speed and pre-bake the photoresist by programmed temperature increase. After pre-bake, the silicon wafer coated with photoresist is moved to a photolithography machine and exposed for 90 seconds. Immerse in a developer for 5 minutes to obtain a photoresist film with a pattern structure. In order to prepare a smooth concave hole array, the positive photoresist pattern structure needs to be further heated to make its cross-sectional edge flow back to an arc cross-section.

[0042] (3) Preparation of PDMS microwell array culture layer: PDMS prepolymer and curing agent were uniformly mixed in a weight ratio of 10:1, degassed in a vacuum oven for 20 min, poured onto the AZ positive resist silicon wafer positive template of the fluid channel layer, pre-cured at 85°C for 2 h, and then cooled. It was slowly peeled off the template and cut to obtain an organoid culture layer with a microwell array structure.

[0043] 1.2 Preparation of an automated drug sensitivity screening device for esophageal cancer organoids

[0044] Design and manufacture an automatically addressable robotic arm loading unit, an automatic sampling unit, and a micropump unit for precisely driving reagents to achieve fully automated distribution and replacement of drug solutions, reduce human errors, and realize high-throughput drug sensitivity screening tests. Figure 2 This is a schematic diagram of the structure of the automated drug sensitivity screening device.

[0045] Example 2

[0046] Establishment of esophageal cancer organoid culture model

[0047] 2.1 Collection of specimens from patients with esophageal cancer

[0048] 2.1.1 Approval was obtained from the Ethics Review Committee of Nanjing Jiangning Hospital. Written informed consent was obtained from the patient before sampling.

[0049] 2.1.2 Collection of materials

[0050] After taking the intraoperative specimen of esophageal cancer, the tissue was immediately divided into three parts. One part was placed in pre-cooled phosphate buffer saline (PBS) and transported to the laboratory at low temperature for tumor cell isolation and organoid culture. The other two parts were placed in liquid nitrogen and 4% paraformaldehyde for sequencing, HE staining and immunohistochemistry detection to complete molecular and histopathological analysis.

[0051] 2.2 Organoid culture, passaging and cryopreservation

[0052] 2.2.1 Culture conditions

[0053] Culture medium preparation: DMEM / F12 basal culture medium was supplemented with 1×B27, 1×N2, 0.01% BSA and double antibody.

[0054] 2.2.2 Cultivation steps

[0055] (1) Rinse fresh tissue with iodine for 10 seconds, and then rinse with saline 4-5 times, each time for more than 3 seconds.

[0056] (2) Cut the tissue into pieces of about 1-3 mm 3 Collect small pieces in a 10 ml centrifuge tube, add 4 ml of digestion solution, and digest at 37°C for 2-4 hours, pipetting every 15 minutes.

[0057] (3) Centrifuge at 1200 rpm x 5 min, discard the supernatant, add 4 ml of digestion solution 2, and digest at 37°C for 5 min.

[0058] (4) Add 5 ml of 1× Hank's balanced salt solution and wash once. Centrifuge at 1200 rpm×3 min and discard the supernatant.

[0059] (5) Treat the cells with 3 ml of red blood cell lysis buffer for 3 min and terminate the reaction by adding 5 ml of Hank's balanced salt solution.

[0060] (6) Centrifuge at 1200 rpm x 3 min and discard the supernatant.

[0061] (7) Add 2 ml Advanced DMEM / F12 to resuspend the cells, centrifuge at 1200 rpm x 3 min, and discard the supernatant.

[0062] (8) Add 5 ml of Advanced DMEM / F12 at 4°C, and then add an equal volume of matrix (the main components of the matrix are laminin, type IV collagen, heparan sulfate proteoglycan (HSPG) and entactin) and mix. This step is performed on ice.

[0063] (9) Add all the cell-gel mixture to the esophageal cancer organoid 3D automated drug sensitivity screening system and place it at 37°C for 30 min to allow the gel to solidify.

[0064] (10) The esophageal cancer organoid 3D automated drug sensitivity screening system was placed in a CO2 incubator and continuously cultured at 37°C for esophageal cancer organoids.

[0065] 2.2.3 Organoid passaging and cryopreservation

[0066] (1) Disassemble the esophageal cancer organoid 3D automated drug sensitivity screening system, add all the mild cell dissociation reagents to the esophageal cancer organoid culture module, and incubate at room temperature (15-25°C) for 1 min.

[0067] (2) Collect the organoid clumps in the esophageal cancer organoid culture module into a 15 mL centrifuge tube.

[0068] (3) Incubate the above 15 mL centrifuge tube at room temperature (15-25°C) on a shaker at 20 rpm for 10 min.

[0069] (4) Centrifuge at 290 x g, 2-8°C for 5 min and discard the supernatant.

[0070] (5) Use a pre-wetted 10 mL pipette to resuspend the cell pellet in 10 mL (2-8°C) DMEM / F-12, then centrifuge at 200 x g, 2-8°C for 5 min and discard the supernatant.

[0071] (6) Passaging: Add an appropriate amount of organoid growth medium (DMEM / F12 basal medium supplemented with 1×B27, 1×N2, 0.01% BSA, and double antibody) to each tube, add an equal amount of matrix (the main components of the matrix are laminin, type IV collagen, heparan sulfate proteoglycan (HSPG) and nestin), and then culture as usual for 2 days.

[0072] Cryopreservation: Add an appropriate amount of organoid freezing solution (purchased from the kit of Shanghai Yisheng Biotechnology Co., Ltd., the amount added is according to the instructions of the kit) to each tube and freeze in a liquid nitrogen tank.

[0073] 2.3 Organoid identification and evaluation

[0074] 2.3.1 HE staining of organoids

[0075] (1) Disassemble the esophageal cancer organoid 3D automated drug sensitivity screening system, rinse the esophageal cancer organoid culture module with PBS, add an appropriate amount of cell recovery solution (the kit is purchased from Shanghai Yisheng Biotechnology Co., Ltd., and the amount added is according to the kit operating instructions) to dissolve the matrix gel, and place it at 4°C for 1-2 h. When the gel is completely dissolved, collect and centrifuge it at 1200 rpm×3 min, and discard the supernatant.

[0076] (3) Fix the cells with 4% formaldehyde for 4-8 h at 1200 rpm x 5 min, and discard the formalin fixative.

[0077] (4) Gently stir the precipitate with 2% agar; place at 4°C for 20 min to allow the cells to solidify in the agar block. Cut the bottom of the centrifuge tube, remove the agar block, and place it in an embedding box.

[0078] (5) Dehydration: 75% alcohol, 45 min; 85% alcohol, 45 min; 95% alcohol, 45 min; 95% alcohol, overnight; 100% alcohol, 1 h; 100% alcohol, 1 h.

[0079] (6) Transparency: Xylene I, 20 min; Xylene II, 20 min.

[0080] (7) Wax immersion: paraffin wax, 60°C, 30 min; paraffin wax, 60°C, 30 min; paraffin wax, 60°C, 60 min.

[0081] (8) Embedding: Use an embedding mold to embed the agar block cells and place it in a -20℃ refrigerator for 10 minutes.

[0082] (9) Prepare room temperature water, unfold the slices, and set the slice unfolding machine to 45°C; slice with a slicer. Use a brush to move the cut slices to room temperature water and 45°C water respectively to unfold them, then place the slices on a slide, place the slide on a rack, and put it in an oven at 65°C for 30 minutes to dry the moisture on the slide and melt the paraffin.

[0083] (10) Dewaxing: xylene I, 15 min; xylene II, 15 min; 100% alcohol I, 1 min; 100% alcohol II, 1 min; 95% alcohol, 1 min; 80% alcohol, 1 min; immersion in running water for 1 min.

[0084] (11) Staining: Stain with hematoxylin for 8 minutes, wash with water for 1 minute; differentiate with 1% hydrochloric acid alcohol for 1-2 seconds, until the color changes from blue to red, rinse with water for more than 15 minutes; stain with 1% eosin for 1-2 minutes, rinse with running water for 1 minute (water-soluble); differentiate with 95% alcohol for 1 minute (alcohol-soluble).

[0085] (12) Dehydration: 95% alcohol, 2 min; 95% alcohol, 2 min; 100% alcohol, 2 min; 100% alcohol, 2 min;

[0086] (13) Transparency: Transparentize with xylene for 2 minutes, take out and dry, then seal with neutral gum.

[0087] 2.3.2 Organoid Immunohistochemistry

[0088] The preliminary preparation was the same as in 2.3.1. After dewaxing, immunohistochemical antibodies (ki-67, CDX2, EpCAM, β-catenin and CK20) were incubated with two different antigen extraction solutions, modified citrate and EDTA, respectively. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide at room temperature. The primary antibody was incubated overnight at 4°C. After rinsing with PBST, the sections were incubated with secondary antibodies for 30 minutes at room temperature.

[0089] 2.3.3 Whole-exome sequencing of organoids

[0090] (1) DNA was extracted using the sodium dodecyl sulfate (SDS) method.

[0091] (2) Through 2.0 fluorometer dsDNA HS kit to detect the concentration.

[0092] (3) Fragment the DNA sample to 200 and 300 bp using the TIANSeq DNA Fragmentation Module kit. Repair the DNA fragments and attach tags to both ends of the fragments after adding adenylate blockers at the 3' end.

[0093] (4) Use PEG / NaCl SPRI solution and magnetic beads to select DNA fragments of target size, perform 9 cycles of polymerase chain reaction (PCR), and purify.

[0094] (5) Whole exome capture was performed using the SeqCap EZ Exome Library Kit. After library amplification, capture probes were added and incubated at 47°C for 16-20 h. The hybridized mixture was amplified through 14 PCR cycles. The final library was sequenced on an Illumina HiSeq X Ten.

[0095] (6) Adapter contamination, reads containing more than 10% ambiguous nucleotides, and paired reads when a single read had more than 50% low-quality (<5) nucleotides were discarded.

[0096] (7) The human GRCh37 / hg19 reference genome was aligned using BWA0.7.10. BAM files were interpreted using SAMtools and Picard tools. Variants were identified using GATK4-4.0.4.0. Variants were analyzed based on the asvsnp150 and 1000Genomes databases, and then annotated using ANNOVAR 17.

[0097] (8) MutSigCV1.41 was used to analyze mutant genes, and maftools was used for mutation spectrum and feature analysis.

[0098] 2.3.4 Organoid Model Evaluation

[0099] Evaluate the ability of organoids to recapitulate the histology, marker expression, and mutational signature of the original tumor tissue, including:

[0100] (1) Whether the organoids show great diversity in growth rate and morphology. If not, redo the experiment. If so, proceed to the next step.

[0101] (2) whether HE staining showed that the tumor-derived organoids exhibited patient-specific heterogeneous morphology, which was considered successful;

[0102] (3) Immunohistochemical marker expression analysis showed whether the organoids and paired primary tumors had similar staining patterns of Ki-67, CDX2, EpCAM, β-catenin, and CK20, which was considered successful if similar;

[0103] (4) Whether the DNA copy number variation between the organoid and the corresponding tumor is similar, which is considered a success;

[0104] (5) Whether the total mutation load and point mutation types between the organoids and the corresponding tumors are similar, which is considered successful;

[0105] (6) Whether the mutation status of common representative genes in esophageal cancer is similar, such as RAS, TP53, NOTCH1, PIK3CA, XRCC1, etc., similarity is considered successful.

[0106] Example 3

[0107] Establishment of a clinical drug screening system for esophageal cancer

[0108] 3.1 Drug screening program

[0109] Paclitaxel + carboplatin regimen;

[0110] Paclitaxel + cisplatin regimen;

[0111] Paclitaxel + fluorouracil regimen;

[0112] Fluorouracil + cisplatin regimen;

[0113] Fluorouracil + oxaliplatin regimen;

[0114] Capecitabine + cisplatin regimen;

[0115] Capecitabine + oxaliplatin regimen;

[0116] Irinotecan + cisplatin regimen;

[0117] Irinotecan + fluorouracil regimen;

[0118] Irinotecan + cisplatin regimen;

[0119] Irinotecan + docetaxel regimen;

[0120] Docetaxel + cisplatin regimen;

[0121] ECF (epirubicin + cisplatin + fluorouracil) regimen;

[0122] ECF (epirubicin + oxaliplatin / cisplatin + fluorouracil) regimen;

[0123] EOX (epirubicin + oxaliplatin + capecitabine) regimen;

[0124] DCF (docetaxel + cisplatin / oxaliplatin / carboplatin + fluorouracil) modified regimen;

[0125] FLO (oxaliplatin + leucovorin + fluorouracil) regimen;

[0126] Trastuzumab + fluorouracil + cisplatin regimen;

[0127] Ramucirumab + paclitaxel regimen;

[0128] Taxane monotherapy;

[0129] Irinotecan monotherapy;

[0130] Ramucirumab monotherapy;

[0131] Trastuzumab monotherapy

[0132] 3.2 Organoid drug sensitivity testing

[0133] 3.2.1 Organoid Drug Screening System

[0134] Drug concentration gradient, taking paclitaxel as an example, its physiological concentration (the peak concentration of the patient's blood drug is 3.3-7.6μM / L), based on which the maximum physiological concentration is passed into the esophageal cancer organoid 3D automated drug sensitivity screening system, and a series of concentration gradients are automatically generated. This concentration gradient can cover the entire drug treatment window. Drug combination scheme concentration: Taking paclitaxel combined with cisplatin as an example, the paclitaxel: cisplatin is 2:1, and other combined doses are converted in proportion according to the clinical treatment plan. The two drugs are passed into the esophageal cancer organoid 3D automated drug sensitivity screening system, and the set compatibility ratio can be generated online.

[0135] 3.2.2 Organoid drug screening steps (1) When the diameter of the esophageal cancer organoids in the esophageal cancer organoid 3D automated drug sensitivity screening system reaches 100 μm, refer to the screening system in step 3.2.1 and continuously input the maximum physiological concentration (i.e., the optimal drug concentration obtained by exploration) into the automated drug sensitivity screening system to form a series of drug concentrations that directly act on the corresponding esophageal cancer organoids.

[0136] (6) Organoids were photographed under a microscope every three days to measure changes in their size, and cell viability was tested every three days: 10% CellTiter-Glo 3D cell viability assay reagent was introduced into the esophageal cancer organoid 3D automated drug sensitivity screening system.

[0137] (7) The system was placed back in the incubator. After incubation for 2-4 h, the cell viability assay reagents (a kit purchased from abcam) at different drug concentration gradients were collected and the cell viability was detected according to the kit instructions.

[0138] 3.2.3 Evaluation of organoid drug screening system

[0139] (1) Through the above screening, the growth rate inhibition metrics (GR) at each concentration were calculated, growth inhibition rate = 1-A570nm value of drug group / A570nm value of control group.

[0140] (2) Fitting dose-response curves (DRC).

[0141] (3) Analysis of GR50 and area under the DRC curve (AUC DRC )

[0142] Experimental Example 1

[0143] Personalized tumor drug sensitivity testing based on in vitro tumor organoid models

[0144] 1.1 Clinical information of the patients

[0145] The patient was diagnosed with primary esophageal cancer metastasis to the retroperitoneal lymph nodes

[0146] Clinicians recommend the following treatments: 1. Paclitaxel polymer micelles, 2. 5-fluorouracil, 3. Gemcitabine, 4. Paclitaxel albumin-bound for injection, 5. Anlotinib hydrochloride, 6. Cisplatin

[0147] Other clinical information: Clinically, radiotherapy, chemotherapy and immunotherapy have been performed. The initial immunotherapy drug was slulizumab, and the patient is currently being treated with Bleize Antilelizumab. The patient felt weak after using Bleize Antilelizumab.

[0148] 1.2 Test items

[0149] Screening Model: Patient-Derived Tumor Organoids (PDOs)

[0150] Technology platform: High-throughput tumor organoid drug sensitivity screening system of the present invention

[0151] Detection technology: Adenosine triphosphate bioluminescent tumor in vitro drug sensitivity detection technology (ATP-TCA)

[0152] 1.3 Results of tumor organoid model construction

[0153] It has a spherical shape with a three-dimensional sense of layering and good light transmittance. There is no black necrotic part in the middle of the sphere, and the edge line of the sphere is clearly visible.

[0154] 1.4 Prediction of drug sensitivity test results

[0155] Table 1 Prediction of drug sensitivity results of each detection scheme

[0156] Serial number Dosage regimen Sensitivity prediction 1 Paclitaxel polymer micelles Drug resistance 2 5-Fluorouracil Drug resistance 3 Gemcitabine sensitive 4 Paclitaxel Albumin-bound for injection Drug resistance 5 Anlotinib Hydrochloride Drug resistance 6 Cisplatin Drug resistance

[0157] Note: Sensitivity and recommendation level are based on:

[0158] Based on the ATP-TCA drug sensitivity screening method, the PDOs drug sensitivity evaluation standard was established to determine the sensitivity of the drug sensitivity detection scheme;

[0159] Evaluation criteria for drug sensitivity of PDOs:

[0160] Sensitive (S): IC50 < 25% PPC

[0161] General (G): IC50>25% and IC90<100% PPC

[0162] Drug resistance (R): IC50>25% and IC90>100% PPC

[0163] PPC is the peak plasma concentration of the drug;

[0164] The recommendation level is determined based on the sensitivity of the in vitro organoid model dosing regimen and the corrected AUC value. The serial numbers in the table are arranged in order according to the corrected AUC value;

[0165] 1.5 Design of drug sensitivity testing scheme

[0166] Table 2 Drug sensitivity test regimen

[0167]

[0168] Dosage regimen source:

[0169] 1: Recommended by a clinician;

[0170] 2: Refer to the "Guidelines for the Diagnosis and Treatment of Esophageal Cancer (2022 Edition)";

[0171] 1.6 Dosage regimen design

[0172] In this testing service, all dosing regimens are set with 6 dosing concentrations, 3 replicates for each dosing concentration, and a blank control group is set up. The adenosine triphosphate bioluminescent tumor in vitro drug sensitivity detection technology (ATP-TCA) is used to detect the activity of organoid cell spheres, draw drug dose-effect curves, calculate IC50 and IC90, and judge drug sensitivity. IC50 indicates the concentration of the drug when the inhibition rate of tumor organoid activity reaches 50%. The smaller the IC50 value, the better the inhibitory effect of the regimen on tumor cells at a lower dosing concentration, that is, tumor cells have a better sensitivity to the regimen; IC90 indicates the concentration of the drug when the inhibition rate of tumor organoid activity reaches 90%.

[0173] 1.7 Calculation of IC50 and IC90 values ​​for designing dosing regimens

[0174]

[0175]

[0176] Note: IC50: It indicates the concentration of the drug when the inhibition rate of tumor organoid activity reaches 50%. The smaller the IC50 value, the better the inhibitory effect on tumor cells can be at a lower dosage concentration, that is, the tumor cells have a better sensitivity to this regimen.

[0177] IC90: The concentration of the drug that achieves a 90% inhibitory effect on the activity of tumor organoids. Cmax: The highest concentration of each regimen in the in vitro drug sensitivity test.

[0178] Cmin: The lowest drug concentration of each regimen in in vitro drug sensitivity testing.

[0179] The dose-effect curve of the inhibitory rate of chemotherapeutic drugs on organoid tumor cells within the detection concentration range is as follows Figure 3 shown.

[0180] 1.8 Results interpretation:

[0181] This drug sensitivity screening was based on a tumor organoid model directly constructed from primary cells extracted from patient tumor tissues, and a total of 6 drug sensitivity tests were performed. The drug sensitivity test results showed that all 6 regimens within the designed concentration range had different degrees of inhibitory effects on the tumor organoid model. The relationship curve between the drug dosage and the inhibitory effect is shown in the figure below: Figure 3 shown.

[0182] Based on the dose-effect curve of the drug inhibition rate on organoid tumor cells obtained from the drug sensitivity test, the IC50 and IC90 of each dosing regimen were calculated, and the sensitivity of the dosing regimen was determined in combination with the PPC concentration of each drug in the body.

[0183] Table 3 Drugs that may benefit patients

[0184]

[0185]

[0186] Note: (1) Corrected AUC value: AUC represents the area under the dose-effect curve and can be used as an indicator of drug efficacy. The corrected AUC value is obtained by normalizing the abscissa of the dose-effect curve of each regimen.

[0187] (2) The treatments in the table are sorted according to the corrected AUC values, but this does not represent a recommended order. Any potentially effective treatment regimen is not ranked in order of priority. The final treatment plan requires a comprehensive judgment by the clinician based on the actual situation. Please follow the doctor's advice for specific treatment.

[0188] According to the above drug sensitivity test results, the corrected AUC values ​​of each dosage regimen were ranked, such as Figure 4 shown.

[0189] The mechanism of action of the regimens that may benefit patients is explained in Table 4.

[0190] Table 4 Interpretation of the mechanism of action of the regimen that may benefit patients

[0191]

[0192]

[0193] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of an esophageal cancer organoid 3D automated drug sensitivity screening system in esophageal cancer drug screening, characterized in that: The system consists of an esophageal cancer organoid microfluidic chip and an automated drug sensitivity screening auxiliary device; The esophageal cancer organoid microfluidic chip comprises a microfluidic chip fluid layer and a microfluidic chip micropore array culture layer; the microfluidic chip fluid layer has a microchannel structure, and the microfluidic chip micropore array culture layer is used for culturing esophageal cancer organoids; The preparation steps of the fluid layer of the microfluidic chip include the following steps: (1) Making a mask plate; (2) Making a SU-8 silicon wafer positive mold; (3) preparing a PDMS fluid layer with a microchannel structure; The steps of preparing the microfluidic chip microwell array culture layer include: (1) Making a mask plate; (2) Making AZ positive photoresist silicon wafer anode film; (3) Fabricating a PDMS microwell array culture layer; The culturing steps of the esophageal cancer organoids include: (1) Fresh esophageal cancer tissue was digested into single cell suspension; (2) adding matrix gel to the single cell suspension to obtain a cell-gel mixture; (3) adding the cell-gel mixture dropwise to the microwell array culture layer of the microfluidic chip for culture; (4) After the culture is completed, a cell dissociation reagent is added to the microwell array culture layer to collect the dissociated esophageal cancer organoid clumps; (5) Washing the esophageal cancer organoid aggregates and then subculturing them.

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