A modeling method of tumor drug sensitivity animal model and use thereof

Abstract

The application provides a modeling method of a tumor drug sensitivity animal model and application thereof, and belongs to the field of animal tumor models. The tumor tissue sample or tumor tissue-derived organoid of a patient is transplanted into the anterior chamber of the mouse eye to quickly construct a tumor drug sensitivity in-vivo model. The model can quickly, dynamically and in real time observe the influence of the targeted drug on tumor colonization, tumor proliferation and vascular formation through in-vivo imaging technology, and can be used for drug rapid screening. The model has high sensitivity, can dynamically and non-destructively evaluate drug efficacy, solves the problems of long drug screening period and low reliability, and provides a new guiding model for clinical tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of animal tumor models. Specifically, it relates to a method for modeling an animal model of tumor drug sensitivity and its application. Background Technology

[0002] Cancer is a serious malignant disease facing humanity today. Treatment varies greatly from person to person, and current clinical practice is mainly based on physician experience, making it difficult to quickly determine the optimal treatment plan, leading to poor treatment outcomes. Therefore, establishing reliable preclinical models to test individual drug sensitivity is crucial for the development of personalized medicine. An ideal preclinical model should be reproducible, highly specific, highly sensitive, rapid, and should fully encompass the biological characteristics of the in situ tumor.

[0003] To date, several preclinical tumor experimental models have been developed, including 2D cell lines, patient-derived xenograft models (PDX models), patient-derived explants (PDE models), animal models (genetically engineered mouse models - gemms), and patient-derived organoids (PDO).

[0004] Two-dimensional (2D) cell lines are the simplest, most affordable, and most commonly used models for drug sensitivity studies. Currently, thousands of tumor cell lines have been generated globally, which can be classified into groups representing different histological subtypes of different tumors. However, these cell lines cannot fully reflect tumor heterogeneity or fully characterize the tumor at the molecular genetic level. Furthermore, comparative genomics and transcriptomics analyses show little correlation between cell lines and clinical samples of the same histological type, and extensive passage of cell lines can introduce genetic alterations with unpredictable biological significance. While cell lines are a valuable alternative tool for basic research and drug screening, the extent to which data generated from cell line studies are clinically meaningful remains unclear.

[0005] The principle behind PDX models is to transplant fresh tumor tissue into immunodeficient mice. Success rates for PDX vary considerably across different studies, but it is generally accepted that xenografts reproduce the histological and genomic profiles of the original cancer tissue. However, generating PDX models is quite time-consuming and labor-intensive, and their passaging is also challenging.

[0006] PDE models are relatively easy to generate because they represent ex vivo cultures of freshly excised tumor tissue. Despite the feasibility of PDE models and their morphological and genetic similarity to tumor tissue, their application in tumor research is limited, primarily due to issues with viability and short-term viability that restrict the potential use of these explants.

[0007] Organoid technology was introduced in 2009 as a pioneering 3D primary tissue culture model and has rapidly developed into a complex and promising preclinical model for cancer research. An organoid is a 3D culture model derived from stem cells (pluripotent or tissue-resident) or differentiated normal or cancer cells that mimics the biological and functional characteristics of healthy or cancerous tissues. Currently, tumor organoids are used in basic research on various cancers (gastrointestinal, pancreatic, liver, prostate, etc.). Although literature shows that organoids, as drug sensitivity models, are fast, highly sensitive, and specific, the lack of a tumor microenvironment limits their clinical application.

[0008] Due to the numerous problems with the aforementioned models, current clinical targeted therapy for patients mainly relies on gene testing results to guide clinical medication. However, several drawbacks remain: 1. Current gene testing takes a long time, with a waiting period of approximately 1-1.5 months before medication; 2. Gene testing is limited by testing technology, potentially resulting in false negatives or false positives, both of which can affect clinical treatment outcomes; 3. Gene testing may also have insufficient coverage, as it cannot cover all genes or regions, and some mutated genes may not be detected. Therefore, gene testing cannot fully reflect the sensitivity of the entire tumor to drugs; 4. If a patient develops resistance to a gene-tested recommended drug, there may be a situation where no treatment is available; 5. After the gene test is conducted, the tissue may become unusable.

[0009] Therefore, there is an urgent need to construct a rapid drug sensitivity model that is more efficient, more sensitive, and more cost-effective than traditional in vivo models. Summary of the Invention

[0010] The purpose of this invention is to provide a modeling method for an animal model of tumor drug sensitivity and its application.

[0011] This invention provides a method for constructing an animal model of tumor drug sensitivity, comprising the following steps:

[0012] a) Obtain tumor tissue samples to obtain tissue specimens;

[0013] b) Cut tissue specimens into pieces to obtain tissue blocks, or digest tissue specimens into single cells and culture them to obtain tumor organoids;

[0014] c) Transplant the tissue block or organoid into the anterior chamber of the animal, and wait for new blood vessels to appear in the anterior chamber graft.

[0015] Furthermore, the tumor tissue includes liver cancer tissue, lung cancer tissue, colon cancer tissue, gastric cancer tissue, pancreatic cancer tissue, thyroid cancer tissue, head and neck tumor tissue, parathyroid adenoma tissue, adrenal tumor tissue, cholangiocarcinoma tissue, and breast cancer tissue.

[0016] Further, in step b), the size of the tissue block is (0.5-2)mm×(0.5-2)mm×(0.5-2)mm; the transplantation quantity is 1-3 blocks.

[0017] Furthermore, the tissue block was transplanted into the anterior chamber of the animal, and new blood vessels appeared in the anterior chamber graft after 3-5 days.

[0018] Furthermore, in step b), the method for preparing tumor organoids is as follows:

[0019] 1) Cut the tissue specimen into tissue fragments, digest the tumor cell tissue with tissue digestion solution, and obtain single cells of tumor tissue;

[0020] 2) Resuspend the cells in matrix gel, inoculate and culture them, and collect them after the organoid proliferation density reaches more than 50%.

[0021] Further, in step 1), the tissue digestion solution is an Advanced DMEM F12 solution with a final concentration of 0.5 mg / ml collagenase XI and 0.2 mg / ml DNase;

[0022] In step 2), the culture medium used for organoids was a DMEM / F12 solution supplemented with 1% vol / vol penicillin and streptomycin solution, 1% vol / vol L-alanyl-L-glutamine, 10 mmol / L 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) solution, 2% vol / vol B27 medium supplement, 1% vol / vol N2 medium supplement, 1.25 μmol / L N-acetyl-L-cysteine, 100 ng / mL epidermal growth factor (EGF), 25 ng / mL recombinant human R-spondin 1 protein, 100 ng / mL fibroblast growth factor 10 (FGF10), 10 mmol / L nicotinamide, and 10.5 μmol / L Y-27632 small molecule inhibitor.

[0023] The substrate gel is Matrigel; the culture conditions are 37°C and 5% CO2.

[0024] Furthermore, organoids with a size of (5-30)μm*(5-30)μm were selected and transplanted into the anterior chamber of the animal's eye; 1-3 organoids were transplanted; after transplantation, neovascularization appeared in the graft after 14-17 days.

[0025] Furthermore, the animal is a mouse, rat, or rabbit; preferably a mouse.

[0026] The present invention also provides an animal model prepared by the above method.

[0027] The present invention also provides the use of the above-mentioned animal model in drug screening, drug toxicity testing or immunotherapy testing.

[0028] This invention provides a modeling method for a tumor drug sensitivity animal model and its application. The model rapidly constructs a tumor drug sensitivity model by inserting tumor organoids, which are derived from surgical or puncture tissue samples or tissue-constructed tumors, into the anterior chamber of a mouse. This modeling method has a high success rate and fast modeling speed.

[0029] The model obtained by the method of this invention can rapidly and dynamically observe the effects of targeted drugs on tumor proliferation (mainly changes in tumor volume) and angiogenesis (changes in the number of new blood vessels and the diameter of blood vessels) in real time by using in vivo imaging technology. This enables rapid drug screening to find the best drugs for tumor treatment and can provide strong support and guarantee for personalized precision medicine for patients.

[0030] The model of this invention has high sensitivity and can perform dynamic and non-destructive evaluation of drug efficacy, solving the problems of long drug screening cycle and low reliability, and providing a new guiding model for clinical tumor treatment.

[0031] This invention can provide doctors with three-dimensional information on tumor morphology and vascular distribution, helping to formulate more precise treatment plans. Furthermore, by constructing organoid models of tumors in vitro and reimplanting tumors in the anterior chamber, the reuse of tumors can be achieved. Simultaneously, this model can also be used to explore the mechanisms by which tumor stem cells directly or indirectly transform into new blood vessels, identify key targets for reversing angiogenesis, and provide in vivo model support for the development of related targeted drugs.

[0032] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0033] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0034] Figure 1 This is an in vivo imaging result of the anterior chamber of a mouse at different time points after lung cancer tissue was implanted in the anterior chamber of the mouse in Example 1.

[0035] Figure 2This is an in vivo imaging result of the anterior chamber of a mouse at different time points after implantation of a papillary thyroid carcinoma organoid into the anterior chamber of mouse in Example 3.

[0036] Figure 3 This is a diagram showing the effect of the antitumor drug osimertinib on neovascularization in the anterior chamber of the mouse model of Example 1, where the neovascularization is connected to the tumor tissue.

[0037] Figure 4 This is a diagram showing the effect of the antitumor drug lenvatinib on neovascularization in the anterior chamber of the mouse model of Example 3, which is connected to the papillary thyroid carcinoma organoid. Detailed Implementation

[0038] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0039] The modeling mouse used in this invention is the SCID mouse.

[0040] Tissue washing solution: phosphate-buffered saline (PBS) + 1% anti-anti solution (Gibco, 15240062).

[0041] Anti-Anti solution (Gibco, 15240062) is an antibiotic resistance agent, primarily used to prevent fungal and bacterial contamination.

[0042] Tissue digestion solution: Advanced DMEM F12 solution with a final concentration of 0.5 mg / ml collagenase XI (C7657; Sigma-Aldrich) and 0.2 mg / ml DNase (17105041; Sigma-Aldrich).

[0043] Advanced DMEM F12 solution: a widely used basal culture medium that reduces serum addition by 50-90% compared to classic DMEM / F-12, without altering cell growth rate or morphology. Purchased from Invitrogen, catalog number 12634010.

[0044] Digestion termination solution: Advanced DMEM F12 solution containing 10% fetal bovine serum.

[0045] Matrigel matrix adhesive, purchased from Corning, product number: 354230.

[0046] Organoid complete culture medium: DMEM / F12 solution supplemented with 1% vol / vol penicillin and streptomycin solution (15140122, Life Technologies); 1% vol / vol L-alanyl-L-glutamine (GlutaMAX, BE-17-605E / U1, Westburg BV); 10 mmol / L HEPES solution (be-17-737E, Westburg BV); 2% vol / vol B27 medium supplement (17504-001, Life Technologies Europe BV); 1% vol / vol N2 medium supplement (17502001, Life Technologies); 1.25 μmol / L N-acetyl-L-cysteine ​​(A7250, Sigma-Aldrich); 100 ng / mL epidermal growth factor (EGF, AF-100-15, PeproTech); recombinant human R-spondin Protein 1 (25 ng / ml, 2593, Prospec); 100 ng / mL fibroblast growth factor 10 (FGF10, 100-26, PeproTech); 10 mmol / L nicotinamide (N0636, Sigma-Aldrich); 10.5 μmol / L Y-27632 small molecule inhibitor (Y0503, Sigma-Aldrich).

[0047] Example 1: Constructing the anterior chamber drug sensitivity model of tumor tissue according to the present invention using tumor tissue.

[0048] I. Modeling Methods

[0049] (a) Preparation of tissue specimens

[0050] 1. This model is applicable to all solid tumors (both benign and malignant) derived from surgery or biopsy, with a size of at least 0.5cm*0.1cm*0.1cm. Taking lung cancer as an example, take a surgical lung cancer tumor tissue sample, approximately 0.5cm*0.5cm*0.5cm in size, place it in UW solution (Biolifesoultion, 101102) and store it on ice, then transport it to the laboratory within 1 hour.

[0051] 2. Select fresh tissue samples, place the tissue samples in sterile culture dishes, wash with tissue washing solution to remove necrotic tissue, calcifications, blood clots, and adipose tissue from non-adipose tissue samples, and obtain tissue specimens.

[0052] (II) Tissue Transplantation

[0053] 1. Place the tissue specimen in a sterile culture dish, add PBS containing 5% serum to keep the tissue moist, and cut it into tissue blocks of 1mm×1mm×1mm in size for later use.

[0054] 2. Mice were generally anesthetized by inhaling 2% isoflurane, and their heads were restrained using a stereotactic head support. A sectioned tissue block was aspirated into a 27-gauge cannula connected to a 1ml Hamilton syringe through a 0.4mm polyethylene tube. Under a stereomicroscope, a 27-gauge needle was used to puncture the cornea near the sclera, and the cannula containing the aspirated tissue block was inserted. The tissue block was then injected into the anterior chamber of the eye and fixed to the iris. One tumor tissue block was transplanted into the anterior chamber of each mouse.

[0055] After successful transplantation, the drug sensitivity model of this invention can be obtained by culturing for 3 to 5 days.

[0056] II. Model Detection

[0057] (I) Detection Method

[0058] Detection of neovascularization using in vivo imaging methods:

[0059] 1. Use 2% isoflurane gas to anesthetize mice, and 1%-1.5% to maintain anesthesia.

[0060] 2. Use a stereotactic head support to restrain the mouse's head and position the eye containing the transplanted tissue upwards to fully expose the eyeball.

[0061] 3. A Leica multi-functional spectral invivo microscopy imaging system was used, featuring a confocal laser with a 405nm single-line laser; 350 continuously tunable lasers ranging from 440-790nm; a two-photon system with a 1040nm single-line laser; and tunable lasers ranging from 680-1300nm. Z-axis invivo imaging was performed using a long-range immersion lens (Leica HXC APO 10×0.3W). PBS was used as the medium between the mouse eyeball and the lens.

[0062] 4. To observe tissue morphology, excitation was performed at 639 nm, and detection was conducted at 613–675 nm to obtain the background reflectance image of the tumor tissue, such as… Figure 1 The reflected light group is shown.

[0063] 5. Observation of blood vessels: Inject fluorescein isothiocyanate-glucan (10 mg / mL) through the posterior orbital sinus. Excitation is performed at 491 nm, and detection is performed at 508–576 nm to obtain a live-cell image of the anterior chamber vessels, i.e., a green fluorescence image of FITC, as shown below. Figure 1 As shown in the green fluorescence group. The background reflectance image of tumor tissue obtained from in vivo imaging and the green fluorescence image of FITC in the anterior chamber vessels are overlaid to obtain the following... Figure 1 The overlapping image set shows the anterior chamber blood vessel vascular map. By observing the vascular map after different culture times and comparing it with the image on the day of transplantation (day 0), it was confirmed whether there was neovascularization (the vascular structure became denser or the blood vessels thickened).

[0064] At 0, 3, 4, 5, 7, 14 and 45 days, the aforementioned in vivo imaging method was used to perform in vivo imaging of the anterior chamber of the modeling mice to obtain the anterior chamber vascular map of the mice and observe whether there were new blood vessels in the graft.

[0065] (II) Test Results

[0066] The test results showed that in this modeling study, all 20 mice developed neovascularization and vascular remodeling between day 3 and day 5 after implantation of lung cancer tissue into the anterior chamber. Furthermore, the vascularity and morphology of the implanted lung cancer tissue were maintained for a long period, with 18 mice maintaining this state for at least 45 days. Two modeling mice died due to early post-transplantation eye infection. The success rate of this modeling study was 90%. The test results of one successfully modeled mouse are shown in the image below. Figure 1 As shown, the mouse's own blood vessels have a loose radial structure from the pupil to the periorbital area, while the neovascularization within the tumor is more dense. Figure 1 (yellow arrow) and the thickening of peritumoral nourishing blood vessels over time ( Figure 1 Red arrow.

[0067] Experimental results show that the method in this embodiment can effectively establish a drug sensitivity model of the anterior chamber of lung cancer tissue, with short modeling time, stable model, and high modeling success rate.

[0068] Example 2: Using tumor tissue to construct the anterior chamber drug sensitivity model of the present invention.

[0069] In addition to the anterior chamber drug sensitivity model of lung cancer tissue shown in Example 1, we also replicated this model in various benign and malignant tumors, using the modeling method of Example 1 to establish anterior chamber drug sensitivity models for other tumor tissues.

[0070] Currently, nearly 200 immunodeficient mice have been used to successfully model various tumors, including parathyroid adenomas, liver cancer, lung cancer, colon cancer, gastric cancer, pancreatic cancer, thyroid cancer, head and neck tumors, parathyroid adenomas, and adrenal tumors. (The benign and malignant tumor tissues, such as parathyroid adenomas, were obtained from surgery or biopsy. The tumor tissues were processed according to the first (a) method in Example 1, and the drug sensitivity model was prepared using the tissue transplantation method in the first (b) method in Example 1.) The overall modeling success rate was 89.5%, with all models successfully established within 3-5 days. The main reason for the 10.5% failure rate in anterior chamber transplantation modeling was early post-transplantation ocular infection leading to mouse death.

[0071] This invention uses a drug sensitivity model constructed from transplanted tumor tissue. The tumor tissue contains immune cells, fibroblasts, tumor cells, etc., thus preserving the microenvironment for tumor development and growth, and providing a model basis for exploring the influence of the microenvironment on tumor cell plasticity during in vivo drug sensitivity testing.

[0072] Experimental results show that the method in this embodiment can effectively establish a drug sensitivity model of the anterior chamber of tumor tissue. The modeling time is short, the model is stable, the modeling success rate is high, and it can be applied to different tumor tissues, showing particularly good clinical application prospects.

[0073] Example 3: Constructing the anterior chamber drug sensitivity model of the tumor organoid of the present invention using organoids.

[0074] I. Modeling Methods

[0075] (I) Specimen Preparation

[0076] Using surgical papillary thyroid carcinoma samples, and otherwise the same as in Example 1, papillary thyroid carcinoma tissue specimens were obtained.

[0077] (II) Constructing Organoids

[0078] 1. Transfer the tissue specimen to a sample collection container and cut the tissue specimen into 1mm pieces with scissors. 3 ~2mm 3 Prepare tissue blocks; add tissue digestion solution three times the volume of the tissue, and incubate the tissue digestion tube in a 37°C cell culture incubator or water bath for 45 minutes; remove the tube every 5-15 minutes and shake it upside down 3-5 times.

[0079] 2. Termination of digestion: Add a digestion termination solution equal in volume to the total volume of the tissue digestion solution used in step 1, and gently aspirate and mix with a pipette.

[0080] 3. Filter the final product of step 2 using a 70μm cell sieve and collect the filtrate. To ensure sufficient cell collection, after the tissue digestion solution is filtered, rinse the cell sieve 2-3 times with tissue washing solution, with each rinse volume not less than 1mL. Collect the washing solution into the above filtrate collection tube, seal it tightly, and place it in a pre-cooled 4℃ benchtop centrifuge. Centrifuge at 300g for 5min, discard the supernatant, and collect the cell pellet.

[0081] 4. When the cell pellet turns red, use erythrocyte lysis buffer to lyse the erythrocytes. Add 1 mL of erythrocyte lysis buffer to the cell pellet, gently pipette to mix, and incubate on ice for 1–2 min; add an equal volume of tissue washing solution, gently aspirate to mix, and terminate the reaction; seal tightly and place in a pre-cooled 4°C benchtop centrifuge, centrifuge at 300 g for 5 min.

[0082] 5. Discard the supernatant, collect the cell pellet, add 1 ml of PBS to resuspend the cell pellet, and gently aspirate and mix with a pipette; seal tightly and place in a pre-cooled 4°C benchtop centrifuge, centrifuge at 300g for 5 min, discard the supernatant, and collect the cell pellet; the washing can be repeated once.

[0083] 6. Add 300 μL of PBS to resuspend the cell pellet, gently aspirate and mix to obtain a homogeneous cell suspension, and take 10-50 μL of the cell suspension for cell counting; after sealing the remaining cell suspension tightly, place it in a pre-cooled 4°C benchtop centrifuge, centrifuge at 300g for 5 min, discard the supernatant, and collect the cell pellet.

[0084] 7. Add matrix gel to the cell pellet obtained in step 6 to make the cell density 1×10⁻⁶. 4 Cells / μL, then resuspend the cell pellet in an ice bath at 0-4℃, and mix the matrix gel and cells thoroughly to obtain a cell / matrix gel suspension.

[0085] 8. Seed the cell / Matrix gel suspension into each well of a cell culture plate, 2.0 μL per well. Incubate the cell culture plate at 37°C with 5% CO2 for 10–15 min. Add 100 μL of organoid complete culture medium to each well; incubate the cell culture plate at 37°C with 5% CO2.

[0086] 9. Once the organoid proliferation density reaches 80%, collect and resuspend the organoids in 5 ml of PBS solution under ice bath conditions at 0-4℃. Select 1-3 tumor organoids with a size greater than 5um*5um under a microscope.

[0087] 10. The organoid was drawn into a 27-gauge cannula through a 0.4 mm polyethylene tube connected to a 1 ml Hamilton syringe.

[0088] (II) Organ transplantation

[0089] Mice were anesthetized by inhaling 2% isoflurane, and their heads were restrained using a stereotactic head support. Under a stereomicroscope, a 27-gauge needle was used to puncture the cornea near the sclera, and a cannula containing the inhaled organoid was inserted. The organoid was then injected into the anterior chamber of the eye and fixed to the iris. One tumor organoid was transplanted into the anterior chamber of each mouse.

[0090] After successful transplantation, the drug sensitivity model of this invention can be obtained by culturing for 14 to 17 days.

[0091] II. Model Detection

[0092] (I) Detection Method

[0093] The detection method is the same as the in vivo imaging method in Example 1 for detecting neovascularization. In vivo imaging (II) was performed on the anterior chamber of the modeling mice at 0, 14, 17, 20, 30 and 60 days to observe whether there was neovascularization in the graft.

[0094] (II) Test Results

[0095] The results showed that neovascularization occurred in the anterior chamber of mice implanted with papillary thyroid carcinoma organoids from day 14 to day 17. Furthermore, the blood vessels and morphology of the mice implanted with papillary thyroid carcinoma organoids were maintained for a long period, currently at least 60 days. A total of 20 mice were used in this modeling study, with 16 successfully modeled, all within 14-17 days post-transplantation, resulting in a success rate of 80%. Three mice failed to model due to early post-transplantation eye infection leading to death, and one mouse failed to develop neovascularization after organoid transplantation. The results of one successfully modeled mouse are shown in the image below. Figure 2 As shown, the mouse's own blood vessels have a loose radial structure from the pupil to the periorbital area, while the neovascularization within the tumor is more dense. Figure 2 (yellow arrow) and the thickening of peritumoral nourishing blood vessels over time ( Figure 2 Red arrow.

[0096] Experimental results show that the method in this embodiment can effectively establish a drug sensitivity model of the anterior chamber of papillary thyroid carcinoma organoids, with short modeling time, model stability, and high modeling success rate.

[0097] Example 4: Constructing the anterior chamber drug sensitivity model of the tumor organoid of the present invention using organoids.

[0098] In addition to the anterior chamber drug sensitivity model of the papillary thyroid carcinoma organoid shown in Example 3, we also replicated this model in various benign and malignant tumors, using the aforementioned modeling method to establish anterior chamber drug sensitivity models of other tumor tissue organoids.

[0099] Currently, our team has used over 100 immunodeficient mice to construct organoid transplantation models, including those for pancreatic cancer, lung cancer, and thyroid cancer. We have also constructed anterior chamber drug sensitivity models for tumor organoids (both benign and malignant tumor tissues, such as pancreatic cancer, were derived from surgery or biopsy, and the drug sensitivity models were prepared using the modeling method described in Part 1 of Example 3). The modeling success rate was 86.3%, with all models successfully established 14-17 days post-transplantation, as shown in Table 1. The main reason for modeling failure was early post-transplantation eye infection leading to mouse death; only 2.1% of failures were due to the lack of neovascularization after organoid transplantation.

[0100] Table 1 shows the animal models for drug sensitivity testing constructed by building various tumor organoids.

[0101] pancreatic cancer 27 25 lung cancer 22 19 thyroid cancer 38 33 Breast cancer 15 11

[0102] Current research suggests that tumor stem cells can lead to tumor resistance and insensitivity to targeted therapies during drug treatment. If candidate drugs can directly inhibit angiogenesis caused by organoids and kill transplanted organoids into the anterior chamber of the eye, it would demonstrate exceptionally high therapeutic efficacy.

[0103] Experimental results show that the method in this embodiment can effectively establish a drug sensitivity model of the anterior chamber of tumor organoids. The modeling time is short, the model is stable, the modeling success rate is high, and it can be applied to different tumors, showing particularly good clinical application prospects.

[0104] The following experimental examples demonstrate the beneficial effects of the mouse model constructed in this invention.

[0105] Experimental Example 1: Verification of Drug Sensitivity Performance in a Mouse Model of Example 1 of the Invention

[0106] I. Experimental Methods

[0107] (I) Experimental Grouping

[0108] Based on in vivo imaging of the anterior chamber, neovascularization was confirmed within the anterior chamber graft in the lung cancer tissue anterior chamber drug sensitivity model of Example 1, indicating successful model establishment. Drug treatment was then conducted to verify drug sensitivity.

[0109] The mice successfully modeled in Example 1 were randomly divided into two groups of nine mice each, and the drug administration regimens were as follows:

[0110] Control group 1: Mice were administered 0.2 ml of PBS solution by gavage once daily for 14 consecutive days. Mice in this group did not receive any drug treatment.

[0111] Experimental group 1: Osimertinib was dissolved in 0.2 ml of PBS solution at a dose of 0.5 mg / kg and administered to mice by gavage once a day for 14 consecutive days.

[0112] (II) Detection of neovascularization using in vivo imaging methods

[0113] Fourteen days later, in vivo imaging was performed on control group 1 and experimental group 1 (the in vivo imaging method was the same as in Example 1). The background reflectance images of the tumor tissues in control group 1 and experimental group 1, as well as the in vivo imaging images of the anterior chamber vessels, i.e., the green fluorescence images of FITC, were obtained. The two images were then overlaid to obtain an overlaid image. Figure 3 ).

[0114] The efficacy of drug treatment was verified by observing changes in neovascularization and tumor volume. For neovascularization, the main observation indicators were changes in the number and diameter of new blood vessels. For tumor volume observation, the short diameter (W) and long diameter (L) of the transplanted tumor were measured using in vivo imaging, and the volume was calculated using the formula V(mm). 3 )=L×W 2 / 2 Calculate the tumor volume, and measure the tumor size once every two days.

[0115] II. Experimental Results

[0116] Compared with control group 1, the tumor volume in experimental group 1 was significantly reduced, by 1.01±0.09 mm. 3 and 0.48±0.14mm 3 The angiogenesis inhibition rate was significantly increased, with 1.70±0.57 in experimental group 1 and 0.48±0.14 in control group 1 (p<0.01). Furthermore, the rate of change in the maximum diameter of new blood vessels also showed a significant difference, with 1.11±0.11 in experimental group 1 and 0.38±0.37 in control group 1 (p<0.01).

[0117] Compared with the control group, the drug-treated group showed very significant changes in tumor volume, number of new blood vessels, and diameter of new blood vessels in the anterior chamber of the model, indicating that the model is highly sensitive to the drug and can be used to determine the therapeutic effect of the drug on the tumor.

[0118] Experimental results show that the anterior chamber drug sensitivity model of tumor tissue of the present invention can be used to rapidly screen drugs in order to find the best drugs for tumor treatment.

[0119] Experimental Example 2 and Example 3 of the present invention: Verification of drug sensitivity performance in mouse models

[0120] I. Experimental Methods

[0121] (I) Experimental Grouping

[0122] Based on in vivo imaging of the anterior chamber, neovascularization was confirmed within the graft of the papillary thyroid carcinoma organoid in Example 3, indicating successful modeling. Drug sensitivity was then verified through subsequent drug treatment.

[0123] The mice successfully modeled in Example 2 were randomly divided into two groups of eight mice each, and the drug administration regimen was as follows:

[0124] Control group 2: Mice were administered 0.2 ml of PBS solution by gavage once daily for 14 consecutive days. Mice in this group did not receive any drug treatment.

[0125] Experimental group 2: Lenvatinib was dissolved in 0.2 ml of PBS solution at a dose of 10 mg / kg and administered to mice by gavage once a day for 14 consecutive days.

[0126] (II) Detection of neovascularization using in vivo imaging methods

[0127] Fourteen days later, in vivo imaging was performed on control group 2 and experimental group 2 (the in vivo imaging method was the same as in Example 1). Background reflectance images of tumor tissue and in vivo images of the anterior chamber vessels of control group 2 and experimental group 2 were obtained, i.e., the green fluorescence image of FITC. These two images were then overlaid to obtain an overlapping image. Figure 4 ).

[0128] The efficacy of drug treatment was verified by observing changes in neovascularization and tumor volume. For neovascularization, the main observation indicators were the time of neovascularization, changes in the number of neovascularizations, and changes in the diameter of neovascularizations. For tumor volume observation, the short diameter (W) and long diameter (L) of the transplanted tumor were measured using a Leica multi-functional spectral in vivo microscopy system, and the volume was calculated using the formula V(mm). 3 )=L×W 2 / 2 Calculate the tumor volume, measuring tumor size every two days. Observe changes in the number of new blood vessels and calculate the angiogenesis inhibition rate.

[0129] II. Experimental Results

[0130] Compared with control group 2, the tumor volume in experimental group 2 was significantly reduced, by 1.04 ± 0.21 mm. 3 and 0.63±0.19mm 3 The angiogenesis inhibition rate also showed a significant change, with experimental group 2 at 1.40±0.31 and control group 2 at 0.67±0.19 (p<0.01). Furthermore, the rate of change in the maximum diameter of neovascularization also showed a significant difference, with experimental group 2 at 1.07±0.05 and control group 2 at 0.57±0.40 (p<0.01). These results indicate that the drug treatment in experimental group 2 has a significant inhibitory effect on tumor growth and angiogenesis.

[0131] The experimental results showed that, compared with the control group, the tumor volume, number of new blood vessels, and diameter of new blood vessels in the anterior chamber of the model were significantly different in the drug-treated group, indicating that the model is highly sensitive to the drug and can be used to determine the therapeutic effect of the drug on the tumor.

[0132] Experimental results show that the anterior chamber drug sensitivity model of tumor organoids of the present invention can be used for rapid drug screening to find the best drugs for tumor treatment.

[0133] This invention provides a modeling method for an in vivo tumor drug sensitivity animal model and its applications. This model exhibits high sensitivity, enabling dynamic and non-destructive evaluation of drug efficacy. It overcomes the problems of long drug screening cycles and low reliability, providing a new guiding model for clinical tumor treatment. This invention not only provides physicians with three-dimensional information on tumor morphology and vascular distribution, aiding in the development of more precise treatment plans, but also allows for the exploration of mechanisms by which tumor stem cells directly or indirectly transform into new blood vessels, identifying key targets for reversing angiogenesis, and providing in vivo model support for the development of related targeted drugs. Its application prospects are very promising.

Claims

1. A method for constructing an animal model of tumor drug sensitivity, characterized in that, Includes the following steps: a) Obtain tumor tissue samples to obtain tissue specimens; b) Cut tissue specimens into pieces to obtain tissue blocks, or digest tissue specimens into single cells and culture them to obtain tumor organoids; c) Transplant tissue blocks or organoids into the anterior chamber of immunodeficient mice. Once neovascularization appears in the anterior chamber graft, the transplant is complete. The tumor tissue sample is a solid tumor derived from surgery or biopsy; The immunodeficient mice were SCID mice; The size of the tissue block is (0.5-2) mm × (0.5-2) mm × (0.5-2) mm; the transplantation quantity is 1-3 blocks.

2. The method as described in claim 1, characterized in that, The tumor tissues include liver cancer tissue, lung cancer tissue, colon cancer tissue, stomach cancer tissue, pancreatic cancer tissue, thyroid cancer tissue, head and neck tumor tissue, parathyroid adenoma tissue, adrenal tumor tissue, cholangiocarcinoma tissue, and breast cancer tissue.

3. The method as described in claim 1 or 2, characterized in that, The tissue block was transplanted into the anterior chamber of the animal, and new blood vessels appeared in the anterior chamber graft after 3-5 days.

4. The method as described in claim 1, characterized in that, In step b), the method for preparing tumor organoids is as follows: 1) Cut the tissue specimen into tissue fragments, digest the tumor cell tissue with tissue digestion solution, and obtain single cells of tumor tissue; 2) Resuspend the cells in matrix gel, inoculate and culture them, and collect them after the organoid proliferation density reaches more than 50%.

5. The method as described in claim 4, characterized in that, In step 1), the tissue digestion solution was an Advanced DMEM F12 solution with a final concentration of 0.5 mg / ml collagenase XI and 0.2 mg / ml DNase. In step 2), the culture medium used for organoids was a DMEM / F12 solution supplemented with 1% vol / vol penicillin and streptomycin, 1% vol / vol L-alanyl-L-glutamine, 10 mmol / L 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) solution, 2% vol / vol B27 medium supplement, 1% vol / vol N2 medium supplement, 1.25 μmol / L N-acetyl-L-cysteine, 100 ng / mL epidermal growth factor (EGF), 25 ng / mL recombinant human R-spondin 1 protein, 100 ng / mL fibroblast growth factor 10 (FGF10), 10 mmol / L nicotinamide, and 10.5 μmol / L Y-27632 small molecule inhibitor. The substrate gel is Matrigel; the culture conditions are 37°C and 5% CO2.

6. The method as described in claim 4 or 5, characterized in that, Organoids with a size of (5-30) μm × (5-30) μm were selected and transplanted into the anterior chamber of the animal; 1-3 organoids were transplanted; neovascularization appeared in the graft after 14-17 days following organoid transplantation.

7. The animal model prepared by the method according to any one of claims 1 to 6 may be used in drug screening, drug toxicity testing or immunotherapy testing.

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