Application of epidermal growth factor in inducing apoptosis of upper urinary tract epithelial cancer organoids

Abstract

The invention provides an application of an epidermal growth factor (EGF) in induction of apoptosis of an upper urinary tract epithelial carcinoma organ (UTUC), and belongs to the technical field of biomedicine. The invention relates to an application of an EGF (Epidermal Growth Factor) or EGFR (Epidermal Growth Factor Receptor) agonist in inducing The EGF induces apoptosis of the upper urinary tract epithelial cancer organoid by activating the STAT1-Caspase 3 pathway and up-regulating the expression level of the EGFR. In view of the fact that the upper urinary tract epithelium carcinoma organoid and the upper urinary tract epithelium carcinoma tissue are highly consistent in form and regulation mechanism, the technical scheme provided by the invention provides a new thought for the medicine for preventing and / or treating the upper urinary tract epithelium carcinoma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to application of epidermal growth factor (EGF) in inducing upper urinary tract urothelial carcinoma (UTUC) organoid to apoptosis. BACKGROUND

[0002] Upper urinary tract urothelial carcinoma (UTUC) includes renal pelvis carcinoma and ureteral carcinoma, and belongs to urothelial carcinoma together with bladder cancer. UTUC is mostly seen in the middle-aged and the elderly, and accounts for 5% to 10% of all urothelial carcinomas. UTUC has many similar clinical and pathological features with bladder cancer, but UTUC itself also has its special points. UTUC is more invasive and has a poorer prognosis, and a high proportion of patients still have intravesical recurrence during follow-up after surgery.

[0003] Many studies have shown that organoids have modeling value in various aspects of biological research. Patient-derived organoids of different cancers can simulate key characteristics of the parent tumor, and can also be used for physiological and pathological research. The organoids are consistent with the parent tumor at the histopathological and molecular levels, and have mutation characteristics consistent with the clonal evolution of the parent tumor, which is the closest to the in vivo physiological environment and the highest in clinical similarity among current research models. Therefore, the survival and apoptosis trend reflected by the upper urinary tract urothelial carcinoma organoid to some drugs can reflect the therapeutic effect of the drugs on the cancer to some extent. Therefore, screening compounds affecting the proliferation and apoptosis of the upper urinary tract urothelial carcinoma organoid as the research object provides a new means for the treatment of cancer. There is no report on drugs for inducing apoptosis of the upper urinary tract urothelial carcinoma organoid. SUMMARY

[0004] Therefore, the present application aims to provide a new application of epidermal growth factor, i.e. application of epidermal growth factor in inducing upper urinary tract urothelial carcinoma organoid to apoptosis.

[0005] The present application provides application of epidermal growth factor or an EGFR agonist in inducing upper urinary tract urothelial carcinoma organoid to apoptosis.

[0006] The present application provides application of epidermal growth factor or an EGFR agonist in constructing an apoptosis model of upper urinary tract urothelial carcinoma organoid.

[0007] Preferably, the epidermal growth factor exists in the form of a cell culture medium additive or a cell culture medium.

[0008] The present application provides application of epidermal growth factor or an EGFR agonist in preparing a drug for preventing and / or treating upper urinary tract urothelial carcinoma.

[0009] Preferably, the treatment of upper urinary tract urothelial carcinoma includes inhibition of growth of the upper urinary tract urothelial carcinoma.

[0010] Preferably, the inhibiting the growth of upper urinary tract epithelial cancer comprises inhibiting the proliferation of upper urinary tract epithelial cancer cells and / or upper urinary tract epithelial cancer organoids and / or promoting the apoptosis of upper urinary tract epithelial cancer cells and / or upper urinary tract epithelial cancer organoids.

[0011] Preferably, the signal pathway for promoting the apoptosis of upper urinary tract epithelial cancer cells and / or upper urinary tract epithelial cancer organoids comprises STAT1-Caspase 3 pathway.

[0012] Preferably, the application further comprises the use of epidermal growth factor in combination with a specific drug for upper urinary tract epithelial cancer in the preparation of a drug for preventing and / or treating upper urinary tract epithelial cancer.

[0013] The application provides an anti-upper urinary tract epithelial cancer drug, and the active ingredients comprise epidermal growth factor and other drugs for treating upper urinary tract epithelial cancer.

[0014] Preferably, the dosage form of the drug comprises injection solution and / or injection powder.

[0015] The application provides the use of epidermal growth factor or an EGFR agonist in inducing the apoptosis of upper urinary tract epithelial cancer organoids. The experiments of the application show that epidermal growth factor not only inhibits the preparation of upper urinary tract epithelial cancer organoids, but also inhibits the growth and survival of the constructed upper urinary tract epithelial cancer organoids. The experiments show that epidermal growth factor gradually changes the morphology of EGF-sensitive upper urinary tract epithelial cancer organoids, the intercellular adhesion of the organoid cells disappears, the low transparency, the irregular edge, and the dissociation into single cells, the cell shrinkage and fragmentation, the cytoplasmic vacuoles, and the transparent circle formed around the organoid are very similar to the typical morphology of apoptotic cells. Through GO, KEGG, and Hallmark database annotation of the transcriptome data of the treated upper urinary tract epithelial cancer organoids and using R language enrichment analysis, it is found that the differential genes are mainly enriched in the cell skeleton, proliferation regulation, and programmed death ligand-receptor binding pathways, and the apoptosis pathway (such as JAK-STAT) is significantly up-regulated. The application further proves that the expression of EGFR of the EGF-sensitive organoids is EGF-dependent, EGF can promote the generation and phosphorylation expression of EGFR; at the same time, the treatment of EGF also heterogeneously activates the effector Caspase 3 / 7 of the apoptosis pathway. It can be seen that EGF induces the apoptosis of upper urinary tract epithelial cancer organoids by activating the STAT1-Caspase 3 pathway and up-regulating the expression level of EGFR. Since the morphology and regulation mechanism of the upper urinary tract epithelial cancer organoids are highly consistent with those of the upper urinary tract epithelial cancer tissue, the technical solution provided by the application provides a new idea for the drug for preventing and / or treating upper urinary tract epithelial cancer. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1Results of EGF inhibiting UTUC organoid growth, A is the dose-effect curve of UTUC organoids after EGF treatment; the numerical value represents the mean ± standard error (n = 3); B is the EGFR gene mutation of UTUC_1_O, UTUC_2_O and UTUC_6_O and the IC 50 value of EGF; C-D are the time-survival curves and white light images on the 0th, 3rd and 6th days after adding 41 ng / mL, 0.45 ng / mL and 50 ng / mL EGF to UTUT_1_O, UTUT_2_O and UTUT_4_O, respectively; scale bar, 100 μm;

[0017] Figure 2 Results of transcriptome analysis of genes differentially expressed in EGF-induced UTUC death; A is a UMAP plot showing the distribution of UTUC_1_O and UTUC_2_O before and after EGF treatment; B is a heat map of differentially expressed genes in UTUC_1_O and UTUC_2_O before and after EGF treatment; C is the intersection of the number of differentially expressed genes in UTUC_1_O and UTUC_2_O after EGF treatment; P < 0.001;

[0018] Figure 3 Results of transcriptome analysis of signaling pathways involved in EGF-induced UTUC death; A is the intersection of differentially expressed genes in UTUC_1_O and UTUC_2_O after EGF treatment; B is the GO annotation and enrichment analysis of differentially expressed genes using R software and R package org.Hs.eg.db (version 3.1.0) to obtain the top 10 gene set enrichment pathways and EGFR pathway enrichment; C is the KEGG annotation and enrichment analysis of differentially expressed genes using R software and KEGG RESTAPI to obtain the top 10 gene set enrichment pathways and EGFR pathway enrichment; D is the Hallmark annotation and enrichment analysis of differentially expressed genes using R software and h.all.v7.4.symbols.gmt subset to obtain the top 10 gene set enrichment pathways and EGFR pathway enrichment; E is the GO enrichment analysis of the top 25 upregulated genes and the top 25 downregulated genes in UTUC_1_O and UTUC_2_O after EGF treatment; all the enrichment pathway analyses above were performed using R package clusterProfiler, p < 0.001;

[0019] Figure 4Figure 6. EGF induces apoptosis in UTUC organoids via STAT1 pathway; A, EGF induces the expression of EGFR and its phosphorylation; B, GSEM analysis of the enrichment of two organoids in the apoptosis pathway; C, GSEM analysis of the enrichment of two organoids in the JAK-STAT pathway; D-E, EGF activates the STAT1 pathway and the expression of related proteins in the downstream apoptosis pathway; EGF treatment concentration: UTUT_1_O: 41 ng / mL, UTUT_2_O: 0.45 ng / mL, UTUT_3_O: 50 ng / mL, UTUT_4_O: 50 ng / mL;

[0020] Figure 5 Figure 7. EGF induces the production of active Caspase 3 to promote apoptosis in UTUC organoids; A-B, fluorescence images of the activity of Cleaved-caspase 3 / 7 in UTUC organoids treated with EGF for 2, 4, and 6 days; scale bar, 50 μm; C-D, quantification of the expression of Caspase 3 and Cleaved-caspase 3 in UTUC organoids treated with EGF for 0, 3, and 6 days; EGF concentration: UTUT_1_O: 41 ng / mL, UTUT_2_O: 0.45 ng / mL. Values represent mean ± standard error, unpaired t-test, *p<0.05. DETAILED DESCRIPTION

[0021] The present application provides the use of epidermal growth factor or an EGFR agonist in inducing apoptosis in upper urinary tract urothelial carcinoma organoids.

[0022] The present application provides the use of epidermal growth factor or an EGFR agonist in constructing an apoptosis model of upper urinary tract urothelial carcinoma organoids.

[0023] In the present application, the epidermal growth factor (EGF) is a polypeptide growth factor composed of 53 amino acids, with a molecular weight of about 6000 Da. EGF is widely present in eukaryotes, and in mammals, EGF is mainly produced by salivary glands, gastrointestinal tract, and skin keratinocytes. EGF is an epidermal-specific growth factor that mainly acts on epidermal cells to promote the proliferation and differentiation of epidermal cells. EGF binds to the EGF receptor (EGFR) on the surface of epidermal cells, activates the EGFR signaling pathway, and thus promotes the proliferation and differentiation of epidermal cells. The epidermal growth factor is preferably present in the form of a cell culture medium or a cell culture medium additive.

[0024] In the present application, the EGFR agonist is a substance that activates EGFR, including a compound, a polypeptide or a protein, etc. The present application does not make special restrictions on the type of EGFR agonist, and the type of EGFR agonist known in the art can be used. As shown in the examples of the present application, the mechanism of inducing apoptosis of the upper urinary tract urothelial carcinoma organoid by epidermal growth factor is to activate the expression of downstream pathways to play an apoptosis role. Therefore, the EGFR agonist can also induce apoptosis of the upper urinary tract urothelial carcinoma organoid.

[0025] In the present application, the method for inducing apoptosis of the upper urinary tract urothelial carcinoma organoid is preferably culturing the upper urinary tract urothelial carcinoma organoid in a system containing epidermal growth factor. The concentration of the epidermal growth factor is preferably not less than 0.45 ng / mL, and can be 41-83.72 ng / mL. The culture condition is preferably a cell culture box at 37°C and 5% CO2. The upper urinary tract urothelial carcinoma organoid preferably includes an epidermal growth factor-sensitive upper urinary tract urothelial carcinoma organoid. The method for constructing the apoptosis model of the upper urinary tract urothelial carcinoma organoid is the same as the method for inducing apoptosis of the upper urinary tract urothelial carcinoma organoid, which is not described here. The application of the apoptosis model of the upper urinary tract urothelial carcinoma organoid in analyzing the mechanism of drug action.

[0026] The present application provides the use of epidermal growth factor or an EGFR agonist in the preparation of a drug for preventing and / or treating upper urinary tract urothelial carcinoma.

[0027] In the present application, the treatment of upper urinary tract urothelial carcinoma preferably includes inhibiting the growth of upper urinary tract urothelial carcinoma. The inhibition of the growth of upper urinary tract urothelial carcinoma preferably includes inhibiting the proliferation of upper urinary tract urothelial carcinoma cells and / or upper urinary tract urothelial carcinoma organoids and / or promoting the apoptosis of upper urinary tract urothelial carcinoma cells or / and upper urinary tract urothelial carcinoma organoids. In an embodiment of the present application, after the upper urinary tract urothelial carcinoma cells or organoids treated by epidermal growth factor are observed morphologically, it is found that epidermal growth factor does not promote the proliferation of UTUC organoids, but inhibits the survival of some UTUC organoids. After the UTUC organoids are treated by epidermal growth factor, the cell proliferation rate slows down, and the morphology of the organoids gradually changes; the intercellular adhesion of the organoid cells disappears, the low transparency, the irregular edge and the dissociation into single cells, the cell shrinkage and fragmentation, the cytoplasmic vacuoles, and the transparent ring formed around the organoids are very similar to the typical morphology of apoptotic cells.

[0028] In the present application, the signal pathway for promoting the apoptosis of upper urinary tract epithelial cancer cells and / or upper urinary tract epithelial cancer organoids preferably comprises the STAT1-Caspase 3 pathway. In another embodiment of the present application, in order to further clarify the specific mechanism of EGF-induced cell apoptosis, the relevant pathways and related genes of EGF-induced cell apoptosis are clarified by transcriptome analysis, and the results show that EGF significantly activates the defensive response of UTUC organoids. The expression levels of immune regulatory factors of UTUC organoids are increased, including SPRR2D, SPRR3, DHRS9, etc., and the high expression of these regulatory genes can enhance the adaptive immunity of UTUC organoids. At the same time, EGF regulates the expression of apoptosis-related proteins in UTUC organoids, thereby causing the programmed death of the organoids. EGF-induced UTUC death involves a series of body defense pathways such as activation of cell inflammation, complement response, immune response, programmed death, etc. In order to further clarify the apoptosis mechanism, the expression level of EGFR and the expression of representative proteins of the apoptosis signaling pathway are also detected, and the results show that the expression of EGFR in the organoids sensitive to EGF is EGF-dependent, and EGF can promote the generation and phosphorylation expression of EGFR. After the expression of EGFR is activated, the downstream JAK-STAT pathway and the apoptosis pathway are significantly enriched in expression, and the expression of STAT1 protein and its phosphorylation degree also gradually increase with the growth of EGF incubation time. The effector Caspase 3 / 7 of the apoptosis pathway is activated heterogeneously after EGF treatment, and the enzyme-cleaved-caspase 3 / 7 with activity is generated, and the activation degree of the apoptosis signaling pathway is higher with the growth of EGF incubation time. It can be seen that after EGF induces the organoids, the expression level of EGFR is significantly increased, and the apoptosis reaction is dominated by Caspase 3 through the STAT1 pathway, thereby inducing the apoptosis of UTUC.

[0029] In the present application, the application preferably further comprises the use of epidermal growth factor in combination with specific drugs for treating upper urinary tract epithelial cancer in the preparation of drugs for preventing and / or treating upper urinary tract epithelial cancer. The specific drugs for treating upper urinary tract epithelial cancer preferably include Jelmyto, aiplatin, cisplatin, etoposide, doxorubicin, nivolumab, pembrolizumab, aserafinib, everolimus, ofatumumab, gleevec, sunitinib, etc.

[0030] The present application provides an anti-upper urinary tract epithelial cancer drug, and the active ingredients include epidermal growth factor and other drugs for treating upper urinary tract epithelial cancer.

[0031] In the present application, the dosage form of the drug preferably includes an injection solution and / or an injection powder. When the injection solution, the excipient of the drug includes a 0.9% sodium chloride aqueous solution. The present application does not have a special limitation on the preparation method of the drug, and the preparation method of the drug known in the art can be used.

[0032] The application of the epidermal growth factor provided by the present application in inducing apoptosis of upper urinary tract epithelial cancer organoids is described in detail below in combination with examples, but they cannot be understood as limiting the protection scope of the present application.

[0033] Example 1

[0034] Construction of upper urinary tract epithelial cancer organoids

[0035] 1) After the sample is removed from the body, wash it twice with pre-cooled PBS, and cut it into four small pieces (particle size <5 mm) with a sterilized scalpel.

[0036] 2) One piece of tissue is soaked in formalin. Two pieces of tissue are snap-frozen and stored at -80°C for subsequent RNA extraction and sequencing.

[0037] 3) The remaining tumor tissue is further chopped into tissue pieces with a particle size <2 mm, and placed in a dissociation solution (5 mg / mL collagenase II, 10 μM Y-27632, 1% DNAase, 2 mg / mL neutral protease) for 30-60 min in a 37°C water bath, until the tissue becomes transparent.

[0038] 4) Centrifuge the digested tissue fragments at 300g for 3 min, and discard the supernatant.

[0039] 5) After incubation with TrypLE Express containing 10 μM Y-27632 at 37°C for 5 min, terminate the digestion by adding 2 times the amount of organoid neutralization solution. The composition of the organoid neutralization solution is shown in Table 1.

[0040] 6) The cell suspension is passed through a 70 μm filter to remove incompletely digested cell clumps.

[0041] 7) Collect the filtrate, centrifuge at 300g for 3 min, discard the supernatant, and wash once with preheated UTUC organoid medium. The composition of the UTUC organoid medium is shown in Table 2. Centrifuge again, and discard the supernatant.

[0042] 8) Resuspend the organoids with pre-cooled Matrigel to prepare an organoid-Matrigel mixture, and then quickly inoculate it into a six-well plate at 30 μL per drop (about 50,000 cells / drop).

[0043] 9) Invert the six-well plate in a 37°C, 5% CO2 cell incubator for 10 min to solidify the Matrigel.

[0044] 10) Add 2.5 mL UTUC organoid medium per well, and 2.5 mL PBS to the blank wells, then place the six-well plate in the cell culture incubator.

[0045] 11) Replace the UTUC organoid medium containing Y-27632 every 3 days, and observe the growth of the organoids using an optical microscope. From the 7th day, culture using UTUC organoid medium without Y-27632.

[0046] Table 1. Components of the organoid medium and neutralization solution

[0047] Reagent name Brand Catalogue number Concentration Advanced DMEM / F-12 Thermo 12634 1× Antibiotic-Antimycotic Thermo 15240- 1× GlutaMAX Thermo 35050- 1× HEPES Thermo 15630 1× FBS Gibco 10099141 20％ Y-27632 Abmole Bioscience M1817 10 mM

[0048] Table 2. Components of the UTUC organoid medium

[0049]

[0050]

[0051] 12) Passage the organoids after 14 days.

[0052] (1) Gently scrape the organoid-matrigel mixture adhered to the bottom of the six-well plate with a 1 mL gun tip, and collect in a 15 mL centrifuge tube, centrifuge at 300g for 3 min, and discard the supernatant.

[0053] (2) Add 2 mL of TrypLE Express containing 10 μM Y-27632, incubate at 37°C for 5 min, and shake 2-3 times during the incubation. Then add 2 times the amount of organoid neutralization solution to terminate the digestion, centrifuge at 300g for 3 min, and discard the supernatant.

[0054] (3) Wash once with UTUC organoid medium, centrifuge again, and discard the supernatant.

[0055] (4) Resuspend the organoids with pre-cooled matrigel to make an organoid-matrigel mixture, and then quickly inoculate them into the six-well plate at a particle size of 30 μL / drop.

[0056] (5) Invert the six-well plate in a cell culture incubator at 37°C, 5% CO2 for 10 min to solidify the matrigel.

[0057] (6) Next, add 2.5 mL UTUC organoid medium per well, and 2.5 mL PBS to the blank wells, then place the six-well plate in the cell culture incubator.

[0058] (7) Replace the fresh UTUC organoid medium every 2-3 days. Passage at a ratio of 1:2 every 2-3 weeks.

[0059] Comparative Example 1

[0060] The preparation of UTUC organoids was carried out according to the method of Example 1, with the exception that the UTUC organoid culture medium was replaced by the bladder cancer organoid culture medium, and the other steps were the same.

[0061] The bladder cancer organoid culture medium is based on the Advanced DMEM / F-12 culture medium, and also contains 1x B27, 500 ng / mL Wnt agonist R-Spindin1, 20 ng / mL fibroblast growth factor 10, 100 ng / mL transforming growth factor beta inhibitor Noggin, 1.25 mM ROS scavenger N-acetyl-L-cysteine, 10 mM nicotinamide, 500 nM activin receptor-like kinase (ALK) inhibitor A83-01, 50 ng / mL EGF, 10 mM p38 mitogen-activated protein (MAP) kinase inhibitor SB202190, 5 ng / mL fibroblast growth factor 2, and 10 mM ROCK inhibitor Y-27632.

[0062] The culture results showed that this culture method failed to successfully construct UTUC organoids. Three organoids grew normally in the first few days, but stopped growing in some cases, and even died.

[0063] The inventors found that EGF was the key factor inhibiting the growth of UTUC organoids by retesting and excluding the growth factor components in the culture medium one by one.

[0064] Example 2

[0065] 1. Organoid viability detection

[0066] 1) Plating: After the above-mentioned passaging preparation of organoids was dissociated and digested into small cell clusters from the matrigel, it was passed through a 100 μm filter membrane, the filtrate was collected and counted. After centrifugation at 300g for 5 min, the supernatant was discarded, and 5% matrigel-containing UTUC organoid culture medium was added to resuspend and prepare 3x10 5 4mL organoid suspension, 80 μL of organoid suspension was taken and added to a 96-well plate, and placed in a cell incubator overnight.

[0067] 2) Drug addition: After plating for 24 h, the corresponding concentrations of EGF drugs were prepared and added to the corresponding wells, and then placed in a cell incubator.

[0068] 3)Detection: Let the 96-well plate to be tested at room temperature for 5 min to restore the temperature of the 96-well plate to room temperature. Then add 80 μL of CellTiter-Glo 3D Cell Viability Reagent to each well, avoid light and incubate on a shaker for 30 min to stabilize the luminescence signal, and then use the microplate reader to detect the luminescence intensity.

[0069] 4)Data analysis: Use GraphPad Prism 8 for data analysis, IC 50 Calculated by non-linear regression (curve fitting) of equation Log (inhibitor) versus normalized response (variable slope).

[0070] 2.Organoid morphology experiment

[0071] 1) After the organoids prepared above are dissociated and digested into small cell clusters from the Matrigel, pass through a 100 μm filter membrane, collect the filtrate, and count.

[0072] 2) After centrifugation at 300g for 5 min, discard the supernatant, add 100 μL of pre-cooled Matrigel to resuspend and configure into 6x10 5 6x10

[0073] 3) After 24 h, discard the old culture medium, then add UTUC organoid culture medium containing different concentrations of EGF to the corresponding wells, and replace the fresh culture medium every 2 days.

[0074] 4) After adding EGF for 0, 3, 6 days, observe and record the morphological changes of the organoids under the microscope.

[0075] The sensitivity of UTUC organoids to EGF is shown in Figure 1 A and B. The results show that EGF does not promote the proliferation of UTUC organoids, and even inhibits the survival of some UTUC organoids. Then, UTUT_1_O and UTUT_2_O were added with EGF-containing culture medium with corresponding IC 50 Concentrations of 41 ng / mL and 0.45 ng / mL EGF. On the first two days after adding EGF, no significant changes in cell morphology occurred. After the third day, the cell proliferation rate slowed down, and the morphology of the organoids gradually changed. The intercellular adhesion of the organoids disappeared, the low transparency, irregular edges, dissociation into single cells, cell shrinkage and fragmentation, cytoplasmic vacuoles, and transparent circles around the organoids were formed, which were very similar to the typical morphology of apoptotic cells. Figure 1UTUC_4_E, Fig. 12C, D). In contrast, UTUC organoids that were insensitive to EGF (UTUC_4_O) did not show any significant changes in organoid morphology and growth status after addition of medium containing 50 ng / mL of EGF.

[0076] 3. Transcriptome sequencing

[0077] 1) Sample collection: Discard old medium, wash once with pre-cooled PBS. Add 1 mL cold PBS, use 1 mL pipette tip to gently scrape the organoid-matrigel mixture attached to the bottom of the well plate and collect it into a sterile RNase-free 1.5 mL EP tube. Centrifuge at 4°C, 2500 g for 5 min, discard the supernatant, and use a pipette to aspirate the residual PBS. Add 1 mL TRIzol reagent, and vortex the solution until no visible organoid clumps are observed and the solution is clear and transparent. Then, place the solution in liquid nitrogen for 5 min, and finally store at -80°C.

[0078] 2) RNA extraction: Thaw the sample stored in TRIzol at room temperature. After adding 0.2 mL chloroform, let it stand for 2 min. Centrifuge at 4°C, 2500 g for 15 min, at this time the solution in the EP tube is divided into three layers, carefully aspirate the upper clear RNA aqueous phase into a new 1.5 mL EP tube with a pipette, discard the middle layer and the white DNA precipitate and pink organic phase at the bottom. Then add 0.5 mL isopropanol, mix well, and stand at -20°C for 30 min. Centrifuge under the same conditions, discard the supernatant. Gently add 1 mL of fresh 75% ethanol to wash the white precipitate, centrifuge, discard the supernatant, and repeat 2 times. Wipe the outer tube wall with a dust-free paper, and dry the precipitate in a 45°C dry metal bath. When only 1-2 μL of solution remains around the precipitate, add 100-200 μL of DEPC water, and gently blow it evenly with a pipette tip. Finally, aspirate 1 μL of RNA solution into a spectrophotometer to obtain the concentration and purity of the extracted RNA.

[0079] 3) Library construction and sequencing: 1 μg of RNA is fragmented and cDNA synthesis is performed using random hexamer primers, M-MuLV reverse transcriptase, polymerase, RNase H, and exonuclease. Then, the cDNA is sequentially subjected to terminal adenylation, fragment purification, PCR amplification, gel electrophoresis separation and recovery of the target DNA to obtain the cDNA library. The library is sequenced using Illumina NovaSeq60 and Illumina SE50.

[0080] 4) Transcriptome data analysis: The raw data in fastq format was processed by perl scripts, reference genome and annotation files were matched to each chip number. The number of reads and FPKM of each gene were calculated, and the gene differential expression analysis between two groups was performed using edgeR in R language.

[0081] 4. Transcriptome analysis of genes and pathways involved in EGF-induced UTUC death

[0082] To explore the mechanism of EGF-induced UTUC death, UTUC_1_O and UTUC_2_O were treated with 41 ng / mL and 0.45 ng / mL EGF for 6 days, respectively. The EGF-treated organoids and the corresponding control groups without EGF treatment were subjected to transcriptome sequencing and comparison.

[0083] Through UMAP dimension reduction and clustering analysis, it was found that the EGF-treated UTUC organoids could not be clustered with the control group, and their gene expression profiles were different Figure 2 A, B) Compared with the control group, there were 1559 differentially expressed genes in UTUC_1_O and UTUC_2_O after EGF treatment Figure 2 C) Among them, the top 5 genes that were commonly up-regulated and down-regulated are shown in Table 5. EGF significantly activated the defensive response of UTUC organoids. The expression levels of immune regulatory factors in UTUC organoids were increased, including SPRR2D, SPRR3, DHRS9, etc. The high expression of these regulatory genes could enhance the adaptive immunity of UTUC organoids. At the same time, EGF regulated the expression of apoptosis-related proteins in UTUC organoids, thereby causing programmed death of the organoids. In addition, the inflammatory response of UTUC organoids was enhanced, indicating that the organoids were in the process of injury repair.

[0084] Next, the changes in signaling pathways of the two UTUC organoids after EGF treatment were further studied. After GO, KEGG, and Hallmark database annotation and enrichment analysis using R language, there were 493 common pathways that significantly changed in the two organoids after EGF induction Figure 3 A) The results of the three database analyses showed that the differential genes were mainly enriched in the pathways of cytoskeleton, proliferation regulation, and programmed death ligand-receptor binding, among which the apoptosis pathway (such as JAK-STAT) was significantly up-regulated Figure 3 B-D) In addition, the GO database showed that the differential genes were also enriched in the pathways of cell metabolism and biosynthesis; the KEGG database showed that there were differential genes enriched in the immune response-related pathways; and the Hallmark database revealed that there were certain degree of differential expression in the regulation pathways of cell inflammatory response and cell invasion and migration.

[0085] Notably, when performing enrichment analysis on the top 25 significantly up-regulated genes and the top 25 significantly down-regulated genes, it was found that about one-fifth of the genes were involved in regulating the death process of cells (including apoptosis) Figure 3 Table 3. Top 5 significantly down-regulated or up-regulated genes after EGF induction

[0086] Table 3. Top 5 significantly down-regulated or up-regulated genes after EGF induction

[0087]

[0088]

[0089] Note: Significance p < 0.00.

[0090] In summary, EGF-induced UTUC death involves epidermal cell stress response, such as activation of cell inflammation, complement response, immune response, programmed death, and a series of body defense pathways.

[0091] 5. Caspase-3 / 7 fluorescence photography

[0092] 1) After the above passaged organoids were dissociated from the substrate glue and digested into small cell clusters, they were filtered through a 100 μm filter membrane, the filtrate was collected and counted.

[0093] 2) After centrifugation at 300g for 5 min, the supernatant was discarded, 100 μL of pre-cooled substrate glue was added to resuspend and configure into 6x10 5 μL of organoid-substrate glue mixture, and then quickly inoculate it into a 12-well plate at 25 μL per drop. After the 12-well plate was inverted to allow the substrate glue to solidify, UTUC organoid culture medium was added, and it was placed in a cell culture incubator.

[0094] 3) After 24 h, the old culture medium was aspirated and replaced with UTUC organoid culture medium containing different concentrations of EGF. Fresh culture medium was replaced every 2 days.

[0095] 4) After 0, 3, and 6 days of EGF addition, the old culture medium was discarded, and fresh culture medium containing Caspase-3 / 7 Green Detection Reagent (1:2000) was added to each well.

[0096] 5) Incubate for 3 h in the dark, discard the old culture medium, add PBS containing Hoechst (1:100), and incubate again in the dark for 10 min. Then wash twice with PBS by gently shaking, and then observe.

[0097] 6. ELISA detection of Cleaved-Caspase-3(Asp175) and Caspase-3 concentration

[0098] 1) Cell lysis: Collect the EGF treated organoids in 15 mL centrifuge tube and perform digestion neutralization, then wash the organoid pellet with cold PBS and centrifuge to discard the supernatant, repeat twice, and the last time use a pipette to remove PBS as much as possible. Add 200 μL of diluted 1 x cell lysis solution, blow several times, and then ice bath for 20 min. Centrifuge at 4°C, 2500g for 20 min, and transfer the supernatant containing Cleaved-Caspase-3(Asp175) and Caspase-3 to a sterile 1.5 mL EP tube.

[0099] 2) Quantify the total protein concentration in the sample using the BCA method:

[0100] a) Gradient dilution of BSA standard: Dilute the BSA standard with RIPA according to the dilution range listed in Table 4.

[0101] Table 4 BSA standard gradient dilution concentration

[0102]

[0103]

[0104] b) Preparation of BCA working solution: Mix reagent A and reagent B in the kit (ratio 50:1) to obtain a green and clear BCA working solution.

[0105] c) If the protein concentration of the extracted sample is large, the protein sample to be tested can be diluted 5-15 times with RIPA first. Add 25 μL of the above prepared BSA working solution and protein sample to each well of the 96-well plate, and set two replicates for each group.

[0106] d) Slowly add 200 μL of freshly prepared BCA working solution to the experimental wells along the well wall.

[0107] e) Shake for 30 min in the dark, and measure the absorbance value of each well at 562 nm wavelength.

[0108] f) Calculate the protein amount: First calculate the average absorbance value of the gradient dilution BSA standard and the protein sample to be tested after blank correction, and draw a standard curve, and obtain the protein concentration of the sample.

[0109] g) Dilute the protein concentration of each sample to 40 ng / μL by adding 1 x RIPA.

[0110] 3) Preparation of diluent (E2) and 1 x washing solution: Prepare 1 x diluent (E2) 3 mL and 1 x diluent 500 mL with triple distilled water, respectively.

[0111] 4) Preparation of Positive Control: After a simple vortex, add 120 μL of E2 to the positive control reagent to prepare the positive control solution (P1). Prepare the positive control according to the dilution range listed in Table 5. Mix well before each transfer of solution.

[0112] Table 5 ELISA Positive Control Gradient Dilution Concentration

[0113] Serial number Volume of positive control solution added (μL) and source Volume of E2 added (μL) P1 0, stock solution 1200 P2 150，P1 300 P3 150，P2 300 P4 150，P3 300 P5 150，P4 300 P6 0 300

[0114] Antigen Incubation: Add 100 μL of sample or positive control to each well of the ELISA plate. Set up two replicates for each group. Incubate in a 37°C incubator for 2.5 h.

[0115] 5) Primary Antibody Incubation: Aspirate the solution and wash each well with 300 μL of lx wash buffer, repeat 4 times, remove as much solution as possible from the wells and invert the plate to drain the wells. Add 100 μL of lx Cleaved-Caspase-3 (Asp175) or lx Caspase-3 to the appropriate wells and incubate at room temperature for 1 h with shaking.

[0116] 6) Secondary Antibody Incubation: Aspirate the solution from the plate as completely as possible and wash each well with 300 μL of lx wash buffer, repeat 4 times, remove as much solution as possible from the wells and invert the plate to drain the wells. Add 100 μL of lx HRP-conjugated anti-rabbit anti-Cleaved-Caspase-3 (Asp175) or lx HRP-conjugated anti-mouse anti-Caspase-3 secondary antibody to the appropriate wells and incubate at room temperature for 1 h with shaking.

[0117] 7) Color Development: Aspirate the solution from the plate as completely as possible and wash each well with 300 μL of lx wash buffer, repeat 4 times, remove as much solution as possible from the wells and invert the plate to drain the wells. Add 100 μL of TMB reagent to each well and incubate at room temperature for 30 min with shaking in the dark.

[0118] 8) Stop: Add 50 μL of stop solution to each well along the wall of the plate and immediately read the absorbance at 450 nm using a microplate reader.

[0119] 7. Western Blot

[0120] 1) Protein Extraction

[0121] a) Take the UTUC organoids that have been treated accordingly and centrifuge at 500 g for 3 min in a 15 mL centrifuge tube. Discard the supernatant.

[0122] b) Add 2 mL TrypLE Express containing 10 μM Y-27632, incubate at 37 °C for 5 min, shake 2-3 times during the incubation. Then add 4 mL to the organoids and mix, centrifuge at 300 g for 3 min, discard the supernatant.

[0123] c) Add RIPA lysis buffer, mix with vortex shaker, ice bath for 30 min.

[0124] d) Centrifuge at 2500 g for 30 min at 4 °C, collect the supernatant into a new 1.5 mL EP tube.

[0125] e) After quantifying the protein concentration by BCA method, according to the amount of protein loading for electrophoresis is 10 μg and the amount of SDS loading buffer added, the total volume of loading is 10 μL. Then denature the protein in a dry metal bath at 100 °C for 5 min, and store at -80 °C.

[0126] 2) Protein electrophoresis

[0127] a) Place the PAGE gel prepared according to the instructions in the designated position of the electrophoresis tank, add electrophoresis buffer to check for leaks. After confirming that the gel is tightly sealed with the electrophoresis tank, add electrophoresis buffer to the scale line.

[0128] b) Before loading the samples, gently shake them first, then sequentially pipette each protein sample into the designated gel loading well. The amount of protein loaded for each group is 10 μg, and the volume of Marker loaded is 3 μL.

[0129] c) Turn on the power, first use constant voltage 60 V electrophoresis, and then use constant voltage 120 V electrophoresis until the bromophenol blue band in the sample reaches about 1 cm from the bottom of the gel.

[0130] 3) Membrane transfer

[0131] a) Prepare fresh 1x transfer buffer and store in the 4 °C refrigerator for use.

[0132] b) Cut a 6 cm x 8 cm size PVDF membrane, immerse in methanol for 5 min.

[0133] c) Place the transfer clip negative side, sponge sheet, filter paper, PVDF membrane, PAGE gel, filter paper, sponge sheet, transfer clip anode side from bottom to top, close the transfer clip. The entire operation should be carried out in 1x transfer buffer and ensure that there are no air bubbles in the transfer clip, then it can be placed in the transfer tank.

[0134] d) Pour 1x transfer buffer to cover the top of the clip, transfer for 2 h in ice bath at a constant current of 220 mA.

[0135] 4) Blocking and antibody incubation

[0136] a) Blocking: PVDF membrane was immersed in 5% BSA blocking solution, and blocked at room temperature for 2h.

[0137] b) Primary antibody incubation: Primary antibody was prepared in PBS at working concentration, and PVDF membrane was cut into appropriate size according to the location of the target protein and placed in the primary antibody working solution, and incubated at 4°C overnight.

[0138] c) Secondary antibody incubation: PVDF membrane was immersed in TBST and washed for 10 min x 5 times using a shaker. Then secondary antibody was prepared in PBS at working concentration, and PVDF membrane was placed in the secondary antibody working solution, and incubated at room temperature for 45 min.

[0139] d) PVDF membrane was immersed in TBST and washed for 10 min x 5 times using a shaker.

[0140] 5) Development

[0141] Fresh development solution was prepared according to the ECL kit instructions, and the water on the surface of the PVDF membrane was gently absorbed with a paper towel, then the development solution was added dropwise to cover the membrane, and the Amersham chemiluminescence gel imager was used for exposure imaging.

[0142] EGF induces apoptosis of UTUC organoids through STAT1-Caspase 3 pathway

[0143] EGFR has multiple phosphorylation sites in the tyrosinase catalytic region on the inner side of the cell membrane. EGFR binds to different ligands, which specifically activates the phosphorylation of EGFR tyrosine sites, thereby initiating the corresponding downstream signal transduction. Different activated phosphorylation sites have different downstream activated signal pathways. Tyr992 is related to PLC-IP3 / DAG pathway; Tyr1173 is related to PI3K-AKT pathway; and Tyr1068 is related to JAK-STAT pathway.

[0144] The results of Western blotting experiment showed that the expression level of EGFR in two cases of UTUC organoids (UTUC_3_O and UTUC_4_O) insensitive to EGF was low, and the addition of EGF could not increase the expression amount of EGFR; on the contrary, under the stimulation of EGF, the expression of EGFR in two cases of EGF-sensitive UTUC organoids (UTUC_1_O and UTUC_2_O) was significantly increased, and the expression of p-EGFR(Y1068) in UTUC_1_O was also significantly enhanced (Fig. A). Figure 4 This indicates that the expression of EGFR in the organoid that may be sensitive to EGF has EGF dependence, and EGF can promote the generation and phosphorylation expression of EGFR.

[0145] When EGFR expression is activated, downstream JAK-STAT and apoptosis pathways are significantly enriched. Figure 4 (B, C). Meanwhile, in the two EGF-sensitive UTUC organoids, the expression and phosphorylation level of STAT1 protein gradually increased with increasing EGF incubation time. Figure 4 D, E).

[0146] Therefore, the transcriptional expression of pro-apoptotic genes (Table 6) and anti-apoptotic genes (Table 7) after EGF treatment was analyzed, and the expression of characteristic proteins of the apoptosis signaling pathway was detected. The results showed that the activated STAT1 pathway promoted the expression levels of pro-apoptotic proteins such as Caspase 7, Caspase 9, and Bax, while the expression of anti-apoptotic proteins such as Bcl-2 was inhibited. Figure 4 D, E).

[0147] As an effector in the apoptosis pathway, Caspase 3 / 7 is heterologously activated within 2-6 days after EGF addition, and is cleaved to generate active Cleaved-caspase 3 / 7. The activation level of the apoptosis signaling pathway increases with increasing EGF incubation time. Figure 5 (A, B). On day 6 of EGF treatment, UTUC organoids produced 4.1–6.6 times more Cleaved-caspase 3 than the untreated group. Figure 5 In C and D), the cleavage effect on the substrate PARP was also enhanced. Figure 4 (D).

[0148] Table 6. Log2|FC| values ​​of pro-apoptotic gene transcription levels after EGF treatment

[0149]

[0150] Note: Significance p < 0.001.

[0151] Table 7. Log2|FC| values ​​of anti-apoptotic gene transcription levels after EGF treatment

[0152]

[0153] Following EGF treatment, the downregulated pathways primarily involve cell cycle regulation, such as changes in chromatin conformation (centromere remodeling and nucleosome dissociation), regulation of cell cycle checkpoints (E2F gene expression, G2M checkpoint), RNA and related enzyme synthesis, and changes in cell structure (such as sister chromatid separation and nuclear membrane disintegration). The upregulated pathways, in addition to involving apoptosis, also involve epidermal cell keratinization, NFκB-mediated inflammatory responses, immune responses, and regulation of proteolytic enzyme activity.

[0154] EGF-induced apoptosis can be related to the expression level of EGFR of the cell. The growth of the UTUC organoids with abnormal expression of EGFR is not dependent on EGF. On the contrary, after the addition of EGF, the organoids will produce a stress response, activate a series of active protection pathways such as immune response, programmed death, etc. After EGF induction, the expression level of EGFR is significantly increased, and the apoptosis reaction dominated by Caspase 3 is cascaded through the STAT1 pathway, thereby inducing the apoptosis of UTUC.

[0155] The above only describes the preferred embodiments of the present application, and it should be pointed out that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Use of epidermal growth factor or EGFR agonist in inducing apoptosis of upper urinary tract urothelial carcinoma organoids.

2. Use of epidermal growth factor or EGFR agonist in constructing an apoptosis model of upper urinary tract urothelial carcinoma organoids.

3. Use according to claim 1 or 2, characterized in that, The epidermal growth factor is in the form of a cell culture medium additive or a cell culture medium.

4. Use of epidermal growth factor or EGFR agonist in the preparation of a drug for preventing and / or treating upper urinary tract urothelial carcinoma.

5. Use according to claim 4, characterized in that, The treatment of upper urinary tract urothelial carcinoma includes inhibition of the growth of upper urinary tract urothelial carcinoma.

6. Use according to claim 5, characterized in that, The inhibition of the growth of upper urinary tract urothelial carcinoma includes inhibition of the proliferation of upper urinary tract urothelial carcinoma cells and / or upper urinary tract urothelial carcinoma organoids and / or promotion of apoptosis of upper urinary tract urothelial carcinoma cells and / or upper urinary tract urothelial carcinoma organoids.

7. Use according to claim 6, characterized in that, The signal pathway for promoting apoptosis of upper urinary tract urothelial carcinoma cells and / or upper urinary tract urothelial carcinoma organoids includes STAT1-Caspase 3 pathway.

8. Use according to any one of claims 4 to 7, characterized in that, The use also includes use of epidermal growth factor in combination with a specific drug for treating upper urinary tract urothelial carcinoma in the preparation of a drug for preventing and / or treating upper urinary tract urothelial carcinoma.

9. An agent against upper urinary tract epithelial carcinoma, characterized by comprising the compound or salt according to claim 1. The active ingredients include epidermal growth factor and other drugs for treating upper urinary tract urothelial carcinoma.

10. The medicament according to claim 9, characterized in that, The dosage form of the drug includes injection solution and / or injection powder.

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