A method for constructing an animal model of peritoneal malignant mesothelioma

Abstract

The present application relates to the technical field of animal model construction method, and particularly relates to a kind of construction method of small molecule drug induced peritoneal malignant mesothelioma animal model.The method uses epidermal growth factor receptor tyrosine kinase inhibitor (Erlotinib or its salt, Erlotinib) to be injected into abdominal cavity to mammal, and peritoneal malignant mesothelioma animal model is induced to form.The technical scheme can solve the technical problem that the construction process of peritoneal malignant mesothelioma animal model in prior art is too long or needs special equipment and conditions.The method of peritoneal malignant mesothelioma animal model constructed by the technical scheme is short in time consumption, high in efficiency, and does not need asbestos exposure and virus gene operation, greatly saves time cost and economic cost, is simple and easy to operate, and is easy to popularize, and the animal model is expected to be used for further revealing malignant mesothelioma mechanism of occurrence and development and screening new therapeutic drugs, and has ideal application prospect.

Description

Technical Field

[0001] This invention relates to the field of animal model construction methods, specifically to a method for constructing an animal model of peritoneal malignant mesothelioma induced by small molecule drugs. Background Technology

[0002] Malignant mesothelioma is a malignant tumor originating from the mesothelial cells of the pleural and peritoneal serosal layers. Pleural malignant mesothelioma accounts for 80%, while peritoneal malignant mesothelioma (MPM) accounts for 15-20%. The occurrence of MPM is closely related to asbestos exposure, with a latency period of up to several decades, generally developing into MPM 30-40 years after asbestos exposure. Approximately 80% of pleural MPMs are associated with asbestos exposure, but only 20% of peritoneal MPMs are, suggesting other factors contributing to MPM development. MPM is highly malignant, has a poor prognosis, and a median survival of only 6-12 months. For some suitable patients, the Peritoneal Surface Oncology Group International (PSOGI) recommends cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) as the standard treatment for peritoneal malignancies, which can extend median survival to 5 years. However, approximately 60% of patients miss the opportunity for CRS+HIPEC treatment due to late-stage tumor discovery and cannot benefit from it. Therefore, new treatment approaches need to be explored to improve the prognosis of patients with peritoneal malignant mesothelioma.

[0003] The molecular mechanisms of peritoneal malignant mesothelioma pathogenesis are not fully understood, necessitating suitable models for studying its pathological mechanisms and screening new drugs. Currently, tumor cell lines derived from patients with peritoneal malignant mesothelioma are the primary models used; however, these cell models have limitations, failing to simulate the heterogeneity and tumor microenvironment of tumors in vivo. Animal models of mesothelioma can effectively mimic the entire process of mesothelioma development and the in vivo microenvironment. A peritoneal mesothelioma model induced by intraperitoneal injection of asbestos has been developed, but this model has a long latency period and low efficiency; approximately 10% of mice inoculated with asbestos develop mesothelioma 20 months later. Previous studies have constructed mesothelioma models by conditionally knocking out tumor suppressor genes closely related to mesothelioma, Trp53, Nf2, Cdkn2a / b, and Bap1. However, the formation of mesothelioma in these gene knockout mouse models is also time-consuming, requiring 8–14 weeks, and exhibits off-target effects, including the development of fibroids, lymphomas, and hepatocellular carcinoma. In addition, transfecting viruses and raising genetically engineered mice requires special equipment and conditions, making it difficult to promote and apply them.

[0004] Therefore, there is an urgent need for a rapid and efficient animal model of peritoneal malignant mesothelioma that does not require asbestos exposure or genetic manipulation, in order to study the pathogenesis of peritoneal malignant mesothelioma and screen for new therapeutic drugs. Summary of the Invention

[0005] The present invention aims to provide a method for constructing an animal model of peritoneal malignant mesothelioma induced by small molecule drugs, so as to solve the technical problems of the existing technology where the construction process of animal models of peritoneal malignant mesothelioma is too time-consuming or requires special equipment and conditions.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for constructing an animal model of peritoneal malignant mesothelioma induced by a small molecule drug involves intraperitoneal injection of an epidermal growth factor receptor tyrosine kinase inhibitor into a mammal to induce the formation of an animal model of peritoneal malignant mesothelioma.

[0008] Furthermore, the epidermal growth factor receptor tyrosine kinase inhibitor is erlotinib or a salt thereof.

[0009] Furthermore, the epidermal growth factor receptor tyrosine kinase inhibitor is injected in the form of a suspension.

[0010] Furthermore, the dosage of the epidermal growth factor receptor tyrosine kinase inhibitor is 50 mg / kg-400 mg / kg, and the administration period is 7-180 days.

[0011] Furthermore, the dosage of the epidermal growth factor receptor tyrosine kinase inhibitor is 200 mg / kg-400 mg / kg, and the administration period is 21-180 days.

[0012] Furthermore, the type of malignant peritoneal mesothelioma is sarcomatous.

[0013] Furthermore, the mammal is a mouse, rat, monkey, dog, cat, rabbit, horse, pig, or cow.

[0014] This technical solution also provides the application of erlotinib and its salts in constructing animal models of malignant peritoneal mesothelioma. Erlotinib or erlotinib hydrochloride is administered intraperitoneally to mammals at a dose of 50 mg / kg-400 mg / kg daily for 7-180 days to induce the formation of an animal model of malignant peritoneal mesothelioma.

[0015] Furthermore, the peritoneal malignant mesothelioma animal model exhibited the inflammatory cell infiltration phenotype of human malignant mesothelioma; erlotinib induced malignant mesothelioma tissues to exhibit a state of loss of tumor suppressor proteins BAP1 and CDKNB and DNA damage.

[0016] Furthermore, erlotinib is used to induce the expression of mesothelioma-specific markers, including WT-1, mesothelin, podocyte pericellular protein, calmodulin, and CK5 / 6; erlotinib is used to induce enhancement of the MAPK, PI3K-AKT, Hippo, STAT3, and Wnt signaling pathways in malignant mesothelioma.

[0017] In summary, the principle and beneficial effects of this technical solution are as follows:

[0018] This technical solution develops a method for constructing an animal model of peritoneal mesothelioma induced by a small molecule drug. Mice were administered daily intraperitoneal injections of erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor. Erlotinib rapidly induced the formation of malignant peritoneal mesothelioma with extremely high efficiency (approximately 100%) within 21 days. Erlotinib-induced malignant mesothelioma exhibited morphological features similar to human sarcomatous malignant mesothelioma and expressed characteristic mesothelioma phenotypes, displaying the characteristic inflammatory cell infiltration phenotype of mesothelioma, and possessing strong tumor invasiveness and metastatic potential. Furthermore, erlotinib-induced malignant mesothelioma showed the absence of tumor suppressor factors BAP1 and CDKN2B, reproducing the gene mutation characteristics commonly found in human malignant mesothelioma. Activation of multiple pro-cancer signaling pathways is involved in the development of malignant mesothelioma, and erlotinib-induced malignant mesothelioma also showed activation of these pathways (MAPK, PI3K-AKT, STAT3, Wnt, and NF-κB signaling pathways). RNA-seq analysis revealed that erlotinib treatment enhanced characteristic genes of malignant mesothelioma. The animal model of peritoneal malignant mesothelioma constructed using this technique is time-efficient, efficient, and eliminates the need for asbestos exposure and viral gene manipulation, significantly reducing time and economic costs. It is simple, easy to implement, and readily applicable. This animal model holds promise for further elucidating the mechanisms of malignant mesothelioma development and screening for new therapeutic drugs.

[0019] Ellotinib is typically used to treat locally advanced or metastatic non-small cell lung cancer (NSCLC), and current technology generally classifies it as a cancer treatment drug. However, in clinical practice, tumor resistance is frequently observed when using epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as ellotinib to treat NSCLC. Previous studies have linked this to gene mutations and abnormal activation of signaling pathways in cancer cells induced by ellotinib use (see: Hrustanovic, Gorjan, Bianca J. Lee, and Trever G. Bivona. 'Mechanisms of Resistance to EGFR-Targeted Therapies'). Cancer Biology & Therapy 14, no. 4 (1 April 2013): 304–14. https: / / doi.org / 10.4161 / cbt.23627; Huang, Lihua, and Liwu Fu. 'Mechanisms ofResistance to EGFR Tyrosine Kinase Inhibitors'. Acta Pharmaceutica Sinica B5, no. 5 (1 September 2015): 390–401. https: / / doi.org / 10.1016 / j.apsb.2015.07.001. Liu, Qian, Shengnan Yu, Weiheng Zhao, Shuang Qin, QianChu, and Kongming Wu. 'EGFR-TKIs Resistance via EGFR-Independent SignalingPathways'. Molecular Cancer 17, no. 1 (19 February 2018): 53. https: / / doi.org / 10.1186 / s12943-018-0793-1. While erlotinib can induce cellular gene mutations and abnormal activation of signaling pathways, it remains unclear whether it can cause normal cells to become cancerous. Therefore, this study aims to explore whether erlotinib can induce cancer in normal mice. This technique uses this cancer-treating drug to induce specific cancer types, offering a new perspective for understanding the significant resistance mechanisms of erlotinib.

[0020] The mesothelioma induced by this technique was small in size 21 days prior to induction, mainly diffusely adhering to the colon, mesentery, and the surface of abdominal organs, making it difficult to detect with the naked eye and often overlooked. Abnormal tumor growth could only be detected through pathological serial section staining and careful microscopic observation of the outer serosal layer of the intestinal lining. Furthermore, erlotinib specifically induces peritoneal mesothelioma, rather than other types of cancer. The tumors adhered to the colon, making them difficult to distinguish visually and easily mistaken for colonic adenomas. Further experiments by the inventors confirmed that these tumor tissues were not colonic adenomas. The induced tumors did not express markers of normal intestinal epithelium and colonic adenocarcinoma, such as cytokeratin 20 (CK20), CDX2, and epithelial cell adhesion molecule (EpCAM). This further confirmed that the erlotinib-induced tumors were not colonic adenocarcinomas but true mesotheliomas. The specificity of erlotinib in inducing peritoneal mesothelioma was unexpected by the inventors before the experiments.

[0021] In summary, this study provides a method for constructing an animal model of malignant peritoneal mesothelioma induced by small molecule drugs. This method enables efficient and rapid construction of a mesothelioma model without asbestos exposure or gene knockout. More importantly, this animal model can be used to accelerate research into other potential molecular mechanisms of malignant peritoneal mesothelioma and for rapid new drug screening. Attached Figure Description

[0022] Figure 1This is a flowchart illustrating the steps involved in constructing an animal model of malignant peritoneal mesothelioma.

[0023] Figure 2 Histopathological features of erlotinib-induced peritoneal malignant mesothelioma (A: Hematoxylin-eosin (H&E) staining images of tumor sections; B: Images of diffuse malignant mesothelioma distributed on the intestinal surface; CH: Immunohistochemical staining images of characteristic markers of malignant mesothelioma, including mesothelin (MSLN), podoplanin, Wilms tumor-1 (WT-1), calretinin, CK5 / 6, and the mesothelial cell lineage marker vimentin; I: Images of Ki-67 staining, a cell proliferation marker in erlotinib-induced malignant mesothelioma; J: Quantitative analysis of the mean percentage of Ki-67 positive nuclei in erlotinib-induced malignant mesothelioma, data presented as mean ± standard deviation (sd); K and L: Immunohistochemical staining images of CD44 and SOX2, a tumor stem cell marker associated with invasiveness and proliferation in malignant mesothelioma).

[0024] Figure 3 Differential diagnosis results between erlotinib-induced peritoneal malignant mesothelioma and colonic adenocarcinoma (A and B: immunohistochemical staining of intestinal epithelial cells and colonic adenocarcinoma marker cytokeratin 20-CK20; C and D: immunohistochemical staining of intestinal epithelial cells and colonic adenocarcinoma marker CDX2; E and F: immunohistochemical staining of intestinal epithelial cells and colonic adenocarcinoma marker epithelial cell adhesion molecule-EpCAM).

[0025] Figure 4 Results of an erlotinib-induced study of the invasiveness and metastasis of peritoneal malignant mesothelioma (A: Sections stained with hematoxylin and eosin (H&E) showing the malignant tumor invading the muscularis propria (ML) and mesenteric adipose tissue (MF); B and C: Immunohistochemical staining of mesothelioma markers CK5 / 6 and podoplanin showing invasive tumor tissue; D: Images of liver metastases; E: Liver metastases stained with H&E; F: Lymph node metastases stained with H&E; G and I: Immunohistochemical staining of mesothelioma markers Wilms tumor-1 (WT-1) and podoplanin showing mesothelioma tissue on the liver surface; H and J: Immunohistochemical staining of mesothelioma markers mesothelin (MSLN) and calretinin showing mesothelioma tissue metastasizing to mesenteric lymph nodes).

[0026] Figure 5The absence of tumor suppressor proteins BAP1 and CDKNB and DNA damage were observed in erlotinib-induced malignant mesothelioma tissues (AJ: immunohistochemical staining of tumor suppressor proteins BAP1, NF2, CDKN2A / 2B and p53; K: immunofluorescence staining of phosphorylated H2AX (p-H2AX) in erlotinib-induced mesothelioma tissues; L: immunohistochemical staining of anti-apoptotic factor BCL-2 in erlotinib-induced mesothelioma tissues).

[0027] Figure 6 The results of a study on the activation of tumor-related signaling pathways in erlotinib-induced malignant mesothelioma tissue (AH: Immunohistochemical staining micrographs of phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated protein kinase B (p-AKT), Yes-associated protein (YAP), phosphorylated YAP (p-YAP), phosphorylated large tumor suppressor protein 1 / 2 (p-LATS1 / 2), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), and β-catenin in erlotinib-induced malignant mesothelioma tissue).

[0028] Figure 7 Results of studies on inflammatory cell infiltration in erlotinib-induced malignant mesothelioma (AD: Immunohistochemical staining images of F4 / 80, CD3, CD4 and NF-κB p65 in erlotinib-induced malignant mesothelioma tissue; E: Real-time quantitative PCR (RT-qPCR) analysis of Tnf, Ifng and Il-6 mRNA levels in erlotinib-induced colon (containing mesothelioma) and control colon, data are expressed as mean ± standard deviation (sd) (n=3), #p<0.05, ##p<0.01, by unpaired t-test; F: Enzyme-linked immunosorbent assay (ELISA) analysis of TNF-α protein levels in erlotinib-induced colon (containing mesothelioma) and control colon, data are expressed as mean ± standard deviation (sd) (n=3), **p<0.01, ***p<0.001, by unpaired t-test).

[0029] Figure 8 Principal component analysis was performed on gene expression profiles of erlotinib-induced colon (including mesothelioma) and control colon (AE: RNA-seq analysis was performed on colon tissue (including mesothelioma) and control colon tissue after 7, 14, 21 and 28 days of erlotinib treatment, and principal component analysis (PCA) was performed on the expression of all genes).

[0030] Figure 9The significant changes in the transcriptome induced by erlotinib treatment were analyzed (AE: differentially expressed genes (DEGs) in colon tissue (including mesothelioma) and control colon tissue at 7, 14, 21 and 28 days of erlotinib treatment. Differentially expressed genes (DEGs) were determined by adjusted p-values ​​(padj) < 0.05 and log2FoldChange > 0.585. Volcano plots showed the number of upregulated, downregulated and unchanged genes; F: hierarchical clustering heatmap of the top 500 DEGs between erlotinib treatment at 28 days (E28D) and control (C7D).

[0031] Figure 10 The upregulation of mesothelioma-related gene transcriptomes induced by erlotinib (AF: heatmap showing upregulation of characteristic mesothelioma genes relative to the control group (C7D) after 7 days (E7D), 14 days (E14D), 21 days (E21D), and 28 days (E28D) of erlotinib treatment; G: real-time quantitative PCR (RT-qPCR) analysis confirming the upregulation of mesothelioma-specific genes between E28D and C7D; H: Western blot verification of the upregulation of mesothelioma-specific proteins (WT1, MSLN, Calretinin, and CK7) between E7D, E28D, and E60D and C7D). Detailed Implementation

[0032] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used are all commercially available. More specifically, the specific antibodies and equipment used in this technical solution are shown in Tables 1 and 2.

[0033] Table 1:

[0034]

[0035] Table 2:

[0036]

[0037] Example 1:

[0038] (1) Laboratory animals

[0039] Specific pathogen-free grade C57BL / 6 female mice, 6-8 weeks old, weighing 17-20g, were purchased from Beijing Spaford Company, China. Animals were housed in a barrier system, and the animal housing environment was maintained at a room temperature of 20℃. 23℃, 50% humidity 70%. The animal room lighting alternates between bright and dark cycles every 12 hours. Animal feed is sourced from Beijing Spaford Feed. Animals have free access to food. Water is supplied via a two-stage reverse osmosis system using an automatic drinking fountain.

[0040] (2) Experimental methods

[0041] (2.1) Preparation of erlotinib injection

[0042] Weigh 200 mg of erlotinib powder (erlotinib, cinnotinib; CP-358774; CAS No.: 183321-74-6; chemical formula see Formula I) using a scale and dissolve it in 10 ml of normal saline injection to prepare an erlotinib suspension. Store protected from light and prepare immediately before use.

[0043]

[0044] Formula I

[0045] (2.2) Animal grouping and model establishment

[0046] Mice were randomly divided into three groups: a control group (n=25), an erlotinib group (n=62), and a high-dose erlotinib group (n=14). The weight of each mouse in the experimental groups was measured using an electronic scale, and the required volume of solution per mouse was calculated based on the ratio of 200 mg / kg erlotinib to 400 mg / kg body weight. C57BL / 6 mice were removed from their cages one by one and placed on a clean bench in the animal room. The injection site (abdomen) was disinfected with an alcohol swab. For mice in the erlotinib group, an erlotinib suspension was drawn up with a 1 ml disposable syringe and injected intraperitoneally, based on a dose of 200 mg / kg erlotinib. For mice in the high-dose erlotinib group, an erlotinib suspension was drawn up with a 1 ml disposable syringe and injected intraperitoneally, based on a dose of 400 mg / kg erlotinib. For mice in the control group, 0.2 ml of physiological saline was drawn up with a 1 ml disposable syringe and injected intraperitoneally. After injection, slowly withdraw the needle to prevent leakage. Injections were administered once daily, and mouse tissue samples were collected on days 7, 14, 21, 28, 60, 150, and 180 post-injection. Samples included colon, peritoneal mesothelioma tissue, liver, and mesenteric lymph nodes. Some tissues were fixed with Carnoy's fixative or paraformaldehyde, while some fresh tissues were preserved in liquid nitrogen for gene expression analysis.

[0047] In practice, erlotinib can be administered at a dosage of 50 mg / kg to 400 mg / kg for 7 to 180 days to induce animal models (although the induction success rate cannot be guaranteed to be 100%, some successfully induced animal models have been obtained for further experimental research). As a preferred option, erlotinib can be administered at a dosage of 200 mg / kg to 400 mg / kg for 21 to 180 days to further improve the induction success rate. More preferably, at a dosage of 200 mg / kg, the administration period is 21 to 180 days; at a dosage of 400 mg / kg, the administration period is 21 to 60 days.

[0048] (2.3) Model Indicator Evaluation

[0049] (2.3.1) HE staining of tissue

[0050] Used to display the basic structure of tissues, such as the cell nucleus and cytoplasm, and to show the morphological structure of mesothelioma tissue.

[0051] (2.3.2) Immunochemical staining and immunofluorescence staining of tissues

[0052] Immunohistochemical staining (IHC) was performed on paraffin-embedded samples. The specific steps were as follows: First, the sections were dewaxed and rehydrated with ethanol at progressively increasing concentrations. To inhibit endogenous peroxidase activity, the sections were incubated in 3% methanol hydrogen peroxide solution for 15 minutes. Antigen retrieval was performed using a pressure cooker boiling method for 10 minutes, followed by citrate buffer (10 mM citrate, 0.05% Tween-20, pH 6) or Tris-EDTA buffer (1 mM Tris, 1 mM EDTA, 0.05% Tween-20, pH 9). Afterward, the sections were thoroughly washed with PBS for 5 minutes. To prevent nonspecific binding, the sections were blocked for 30 minutes with either 10% goat serum (Boster Biological, China) or 1% bovine serum albumin (Boster Biological, China). Subsequently, the sections were incubated overnight at 4°C with primary antibodies. Details of the primary antibodies are shown in Reagent Table 1. The primary antibodies were detected using HRP-labeled secondary antibodies (Seville, China). DAB (Seville, China) was used for staining, and cell nuclei were stained with hematoxylin. For immunofluorescence (IF) staining, primary antibodies were detected by secondary antibodies (Boster, China) conjugated to fluorescent probes, and cell nuclei were stained with DAPI. All images were captured using an Olympus BX63 optical and fluorescence microscope.

[0053] (2.3.3) Immunoblot analysis

[0054] Proteins were extracted from fresh frozen colon containing mesothelioma. The lysis buffer used contained 0.5% NP-40, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM sodium alum, 10 mg / mL leucopeptidase, and 10 mg / mL aprotinin. Extraction involved brief sonication, followed by protein separation by SDS-PAGE. The proteins were then transferred to a PVDF membrane (IPVH00010, Immobilon®-p, Millipore, Germany) by electrophoresis. The membrane was pre-incubated in TBS-T containing 5% skim milk. Primary and secondary antibodies were then applied, and protein bands were detected by enhanced chemiluminescence (BeyoELC Moon, China). Scanning was performed using a ChemiDoc imaging system (BIO-RAD, USA). The primary antibodies used are listed in Table 1.

[0055] (2.3.4) Enzyme-linked immunosorbent assay (ELISA)

[0056] The levels of TNFα in the colon of mesothelioma-containing tissue from erlotinib-treated mice and in the colon of control mice were analyzed using ELISA kits (Servicebio® Mouse TNF-α ELISA Kits) provided by Wuhan Servicebio Biotechnology Co., Ltd. (China). Specifically, colons containing mesothelioma were collected from three mice treated with erlotinib for 7, 28, and 60 days, and three control mice. Tissue samples were first weighed and mechanically homogenized with 0.9% saline under ice-water bath conditions. Subsequently, the homogenate was centrifuged, and the supernatant was analyzed using the ELISA kit according to the manufacturer's instructions. Results were quantified in picograms per milliliter (pg / ml) of tissue.

[0057] (2.3.5) Quantitative Real-Time PCR (qPCR) Analysis

[0058] Total RNA was extracted from colon samples containing mesothelioma using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The isolated total RNA was reverse transcribed using RT Master Mix II (gDNA digester plus) (HY-K0511A, MCE, USA) to convert the RNA into complementary DNA (cDNA) for further analysis. Quantitative real-time PCR (qPCR) analysis was performed using SYBR GREEN qPCR Master Mix (HY-K0523, MCE, USA) on a CFX96TM real-time system (BIO-RAD, USA). The relative expression levels of the target gene were normalized to the expression of the internal reference gene GAPDH. The specific primer sequences used for qPCR are shown in Table 3. The primers in Table 3 are numbered from top to bottom as SEQ ID NO.1-SEQ ID NO.16.

[0059] Table 3:

[0060]

[0061] (2.3.6) RNA sequencing analysis

[0062] Colon samples containing tumors and from control groups were collected, rapidly frozen without fixation, and prepared for RNA sequencing by Novogene (Beijing, China). Total RNA was isolated using TRIzol reagents according to the manufacturer's instructions and used as input material for RNA sample preparation. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Preparation Kit, as recommended by the manufacturer. Library sequencing was performed using the Illumina NovaSeq 6000 platform. Sequencing data were aligned to a reference Ensembl annotated mouse transcriptome (Mus_musculus_Ensemble_94) using Hisat2 (v2.0.5). The number of reads mapped to each gene was calculated using FeatureCounts (v1.5.0-p3). Expression values ​​for each gene were normalized using fragments per thousand bases per million mapped reads (FPKM). Differential expression analysis between the erlotinib-treated and control groups was performed using the DESeq2R package (1.20.0). The p-values ​​of the results were adjusted using the Benjamini and Hochberg methods to control for false discovery. Genes with significant differential expression (DEGs) were identified by padj < 0.05 and |log2(fold change)| > 0.585. Heatmaps and PCA plots were generated in R. ClusterPro was used. The ler R package (v3.8.1) performed gene ontology (GO) enrichment analysis, KEGG and Reactome signaling pathway enrichment analysis on DEGS, with a corrected P value of less than 0.05 as significant enrichment.

[0063] Gene set enrichment analysis (GSEA) is used to identify upregulated or downregulated biological terms, pathways, and processes in different samples. Genes are initially ranked according to the degree of differential expression between two samples, and then a predefined gene set is examined to see if it is enriched at the top or bottom of the list. The GSEA tool on the Broad Institute website facilitates the analysis of gene sets from the GO, KEGG, and Reactome databases.

[0064] (3) Experimental results

[0065] (3.1) Intraperitoneal injection of erlotinib leads to the rapid development of malignant mesothelial carcinoma.

[0066] We administered the first-generation EGFR-TKI erlotinib intraperitoneally to adult mice and investigated its effects. Erlotinib was injected intraperitoneally into the experimental group mice at doses of 200 mg / kg and 400 mg / kg daily. Samples were collected from a number of mice on days 7, 14, 21, 28, 60, 150, and 180 after injection (see schematic diagram). Figure 1 Control group mice received intraperitoneal saline injections according to the same protocol, and tissue samples were collected in batches at the same time points. Histopathological analysis showed that, 21 days after injection, all mice treated with erlotinib developed peritoneal tumors near the colon. Figure 2 A and Figure 2 (B) In addition, 6 out of 16 mice treated with erlotinib for 14 days developed peritoneal tumors adjacent to the colon. We counted the number of mice that developed tumors at different time points, as detailed in Table 4.

[0067] Table 4: Tumor incidence at different treatment times with erlotinib (E7D, E14D, E21D, E28D, E60D, E150D, E180D represent mice receiving erlotinib at a dose of 200 mg / kg for 7, 14, 21, 28, 60, 150, and 180 days; hE21D, hE28D, hE60D represent mice receiving erlotinib at a dose of 400 mg / kg for 21, 28, and 60 days)

[0068]

[0069] These tumors vary in size: some can only be detected by H&E staining, while others are visible to the naked eye. Figure 2B, black arrow). Histologically, these induced peritoneal tumors primarily originate from the mesothelial cells of the intestinal serosa (B). Figure 2 A, the black dashed line shows the interface between the muscularis propria and mesothelium. Erlotinib-induced tumors exhibit typical mesothelioma morphology. Figure 2 A). Based on cellular composition and biological behavior, mesothelioma can be divided into three main histological subtypes: epithelioid (accounting for 50%-70% of cases), sarcomatoid (accounting for 10%-20% of cases), and biphasic (accounting for approximately 30% of cases). In our experiments, most erlotinib-induced tumors were sarcomatoid, lacking the epithelioid subtype. Figure 2 A). These sarcomatoid cells exhibit a short, spindle-shaped appearance, forming dense spherical masses. Figure 2 A, the black arrow).

[0070] To further confirm the tumor, we performed immunohistochemical (IHC) and immunofluorescence (IF) staining analyses on tumor sections. The induced tumors showed positive staining for mesothelioma-specific markers, including Wilms tumor-1 (WT-1), mesothelin (MSLN), podoplanin, calretinin, and CK5 / 6 (…). Figure 2 CG). Furthermore, the mesothelial cell lineage marker Vimentin is also expressed in tumor tissues ( Figure 2 H). Most mesotheliomas proliferate very rapidly. We assessed the expression of the proliferation marker Ki-67, and tumor tissue showed abundant positive Ki-67 staining (H). Figure 2 I and Figure 2 J), indicating that the tumor has a strong proliferative capacity. Population differentiation antigen 44 (CD44) is a key adhesion / relocation molecule and a major receptor for hyaluronic acid (HA), which is associated with mesothelioma invasion and proliferation. In erlotinib-induced mesothelioma, CD44 is strongly positive ( Figure 2 K), indicating a high proliferative and invasive capacity of the tumor. Pluripotent SOX2-positive cancer stem cells are found in mesothelioma ( Figure 2 L).

[0071] We conducted further differential diagnosis. Because the tumors adhered to the colon, they were difficult to distinguish visually. To confirm that these tumor tissues were not colonic adenomas, we performed immunohistochemical staining for differentiation. We found that the induced tumors did not express markers of normal intestinal epithelium and colonic adenocarcinoma, such as cytokeratin 20 (CK20), CDX2, and epithelial cell adhesion molecule (EpCAM). Figure 3 These findings indicate that the tumors induced by erlotinib are not colonic adenocarcinomas, but rather true mesotheliomas.

[0072] Mesothelioma is highly invasive and metastatic. In our study, erlotinib-induced tumors also frequently invaded adjacent adipose tissue. Figure 4 A, red rectangle) and intestinal smooth muscle tissue ( Figure 4 A, black rectangles), these can be identified by HE staining and immunohistochemical staining with mesothelioma-specific markers ( Figure 4 AC). This indicates that erlotinib-induced mesothelioma is highly invasive. Furthermore, we found mesothelioma tissue on the liver surface and in the mesenteric lymph nodes, confirmed by HE staining and immunohistochemical staining. Figure 4 These results demonstrate that erlotinib-induced mesothelioma has the ability to metastasize to distant organs.

[0073] (3.2) Ellotinib-induced mesothelioma showed loss of tumor suppressor genes BAP1 and CDKN2A / 2B and DNA damage.

[0074] Tumor suppressor genes BAP1, NF2, and CDKN2A / B play crucial roles in the pathogenesis of human malignant mesothelioma. We performed immunohistochemical staining to examine the expression of proteins encoded by these major mesothelioma tumor suppressor genes: BAP1, NF2, and CDKN2A / B. Ellotinib-induced mesothelioma showed loss of BAP1 nuclear expression. Figure 5 A). In the control colonic epithelium (Ctr), weak nuclear BAP1 staining was observed, and immune cells in the lamina propria showed obvious nuclear and cytoplasmic BAP1 staining. Figure 5 B). The induced mesothelioma tumors retained NF2 expression ( Figure 5 B and Figure 5 D). CDKN2A is not expressed in normal mesothelial and mucosal epithelial cells. CDKN2A is also not expressed in erlotinib-induced mesothelioma. Figure 5 E and Figure 5 F). CDKN2B is expressed in normal mesothelial and mucosal epithelial cells, as well as immune cells. Figure 5 H), but not expressed in erlotinib-induced mesothelioma. Figure 5 G). This indicates that CDKN2B is also lost in erlotinib-induced tumors. These results suggest that erlotinib-induced mesothelioma exhibits loss of BAP1 and CDKN2B proteins. Acquired loss of BAP1 and CDKN2B proteins indicates that erlotinib causes DNA damage. Previous studies have reported that the tumor suppressor protein p53 can respond to asbestos-induced DNA damage in mesothelioma. We examined p53 protein expression in erlotinib-induced mesothelioma and observed a significant increase in p53-positive cells in tumor tissue compared to control colon tissue. Figure 5I) indicates the presence of DNA damage. Furthermore, elevated levels of the DNA damage-related γ-H2AX marker were detected in the tumor ( Figure 5 K). The anti-apoptotic marker BCL-2 was also upregulated in tumors ( Figure 5 These findings indicate that erlotinib-induced mesothelioma exhibits the loss of BAP1 and CDKN2B and DNA damage.

[0075] (3.3) Enhanced MAPK, PI3K-AKT, Hippo, STAT3 and Wnt signaling pathways in erlotinib-induced malignant mesothelioma.

[0076] Besides the loss of tumor suppressor genes, multiple oncology-related signaling pathways, including MAPK, PI3K-AKT, Hippo, STAT3, and Wnt pathways, play crucial roles in the development and progression of malignant mesothelioma. Tumors show weak or no staining for phosphorylated EGFR (p-EGFR). Figure 6 A), which is consistent with the inhibitory effect of erlotinib on EGFR. However, we observed that downstream activated phosphorylated ERK1 / 2 (p-ERK1 / 2) stained strongly positive in erlotinib-induced mesothelioma tissue (A). Figure 6 B). These findings indicate that although EGFR is suppressed, the downstream MAPK signaling pathway is still activated in tumor tissue, suggesting that there may be an alternative pathway activating the MAPK signaling pathway. Expression of phosphorylated AKT (p-AKT) in tumor specimens demonstrates activation of the PI3K-AKT pathway. Figure 6 C). Inhibition of the Hippo-YAP signaling pathway is involved in the pathogenesis of MM. In tumor specimens, we observed a large number of YAP-stained cells (C). Figure 6 D). Conversely, only a few cells showed phosphorylated YAP (p-YAP) staining (D). Figure 6 E). p-LATS1 / 2 is a YAP-phosphorylated kinase, and its immunostaining levels are also low in induced mesothelioma tissues. Figure 6 F). This indicates that erlotinib inhibits the Hippo-YAP pathway. STAT3 activation promotes the development of malignant mesothelioma. In erlotinib-induced malignant mesothelioma, a large number of cells showed positive staining for phosphorylated STAT3 (p-STAT3). Figure 6 G). Activation of the Wnt / β-catenin signaling pathway promotes the proliferation of mesothelioma cells. In induced mesothelioma cells, β-catenin was detected as strongly positive in the cell nucleus. Figure 6 The results indicate activation of the Wnt signaling pathway. These findings suggest that these oncology-related MAPK, PI3K, Hippo, STAT3, and Wnt signaling pathways are enhanced in erlotinib-induced malignant mesothelioma.

[0077] (3.4) Elotinib-induced malignant mesothelioma exhibits an inflammatory cell infiltration phenotype similar to that of human malignant mesothelioma.

[0078] Studies have shown that chronic inflammation caused by asbestos exposure is closely related to the pathogenesis of multiple myeloma (MM) in humans. Interestingly, erlotinib-induced malignant mesothelioma also exhibits a similar inflammatory phenotype. We observed macrophage infiltration in erlotinib-induced malignant mesothelioma, which was confirmed by immunohistochemical staining with the macrophage marker F4 / 80. Figure 7 A). Similar to human mesothelioma, erlotinib-induced tumors also showed infiltration of T lymphocytes, and immunohistochemical staining for CD3 and CD4 confirmed the presence of these immune cells. Figure 7 B and Figure 7 C). Activation of the NF-κB signaling pathway is an important factor in inflammation. In erlotinib-induced mesothelioma, immunohistochemical staining showed a large amount of positive expression of NF-κB p65. Figure 7 D). Elotinib treatment increased the mRNA and protein expression levels of tumor necrosis factor (TnF) in the colon (including mesothelioma). Figure 7 E and Figure 7 F). These results indicate that erlotinib-induced malignant mesothelioma exhibits an inflammatory cell infiltration phenotype similar to that of human malignant mesothelioma.

[0079] (3.5) Ellotinib promotes upregulation of the transcriptome of genes related to malignant mesothelioma

[0080] To explore the transcriptional alterations induced by erlotinib, we employed RNA sequencing (RNA-seq) for gene expression profiling. Isolating mesothelioma tissue and monolayer mesothelial cells from the intestine is challenging due to the widespread extraintestinal spread of erlotinib-induced mesothelioma on the serosa. Therefore, we collected colon tissue from mice with mesothelioma directly from erlotinib-treated mice and compared it with colon tissue from control mice. We then performed RNA-seq analysis on these samples. We performed differential gene expression analysis on erlotinib-treated tumor-bearing colon at days 7, 14, 21, and 28 post-treatment, comparing it with control colon. First, we compared gene expression changes between colon treated with erlotinib for 28 days (E28D) and control colon on day 7 (C7D). Principal component analysis (PCA) of the RNA-seq data revealed significant clustering between E28D and C7D colons. Figure 8D). The E28D colon showed greater dispersion, indicating heterogeneity after erlotinib treatment. Of all genes, 1609 were identified as differentially expressed genes (DEGs) (padj < 0.05 and |Log2Foldchange| > 0.585, equal to a 1.5-fold change), of which 1035 were upregulated and 574 were downregulated. Figure 9 D). Unsupervised hierarchical clustering of the first 500 DEGs further confirmed the separation between the E28D and C7D colons ( Figure 9 F). Similarly, PCA analysis of the colons with tumors from E7D, E14D, E21D, and hE28D (treated with high doses of erlotinib for 28 days) showed significant separation from the C7D colon. Figure 8 AC and E), volcano plots show that the colons with tumors in E7D, E14D, E21D, and hE28D have a large number of DEGs compared to the C7D colon (AC and E). Figure 9 Interestingly, earlier erlotinib treatments (E7D and E14D) induced more gene expression alterations than longer erlotinib treatments (E21D and E28D), suggesting that tissue cells may gradually adapt to erlotinib treatment. Overall, erlotinib treatment induced a large number of transcriptomic alterations.

[0081] To specifically investigate transcriptional alterations in mesothelioma-specific genes following erlotinib treatment, we focused on several well-known mesothelioma-specific genes. These genes—Wt1, Msln, Pdpn (encoding podoplanin), Calb2 (encoding calciretinin), Krt7 (encoding CK7), and Muc16—showed significant upregulation in the E28D colon compared to the C7D colon. Figure 10 D). RT-qPCR validated the differential expression of these genes in the E28D and C7D colons ( Figure 10 F). We further analyzed the expression changes of these specific genes at different time points after erlotinib treatment. Seven days after erlotinib treatment (E7D vs C7D), Wt1, Msln, Pdpn, Calb2, Krt7, and Muc16 showed significant upregulation. Figure 10 A). Similarly, after 14 days of treatment (E14D vs C7D), Wt1, Msln, Calb2, Krt7, and Muc16 showed significant upregulation. Figure 10 B). Finally, after 21 days of treatment (E21D vs C7D), Wt1, Msln, Calb2, Krt7, and Muc16 showed significant upregulation ( Figure 10C). Western blot analysis confirmed elevated levels of WT1, MSLN, Calretinin, and CK7 proteins at various time points in the colon treated with erlotinib. Figure 10 F). Even before tumors appeared 7 days after treatment, the upregulation of mesothelioma-specific genes began, suggesting that erlotinib may rapidly induce precancerous lesions. Overall, these findings indicate that erlotinib rapidly upregulates the expression of mesothelioma-specific genes within 7 days and maintains this effect.

[0082] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing an animal model of malignant peritoneal mesothelioma induced by a small molecule drug, characterized in that: An animal model of malignant peritoneal mesothelioma was induced by intraperitoneal injection of an epidermal growth factor receptor tyrosine kinase inhibitor into mammals. The epidermal growth factor receptor tyrosine kinase inhibitor is erlotinib or its salt; the dosage of the epidermal growth factor receptor tyrosine kinase inhibitor is 50 mg / kg-400 mg / kg, and the dosing period is 7-180 days; The mammal in question is the mouse.

2. The method for constructing an animal model of peritoneal malignant mesothelioma induced by a small molecule drug according to claim 1, characterized in that: The epidermal growth factor receptor tyrosine kinase inhibitor was injected in suspension form.

3. The method for constructing an animal model of peritoneal malignant mesothelioma induced by a small molecule drug according to claim 2, characterized in that: The dosage of the epidermal growth factor receptor tyrosine kinase inhibitor is 200 mg / kg-400 mg / kg, and the dosing period is 21-180 days.

4. The method for constructing an animal model of peritoneal malignant mesothelioma induced by a small molecule drug according to claim 3, characterized in that: The type of malignant peritoneal mesothelioma is sarcomatous.

5. The application of erlotinib and its salts in constructing animal models of malignant peritoneal mesothelioma, characterized by: Mice were induced to develop a malignant peritoneal mesothelioma animal model by intraperitoneal injection of erlotinib or erlotinib hydrochloride at a dose of 50 mg / kg-400 mg / kg daily for 7-180 days.

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