Screening And Mechanism Study Of Lead Compounds Of Anticancer Traditional Chinese Medicine Based On Lung Cancer Organoid Platform

Lung cancer is a major disease that endangers the health of the Chinese people and is the most common respiratory malignancy in the world.leading to the largest number of deaths.The discovery of new drugs for lung cancer has always been a hot spot in the pharmaceutical industry and clinical research.Although new drugs with EGFR.ALK and other targets are widely used in clinical practice,most patients with lung cancer and drug resistance need to find more new drug molecules.New drug lead compound discovery is a key step in new drug research and development,good new drug lead compound discovery comes from the establishment of highly clinically relevant assays and high-quality drug compound libraries.Tumor organoids technology is a new type of in vitro 3D cell culture technology,which is considered to be a major breakthrough in cancer stem cell technology in the past decade.Nature Methods rates organoids technology as the 2017 annual technology.Tumor organoids cultured in vitro can not only establish tumor models from tumor tissues with high success rate,but also have tissue structure and genetic background similar to tumor tissues,thus retaining tons of tumor biological information and a large number of tumor sensitivity characteristics of drugs,making it an important tool for personalized medical treatment of tumors and development of new anticancer drugs.Establishing a lung cancer tumor organoid library from lung cancer tissue and a drug screening model from this kind of organ library has the characteristics of high individual diversity,high clinical relevance,phenotypic detection,and high drug screening throughput.The compounds screened from the lung cancer organoid library can be used to simulate the response of the drug in clinical application.Although the pharmaceutical industry and medicinal chemist have created many compound synthesis techniques and also constructed a huge library of synthetic compounds,it is still very small relative to the scale of compounds in nature.Traditional Chinese medicine is the treasure of the Chinese nation,the scale of a good deal of compounds in traditional Chinese medicine is much higher than the existing human can synthesize ingredients.However,due to the complex composition of traditional Chinese medicine and the lack of scientific evaluation methods,traditional Chinese medicine cannot be the mainstream of modern medicine system.As for artemisinin and other Chinese herbal extracts,not be recognized by the world until the scientific system of Western medicine accept it.Modernization of traditional Chinese medicine(TCM)is an ideal and method of modern new drug research and development.Screening.study the development of TCM and its natural extracts through modern tumor detection technology model and high-throughput screening technology.All of this will provide new ideas for TCM modernization.Purpose:Based on the thinking and methods of modern biomedical research and development,this study explores the new anticancer drug discovery model for screening and studying natural compounds extracted from traditional Chinese medicine by using tumor organoid specimen bank,natural compound specimen bank,high-throughput drug screening,tumor cell biology and tumor signaling pathway research and other technology platforms.Methods and Results:Part Ⅰ:Establishment of tumor organoid model of lung cancer1.Acquisition of Lung Cancer Tumor SpecimensThe lung cancer tumor tissues used in this study rely on the investigator-initiated clinical trials,including the "Clinical Correlation Study of Tumor Organoids in Non-Small Cell Lung Cancer",and the ethical approval number of the Ethics Committee of Beijing Chest Hospital is No.48,2018,the US Clinical Trial Registry No.03453307.According to the inclusion criteria set in the clinical study,after the enrolled patients signed the informed consent,surgery or biopsy tissue was transferred to the laboratory for tumor organoid modeling.2.Establishment and Culture of Tumor Organoid ModelsAfter tumor tissues were incubated with protease complexes,single-cell suspensions were obtained by enzymatic hydrolysis.The tumor cells were embedded in Matrigel with the upper layer medium containing multiple growth factors.After 5-8 days of culture,first generation tumor organoids were formed.These tumor organoids can continue to be passaged,cryopreserved,or used for research or new drug screening.In January 2018 to January 2019 from Beijing chest hospital affiliated to the capital university of medical sciences,thoracic surgery in lung cancer surgery patients collected specimens of 27 cases,use of its culture conditions,screening and optimizing the system successfully establish the lung cancer cells in 3 d class organ culture and build the condition of the kind of organ model of 24 patients with lung cancer,including 13 cases of male patients,There were 11 female patients,13 adenocarcinoma patients,11 squamous cell carcinoma patients,aged 47-68 years.7 stage Ⅰ patients,11 stage Ⅱ patients.6 stage Ⅲ patients.13 patients with tumor diameter of 1-3cm,7 patients with tumor diameter of 3-5cm,and 4 patients with tumor diameter greater than 7cm.The overall success rate of modeling was 88.9%,and the modeling cycle was stable in about 3-4 weeks.3.Detection of tissue stainingTo verify that tumor organoids cultured in vitro have a tissue structure parallel to that of tumors in vivo,we observed the internal tissue structure of cultured tumor organoids by tissue staining.Lung cancer tumor organoids were expanded to a total volume of 1 x 10 7 Total cells of grade.After subculturing.tumor organoids were grown to a size of 100-800 microns,collected by low-speed centrifugation at 100 g and fixed with 4%paraformaldehyde,dehydrated,embedded in paraffin,sectioned,then stained with hematoxylin and eosin stain,last photographed under a microscope.It can be observed that tumor organoids have abundant tumor histological features and individual diversity.Histological staining was performed on 24 lung cancer organoid models and corresponding patient tissues.The results showed that the organoid model of lung cancer had a high degree of morphological and histological agreement with the original patient tissue.4.In vivo tumorigenicity assayTo verify the tumorigenicity of tumor organoids cultured in vitro,tumor organoids were implanted into NOG mice,a highly immunodeficient model.After 20-30 days of subcutaneous growth of tumor organoids in NOG mice,a significant increase in size was observed.Demonstrated that tumor organoids of tumors cultured in vitro are very tumorigenic.In this study,3 of 7 cases of nude mice were successful,with a success rate of 42.9%.Among them,adenocarcinoma formation was successful in 1 of 3 cases,the success rate was 33.3%.2 out of 4 patients with squamous cell carcinoma were successful,the success rate was 50%.5.Detection of EGFR mutations in tumor organoids by ARMS PCRTo investigate whether tumor organoids cultured in vitro still have the oncogenic driver genes carried by the primary tissue.EGFR L858R mutation is known to be detected in primary tumor tissues.We extracted genomic DN A from tumor tissues and paired tumor organoids,respectively.Through ARMS PCR,we detected that the oncogenic driver genes carried in tumor tissues were still retained in tumor organoids.The EGFR mutations detected in tumor-like organs were consistent with those detected in tumor specimens.6.Second generation sequencing of tumor tissues and tumor organoidsTo investigate the genomic similarity of tumor organoids to tumor tissues in vitro.we examined mutations in 113 tumor-associated genes by second-generation sequencing,which liquid chip capture sequencing target region gene detection protocol is used to synthesize effective specific probes and perform liquid phase hybridization with genomic DNA to capture and enrich the target region sequence.and then perform high-throughput sequencing using Illumina sequencing platform.It was found that tumor organoids cultured in vitro retained the gene mutation profile in paired tumor tissues.In vitro established lung cancer tumor organoids have a high degree of concordance from the genetic level and the primary tumor.Part Ⅱ:Functional screening platform for tumor organoids1.Establishment of high-throughput drug screening(HTS)technologyTo make lung cancer tumor organoids as a suitable platform for high-throughput drug screening requires ensuring the establishment of microwell culture system and the differences between two microwells in detection methods,and optimization of signal to noise ratio.We evaluated the marginal effects,inter-assay errors,and positive control selection in 96-well microplates.In particular,the convenience and stability of the comparative detection method,the setting of positive control,the adjustment of organoid cell seeding volume,and the use of Z factor for signal window and noise evaluation.A stable Z factor of>0.5 was achieved to meet the technical requirements for high-throughput screening.Based on the requirements of high-throughput drug screening,the high-throughput drug screening platform established through the tumor organoid model adopts the mode of primary screening and secondary screening.In the primary screening,the drugs to be tested are in the form of single concentration,60 compounds are tested in each well plate,and the testing of each tumor organoid can quickly complete the testing of 1000 compounds in 16 96-well plates.2.CellTiter-Glo Cell Viability AssayTo achieve the stability and convenience of high-throughput test,we chose to use the high-throughput cell viability test technology introduced by Promeg,USA.Within the 96-well system,it is possible to achieve.3.Natural Compound LibraryCompound library is the core component of high-throughput drug screening.Only high-quality libraries of pharmaceutical compounds can be used to analyze molecules with druggable potential.The natural compound library in this study was purchased from MedChemExpress’s natural compound library and contains more than 1000 natural compounds with pharmacophoric effects.4.Validation of screening the efficacy of solamargine in lung cancer cel]linesRepresentative natural compounds screened in lung cancer tumor organoids were further validated for their anticancer activity in lung cancer cell lines.Different from the preliminary screening,in the lung cancer cell line,solamargine was added into the tumor cell line by gradient dose dilution.After 3-day drug treatment,the effect of drug on cell viability was detected by CellTiter-Glo cell viability assay,and half maximal inhibitory concentration(IC50)was calculated by software fitting.Part Ⅲ:Mechanism of Antitumor Activity of solamargineModern biomedical research and development often start from the mechanism.By establishing the detection methods for certain specific targets,high-throughput screening techniques are used to find small molecules or macromolecular drugs that can inhibit this target.However,due to the complexity of life itself,there is a scarcity of single target targets for the treatment of tumors,thereupon combination of drugs has become a trend.Due to its rich structure,natural compounds of traditional Chinese medicine can inhibit tumor growth by inhibiting multiple targets at the same time.Therefore,it is more reasonable to screen natural compounds of traditional Chinese medicine through phenotypic screening.Study the functional mechanism after we finish the phenotypic screening.In this study,we screened the molecular signaling pathway of the typical natural compound solamargine to find the possible signaling pathway of inhibiting the growth of lung cancer cells.1.Construction of the Reporter Gene SystemReporter gene system is a method connecting a transcription factor binding promoter sequence in front of luciferase to detect the activation of a signal pathway by artificially concatenating and amplifying a promoter sequence.This study constructs a reporter gene system for more than 100 signaling pathways.Taking the hedgehog pathway as an example,reporter gene clones were constructed by concatenating the Gli-binding regions and transfected into cells to construct stable cell lines.In this cell line,it was possible to test the inhibitory effect of natural compounds on this pathway.2.Real-time PCR(Real time PCR)To verify the inhibitory effect of solamargine on the hedgehog pathway,a fluorescent quantitative PCR method was used to verify the reliability of the assay.Because activation of the hedgehog pathway upregulates Glil expression levels,the detection of Gli1 mRNA levels can effectively evaluate the activation of the hedgehog pathway.In this study,fluorescent quantitative PCR primers for GLI1 were designed to test the inhibitory effect on the mRNA level of GLI1 after drug treatment,verifying the conclusions obtained with the reporter gene system.3.BODIPY-cyclopamine competition binding assayIn this study,we further searched for target proteins of the hedgehog pathway inhibited by solamargine.Because Smoothened(SMO)proteins are homologous to G protein-coupled receptors,they are made of a single peptide chain of seven transmembrane regions.Drug binding to SMO can inhibit the hedgehog pathway,and this study utilized a fluorescently labeled probe,BODIPY-cyclopamine,to probe molecules that may bind to the cyclopamine-bound position on SMO.By fluorescence binding assay,it was found that solamargine could inhibit the binding of BODIPY-cyclopamine to SMO,which suggested that the inhibition of SMO protein by solamargine was one of the possible mechanisms for the inhibition of hedgehog pathway.Conclusion:China has a large number of clinical practices of traditional Chinese medicine accumulation,but it is difficult for traditional Chinese medicine get into the system of modern medicine,how to achieve the modernization of traditional Chinese medicine is the subject of traditional Chinese medicine.In this study,through the breakthrough tumor organoid culture technology in recent years,a high-throughput screening method was established to screen natural compounds in traditional Chinese medicine and evaluate their anticancer activity in lung cancer organoids,providing lead compounds for drug development.By cell biology and molecular biology methods,the pharmacodynamics and mechanism of signal pathway were studied.The preliminary work of this study provides only a superficial exploration of the mode of screening anticancer lead compounds from natural compounds of traditional Chinese medicine.