Establishment Of Nasopharyngeal Carcinoma Organoid Culture System And Preliminary Exploration Of Drug Sensitivity Test In Vitro

Introduction and objective:Nasopharyngeal carcinoma is a high incidence of malignant tumors in China,accounting for the highest incidence of head and neck tumors.Nasopharyngeal carcinoma is mainly poorly differentiated and undifferentiated.More than half of the patients are diagnosed with advanced disease at the time of diagnosis,and 10%of patients have distant metastases.Primary NPC is sensitive to radiotherapy and chemotherapy,but there are still some refractory NPC resistant to the first-line chemotherapy drugs,leading to a relapse.The treatment of recurrent NPC is difficult,and both the traditional in vitro cell line and PDX models have obvious shortcomings.Therefore,it is imperative to establish better disease models,to further study the mechanism of recurrent NPC,and to explore a more effective treatment method.Tumor organoids are tumor micro-organs in which stem cells are cultured in 3D in vitro.They have been verified by histopathology,molecular biology and genomics,and can retain most of the biological characteristics and tumor heterogeneity of primary tumors,which could be the best models for basic and clinical researches.This study aimed to culture and identify NPC organoids,conduct drug-sensitivity test,and to establish a NPC organoid biobank using NPC biopsy and resected tissues.Materials and methodsFifteen fresh nasopharygeal cancer tissue samples(diagoised by pathology)were collected from NanFang hospital for 3D culture from June 2018 to December 2019.NPC organoids were collected,fixed with 10%formalin,embedded in paraffin,sectioned,and stained with hematoxylin and eosin.The expression of CD133/CD44/CD47/BMI-1/EBER were detected by immunohistochemistry and in situ hybridization.Organoids were tested for sensitivity of chemotherapeutic drugs and targeted drugs,and the results were compared with the treatment response of the enrolled patients.Results1.Successful establishment of nasopharyngeal carcinoma organoid culture system.A total of 50 NPC samples were collected,and 29 organoids were successfully cultured,with a success rate of 58%.There were 16 primary NPC organoids and 13 recurrent NPC organoids.The success rate of culture of primary and recurrent NPC organoids was 47.06%and 81.25%,respectively.The success rate of culture was mainly related to tissue quality and size,specimen pollution,etc.(P=0.002).2.The 3D cultures were identified as NPC organoidsThe pathological characteristics of HE staining of 3D NPC cultures were consistent with those of parental tumor tissues,which had typical nuclear atypia.The markers,CD133,CD44,CD47,BMI-1 and EBER,were all positivelv expressed in both 3D cultures and parent tumor tissues.3.Preliminary establishment of drug-sensitivity platform of NPC organoids In this study,ATP cell activity analysis was used for in vitro drug-sensitivity test of tumor organoids in 23 patients.It’s successful in 18 patients,with a success rate of 78.26%.The main causes of failure were that the organoids were excessively digested and polluted during the process of drug-sensitivity test.As a method of tumor diagnosis,the drug-sensitivity test result of organoids has a consistency of 72.22%with the clinical efficacy of patients.There were a sensitivity rate of 50%,a specificity rate of 90%,an accuracy rate of 72.22%,a positive predictive rate of 80%,and a negative predictive rate of 69.23%.The Kappa consistency test showed a value of 0.42.The consistency rate of the targeted drugs was 100%,respectively.The cetuximab had the highest coincidence rate of 100%(4/4),while the gemcitabine showed the lowest coincidence rate of 33.33%(2/6).4.Preliminary establishment of NPC organoid biobankIn this study,the cell number of the 6 NPC organoids reached 106,and they can be stored frozen,resurrected and subcultured continuously,which was in line with the standard of database establishment.The organoids was frozen in storage,and the quality control standards as well as the rules and regulations of the sample database were preliminary drafted.ConclusionWe have successfully established a NPC organoid model and a drug-sensitivity detection platform,which provides a good reference for future study of the molecular mechanism of NPC,and its clinical precision medicine.