| Background:At present,the commonly used tumor research models include: 2D cell lines,Patient-Derived Xenografts(PDX)models and 3D cultured Patient-Derived Organoid(PDO)models.The cell lines commonly used in traditional tumor research models lack the original tumor phenotype and genetic heterogeneity.PDX model has a long construction period,and the tumor microenvironment will be gradually replaced by mouse extracellular matrix components.As a new technology system and research method,PDO makes up for the shortcomings of 2D tumor cell lines in vitro and PDX models in vivo,and becomes an important bridge connecting 2D cell culture and live animal models.It is also a very potential translational model in clinical medicine and basic research.Salivary gland tumor cells are difficult to form stable cell lines in vitro,so far,the salivary gland tumor cell lines that can be used for in vitro research are very limited.Organoids can be expanded relatively quickly,facilitating preservation and genetic modification.At present,a variety of tumor organoid libraries have been established,including breast cancer,colon cancer,and pancreatic cancer.With the development of biological sequencing technology,the intrinsic biological characteristics of tumors have been gradually discovered.The study of multi-omics such as genome,transcriptome and proteome of tumor tissues is also a hot topic in tumor research.Although technical means such as microdissection can remove excessive stromal components in tumors to a certain extent,reducing the impact on the representability of sequencing data.However,it is difficult to accurately reflect the actual situation of simple tumor cells by using only tumor tissue samples for sequencing analysis.With the development of organoid technology,the culture of PDO model selectively expands tumor-associated epithelial cells,eliminates the influence of tumor stromal components on tumor biological information components,and has a high purity of tumor cells,focusing on the characteristics of tumor epithelial cells,and at the same time,the basic tumor information such as gene mutation,proliferation and self-renewal rate of patient-derived tumor cells.They can also migrate to organoid research platforms and further expand the basic information of these tumor cells through the proliferation of organoids.Salivary gland tumor(SGT)is a large group of tumors occurring in the head and neck,including a variety of types,and the heterogeneity and overlap between tumor cells often bring difficulties in diagnosis.Therefore,there is an urgent need to combine sequencing technology and bioinformatics analysis to help us to thoroughly explore the cellular and molecular mechanisms of heterogeneity,cellular and structural overlap among different SGT types,and to explore specific diagnostic biomarkers between SGT types.In this study,we attempted to construct a salivary gland tumor organoid library,and verify the consistency of the organoid library with the original tumor tissue at the histomorphology and molecular levels.The specific gene expression of each tumor was analyzed by transcriptomics,and the relevant specific biological markers were further verified by RNA expression and immunohistochemistry staining.Objective:Explore the salivary tumor organoid culture protocol,and construct an organoid library containing SGT and normal salivary gland(NSG)with different pathological types.The similarities and differences between SGT organoids and SGT tissues were characterized by histomorphological and transcriptomic analysis.Combined with bioinformatics methods,the transcriptional molecular characteristics of different types of SGT were analyzed,and the heterogeneous and overlapping cellular molecular mechanisms between subtypes were understood,in order to discover tumor-specific molecular markers and help guide the differential diagnosis of common salivary gland tumors in clinical pathology.Methods:1.Collect salivary gland tumor tissue samples obtained by surgery or biopsy.2.Select the tumor tissue and normal glands of the 6 most common SGTs for organoid culture.3.Statistical analysis of cultured salivary gland tumor organoids and general clinical information of corresponding clinical patients.4.Histological evaluation of related organoids by inverted microscope observation,H&E and immunofluorescence staining(selecting phenotypic markers commonly used in clinical research),statistically analyzing the growth status of organoids of various salivary gland tumors and the morphological and molecular marker characteristics of organoids compared with the corresponding original tissues.5.Collect SGT tumor tissue,normal glands and cultured tumor organoids,normal salivary gland organoids for transcriptome sequencing,analyze the similarities and differences of various types of salivary gland tumor tissues and corresponding cultured organoid transcriptome data through various bioinformatics analysis methods such as dimensionality reduction analysis,consensus clustering and hierarchical clustering,and clarify the composition and structural characteristics of SGT cells and the molecular mechanism of formation.6.Through the comparative analysis of transcriptome data series of six common SGTs and their matching normal organoids,potential specific diagnostic markers for each type of salivary gland tumor were screened.7.Two biomarkers of salivary gland malignancy-mucoepidermoid carcinoma(MEC)with the highest clinical incidence were selected for q PCR evaluation,and immunohistochemical staining was confirmed using tissue microarrays(TMA)to evaluate the accuracy of sequencing results and detect the expression distribution of gene proteins in a variety of SGTs.8.Immunohistochemical staining was used to examine the protein expression of candidate MEC biomarkers in the cultured six SGT organoids and their derived tumor tissues.Results:1.We generated an organoid biobank of patient-derived salivary gland tumors,containing6 common salivary gland tumors,including 21 benign SGTOs: 11 cases of pleomorphic adenoma(PA)and 10 cases of basal cell adenoma(BCA).There were 24 malignant SGTOs: 6 cases of mucoepidermoid carcinoma(MEC),10 cases of adenoid cystic carcinoma(ACC),4 cases of acinar cell carcinoma(Aci CC),4 cases of salivary ductal carcinoma(SDC),and 26 cases of normal glandular organoids.Most types of PDOs were in the rapid growth phase in the first week,forming organoid morphology with more mature structures within 14 days,and then entering the growth plateau phase at about 21 days.Except for the low proliferation rate of Aci CC,most organoids can be expanded for5 passages.The success rate of SGT organoid culture is 71/85(84%).2.PDOs reflect the morphological and transcriptional characteristics of parental tumors,reproduce the tissue structure characteristics of parental tumors and the expression of specific diagnostic markers.Overlapping and heterogeneous growth patterns of cell types were observed in different organoid types,and were more consistent with parental tumors.3.Compared with the general tumor tissue,the cluster analysis of PDOs significantly revealed the epithelial characteristics of SGT,and analyzed the intrinsic relationship between different types of salivary gland tumors from the transcriptomic level,and visually demonstrated the similarities and differences between SGT tissue and corresponding organoids by bioinformatics.4.After comparative analysis by bioinformatics method,6 specific molecular markers of common SGT were screened.5.q PCR technology and TMA section staining verify that PTP4A1 is expected to become a potential biomarker for MEC diagnosis.6.SGT organoids maintained and reproduced the expression of parent tumor tissue,MEC candidate biomarker PTP4A1 and NEFL protein.Conclusion:We established the first organoid biobank containing multiple salivary gland tumors.The PDOs of SGT recapitulates the morphological and transcriptional features of the primary tumor,revealing the molecular signatures between the different types of SGT and their specific biomarkers.PTP4A1 can be used as a specific diagnostic biomarker for mucoepidermoid carcinoma. |
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