Based On Organoid Model To Investigate EN1 Specifically Regulating ACC Proliferation And Infiltration

Objective: Adenoid cystic carcinoma(ACC)is a common salivary gland malignant tumor in the head and neck.It is characterized by slow growth,strong local invasion,often accompanied bylocal nerve invasion symptoms,easy recurrence and distant metastasis after operation.ACC has three tissue subtypes: cribriform pattern,tubular pattern and solid pattern.The cellular morphology and structural composition of ACC overlap with other salivary gland tumors.Basal cell adenoma(BCA)is a relatively common benign salivary gland tumor,which has histological subtypes such as trabecular,tubular,solid and cribriform.Among them,cribriform basal cell adenoma and cribriform adenoid cystic carcinoma have similar cell composition and morphological structure,so the differential diagnosis is difficult.However,BCA(benign tumor)and ACC(highly aggressive malignant tumor)have dramatic differences in surgical methods and adjuvant treatment.Therefore,it is necessary to investigate the pathogenesis of cribriform ACC and BCA and explore the related biomarkers to differentiate them.EN1(Engrailed-1)is a homeobox transcription factor that plays an important role in embryonic development.After embryogenesis,EN1 is silenced in most cells.The transcriptomics study of our group has found that EN1 is specifically expressed in ACC,but its role as a differential diagnostic marker and its mechanism are still unclear.This study aims to evaluate the properties of EN1 as a marker for differential diagnosis of ACC by exploring the regulatory mechanism and biological function of EN1 in ACC tumorigenesis.Methods: Patient-Derived Organoids(PDOs)of ACC and BCA tumors were constructed in a three-dimensional culture system.The growth characteristics of the organoids were observed by inverted microscope,and the histomorphology of the organoids was identified by H&E and immunofluorescence(IF)staining.The tissue samples of ACC,BCA and NSG were collected for transcriptome sequencing.The differentially expressed genes(DEGs)in ACC and BCA tissues were analyzed by bioinformatics and the enrichment of differentially expressed gene pathways was analyzed.Clinical samples were collected and Tissue microarray(TMA)sections were made.Immunohistochemical(IHC)staining,real-time fluorescence quantitative PCR(qRT-PCR)and Western blot(WB)were used to detect the expression of EN1.The expression changes of TGFβ in ACC and BCA were analyzed by transcriptome data.The expression of TGFβ1 in relevant clinical samples was examined by qRT-PCR and IHC staining.Human ACC(SACC-83 and SACC-LM)cell were used as the research objects,and the regulatory effect of TGFβ signaling pathway on EN1 expression in ACC cells was detected by qRT-PCR.Meanwhile,the effect of TGFβ on the morphology and migration of PDOs and the expression of EN1 were observed.Results: The PDOs models of ACC and BCA tumors were successfully established.HE and IF staining results showed that PDOs basically simulated the morphological and molecular characteristics of their parental tissues.Transcriptome data analysis and qRTPCR,WB and IHC staining showed that EN1 was specifically highly expressed in ACC and lowly expressed in BCA.These results indicate that EN1 can be used as a differential diagnostic marker for ACC and BCA,especially for the cribriform subtype.In the PDOs model study,EN1 was found to be highly expressed in ACC but not in BCA.In addition,TGFβ signaling was found to regulate EN1 expression in ACC cells and PDOs models.In the PDOs model,TGFβ induced EN1 expression,which could promote tumor clonal budding of ACC,and was highly expressed in the budding part.Conclusion:1.EN1 can be used as a differential diagnostic marker for ACC and BCA.2.TGFβ-induced EN1 facilitates the tumor budding of ACC,which may be related to the proliferative and invasive mechanism of ACC.