Effects Of Tunicamycin-induced Endoplasmic Reticulum Stress On Chemoresistance Of Head And Neck Squamous Cell Carcinoma In Three-dimensional Cultures

Objective: Chemotherapy resistance is one of the main reasons for poor treatment of advanced head and neck squamous cell carcinoma(HNSCC).Effectively inhibiting the drug resistance of HNSCC cells is an urgent problem to be solved in clinical treatment.Endoplasmic reticulum(ER)stress is closely related to tumor progression.Evidence suggests that the activation of ER stress pathway can promote the anti-tumor effects of certain drugs.The collagen matrix can significantly improve the stemness and drug resistance of tumor cells,which is an ideal model to study the drug resistance of HNSCC.In this thesis,a three-dimensional(3D)collagen type Ⅰ rat tail culture model in vitro was established to simulate the drug resistance of HNSCC cells and investigate the influence and regulation of ER stress induced by tunicamycin(TM)on HNSCC resistance,providing new ideas,new methods and new strategies for clinical treatment of HNSCC.Methods: At the cellular level,collagen type Ⅰ rat tail was used to establish a 3D culture model of FaDu hypopharyngeal carcinoma cells in vitro.In this 3D model,the stem cell marker was detected by flow cytometry and western blot(WB),and drug sensitivity of cells was detected by CCK-8 assay before and after ER stress induced by TM to investigate the effect of ER stress activation on stemness and drug resistance of FaDu cells.At the animal level,FaDu cells cultured in 3D were inoculated into BALB/c nude mice,then corresponding drugs were given after tumorigenesis to explore the effect of ER stress on tumor growth.Enzyme-linked immunosorbent assay(ELISA)and WB were used to explore the relevant mechanism.Results: The 3D culture model in vitro established with collagen type Ⅰ rat tail enhanced the stemness and drug resistance of FaDu cells.It raised the secretion of activated transforming growth factor beta 1(TGF-β1),promoted the activation of TGF-β1/Smad2/3 signaling pathway and the maturation of integrin beta 1(integrin β1).In this model,TM-induced ER stress preferentially attenuated the stemness and chemoresistance of FaDu cells,down-regulated the concentration of activated TGF-β1and the expression of downstream Smad2/3 and Phospho-Smad2,resulted in deglycosylation of integrin β1.In the mouse xenograft model,TM combined with paclitaxel showed significant tumor growth inhibition compared to monotherapy and TM enhanced the antitumor effect of paclitaxel in animals.For TGF-β1 and integrin β1,the analysis of clinical data from The Cancer Genome Atlas Program(TCGA)database showed that the expression level of HNSCC tissues was significantly higher than normal tissues.Conclusion: TM-induced ER stress inhibits the stemness and drug resistance of FaDu cells in 3D collagen type Ⅰ rat tail culture and enhanced the anti-tumor effect of paclitaxel in animals.Mechanistic investigation revealed that this effect is regulated by TGF-β1/Smad2/3 signaling pathway and may be related to integrin β1 deglycosylation.