Oscc-derived Cfdna Promote Stemness And Migration Of Oscc Cell Line By Inducing M2 Macrophage Polarization

Objective:In recent years,CFDNA has attracted extensive attention as a tumor liquid biopsy marker.Exploring the effect of CFDNA derived from oral squamous cell carcinoma on macrophages and clarifying the factors affecting macrophage polarization in the tumor microenvironment is conducive to find new targets for oral cancer immunotherapy thereby enhancing the effect of tumor immunotherapy.Therefore,this study investigates OSCC-derived CFDNA effect on stemness and migration of OSCC cell line by polarizing macrophage.Methods:1.The expression of M2 macrophage related marker CD163 in normal head and neck tissues and HNSCC was analyzed by GEPIA database.IHC and IF was used to analyze macrophage marker CD68 and M2 macrophage marker CD163 in OSCC/normal oral tissues/oral dysplasia.2.CAL-27 CM was concentrated by Ultra centrifugal filter device.MagMAX^TMCell-Free DNA Isolation Kit was used to isolate CFDNA from concentrated CAL-27 CM.Nanodrop,Quant-i T^TM Pico Green（?）ds DNA Reagent and Kits and agarose gel electrophoresis was used to detect and quantify CFDNA.3.THP-1 was firstly induced to macrophages by PMA and then treated by CFDNA.Morphological characteristics were observed under microscope.Expression levels of M1 macrophages related genes（CD86,TNF-α,IL-6）and M2 macrophages related genes（CD163,Arg-1,CD204）were determined by RT-qPCR.4.CAL-27 was treated by CFDNA-CM and expression levels of stemness related genes（OCT-4 and SOX2）were determined by RT-qPCR.Scratch assay showed the migration ability of CAL-27 after treated by CFDNA-CM.Results:1.The GEPIA database showed that compared with normal head and neck tissues,HNSCC highly expressed CD163（P<0.05）.IHC and IF showed that there were more M2 macrophages in the deep connective tissue of the dysplasia oral epithelium and the stroma of oral squamous cell carcinoma compared with the normal oral tissues（P<0.05）.2.OSCC cell line CAL-27 could secret CFDNA length between 10000-15000 bp and release of CFDNA was characterized over time,reaching a maximum after 72 hours.3.Macrophages treated by CFDNA were elongated and RT-qPCR showed its m RNA expressions of M2 macrophage related genes（CD163,CD204 and Arg-1）were significantly increased（P<0.05）.4.RT-qPCR showed that after treated by CFDNA-CM,the m RNA expressions of stemness related genes（SOX2,OCT-4）in CAL-27 was significantly increased（P<0.05）.Scratch assay showed the promoted migration ability of CAL-27 after treated by CFDNA-CM（P<0.05）.