Noninvasive focused ultrasound non-specifically activating spleen immunological function to universally suppress tumor proliferation: a new strategy for cancer immunotherapy -NK cells

NK cells were sorted from the spleen using the Beckman Kurt MoFlo Astrios ultra high speed flow cytometry sorting system. Subsequently, general transcriptome sequencing was performed when the positive cell rate was greater than 90% detected by flow cytometry. The cDNA library construction and sequencing of all RNA samples were performed by Shanghai Ouyi Biomedical Technology Co., LTD. Transcriptome sequencing was performed to screen differentially expressed genes (DEGs). Then, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were applied to annotate the function of the DEGs. The results revealed that FUS firstly regulated calcium-related signaling pathways to further modulate other signaling pathways, such as ECM-receptor interaction, PI3K-AKT signaling pathway, Rap1 signaling pathway and Hippo signaling pathway to promote immune cell proliferation, migration and activation to suppress cancer cell deterioration. NK cells (CD45+ CD3- NK1.1+) were sorted from the spleen using the Beckman Kurt MoFlo Astrios ultra high speed flow cytometry sorting system (MoFlo Astrios EQ, Beckman Coulter, lnc, USA). Subsequently, general transcriptome sequencing was performed when the positive cell rate was greater than 90% detected by flow cytometry (CytoFLEX LX, Beckman Coulter Life Sciences, USA). RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then the libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).