CAFs Targeted Ultrasound-responsive Nanodroplets Loaded V9302 And GLULsiRNA To Inhibit Melanoma Growth Via Glutamine Metabolic Reprogramming And Tumor Microenvironment Remodeling

Objective:Metabolic therapies have made rapid progress over the past decade,but their efficacy in melanoma has been modest,largely due to the interaction of cancer-associated fibroblasts(CAFs)with cancer cells to regulate their growth.Modifying the tumour microenvironment(Tumor microenvironment,TME)can be challenging and elusive.The tumour microenvironment is a complex metabolic system and therefore the metabolic vulnerability of non-cancerous and cancerous cells in the TME needs to be taken into account.CAFs are critical for glutamine deprivation survival in melanoma.Co-targeting the metabolism of stroma and tumour cells to enhance remodelling of the tumour microenvironment is a novel and exciting approach to address these barriers.Many investigators have reported the ability of various nanocarriers in synergistic therapy.Unlike other types of nanocarriers that have been extensively studied to date,few studies have focused on the use of nanoscale ultrasound contrast agents as therapeutic agents for the tumour microenvironment and tumours.Nanoscale ultrasound contrast agents have the competitive advantage of high efficiency and low toxicity due to their ultrasound imaging capabilities and targeted delivery of anti-cancer drugs and genes.Here,we successfully constructed controlled-release tumour-associated fibroblasts(CAFs)targeting nanodroplets loaded with V9302,an ASCT2 inhibitor that inhibits glutamine uptake,and GLULsiRNA,an siRNA that inhibits glutamine synthesis,and used ultrasound-targeted microbubble disruption(UTMD)to facilitate drug and gene delivery.Together,they block the metabolism of reactive stromal cells and tumour cells,reprogramming glutamine metabolism and facilitating tumour therapy.Methods:FH-V9302-siGLUL-NDs were prepared by ultrasonic emulsification;characterization of FH-V9302-siGLUL-NDs was verified by transmission electron microscopy,nano-laser particle size measurement,gel electrophoresis and UV spectrophotometry;nutrient deprivation assay was performed to verify the dependence of melanoma cells on glutamine metabolism;in vitro experiments were performed using Western Blot and cell viability assay to verify the successful induction of CAFs.Western Blot and cell viability assays were used to verify the successful induction of CAFs;in vitro flow cytometry and fluorescence microscopy were used to detect the in vitro targeting of FH-NDs;in vivo frozen sections and fluorescence live imaging were used to assess the in vivo targeting of nanodroplets;Western Blot was used to verify the silencing efficiency of siGLUL-loaded nanodroplets;Western Blot was used to verify that nanodroplets loaded with different The in vivo targeting of nanodroplets was assessed using frozen sections and fluorescence live imaging.The anti-tumor effects of FHV9302-siGLUL-NDs in vivo were investigated using Western Blot and fluorescence microscopy to determine the effects of nanodroplets on the tumor microenvironment.Results:(1)The prepared FH-V9302-siGLUL-NDs were small and homogeneous in size,with a ζ potential of +37.67 ± 10.23 mV.(2)The optimal v/v for loading siGLUL was determined to be 20:1.(3)Ultrasound irradiation promoted drug release from FH-V9302siGLUL-NDs(p<0.05).(4)B16F10 is glutamine metabolism dependent.(5)NIH/3T3 cells were successfully induced as a cell model for CAFs cells.(6)In vitro assays FH-NDs had good CAFs targeting ability(p<0.05).(7)In vivo assay FH-NDs had good tumour targeting ability(p<0.05).(8)Nanodrops loaded with siGLUL have good gene silencing efficiency.(9)Nanodrops loaded with siGLUL reduced stromal secretion,altered the cell morphology of CAFs and improved the tumour microenvironment.(10)FH-V9302-siGLUL-NDs exhibited good hyperechoic signals in both in vivo and in vitro ultrasonography modes.(11)Ultrasound combined with metabolic blockade treatment significantly inhibited tumour growth viability,migration and invasion.(12)FH-V9302-siGLUL-NDs were able to stay in the tumour for a long time and reached maximum enrichment at 4 hours after injection.(13)A 2:1 ratio of CAFs to B16F10 was determined to mimic the tumour microenvironment in vivo.(14)In the in vivo experiments,the FH-V9302-siGLUL-NDs+US group had the slowest tumour growth rate and the lowest tumour weight.Histological section staining showed the highest level of apoptosis and lowest proliferation within the tumour of the FH-V9302-siGLUL-NDs+US group.(15)The FH-V9302-siGLUL-NDs+US group could significantly reshape the tumor microenvironment in mice.(16)FH-V9302-siGLUL-NDs had a good safety profile and no significant effects on the major organs of mice.Conclusion:FH-V9302-siGLUL-NDs combined with UTMD had strong synergistic antitumour effects,i.e.low frequency ultrasound irradiation combined with glutamine metabolism The combination of low-frequency ultrasound irradiation and glutamine metabolism blockade could synergistically inhibit tumor cell viability,promote apoptosis and significantly slow down tumor growth in tumor-bearing mice.In addition,this strategy can significantly alter the tumour microenvironment and inhibit stromal production.It holds great promise for future clinical translation and has a potentially bright future in comprehensive diagnostic therapy.