Functional Blockage Of Cancer-Associated Fibroblast With Ultrafine Gold Nanomaterials Takes Anti-tumor Effect On Oral Squamous Cell Carcinoma

[Purpose] Cancer-associated fibroblasts(CAFs)were one of the most important cellular components in the tumor microenvironment of Oral Squamous Cell Carcinoma(OSCC).CAFs plays an important role in the in the proliferation,invasion and metastasis of tumors.With the development of nanomedicine in recent years,the design of nanomaterials with CAFs cells as therapeutic targets has become a new treatment strategy for malignant tumors.In this study,we aimed to explore the size-dependent effects of GNPs on the the biological function of CAFs and further on the OSCC tumor cells.It is expected to provide theoretical basis for the application of gold nanoparticles in the treatment of OSCC.[Methods] 1.Gold nanoparticles of different size from 3-nm to-80-nm were synthesized by citrate and DMSA reduction of HAu Cl4 and characterized by TEM,UV-Vis,DLS and Zeta potential.2.CAFs and normal fibroblasts(NFs)were primary cultured using tissue adhesion methods from tissues from patient with OSCC;The protein expression of ?-SMA,FSP-1,Vimentin,N-cadherin were detected by immunofluorescence and q RT-PCR;After CAFs cultured with 15 ?g/m L various sizes of GNPs,the cellular uptake of GNPs by CAFs was detected by inductively coupled plasma mass spectroscopy(ICP-MS)and the cellular location of GNPs in CAFs were detected by TEM;the proliferation of CAFs were detected by CCK-8 assay;the cytoskeleton was stained with ghostpen cyclic peptide;the migration of CAFs were detected by Transwell assay;the protein expression of CAFs were detected by immunofluorescence and q RT-PCR,the cytokine profiles of CAFs were detected by ELISA assay.3.After cultured with conditioned medium(the mixture of fresh medium with supernatant from CAFs treated with 15 ?g/m L various sizes of GNPs),the proliferation and migration of OSCC cells were evaluated by CCK-8 and Transwell assay;Murine OSCC model were constructed by subcutaneous transplantation of OSCC cells and CAFs.GNPs of various sizes of GNPs were injected intratumorly and subcutaneously around the tumor every 3 days for 5 times.The weight and tumor volumes were recorded per 3 days.The expression of ?-SMA,FSP-1,Vimentin,N-Cadherin in CAFs,Ki-67 in tumor cells and IL-6,IL-8,HGF,VEGF,TGF-?1,and PDGF-?? were detected by immunohistochemical assay.[Results] 1.3-nm,15-nm,30-nm,50-nm,80-nm GNPs were successfully synthesized by citrate and DMSA reduction of HAu Cl4.2.TEM results showed the cellular location of GNPs in CAFs were mostly in vesicle.ICP-MS results showed 50-nm GNPs was the most quality uptake by CAFs,but 3-nm was the most quantity uptake by CAFs;Difference sizes of GNPs have no influence in the proliferation rate and cytoskeleton structure of CAFs;The migration of CAFs was decreased after treated with 15 ?g/m L 3-nm GNPs;Immunofluorescence and q RT-PCR results showed that the protein expression of ?-SMA,FSP-1,Vimentin,N-Cadherin were significantly decreased after treated with 3-nm GNPs.Elisa results showed that the cytokines IL-6,IL-8,HGF,VEGF,TGF-?1,and PDGF-?? secreted by CAFs were decreased after treated with 3-nm GNPs.3.After treated with conditioned medium(3-nm and 15-nm),the proliferation and migration of OSCC cells(R-cab and SCC9)has been suppression.In animal study,3-nm GNPs could significantly inhibited the volume of tumor.Immunohistochemical assay showed that 3-nm GNPs could inhibit the expression of ?-SMA,FSP-1,Vimentin,N-cadherin in CAFs and Ki-67 in tumor cells.The cytokines IL-6,IL-8,HGF,VEGF,TGF-?1,and PDGF-?? secreted by CAFs were also decreased after treated with 3-nm GNPs.The weight change of mice and H&E section of major organ results showed intratumorly and subcutaneously injection of GNPs has no obvious toxicity.[Conclusion] We have shown that GNPs could reprogram the protein expression and cytokine secretion profile of CAFs in a size-dependent manner and further inhibit the growth of OSCC tumor.Among various sizes of GNPs,ultra-small(3-nm)GNPs had the greatest effect.Ultra-small(3-nm)GNPs could potentially be utilized as a novel method to interrupt the communications in the tumor microenviro-nment and inhibit the growth of malignant tumor.