The Construction And Investigation Of Head And Neck Squamous Cell Carcinoma Three-Dimensional Metastasis Model Based On Fine Modified Chondroitin Sulfate

Background: Metastasis is an important reason for the high mortality of head and neck squamous cell carcinoma(HNSCC).Constructing a cell model that highly mimics the characteristics of HNSCC metastasis in vivo can help to understand the mechanism of HNSCC metastasis.The expression of chondroitin sulfate(CS)in the HNSCC microenvironment is significantly different from that of normal tissues.The expression of chondroitin sulfate proteoglycan 4(CSPG4)in HNSCC tissues is significantly increased and is closely related to HNSCC metastasis and poor prognosis,but its molecular mechanism is not clear.In our previous work,we found that the three-dimensional(3D)composite scaffold containing CS could enhance the metastatic ability of HNSCC cells and promote the high expression of CSPG4 in cells,which was similar to HNSCC cells in vivo.It suggested that abnormally expressed CS in HNSCC tissues participated in regulating tumor cell metastasis.Objective: A new 3D metastatic model regulated by CS was constructed based on the characteristics of CS in the HNSCC microenvironment in vivo.And the role of CS content on the characteristics of HNSCC metastasis was systematically studied.Further,the molecular mechanism of abnormally expressed CS-regulated metastasis was clarified base on the optimized 3D cell model.Methods:(1)Alginate calcium-chondroitin sulfate composite gel microspheres(ALG-CS)with different CS mass ratios were prepared.Effects of CS mass ratios on the physical properties,including the morphology,diameter,stability and strength,and biocompatibility were investigated to determine the optimization of CS mass ratios;(2)The effects of CS mass ratios on the metastasis ability of human HNSCC cell line SAS was investigated by detecting the expression of matrix metalloproteinases(MMPs);(3)The effects of CS mass ratios on SAS cell metastasis ability was explained by detecting the expression of metastasis-associated pathway proteins,and the related molecular mechanisms were clarified.Results:(1)When the mass ratio of CS was 0-20%,ALG-CS had good sphericity,uniform particle size,high stability and strength.It was a better microsphere preparation condition for cell culture;(2)SAS cells proliferated and remained viable in ALG-CS;(3)Compared with 2D cells,3D cultured SAS cells in ALG-CS highly expressed activated MMP2,MMP9.Although 3D-cultured cells showed increased activation of MMP2 and MMP9 with the increasing CS mass,no statistical difference between these groups was detected;(4)By detecting the protein expression of multiple signaling pathways related to metastasis,it was found that the activation level of ERK1/2 signaling pathway increased as the increasing of CS mass ratio.No different expression of signal molecules related to PI3K/AKT pathway was found;(5)The expression of programmed cell death ligand 1(PD-L1)increased with increased CS mass ratio,no statistical difference between these groups was detected.Conclusion: According to the physical properties and biocompatibility,the maximum mass ratio of CS in ALG-CS was determined to be 20%.The expression of activated MMPs in HNSCC cells increased with increasing CS mass ratio,which suggested enhanced metastatic ability.The ERK1/2 signaling pathway might be involved in regulating this process.In addition,the increasing of CS mass ratio stimulated cells to highly express PD-L1,which provided a new idea for immunotherapy of HNSCC.The new metastasis model constructed in this study can reflect the regulation of HNSCC cell metastasis by the fine changes of CS in the microenvironment of HNSCC,and provide new ideas and technologies for the research of HNSCC metastasis.