Dynamic Immune Response Of Macrophages In Three-Dimensional Tumor Microenvironment Revealed By Single-cell Analysis

The immune response is a key factor to study the development of tumors and improve therapeutic efficacy.Immune response is a response caused by the interaction between immune cells and tumor cells and immune cells,such as secretion of a variety of functional proteins.Macrophages are the main components of natural immune cells in the tumor microenvironment.The number ratio and functional phenotype of macrophages play an important role in different stages of tumor development.However,due to the high heterogeneity of both tumor cells and macrophages,their in-depth study needs the help of single-cell analysis technology.Currently,single-cell protein analysis techniques mainly include intracellular staining-based(such as fluorescence flow cytometry,mass spectrometry flow cytometry)and microfluidic-based microengraving and single-cell barcode technology.Secretory protein analysis is the detection of proteins secreted into the extracellular environment,which truly reflects the immune response of cells after affecting the microenvironment,and the dynamic detection of extracellular secretion can track continuous immune changes.But at present,these techniques are all based on a two-dimensional culture model.Tumor is a complex three-dimensional(3D)microenvironment.In addition to tumor cells,there are many immune cells and matrix effects.Therefore,how to build a 3D tumor microenvironment and study highly heterogeneous tumor immune cells at the single-cell level is an unsolved problem.The complex 3D microenvironment of the tumor establishes dynamic interaction with surrounding tissues through complex chemical signals.At present,tumor three-dimensional culture based on microfluidic chip has the advantages of low sample cost,high flux,and high integration level.It is widely used in tumor three-dimensional microsphere formation,culture,and tumor research.However,the development of simple and easy to control high-throughput tumor microsphere culture method is still the research focus.Based on the above background,we have carried out the following work:1.Based on microfluidic technology,a simple and high-throughput method of 3D tumor microsphere spheroidization was developed,and a long-term culture was carried out in this chip.With PDMS as the material,we made 10800 microporous array chip,and cultured tumor cells in the medium containing surfactant F68,which can promote tumor spheroidization.We investigated the effects of F68 concentration on the proliferation and protein secretion of human breast cancer(MCF-7),human oral squamous cell carcinoma(SCC25),primary oral squamous cell carcinoma(OSCC)cells.It was found that the F68 concentration of 0,04%had no significant effect on cell proliferation and protein secretion.On this basis,we analyzed the relationship between the number of tumor cells and the area of tumor microspheres and the growth of microspheres for a long time(12 days,with good cell viability)under 0.04%F68 culture conditions.The results show that we have successfully developed a simple and easy-to-use high-throughput 3D tumor microsphere method,which can be used for the long-term culture of 3D tumor microsphere with good cell activity.2.Under the condition of SCC25 tumor microspheres,the dynamic immune response of macrophages differentiated from human tissue lymphoma cells(U937)was investigated.Under the condition of the co-culture of the SCC25 tumor microsphere and macrophage for 12 hours,we found that the SCC25 tumor microsphere inhibited TNF-a and IL-6 secretion of single U937 differentiated macrophage,but the change of IL-8 secretion was not significant.The trend of three secretory proteins of multiple macrophages is consistent with that of single macrophages.In two successive tests,it was found that SCC25 microspheres further inhibited the secretion of TNF-a and IL-6 under the stimulation of LPS.These results indicate that the three-dimensional tumor microenvironment suppresses the immune response of macrophages.