Study Of Nano-caged Material Synergistic With Matrigel For Capturing And In Situ Culture Tumor Cells

Circulating tumor cells（CTCs）refer to tumor cells shed from primary tumor lesions and enter the peripheral blood circulation system,which is an essential condition for distant metastasis of cancer.A circulating tumor cell clusters（CTC cluster）refer to a cell cluster composed of≥2 CTCs with higher cancer metastasis potential.Molecular information such as genes and proteomes of CTCs and CTC clusters in the blood can reflect the degree of tumor burden in patients,which is helpful for the diagnosis,treatment,and prognosis of cancer,and is of great significance for personalized treatment.However,because the content of CTCs and CTC clusters in peripheral blood is extremely rare and cannot meet the quantitative requirements of downstream molecular analysis,after separation and enrichment of CTCs in blood,it is necessary to further expand the number of cells in vitro culture technology.There are two existing in vitro expansion technologies for CTCs.One is to release the captured CTCs from in situ based on enzymatic degradation and then culture them in vitro,but it will cause cell damage,resulting in a low success rate of culture.The other is in situ culture after capture.The current culture environment is mainly the liquid medium,which is very different from the in-vivo tumor survival environment.To realize the non-destructive and efficient expansion and cultivation of CTCs in vitro,this paper developed a new technology for CTCs capture/cultivation integration,using the nanocage materials developed by the research group in the early stage to efficiently capture single tumor cells and cell spheroids,and cooperate with the matrix.The glue realizes the three-dimensional in situ culture of the captured cells.The specific studies are as follows:1)Preparation of porous materials imitating natural nanocage structures with flexible structures.The template was prepared by the particle stacking method.After filling the Ti O₂ gel into the template gap,the template particles were removed by high-temperature calcination.Finally,an artificial nanocage structure with a surface pore size of 385 nm and a lower through-hole pore size of 110 nm was prepared,which solved the problem of the difficult-to-control nanocage structure of the pollen.2)Capture and in situ two-dimensional culture of single tumor cells by nanocage materials.A pollen-derived natural nanocage material was used to capture single tumor cells efficiently,and the captured cells were cultured in situ,and the effect of medium types on cell expansion in vitro was studied.The results show that,compared with the liquid medium,Matrigel can provide three-dimensional support space for cell culture.It is conducive to the rapid proliferation of cells on the surface of the nanocage material,thereby realizing the in situ two-dimensional culture of single tumor cells.3)The capture and in the situ three-dimensional culture of CTC cluster-like cell spheres by nano-cage materials.Cell spheroids were prepared by a hanging drop method.The results showed that when the volume of hanging drop was 20μL and the cell density was 5×10³cells/m L,cell spheroids with diameters similar to in vivo CTC cluster（～150μm）could be prepared.Further research showed that the capture rate of the nanocage material for the imitation CTC cluster cell spheroids was more than 80%.The captured cell spheroids were cultured in situ in cooperation with Matrigel,and the cell spheroids would gradually migrate into Matrigel,realizing the original function of the cell spheroids.three-dimensional culture.This paper constructs a new method integrating efficient capture of CTCs and in vitro amplification,which achieves a high capture efficiency without damage to cells.It can realize in situ three-dimensional culture of captured cell spheroids,and can be used for downstream CTCs gene and proteome analysis,which contributes to the diagnosis,treatment and prognosis monitoring of cancer.