Construction Of 3D Cancer Microenvironment And Study Of Cell-Microenvironment Interaction

Accumulating evidence has shown that the three-dimensional(3D)cell microenvironment cooperates with genetic codes to determine cancer progression including tumorigenesis and metastasis.However,current researches are mostly based on two-dimensional(2D)cancer models,which do not recapitulate the 3D cancer microenvironment in vivo such as cell-cell and cell-ECM interaction.Thus it is important to develop convenient technologies capable of engineering 3D models for cancer study.Additionally,similar to genetic profile,cancer cell microenvironment is dynamically evolving during cancer progression,such as the stiffening of the matrix.However,how cancer cell remodels its surroundings and promotes progression remain largely unknown.To solve these problems,with advanced micro/nano manufacturing technology,we propose a variety of methods to construct 3D cancer microenvironment in a convenient manner.And on this basis,we quantitatively investigate the remodeling process of cancer cells to their 3D microenvironment,and its underlying mechanism as well as implications.First,to recapitulate the cell-cell interaction in vitro,we develop a hanging drop cell culture plate to fabricate 3D cancer multicellular spheroid.The hanging drop cell culture plate is designed with units that are able to control the surface tension between droplet and plate,which significantly improves the droplet formation efficiency and increases droplet volume.The designed plate is easy to use for the user from the field thus holds great potential to be widely used for 3D multicellular spheroid culture.In addition,in order to avoid the tedious process in preparing hanging droplets,we propose a biomimetic structure that is capable of high-throughput and rapid generation of hanging droplets.Theoretical and experimental studies uncover the fundamental principles and structural effects in the droplet generation process,which is useful for designing next-generation 3D cell culture plates.Second,to recapitulate the cell-ECM interaction in vitro,we use highly biocompatible hydrogel to mimic ECM,and propose a series of approaches for constructing the cellular microenvironment based on cell encapsulation.We develop the Biopen for rapid fabrication of 3D cancer model.Because of its simplicity,flexibility and low cost in building cellular microenvironment,it has great potential to promote utilizing 3D cell cultures in cancer research.In addition,taking advantage of the electrostatic interaction between positive and negative charges,we assemble the charged microgels into complex 3D structures.Simulation study reveals the self-assembly process tends to minimize the total free surface,which is useful for controlling final assembled construct by designing the shape of microgels.Experimental results show the charged hydrogels have good biocompatibility and are capable of building highly complex 3D spherical structures,holding great promise to construct physiologically-relevant microenvironment.Third,based on the 3D cancer model,we,for the first time,quantitatively measure the local stiffness of collagen matrix around a single cancer cell and find that the stiffness of collagen around the cell(MDA-MB-231 cell line)increases 100 times compared with the collagen that are far away(above 100 ?m)from the cell.Further,we find that the stiffening effect is mainly due to the strong contraction force applied by cancer cell,and the collagen fiber becomes stiffer after being streched by the cell;therefore the stress field applied by the cell is able to be calculated according to the extent of collagen being stiffened.We find that the stress decreases with distance to the cell and follows a power-law form,? ~ d-2.Finally,the buckling of collagen fibers near the cell is observed by real time imaging,and the curvatures of the fiber are analyzed as a function of distance.These findings uncover the fact that there exits a strong stiffness gradient around a MDA-MB-231 cancer cell in 3D collagen matrix,which may provide a mechanically communicating approach by attracting other cells using stiffness gradient.Finally,we examine the effect of stiffened microenvironment on the growth of cancer cell.We find that the stiffness of the matrix governs the phenotype transition of tumor growth.Particularly,stiffened microenvironment turns the benign MCF10 A cells into a malignant phenotype,characterized by an uncontrolled proliferation and invasive manner.Further,we,for the first time,study this transition process from a physical point of view and reveal the cell volume distribution in the spheroid during cancer growth.Real-time cell tracking results show that a few cells in the spheroid migrate both from the center to the boundary and in the opposite direction,and their volume changes obviously during the migration process.In summary,we present a variety of new methods that are capable of rapidly and accurately engineering 3D cancer microenvironment,providing useful tools for tumor biology and drug screening studies.And based on 3D cancer models we investigate the interaction between cancer cells and extracellular matrix from a mechanical point of view,uncovering how cancer cell mechanically remodels its surroundings and the cell volume distribution during cancer growth.This study provides a new perspective for understanding cancer disease,which is promising for developing new strategies in prevention and treatment of cancer.