Construction Of Microenvironment With Different Dimensions And Controlled Stiffness For Analysis Of Tumor Cell Behaviors

Over the past decades,increasing studies have demonstrated that most adherent cells are mechanosensitive,suggesting that the mechanistic properties of the microenvironment have profound impacts on the behavior of the vast majority of cells.Exploring the molecular mechanisms behind mechanistic messaging in cancer cells can provide mechanobiological insights into the development of disease,which in turn can lead to the development of more targeted therapeutic approaches and provide an important foundation for cancer prevention,detection and treatment.The mechanistic properties of tumor tissues are unique,so the simulation of in-vivo physiological properties is crucial for the accuracy of in vitro studies.However,the tumor microenvironment is complex and variable with a wide range of mechanical properties,so the development of tumor bionic platforms with different dimensions to well simulate the mechanical microenvironment of different tumor is a hot topic.In this paper,we use microfluidics combined with hydrogel materials with high biocompatibility and stable mechanical properties to simulate tumor mechanical microenvironments in different dimensions,thus conducting a series of studies on important tumor cell behaviors.The main studies are as follows.1.A two-dimensional biomimetic multi-factor stimulation platform was developed and fabricated,which can easily create a two-dimensional matrix with controllable stiffness and can be easily introduced with biochemical factor stimulation by adding exogenous substances.The results show that extracellular matrix(ECM)stiffness enhances cell stretching and further strengthens cell-matrix adhesion to promote cell proliferation.The response of cellular endocytosis efficiency to matrix stiffness also differed significantly:nanoparticles of the same size and shape,but with different charges,are endocytosed more on harder substrates.In addition,inorganic polyphosphates(polyP),which act as energy stores and producers in the extracellular space,were shown for the first time to synergistically promote cell stretching,adhesion,proliferation and endocytosis behavior by increasing ATP metabolism.These results explore the impact of microenvironmental properties on the mechanistic response of glioma and provide strong evidence that this bionic multifactor stimulation approach can provide valuable inspiration for in vitro bionic cell culture and nanoparticle-like drug phagocytosis studies.2.Based on the successful construction of a two-dimensional platform,a method to prepare three-dimensional gel microspheres with widely adjustable stiffness was developed,which provides an important basis for deep analysis of tumor metastasis.By mixing biodegradable polylactic acid(PLA)nanofibers with modified alginate with different concentrations of Ca2+,the range of stiffness of the microgels was significantly increased while maintaining the pore size,which could effectively mimic the tumor microenvironment.The results showed that the Young’s modulus of the modified alginate increased more than threefold when 2.0 w/v%PLA short nanofibers were added,and the prepared microgels were biocompatible.The cell behavior of two typical breast cancer cell lines,MCF-7 and SUM-159,was further analyzed by encapsulating the cells in three-dimensional microgels that simulated the mechanical properties of breast tumors.MCF-7 and SUM-159 cells in soft microgel spheres exhibited strong migration ability in addition to maintaining high survival and proliferation rates,and RT-qPCR analysis revealed potential changes at the genetic level.The results of the systematic study suggest that the method can effectively create a broadly adjustable three-dimensional mechanistic microenvironment,and the results of the cell behavior analysis indicate that the method has promising applications in tumorigenesis and progression.3.Based on the aforementioned study of 3D matrix stiffness microgel model,an innovative strategy to mechanically differentiate different regions of the 3D microenvironment is further devleoped.We have used modified alginate hydrogels with concentration gradients combined with microfluidic methods to fabricate core-shell and triple core-shell structures with stiffness differences,which achieve layer-by-layer increase or decrease of mechanical stiffness in a microscale three-dimensional space with high throughput and high homogeneity.For biological applications,we encapsulated MCF-7 breast cancer cells in different regions of the constructed soft-core,hard-shell and hard-core,soft-shell hydrogel microspheres and observed high cell viability within 10 days.This three-dimensional mimicry with local mechanical microenvironmental differences was demonstrated to be an ideal model for studying the behavior of non-uniform mechanical cues on their internal cellular correlations.It provides a basis for subsequent in vitro simulations of non-uniform stiffness tissues.