The Correlated Mechanisms Studies Of Cancer Invasion And Metastasis Based On Microfabrication

Cancer is a fatal disease.The most important cause of its fatality is the metastasis of cancer cells.The growth of this tumor or metastasis at a fatal site leads to life-threatening crisis.Therefore,research on cancer treatment is a major concern for humans.With the rapid development of various disciplines and the integration of each other,each discipline is no longer a separate existence,but through the collision to produce a different spark.And the cancer research has no longer been limited to medical scientists or biologists,on cancer research model is no longer limited to live experiments such as the model of mice.The experimental model of cancer treatment in vitro has gradually entered the stage of history.Since then,it has become an important tool for cancer research.Even in the near recent years,it even leads the research boom of "organ on a chip".The diameter of a single cancer cell is about 20 ?m.This meso-scale determines that the in vitro model of cancer research can not be separated from the micro-processing technology.A variety of sophisticated micro-machining technologies have also emerged,such as lithography,soft etching,and light curing technology,chemical solid technology,microfluidics,etc.With the continuous development of microfabrication technology,a variety of in vitro models of cancer research have also been published,and a large number of achievements have been made on cancer research.The detail is elaborated in Chapter 2.Especially for the construction of in vitro three-dimensional extracellular matrix(ECM)models provide a pivotal role.The cancer cells in the living body are in the three-dimensional matrix environment,so the traditional two-dimensional surface culture model is far from meeting the needs of people to study cancer,and the quasi-three-dimensional and three-dimensional in vitro culture models of cancer cells are developed following the rapid development of microfibracation technology.The largest difference between quasi-three-dimensional/three-dimensional in vitro culture models and two-dimensional in vitro culture models is the existent of extracellular matrix(ECM).In the two-dimensional culture system,there is no ECM,but the cells are directly adhered to the surface of the dish,and then the medium is added.For example,the cells are cultured in petri dishes,while the quasi three-dimensional or three-dimensional culture system not only provide cells with the necessary medium for their growth and proliferation,but also ECM is provided for surrounding them.The quasi three-dimensional or three-dimensional culture environment is even better because of the similarity to the real micro-environment in the living body.After carefully survey the real micro-environment of cancer cells in vivo and the current quasi-three-dimensional and three-dimensional models concerning cancer research in vitro,this project established two distinctive models of cancer research.The quasi three-dimensional model described in Chapter Three.And the fourth chapter describes three-dimensional research models.Using the two research models as the tools,the study focused on the mechanism of invasion and metastasis of cancer cells.It has been found that the invasive front of the collective cancer cells has a variety of morphologies.The reasons for the formation of these frontal morphologies are still unclear.In order to answer this question,the quasi-three-dimensional in vitro model is established with a series of geometric space constraints and different hardness of ECM micro-environments(see Chapter 3 for the detail of the process).It was used to investigate the relationship between the morphology of the invasion front and the two factors in the invasion process of the collective cancer cells.For the diversity reports on the invasion front of cancer cell populations,there is no quantitative definition for various topographies.This topic identifies four topographies in geometric form when recognizing and judging the various front topographies of stream,blunt tip,cone tip and lumen shape with giving a clear definition.Through the statistical work of clinical slices,the experimental verification of quasi-three-dimensional models in vitro,and the calculation and verification of theoretical models,it was found that the invasion behavior of cancer cells against ECM was inextricably linked with the geometric constraint and the ECM hardness.And based on experimental data,the phase diagram of the invasive frontal morphology of cancer cell population with geometric space and ECM hardness changes was obtained.The change pattern of this phase diagram has a high degree of consistency with the trend of changes in the statistical results of clinical slices,that is,when the geometric space is extremely narrow,the cancer cell population invades with a streamlined front due to the limitations of space;When the geometric space is extremely large,with the ECM hardness is large,the cancer cell population mostly invades outwards with a cone shape.In the middle condition between the above,the invasive front of the cancer cell group is mostly blunt tip.This result has also been more consistently verified by the theoretical model.Three-dimensional model is an important system for cancer research.It has become an ideal cancer research model based on the high similarity between the ECM in the model and the ECM microenvironment in vivo.This project has designed a three-dimensional cancer research model that can provide complex nutrition or drug gradients.The central of the three-dimensional model is an ECM made by mixing collagen and matrix(Matrigel)with a thickness of 100?m.The thickness is suitable for the optimal observation range of various microscopic instruments;the micro-chamber array is arranged in the ECM.The small chamber can be used to culture cells.The shape of the small chamber can be set according to the needs;four sides of the ECM are four independent injection channels,that is,four different types of nutrition or drug can be injected through four independent injection channels and then enters the ECM through diffusion,producing different concentration gradients in different regions of the ECM.The three-dimensional cancer research model can be used to study the invasion of ECM by cancer cells,the invasion of tissue structures by cancer cells,the intercellular game,the research of ECM orientation,drug screening,and the like.For the study of cancer,a three-dimensional model with strong plasticity and great application potential was constructed.In this study,the three-dimensional model was used to study the orientation of ECM,the invasion of cancer cells to ECM,and the invasion of mammary ducts by breast cancer cells.It was found that the distribution and orientation of collagen fibers in ECM were affected by the fiber density and strain.It was found that in the early stage of invasion of ECM,the cancer cells firstly protruded out of the highly deformed antennae,and the orientation of the antennae was surprisingly consistent with the collagen fiber.It was initially judged that collagen fibers had a guiding effect on the invasive behavior of cancer cells to the ECM.The process of breast cancer cells breaking through the mammary duct was reemerged,and it was found that the cancer cells first deformed a large number of filaments when they invaded the duct.The filaments first thread the duct into the ECM,after which the main part of the cancer cell body invades along the gap where the filament structure is located,destroying the duct structure.Several follow-up designs were performed on this three-dimensional cancer model,including putting spheroid in the ECM chamber,which more realistically simulates the process of cancer cells invading from the location to ECM,finding the critical point of invasion and metastasis.The model can be used to find out the conditions for inhibiting the production of the qualitative change point,to control the cancer state effectively.Another example is that based on a high-intensity magnetic field is known can change the orientation of the collagen fibers,different external magnetic fields are designed to purposefully control the orientation of the collagen fibers in the ECM to create a specific three-dimensional model.