Quantitative Study Of The Critical Point For Tumor Spheroids Invasion

The lethality of cancer is that it has the strong metastasis/invasion.Tumor from proliferation to metastasis/invasion is a mutational process,therefore it is crucial to find the key factors when tumors maintain quasi-steady state and begin to mutate.To understand the mechanism of cancer metastasis more clearly,gene sequencing,gene knockout,fluorescent protein mark,protein expression,etc.,have studied by researchers.An increasing number of animal models and cells in three-dimensional(3D)in vitro culture are used as research methods,multi-cellular tumor spheroid(MCTS)are extensivly used in in vitro tumor studies compared to traditional two-dimensional(2D)cultures.From the perspective of morphology,spheroids will have many branches on the edge of invading,which will release many abnormal signals.To accurately capture these abnormal signals in a short period of time,we used three different spheroid models in experiment,used a self-made living cell system to track status of spheroids over a long time,and established automated quantitative analysis method for spheroid morphology parameters.We applied this approach to several tumor spheroid models,and demonstrated its capability as excellent references to evaluate the proliferation-to-invasion transition time and to compare invasion potential of tumor cell.This enable our quantitative approach has potential application to the field of cancer metastasis investigations and clinic diagnostics.The main works are as follows:(1)Home-built of living cell system:A living cell system for in vitro imaging of tumor cells was constructed,it includes two parts,incubator and the control system.The incubator is used to provide a favorable environment for cell growth,such as uniform temperature,a range of humidity,stable carbon dioxide concentration,etc.the control system is used for temperature control of the incubator lid,bottom,water bath heating,and objective lens warming,as well as carbon dioxide concentration control.This living system is low cost,temperature uniform,and provides a good growth microenvironment for the spheroids.(2)Spheroid formation and invasion experiments of U87 cells,H1299 cells,and MDA-MB-231 cells:Multicellular tumor spheroids were used to mimic organ type models of normal and solid tumor tissues.Traditional techniques of spheroids formation methods,for example,lab on a chip method,spinner flasks,micro-patterned plates,ultra-low attachment plates,hanging drop method,etc.Each method has its own advantages and disadvantages.In this study,an ultra-low attachment plate method was used for the spheroid formation experiment.The 1.0×10~4 cells/mL of cell density was added into a 96-well ultra-low attachment round bottom plate.Next,it was placed in an incubator for 4 days.After 4 days,the compact and uniform spheroids formed.Subsequently,spheroids were transferred from the 96-well ultra-low attachment round bottom plate to the 96-well ultra-low attachment flat bottom plate,adding the culture medium containing 2.0 mg/mL collagen in invasion experiment.(3)Morphological multi-parameter quantitative analysis of spheroids:The key of tumor lethality is its metastasis,which is the critical point of tumor to cancer transition.In this study,we innovatively use the critical behavior of steady state transition(or phase transition)in physics to develop a quantitative standard for the invasion of collective cancer cells,and to study its triggering conditions and mechanisms.We established four morphological parameters to quantify the critical point of invasion of spheroids,and to find a way to inhibit tumor metastasis.This method can provide the new approaches and solutions for the diagnosis and treatment of cancer/cancer,reducing or delaying the death of cancer patients.