The Effect Of Stromal Cells On Tumor Cells Invasion And Secretion Of Extracellular Vesicles On Microfluidic Chips

Objectives: We aimed to construct a microfluidic system,integrating threedimensional(3D)co-culture of tumor and stromal cells,and on-line capture and detection of extracellular vesicles(EVs)secreted by tumor cells.In addition,we want to know whether stromal cells have an impact on the secretion of EVs by tumor cells.Materials and methods: Salivary adenoid cystic carcinoma cell lines,SACC-83 and SACC-LM,were used in this study.Carcinoma-associated fibroblasts(CAF)were obtained by isolating stromal cells from SACC tumor tissues.Cells were cultured with EVs-free-serum medium to collect condition medium(CM)after 48 hours incubation.After removing the cell debris,apoptotic bodies and large vesicles by differential centrifugation,the supernatant was retained.Chip templates were prepared by SU8 on a wafer and patterned by photoresist technology,and the microfluidic chips were made of Polydimethylsiloxane(PDMS)and slide.Firstly,we characterized the EVs capture and detection chip.After the treatment of chip channel’s surface by CD63 antibody,CM of SACC was added into the chip.According to the principle of antigen-antibody specific binding,EVs could be recognized and captured by CD63 antibody.We further scanned the EVs sample captured in the microfluidic chip by Atomic Force Microscope.Then the CM was divided into 4 concentration gradients(stock solution,1:2 diluent solution,1:4 diluent solution and 1:8 diluent solution)and added into the chip to capture EVs.The captured EVs were labeled and detected by detection antibodies based on immunofluorescence method.At the same time,EXOCET standard kit was used to detect the amount of EVs in corresponding CM.The amount of EVs in CM detected by the chip and EXOCET kit were compared to verify the feasibility of chip.Then we constructed a perfusion 3D cell co-culture model based on microfluidics technology.This chip consists of 5 channels(C1 to C5)separated by micropillars.C1 and C5 are the medium perfusion channels,C2 and C4 are 3D cell culture channels,and C3 is the matrix channel.The 3D co-culture chip was connected with a syringe pump and EVs capture and detection chip by microtubes.Cells were continuously cultured at the perfusion speed of 0.2 μL/min for2 or 4 days.After the perfusion culture,cells were stained with Hoechst33342,Rh-123 and PI to assess the cellular viability and invasion.Finally,we detected the tumor cell-derived EVs on-line by CD63 and MMP2 antibody.Results: We constructed an EV capture and detection chip based on microfluidics: AFM scanning images showed that the captured EVs’ size was mostly at the range of 30 nm to 200 nm which Conforms to the currently accepted range of exosomes.After immunofluorescence labeling,a large number of EVs could be observed on the chip.The fluorescence intensity of EVs captured on the chip was positively correlated with the EVs concentration in CM.Secondly,we constructed a3 D cell co-culture chip based on microfluidics.After perfusion culture for 2 days or 4days,almost all the cells were viable and aggregated into clusters.The invasion area of SACC cells is greater when they co-cultured with CAF.This indicates that CAF cells have the effect of promoting SACC invasion.Then we integrated the EV capture and detection chip with the 3D cell co-culture chip.Using the microfluidic system,it was found that the amount of EVs was positively correlated with culture time and CAF cells could promote SACC cells to secrete more EVs.Conclusion:In this study,we constructed a microfluidic system by integrating a 3D cell co-culture chip with an EVs capture and detection chip.This system allowed to co-culture tumor and stromal cells and further detect EV secretion online by tumor cells.Using this system,we demonstrated that stromal cells significantly promoted the invasion and EVs secretion of tumor cells.