A Lung Cancer Model In Vitro Was Constructed Based On 3D Bioprinting

IntroductionLung cancer is now the leading cause of cancer incidence and the second leading cause of death worldwide.Although the treatment methods and means of lung cancer have made great progress,the recurrence rate and mortality rate are still high.The lack of an effective in vitro model leads to limited progress in lung cancer research.Most research models of lung cancer are 2D cell line lung cancer models or animal models of patient-derived xenografts.The 2D cell line model is widely used in experimental research because of its low cost,simplicity,and rapidity.However,the 2D cell line model can only represent a subgroup of tumors,and in the long-term 2D growth and proliferation process in vitro,tumor cells lack the environment for growth in vivo,which makes it difficult to maintain some biological functions of the original tumor in vivo.Compared with the 2D cell line model,animal models of patient-derived xenografts are closer to the real human tumor growth microenvironment.However,the high cost and low time cost efficiency of animal models of patient-derived xenografts also limit its application.Because of the above,this study intends to explore the construction of a 3D in vitro model of lung cancer through 3D bioprinting technology and pay attention to the growth microenvironment of tumor cells in addition to tumor cells.The 3D lung cancer model constructed in vitro is more suitable for the research and development and screening of anti-tumor drugs and provides a better verification platform for the personalized treatment of cancer patients.Part Ⅰ:Research of lung cancer model in Vitro based on 3D bioprinting technique and RNA-sequencing technique Objective:To construct an in vitro model of lung cancer by 3D bioprinting and evaluate the feasibility of the model.Transcriptome sequencing was used to compare the differential genes and functions of 2D and 3D lung cancer cells.Methods:1.A549 cells from a non-small cell lung cancer cell line were mixed with sodium alginate/gelatin/fibrinogen as a 3D-printed bio-ink to construct a hydrogel scaffold for in vitro lung cancer model.Ca Cl2/ TGase/thrombin was used as a crosslinking agent.Among them,Ca Cl2 is used for crosslinking sodium alginate,transglutaminase for crosslinking gelatin,and thrombin for crosslinking fibrinogen.2.3D printing bioprinting in this experiment was carried out with multi-nozzle 3D bioprinting.The 3D bio-printer was loaded with cellular alginate sodium/gelatin/fibrinogen bio-ink containing A549 cells,and a 50×50mm crisscrossed grid hydrogel scaffold was printed by extrusion printer after the predetermined printing route was set by the computer.The scaffold is printed on a pre-cooled platform,and the thermosensitive material produced by the 3D bio-printer will undergo a simple molding on the platform with low contact temperature.At this time,the hydrogel scaffold is not strong,and then through the Ca Cl2/ TGase/ thrombin cross-linked hydrogel scaffold,to form a stable tumor model of A549 cells in 3D space with multiple pores.3.Conduct a follow-up study on the in vitro tumor model after printing and crosslinking,and observe the 3D in vitro lung cancer tumor model with a microscope and electron microscope.The biological evaluation was carried out on the tumor model of lung cancer in vitro.Live/dead reagents were used to detect the live/dead rate of A549 cells in the printed model.Alamar Blue was used to measure the growth capacity of lung cancer models in vitro.4.A549 cells cultured in 2D and 3D tumor models in vitro were collected and bioinformatics analysis was conducted by RNA-seq.conclusion :In this part of the study,the continuous growth of lung cancer in vitro tumor model was constructed by using 3D bioprinting technology.Through observation,the in vitro model is closer to the real human tumor growth environment than the 2D model.The survival rate of A549 in the 3D lung cancer model was not significantly different from that in the 2D group.The proliferation rate of A549 in the lung cancer model in vitro did not decrease significantly with the change in a growth environment.RNA-seq results showed that the gene expression levels of A549 cells cultured in the two environments were different in the 2D and 3D lung cancer models,and they had their gene expression profiles.Enrichment analysis showed that differential genes were related to cell cycle,NF-KAPPA-B,PI3K-AKT,and other signaling pathways.Demonstrated that a 3D growth environment promoted the development of lung cancer cells.3D bioprinting may provide a new research platform for studying the mechanism of lung cancer tumor microenvironment and anti-cancer drug screening.Part Ⅱ: Construction of in vitro model of lung cancer by coaxial 3D bioprintingObjective: To construct a shell/core lung cancer model in vitro using coaxial 3D bioprinting technology,evaluate the feasibility of this model for the culture of A549 cells,and compare the differences in biological function and drug resistance of A549 cells in 2D and coaxial 3D models,to provide a basis for subsequent studies on cell-cell interaction.Methods: 1.Shell/core hydrogel was constructed by a 3D bio-printer to construct a lung cancer model in vitro.Two separate channels could be printed by using a special coaxial printing needle.At the initial stage of printing,after the two-channel hydrogels contact together,Ca2+ in the core channel and sodium alginate in the shell channel immediately cross-linked,forming a cross-linked wall between the two channels,and the cells in the core can freely pass through the inner axis.After the printing,Ca Cl2 was used to crosslink the hydrogel as a whole to make the whole in vitro lung cancer model stable and firm,and the shell/core A549 hydrogel scaffold was completed.2.Optical microscopy,fluorescence microscopy,electron microscopy,and HE staining were used to observe the lung cancer body model;live/dead reagents were used to detect the overall cell survival rate after printing;Alamar Blue was used to detect the overall growth capacity after printing and the drug resistance after cisplatin treatment.Cell scratch assay and Transwell assay were used to detect the invasion and migration ability of lung cancer models in vitro.Results: 1.Coaxial 3D bioprinting could construct a shell/core lung cancer model in vitro.In this model,cells can grow freely in the 3D environment of the channel in the core,and form a porous mesh structure in the shell,which can exchange nutrients and release metabolites with the external medium.2.The survival rate of A549 cells in the coaxial 3D group and 2D group was 86.6±2.3% and 91.77±3.2% respectively on the 10 th day of culture.There was no significant difference between the two groups,P> 0.05,and no statistical difference.3.On the day after coaxial printing of the in vitro model,cells were scattered individually in the hydrogel axis.On day 3,the cells appeared to assemble autonomously.By day 10,the shell-core hydrogel scaffolds were still intact,and cells filled the entire inner axis.Alamar Blue measured the relative proliferation rate of the 2D group and the 3D group on day 7.Compared with the cells on day 1,the cells in the 3D and 2D groups increased by 2.34 times and 2.13 times on day 7,P<0.05,with a statistical difference.4.A549 cells in coaxial 3D printing lung cancer models were more resistant to drug treatment.After being treated with 32ug/ml DDP for 48 h,the relative survival rate of the 2D group was 7.92±3.2%,and that of the 3D group was 19.54±2.7%,P< 0.05,with a statistical difference.5.In the scratch experiment,the relative migration distance of the 3D group and the 2D group was 0.374 and 0.217 respectively,P<0.05,with a statistical difference;In the Transwell migration experiment,1104 cells were migrated in the 3D group,and 524 in the 2D group,P< 0.05,with a statistical difference;In the Transwell invasion assay,942 cells penetrated the cells in the 3D group and 349 in the 2D group,P<0.05,with a statistical difference.Conclusion: The shell/core hydrogel scaffolds developed by coaxial 3D bio-printing in this part of the study are an excellent and stable model of lung cancer in vitro,and the survival rate and proliferation ability of A549 cells cultured under this model are good.Compared with the 2D group,the activity,and proliferation of A549 cells in shell/core A549 hydrogel scaffolds were not significantly different.Compared with the 2D group, A549 cells in the coaxial 3D group had stronger invasion and migration ability,and stronger drug resistance.At the same time,the in vitro model of lung cancer produced by coaxial 3D bioprinting may be used as a new drug screening platform.Part Ⅲ: Coaxial 3D bioprinting studies the interaction between lung cancer cells and mesenchymal stem cellsObjective: In this study,a shell MSC/ core A549 model of lung cancer in vitro was constructed using coaxial 3D bioprinting technology to simulate the microenvironment of lung cancer in vivo,to study the durability of A549 cells in 3D lung cancer in vitro model,and to understand the mechanism of the interaction between A549 and MSC in vitro lung cancer model.Method: 1.Shell MSC/ core A549 hydrogel scaffold was constructed by coaxial 3D bioprinting technology using gelatin/sodium alginate as raw materials.2.A549 cells in 2D-A549,shell/core A549,and shell MSC/ core A549 models were detected by q PCR to detect the relative expression levels of CD44,CD133,SOX2,and NANOG related to the stemness of A549 cells,and N-Cadherin related to EMT.The relative expression of E-Cadherin,fibronectin,Twist1,and Snail1,and the relative expression of MMP2 and MMP9 associated with cell invasion.3.In vitro tumor models of lung cancer were cultured using 2D-A549 and 3DMSC co-cultured Conditioned medium(CM1)and A549/MSC conditioned medium(CM2)respectively,A549 cells were collected and evaluated for tumorigenicity using q PCR.4.The expression levels of IL-10,TGF-β1 and CXCL12 classical tumor enhancers in 2D-MSC,3D-MSC and shell MSC/ core A549 were detected by q PCR.5.The tumorigenic ability of A549 cells in vitro lung cancer model was verified by the xenograft tumor model in nude mice.Result: 1.Coaxial printing can construct a shell MSC/ core A549 in vitro lung cancer model,which can be used as a new platform to study cell-cell interaction.2.The cell dryness of A549 in the shell MSC/ core A549 model was detected by q PCR.The results showed that the relative expressions of CD44,CD133,SOX2,and NANOG of A549 cells in the shell/core A549 model on day 15 were 4.8,3.8,3.1,and 14.7 times,respectively,compared with 2D-A549 group on day 5.On day 15,the relative expressions of CD44,CD133,SOX2,and NANOG in A549 cells were 5.1,5.3,3.4,and 18.7 times in the shell MSC/ core A549 model,respectively.All the above P< 0.05,with a statistical difference.3.q PCR was used to detect EMT markers of A549 in shell MSC/ core A549 model printed coaxial.The results showed that compared with the 2D-A549 group on day 5,the relative expression of N-cadherin,E-cadherin,and Fibronectin in shell/core A549 cells on day 15 was 4.8 times,0.23 times,5.5 times,respectively.0.05,with statistical difference;The relative expression of Snail1 and Twist1 did not change significantly.On day 15,the relative expressions of N-cadherin,E-cadherin,Fibronectin,Snail1,and Twist1 of A549 cells in shell MSC/ core A549 model were 5.1,0.20,6.2,4.9 and 5.8,respectively.P<0.05,with a statistical difference.4.Markers of migration ability of A549 cells in shell MSC/ core A549 model were detected by q PCR.The results showed that compared with the 2D-A549 group on day 5,the relative expression of MMP2 and MMP9 in shell/core A549 on day 15 was 3.1 and 2.7 times,respectively.The relative expression of MMP2 and MMP9 in shell MSC/ core A549 on day 15 was 5.7 and 5.8 times,respectively.All the above P<0.05,with a statistical difference.5.Shell/core A549 cells were treated with the conditioned medium,and it was found that the tumorigenic ability of shell/core A549 cells was enhanced.Compared with 2D-A549,the relative expressions of MMP9,CD133,and NANOG of MSC/ core A549 without conditioned medium treatment were 10.2,3.21 and 4.45 times,and 13.3,3.9,and 10.8 times,respectively,after CM1 treatment.The relative expressions of CM2 were 13.6,4.5,and 11.6 times,respectively.All the above P< 0.05,with a statistical difference.6.The expression of classical tumor enhancer IL-10,TGF-β1 and CXCL12 of MSC in 3D model was detected by q PCR.The results showed that the relative expression of IL-10,TGF-β1,and CXCL12 in 2D-MSC was 7.5,3.3,and 3.5 times,respectively,compared with that in 2D-MSC.The relative expressions of IL-10,TGF-β1,and CXCL12 in shell MSC/ core A549 were 7.96,3,and 4.3 times,respectively.All the above P< 0.05,with a statistical difference.7.Shell MSC/ core A549 and shell/core A549 cells in shell/core A549 models showed stronger tumor-causing ability compared with 2D.On day 24,the tumor volume of the 2D group was 255.3 mm3,that of the shell/core-A549 group was 585.4 mm3,and that of the shell MSC/ core-A549 group was 661.071 mm3.P<0.05,with a statistical difference.Conclusion: The in vitro lung cancer model using coaxial 3D bioprinting as a research platform confirmed that the interaction between lung cancer cells and MSC cells has stronger tumorigenicity.A549 interacts with MSC to produce tumor enhancers secreted in the medium,thus enhancing the tumor-causing ability of lung cancer.The nude mouse transplanted tumor model also verified that A549 cells were more tumorigenic after coculture,providing a valuable research platform for future in vitro tumor microenvironment models and the mechanism of cell-cell interaction.