Hyaluronan Enrichment And Mechanically Matched GelMA/HAMA Hydrogel-Based 3D Pancreatic Ductal Adenocarcinoma Model

Objective A three-dimensional in vitro culture model constructed by methacrylated gelatin(Gelma)and hyaluronic acid with methacrylate(HAMA)simulating the mechanical properties of tumor microenvironment(TME)and the enrichment of hyaluronic acid(HA)in pancreatic ductal adenocarcinoma(PDAC),Further investigate the biological behavior of pancreatic cancer cells in GelMA&HAMA hydrogel model.Methods The specimens of pancreatic ductal adenocarcinoma and adjacent tissues diagnosed by the Department of general surgery of the Affiliated Hospital of Nantong University were collected to evaluate the mechanical properties of PDAC tissue.The differential expression of hyaluronic acid synthase(HAS)in PDAC tissues to verify the enrichment process of hyaluronic acid based on TCGA＿GTEX＿PAAD database;Gelma matched with stiffness of PDAC tissue combined with 0.5%,1% and 2% HAMA to simulate the HA enrichment process in the progress of PDAC.The rheological behavior,swelling rate and contact angle of GelMA&HAMA hybrid hydrogels and GelMA gels were evaluated.Scanning electron microscopy(SEM)was used to observe the micromorphology of hydrogel,decellularized and non-acellular pancreatic cancer and the adjacent.Pancreatic cancer cell line PANC-1 and MIA-PACA2 on hydrogel at day 4,morphological changes were observed under microscope.qRT-PCR and immunofluorescence were used to evaluate dry(NANOG,OCT4),drug resistance(ABCB1,ABCC1,ABCG2)and metastasis(Ecadherin,N-cadherin,MMP7,and E-cadherin)related genes.The effects of GelMA&HAMA on migration,invasion,drug resistance,proliferation and tumorigenic ability of pancreatic cancer cells were evaluated in vitro by Transwell,invasion,scratch healing,CCK-8 test,colony formation assay and in vivo nude mice tumorigenesis test.Two-dimensional(2D)culture as control,RNAseq showed the differentiated expressed genes(GEGs)between PANC-1 cells “educated” by GelMA&HAMA.Further GO/ KEGG analysis showed the enrichment of biological process(BP),molecular function(MF)and cellular components(CC);Western Bolt verified potential regulatory pathways in GelMA&HAMA hydrogels.Based on TCGA＿PAAD database analyzed the expression difference of NOS2 and PLPP4 in PDAC tissues and evaluated their characters of prognosis in PDAC according to Kaplan Meier analysis and ROC curve.Results Young’s modulus of PDAC tissue was about 44.31 ±3.23 k Pa,which was significantly higher than the adjacent(3.09 ±0.96 k Pa,p < 0.05).The expressions of HAS1,Has2 and HAS3 in PDAC tissues were significantly higher than the adjacent(p < 0.05).Young’s modulus of 7.5%(w/v)GelMA was about 37.30 + 7.89 k Pa,and there was no significant difference compared with PDAC tissues(p > 0.05).Young’s modulus of 0.5%(w / v),1%(w /v)and2%(w / v)HAMA mixed with 7.5%(w /v)GelMA were 47.41 ±7.11 k Pa,45.46 ± 4.65 k Pa and 44.89 ± 4.56 k Pa respectively.They were all matched with the stiffness of PDAC tissues.Rheological tests showed that the storage modulus(G’)of hydrogel was far greater than loss modulus(G”),indicating that GelMA and GelMA&HAMA hydrogels had excellent gelling properties.Swelling results showed that the water absorption of GelMA hydrogel was negatively correlated with the concentration,and the absorbency of 7.5%(w/v)GelMA&HAMA decreased accompanied with concentration of HAMA.The contact angle of hydrogel in all group was about 90 °,indicating that all hydrogels had consistent hydrophobicity.The results of scanning electron microscope(SEM)showed that all hydrogels had porous structure for cell attachment and growth,while PDAC tissues and the adjacent with or without decellularization displayed more dense structure.PDAC cells formed a three-dimensional structure after four days of hydrogel culture.The single cell morphology of PANC-1cells and MIA-PACA2 cells reconstituted from the flat spindle shape on monolayer culture to spherical and cluster state.GAPDH as internal reference,qRT-PCR results showed that PANC-1 cells and MIA-PACA2 cells were highly expressed with stemness(NANOG,OCT4),drug resistance(ABCB1,ABCC1,ABCG2)and metastasis related genes(E-cadherin,N-cadherin,MMP7,MMP9)compared with traditional two-dimensional plane culture.The results of NANOG,ABCB1,ABCG2 and E-cadherin evaluated by immunofluorescence were consistent with the results of qRT-PCR.The results of Transwell,invasion experiment and Wound healing assay showed the migration and invasion ability of PANC-1cells and MIA-PACA2 cells on three-dimensional structure were significantly enhanced compared with 2D culture(p < 0.05).The evaluation of gemcitabine resistance showed that the IC50 values of PANC-1 cells in 3D culture were 469.60,358.30 and 36.77 μ g / ml respectively on the 2nd,3rd and 4th days,which were significantly higher than those in two-dimensional culture,205.30,24.71 and22.93 μ g / ml respectively(p< 0.05).On the 3rd,4th and 5th day,the IC50 of MIA-PACA2 cells were 248.40 vs 72.97,70.68 vs 16.92 and 12.72 vs 2.05 μ g / ml,respectively(p< 0.05).7.5%(w / v)GelMA & 1%(w /v)HAMA enhanced the colony formation and the tumorigenesis ability in nude mice of PANC-1 cells and MIAPACA2 cells.RNA-seq of PANC-1 cells cultured with 7.5%(w / v)GelMA and 1%(w / v)HAMA showed 2255 differentially expressed genes(DEGs);Go analysis based on DEGs revealed BP containing collagen activated signal pathway and oxygen homeostasis,CC containing extracellular matrix components and I-kappa B/NF kappa B complex,and MF containing collagen receptor activity and collagen binding.KEGG analysis showed the top 50 enrichment pathways,and the activation of PI3K-Akt pathway was verified by Westen Bolt.GSEA showed monooxygenase activity and phosphatidic acid phosphatase activity were significantly enriched in PANC-1 cells on 3D culture.Overexpression of NOS2(involved in monooxygenase activity)and PLPP4(involved in phosphatidic acid phosphatase activity)in pancreatic cancer predict poor prognosis with great performance.Conclusions In our research,interpenetrating GelMA and HAMA hydrogels was prepared to reproduce the variation of stiffness and HA content during PDAC progression.The physical and chemical properties of mechanical stiffness,rheology,swelling behavior,hydrophilicity and micromorphology were characterized.The behavior of “educated” PANC-1 cells and MIA-PACA2 cells in terms of stemness,gemcitabine-resistance and metastasis were also implemented.RNA-seq and bioinformatics analysis were carried out to illuminate the potential mechanism of 3D v.s.2D,while the value of diagnostic was evaluated based on TCGA-PAAD database.This study might provide new insights into rational design of 3D scaffolds for PDAC engineering,to clarify the profound mechanism and optimize the anti-cancer therapeutic strategies.