Construction Of 3D Cell Spheroids Based On Microenvironment Of Pancreatic Cancer And Their Screening Of 9FU-AS1411

Drug screening can quickly eliminate inappropriate lead compounds in the early stages of research,reducing unnecessary time and cost,thereby identifying and optimizing lead compounds before clinical trials to obtain candidate drugs.Therefore,it is crucial to establish a disease model that accurately replicates pathophysiological behavior and responds to lead compounds.Traditional anti-tumor drug screening models mainly include patient derived cancer cells（PDC）and patient derived xenografts（PDX）.The former lacks diversity in cell types,spatial structure,and microenvironment,making it difficult to simulate the real environment in the body.The latter has the problem of individual origin,which cannot fully represent the unique characteristics of each cancer patient.In addition,there have been ethical issues regarding the visibility of blood vessels in animal models and the reproductive speed of experimental animals.The recently rising 3D cell spheroids has arose extensive attention among researchers which are similar to changes in tumor morphology,microenvironment,volumetric growth kinetics,nutrient distribution,oxygen concentration,cell proliferation,and drug resistance in vivo,and are more simulative.Pancreatic cancer is a highly malignant and deadly cancer.Gemcitabine and fluorouracil are first-line chemotherapy drugs for pancreatic cancer.However,the canceration structure of pancreatic cancer is protected by a dense matrix barrier,which hinders the penetration of chemotherapy drugs.The problem of how to penetrate the matrix and target the delivery of drugs to tumor sites urgently needs to be resolved.In this dissertation,we conjugate 5-Fluorouracil（FU）,the first-line drug for pancreatic cancer,with the nucleolin aptamer AS1411,which can target the nucleolin protein on the surface of tumor cells,to form a nucleolin aptamer-fluorouracil conjugate（FU-AS1411）,so as to improve its ability to penetrate the matrix and target delivery to pancreatic cancer.At the same time,a 3D cell spheroids model based on pancreatic cancer microenvironment was constructed which using HUVECs,Gel MA hydrogel and fibroblasts as the matrix.Next,the selectivity and anti-tumor activity of the conjugate 9FU-AS1411 on tumor cells were systematically investigated in 2D model,3D cell spheroids model and 3D cell spheroids model based on tumor microenvironment.The main content and results are as follows:1.Construction a 3D cell spheroids model and a 3D cell spheroids model based on tumor microenvironment.（1）Cell spheroids which were prepared using agarose molds have a uniform size（200μm）,a rounded appearance,and a transparent spheroids.Miapaca-2 and HSFs were used to construct a 3D cell spheroids model of pancreatic cancer.The outside was wrapped by fibroblasts,and pancreatic cancer cells were concentrated inside the spheroids or scattered around the spheroids.After 7 days of culture,the cell ball activity was>90%.（2）Through light crosslinking of HUVECs,HSFs and Gel MA hydrogel,a relatively mature and stable microvascular network was rapidly formed,and the activity of the vascular network was still more than 80%after 7 days.（3）Based on（1）and（2）,the tumor vessel microenvironment is jointly constructed to form a 3D cell spheroids model based on the tumor microenvironment.2.Research on tumor selectivity and antitumor activity of 9FU-AS1411 in 2D model,3D cell spheroids model,and 3D cell spheroids model based on the tumor microenvironment.（1）FAM-9FU-AS1411 has good selectivity for tumor cells in 2D models,3D cell spheroids models,and 3D cell spheroids models based on tumor microenvironment,and has extremely low binding rates with tumor microenvironments composed of HUVECs and HSFs.（2）After subcutaneous tumorigenesis and administration in mice,FAM-9FU-AS1411 can be highly aggregated at the tumor site,with no aggregation in the heart,spleen,and lungs,and a small amount of aggregation in the liver and kidneys.（3）After FU and 9FU-AS1411 treated 3D cell spheroids of pancreatic cancer and normal cells for 72 hours,9FU-AS1411 had strong effects on pancreatic cancer and weak toxicity on normal cells at the same dose compared with FU.For pancreatic cancer cells in 2D state,the IC₅₀value of FU is 5～10 times that of 9FU-AS1411.（4）After 9FU-AS1411 treatment,the apoptosis rate of pancreatic cancer Miapaca-2 and BXPC-3 in 2D and 3D was 20%～50%higher than that of FU.Panc-1 had the similar trend,but there was no significant difference;After 9FU-AS1411 treatment,the apoptosis rate of 2D cells was 10%to 30%higher than that of 3D cell spheroids;9FU-AS1411 significantly blocked cells in the G1 phase,while FU blocked cells in the S phase.（5）The expression of Ki-67 after 9FU-AS1411 treatment of cell spheroids was lower than that of FU.In summary,the 3D cell spheroids model based on the tumor microenvironment prepared in this dissertation can be used for drug screening;The designed nucleolin aptamer-fluorouracil conjugate 9FU-AS1411 has excellent selectivity and antitumor activity for tumor cells in 2D models,3D cell spheroids model and 3D cell spheroids model based on tumor microenvironment.This study will provide experimental data and theoretical basis for preclinical screening of anti pancreatic cancer drugs.