Effects Of Three-dimensional Tumor Decellularized Scaffolds With Different Stiffness On The Drug Resistance Of Breast Tumor Cells

Breast cancer is the most common malignancy in women.Although the continuous improvement of early diagnosis and treatment has significantly reduced the mortality of breast cancer,some patients will eventually suffer from cancer metastasis and recurrence.The resistance of breast tumor cells to chemotherapeutic drugs is one of the causes of shortened survival and postoperative recurrence of breast cancer patients,which may be caused by a variety of factors,expectially the tumor microenvironment plays an important role.In three dimensional(3D)tumor microenvironment,proliferation,drug resistance,and malignant phenotype of tumor cells are associated with matrix stiffness.Therefore,preparation of a 3D tumor model in vitro with suitable stiffness is of great significance for studying drug resistance of breast tumor cells.The decellularized extracellular matrix(DECM)of tumor tissue retains the components and structure of the native tissue,which could better mimic the tumor microenvironment in vivo compared with other 3D tumor culture models.In the previous study of our lab,MDA-MB-231 cells expressing different abundances of extracellular matrix-modifying enzyme lysyl oxidase(LOX)were successfully constructed,and then the ECM of tumor tissues was covalently cross-linked by different levels of LOX enzymes expressed by the cell,thus successfully prepared three kinds of tumor DECM scaffolds(Φ10 mm × 2.0 mm)with different stiffness(low(0.74 ± 0.10 kPa),medium(1.6 ± 0.14 kPa),and high(2.0 ± 0.19 kPa)).Previous experiments have confirmed that the scaffolds could maintain the adhesion and growth of MDA-MB-231 cells.However,the effect of these scaffolds on the malignant phenotypes such as migration and drug resistance of tumor cells remains unknown.This paper further explored the effects of 3D tumor DECM scaffolds with different stiffness on the drug resistance of MDA-MB-231 cells.The main research contents and results were as follows:(1)Effects of tumor DECM scaffolds with different stiffness on the drug resistance of breast tumor cells.The recellularization of scaffold was evaluated by hematoxylin-eosin(HE)staining,laser confocal microscopy 3D reconstruction and DNA quantitative assay.The results showed that MDA-MB-231 cells could infiltrate into the DECM scaffolds.The scaffold with low stiffness could better promote cell infiltration compared with scaffolds with medium and high stiffness.And there were no significant difference in the cell seeding efficiencies and the cell counts after recellularization for 10 days among three groups.The cell viability,apoptosis and cell cycle of MDA-MB-231 cells treated with cisplatin(DDP)were detected by MTS assay and flow cytometry.The results showed that after treated with 20 ?M DDP,the cells cultured in the DECM scaffolds were more active and less apoptotic than the two dimensional(2D)culture.The cell viability and cell proliferation ability in the high stiffness group were both significantly higher than those in the low stiffness group,while the apoptosis rate in the high stiffness was 11.98 ± 2.95%,which was significantly lower than that in the low stiffness group(40.35±3.62%).The expression of drug resistance-related genes and proteins in recellularized scaffolds was detected by qPCR and Western blot,respectively.The results showed that the high stiffness group could promote expression of B-cell lymphoma-2(Bcl-2)and ATP-binding cassette subfamily B member 1(ABCB1)at both the mRNA and proteins levels in MDA-MB-231 cells compared with the low stiffness group and the medium stiffness group.(2)Effect of ABCB1 on the drug resistance of MDA-MB-231 cells in DECM scaffolds with different stiffness.Expression of ABCB1 protein in MDA-MB-231 cultured in DECM scaffolds with different stiffness after being treated with elacridar was detected by Western blot.The results showed that elacridar treatment significantly down-regulated the expression of ABCB1 protein in MDA-MB-231 cultured in DECM scaffolds with different stiffness,without significant difference in ABCB1 protein expression among the three groups.Cell viability,apoptosis and cell cycle were detected by MTS and flow cytometry after elacridar and DDP treatment.The results showed that the IC50 values of each group were reduced to some extent after elacridar treatments,without significant difference in IC50 values among the groups.After treatment with the elacridar,the percentage of proliferative cells was significantly decreased in the high stiffness group,with the apoptosis rate increased from 11.98 ± 2.95% to 48.89 ± 5.48%.In summary,the study proved that 3D matrix stiffness could effect the drug resistance of breast tumor cells.MDA-MB-231 cells in 3D tumor DECM scaffold with high stiffness(2.0 ± 0.19 kPa)showed high resistance to DDP,a commonly used chemotherapeutic drug,and the phenomenon may be related to the inhibition of ABCB1 to DDP-induced apoptosis.The findings could provide a certain theoretical basis for subsequent 3D culture,and provide a new research tool for tumor drug screening,angiogenesis and epithelial-mesenchymal transition.