Enrichment Of Ovarian Cancer Stem-like Cells By Hydrogels Based On Poly(Methyl Vinyl Ether-alt-Maleic Acid)

Cancer stem cells(CSCs)are associated with the cell behaviors of cancer metastasis,invasion,and malignant transformation and are widely believed to be the crucial root of chemo/radio-resistance and cancer recurrence.However,the study of CSC mechanisms requires that these CSCs should be isolated or enriched.In recent years,the development of 3D cell culture techniques has made hydrogels a hot research area because of their broad range of biomedical applications.The extracellular matrix is a multi-component gel system.In this paper,three kinds of controllable P(MVE-alt-MA)-based hydrogel systems were constructed by using poly(methyl vinyl ether-alt-maleic acid)[P(MVE-alt-MA)]as the main raw material.The constructed hydrogels mimic the structure and properties of extracellular matrix.The stiffness,topography,components,and adhesiveness of the matrix were adjusted to reveal the effects of these factors on CSC stemness.The structure of the hydrogels was fully characterized by scanning electron microscopy,infrared spectroscopy,swelling rate,and rheology.The simplest polyethylene glycol(PEG)-crosslinked P(MVE-alt-MA)hydrogels(abbreviated as PEMM)was first constructed,and the arginine-glycine-aspartic acid(RGD)was added to mimic the properties of collagen in the extracellular matrix.In addition,alginate was added to mimic the hyaluronic acid in the extracellular matrix,further mimicking the properties of the extracellular matrix.These controllable hydrogel systems mimicking the extracellular matrix were used to regulate the single factor accurately and independently,and their effects on CSC stemness were studied.Cells with high expression of CSC markers were screened for in vivo tumorigenicity study,and a preliminary exploration of the mechanisms of signaling pathways involved in tumor invasion,migration,and malignant transformation was carried out.The main contents of this paper are divided into the following 4 parts:(1)Effect of the elastic modulus of PEG-crosslinked P(MVE-alt-MA)hydrogels on the enrichment of ovarian cancer stem-like cells.Two kinds of PEMM hydrogels with different stiffness(12 k Pa and 97 k Pa)were prepared by controlling the crosslinking density.The network structure of the hydrogels became more compact with the increase of crosslinking degree,leading to a greater elastic modulus and a lower swelling rate.Cell proliferation and viability assays confirmed the good biocompatibility of the hydrogels,and the matrix properties were beneficial for spheroid formation of SK-OV-3 ovarian cancer cells.A combined analysis of multiple CSC markers and drug resistance showed that the cell pheroids cultured in the hydrogels,and especially in the softer hydrogel,acquired certain SC-like properties that enhanced their drug resistance.Meanwhile,the expression of the umor invasion,migration,and malignant transformation signaling pathway-associated genes revealed that matrix stiffness was involved in the regulation of several signaling pathways,including the epithelial-mesenchymal transition(EMT),interleukin-6(IL-6),and Wnt pathways that affected the invasiveness of the ovarian cancer,the EMT,and the conversion of non-CSCs into CSCs.The stemness and malignancy of ovarian cancer cells were higher when cultured in a soft hydrogel(12 k Pa)with a macroporous structure and a high swelling rate than on a 2D plate or in a stiff hydrogel(97 k Pa).This research lays the foundation for follow-up work,and provides a platform for studying the effect of matrix factors on the enrichment of CSC-like cells.(2)Effect of the RGD content in PEG-crosslinked P(MVE-alt-MA)hydrogels on the richment of ovarian cancer stem-like cells.Hydrogels were prepared with various dhesive properties by controlling the RGD concentration in a PEMM hydrogel.The RGD oncentration on the substrate surface affected cell behavior.The cell aggregates cultured n the hydrogel surface at a lower RGD concentration acquired certain CSC-like properties d therefore showed enhanced drug resistance.By contrast,the cells on surfaces with igher RGD concentrations showed obvious cell spreading and increased drug sensitivity.The low RGD concentration did not alter the effect of matrix stiffness on CSC enrichment; owever,the effect of stiffness differences on cancer cell behavior was no longer obvious n the hydrogels with the higher RGD concentration.Moreover,the presence of RGD in the tiff hydrogels had less effect on cancer cell behavior than did the presence of RGD in the ft hydrogels,indicating an effect of matrix stiffness on the function of RGD.The binding f ovarian cancer cells to the RGD on the substrate surface is involved in the cell adhesion ffects on cell morphology and differentiation,and this,in turn,affects the expression of ancer cell stemness and the EMT.The downregulation of Snail,MMP9,IL-6,and Wnt1 n a higher RGD concentration surface affects the invasiveness of ovarian cancer,the cquisition of cancer cell stemness,and the signaling pathway for converting the non-CSCs nto CSCs.The results suggest that the RGD content and matrix stiffness can haveynergetic effects on the expression of cancer cell stemness and the EMT,IL-6,and Wnt athways.(3)Enrichment of ovarian cancer stem-like cells in PEG-crosslinked P(MVE-alt-MA)and lginate double-network hydrogels.Three kinds of double-network hydrogels(PEMM/Alg),onsisting of PEMM(network 1)and cations(Sr²⁺,Ca²⁺,or Fe³⁺)crosslinked alginates network 2,denoted as Sr Alg,Ca Alg,or Fe Alg),were prepared.The stiffness,morphology,nd components of the PEMM/Alg hydrogels were systematically investigated to nderstand their effects on CSC enrichment.Only proved the PEMM/Fe Alg hydrogels a apable of supporting the long-term growth,proliferation,and spheroid formation of SK- V-3 ovarian cancer cells.The cell spheroids cultured in the PEMM/Fe Alg hydrogels,and specially in the PEMM-I/Fe Alg hydrogel,acquired certain CSC-like properties and howed enhanced drug resistance.The changes in CSC markers at different times also ndicated their roles in corresponding stages.The results suggest that the matrix components, tiffness,and morphology act together to regulate the EMT,IL-6,and Wnt pathways, hereby affecting the invasiveness of ovarian cancer and the conversion of the non-CSCs nto CSCs.The addition of alginate to the PEMM hydrogels did more than just change the atrix components;it also introduced cations and altered the matrix stiffness and opography by modulating the double-network structure,making these hydrogels an ideal esearch model(4)Evaluation of the xenogeneic tumorigenicity of ovarian cancer stem-like cells after nrichment by culture in P(MVE-alt-MA)-based hydrogels.Studying the effects of the ydrogel components,stiffness,topography,and adhesive properties on the enrichment of varian cancer stem-like cells served as a preliminary screening of the culture environment hat would favor CSC enrichment.The cells cultured in either the PEMM-1 or the PEMM-/Fe Alg hydrogels showed high expression of CSC markers,enhanced drug resistance,and ignificant activation of malignant pathways and were selected for evaluation of umorigenicity.The number of injected cells,the time required for tumor formation,and the eight and volume of the developed tumors showed that even though the number of injected ells from the hydrogel groups(1×10⁵)was one order of magnitude smaller than that from he 2D control(1×10⁶),the tumor formation was faster and more expansive and the xpression of the CSC markers was increased,thereby confirming the increase in the roportion of CSCs in the CSC-like cells enriched by culture in P(MVE-alt-MA)-based ydrogels.In conclusion,this study constructed different kinds of hydrogel systems that mimic extracellular matrix,systematically explored the effects of various factors of hydrogels on tumor cell behavior,and thus achieved the enrichment of CSCs,providing more research models and research platforms for in vitro tumor pathological analysis.