Inhibitory Effect And Mechanism Of Mesenchymal Stem Cells Cultured In 3D System On Hepatoma Cell HepG2

Aim Mesenchymal stem cells(MSCs)have the ability of self-renewal,high proliferation and multi-lineage differentiation potential.In addition,MSCs have been tried to be used for varied tumor therapy due to MSCs can home to sites of tumorigenesis and participate in the development of tumor.However,MSCs are commonly cultured as monolayers by conventional two-dimensional(2D)culture techniques at present.In 2D conditions,cells would gradually loss some in vivo properties that are important for structures,proliferation and cellular functions.The experiment in vivo has a variety of uncontrollable factors,which is not conducive to exp lore specific mechanisms.The three-dimensional(3D)culture approach has taken us a step closer to the conditions in vivo.A major advantage of the 3D over the 2D approach is that 3D system decrease the gap between cell cultures system and the cellular physiology,and more beneficial to study the inhibitory effect of MSCs on the tumor.Therefore,this research estab-lished the 3D culture system of MSCs and explored the inhibitory effect and mechanism of 3D-cultured MSCs on HepG2 proliferation,which provides a simpler,safer and more reliable ap-proach for MSCs suppressing tumor cells,and provides effective experimental data for clinical treatment of tumor,and expands application prospect for stem cells.Method Firstly,we used collagen/Matrigel scaffold to culture MSCs and evaluated growth pattern and expression levels of selected signaling molecules of MSCs in 3D cell culture system and 2D cell monolayers by AO/PI staining,CCK-8 and transmission electron.Secondly,we detected the inhibitory effect of 3D-and 2D-cultured MSCs through secreting cytokines on HepG2 cells by CM experiment in vitro.In the same time,the initial time of tumor formation and the tumor size were measured by animal transplantation experiment in vivo.Then,histo-pathological examinations were conducted by hematoxylin and eosin(H&E)staining.To evi-dence the modifications in the transcriptome of MSCs induced by the 3D cell culture system,gene expression profiles of 3D-and 2D-cultured MSCs were analyzed,which showed that the expression of IL-24 in 3D system was most prominently upregulated.The expression of IL-24 in 3D system was verified by reverse transcription polymerase chain reaction(RT-PCR),Quantitative Real-time PCR and enzyme linked immunosorbent assay(ELISA).Then the expression of IL-24 receptors,IL-20R1,IL-20R2 and IL-22R1,were detected.At last,the ex-pression of proteins related to JAK-STAT signaling in HepG2 cells were detected by Western Blot.Results First of all,AO/PI staining results showed that MSCs survived well,and there were very few dead cells both in 3D cell culture system and 2D cell monolayers.And 3D-cultured MSCs showed higher proliferation ability by CCK-8 method.Since the 4th day,proliferation ability of MSCs indicated very significant differences between 3D and 2D culture systems(***P<0.001).MSCs in 3D collagen/Matrigel scaffold system continued to proliferate to the 11th day and reached 6.71 times increment,while cells cultured in 2D system increased 2.66 times at the 11th day.Then,TEM results showed that MSCs cultured in 3D scaffold exhibited fewer microvilli and possessed more rough endoplasmic reticulum than that in 2D culture sys-tem,which might be related to protein biosynthesis capacity.Secondly,the CM experiments showed that both 3D-and 2D-cultured MSCs CM were able to inhibit the proliferation of he-patoma cells HepG2,moreover,3D-cultured MSCs CM were more effective in vitro.What's more,the inhibitory rates increased along with the concentration of CM and the treatment time prolonging.The highest inhibitory rate of 3D and 2D MSCs CM group reached 36.10%and 23.63%respectively when HepG2 cells were cultured in 100%MSCs CM for 72 hours.In vivo,animal transplantation experiment showed that MSCs cultured in 3D system were able to more significantly delay the tumor initiation of HepG2 cells for 12 days,and MSCs cultured in 2D system delay the tumor initiation for 8 days.And the tumor volumes in 3D or 2D MSCs-CM treated groups were significantly smaller than those in untreated control groups(***p<0.001).Affymetrix GeneChip(?)Prime ViewTTM Human Gene Expression Array found that the most upregulated gene of 3D-cultured MSCs was IL-24.This result was further verified by RT-PCR,Real-time PCR and ELISA assay.RT-PCR analysis showed that HepG2 cells expressed IL-24 receptor IL-22R1?IL-22R1 and IL-20R2,and didn't expressed IL-20R1,which suggested that IL-24 signaling was mediated by IL-22R1/IL-20R2 in HepG2 cells.Western blot results showed that the expression levels of the p-JAK1 and p-STAT3 in HepG2 cells cultured in 3D MSCs CM significantly increased(***p<0.001).Conclusion:MSCs could successfully expand and propagated in 3D collagen/Matrigel scaffolds and had higher proliferation ability and secreted more antitumor factors than 2D-cul-tured MSCs.Both 3D-and 2D-cultured MSCs CM were able to inhibit the proliferation of HepG2 cells in vitro and vivo,but 3D-cultured MSCs CM were more effective.The inhibitory mechanism was further investigated and showed that MSCs cultured in 3D cell culture system secreted more IL-24 to activate JAK1-STAT3 signaling by receptor IL-22R1/IL-20R2 and caused HepG2 cell apoptosis,and further inhibited HepG2 cells in vitro and vivo.This study has broken through the traditional study of 2D MSCs culture,which provides a simpler,safer and more reliable approach for MSCs suppressing tumor cells,and provides effective experi-mental data for clinical treatment of tumor,and expands application prospect for MSCs.