Semaphorin3F Sensitizes Multicellular Tumor Spheroid To Chemotherapy Through Downregulation Integrinαvβ3 In Colon Cancer Cells

Background and purposeThe three-dimensional(3-D) multicellular spheroid model exhibits a phenomenon known as "multicellular resistance"(MCR),which becomes less sensitive to anticancer drugs than traditional two-dimensional(2-D) cell culture model.MCR can better simulate the patterns of drug resistance observed in vivo.The 3-D culture model has been made through innovative approaches of Dr Kerbel's group who have shown intrinsic resistance to cytotoxic drugs and radiation by culturing cell lines as spheroid.We also confirmed much cancer cell lines including pulmonary carcinoma,colon carcinoma and gastric carcinoma, when they grown in 3-D culture,IC50s of several chemotherapeutic agents are much higher than 2-D cell culture.MCR is mainly focused the signal transmission-based network on the cell-cell and cell-matrix interaction and their special microenvironment.Importantly,when grown as spheroids,cells secrete more cell-cell and cell-matrix interaction molecules such as E-cadherin and Integrins.The amount of interaction molecules by culturing cell lines as spheroids,assist tumor cells for survival and induce anti-cancer drug resistance.This interaction molecules-dependent resistance is reversible and disappears as soon as cells interactions are disrupted.Among the inhibitors of cell interactions,the anti-integrin antibodies including againstαv orβ1 subunit could inhibit cell-matrix interactions,induce apoptosis and sensitize the MCR.Semaphorin3F(Sema3F) is a kind of trans-membrane molecules,which regulates nerves anchorage and guides intracellular signals for cell growth,migration and angiogenesis.Previous study has proved that Sema3F could downregulate Integrinαvβ3 in the endothelial cells.We presumed that Sema3F could sensitize MCR through down regulation of Integrinαvβ3 expression and raise the pharmacologic possibility to sensitize MCR for therapeutic potentials.Therefore,we proved Sema3F could downregulate the expression of cell adhesion molecule Integrinαvβ3,which may represent a feasible therapeutic agent to sensitize MCR.Methods1.We established human colorectal adenocarcinoma HT29 3-D culture model and detected the construction of multicellular spheroid with scanning and transmission electron microscope,then we compared the chemosensitivity of 3-D multicellular spheroid and 2-D cultured cells and analysed the protein expression level of Sema3F in five kinds of human colon cancer cell lines by Western blotting.2.We detected the mRNA and protein expression levels of Integrinαv and Integrinβ3 between 3-D multicellular spheroid and 2-D cultured cells by RT-PCR,Histochemistry,Immunoflourescent and Western blot analysis.And then we compared the sensitivity to chemotherapy of 3-D multicellular spheroid cells,blocking with the Integrinαvβ3 antibody of 3-D multicellular spheroid cells and 2-D cultured cells.3.We detected the effect of Sema3F on Integrinαvβ3 expression and the sensitivity of colon cancer cells to chemotherapy by MTT assay and Radial outgrowth assay.Results1.Successful establishment of 3-D culture model of human colorectal adenocarcinoma HT29(Figure1).Scanning elecrtron microscope(Figure2) and Transmission electron microscope(Figure3) of multicellular spheroids displayed the spheroids were irregular in diameter with 1.0-2.0mm.Transmission electron microscope of 3-D multicellular spheroid (×15000).Tight cell junctions,desmosome junctions and intermediate junctions were observed in 3-D multicellular spheroid.HT29 and LoVo cell lines have low mRNA (Figure17) and protein(Figure18) expression of Sema3F than the other cell lines. 2.The mRNA(Figure8) and protein(Figure9 and Figure 10) expression levels of Integrinαv and Integrinβ3 in 3-D multicellular spheroid were much higher than that in 2-D cultured cells.Sensitivity to chemotherapy of 3-D multicellular spheroid was lower than 2-D cultured cells.(Figure7).Treatment with the Integrinαvβ3 antibody can sensitize 3-D multicellular spheroid to chemotherapy.3.Successful establishment of stable Sema3F-transfected HT29 3-D multicellular spheroid.After treated with 5-FU(0,125,250,500,1000,2000μg/ml) for 48h,the sensitivity to chemotherapy of stable Sema3F-transfected cells was tested via MTT assay(Figure24), Radial outgrowth assay(Figure 25),Flow Cytometry testing(Figure4,5,13) and Colony formation(Figure26) assay in 2-D cultured and 3-D multicellular spheroid cells.We detected Integrinαvβ3 protein expression level of Sema3F-transfected HT29, HT29-Neo and HT29 cells and demonstrated the enhancement of Sema3F protein expression in Sema3F-transfected cells could increase the HT29 cell sensitivity to chemotherapy through downregulation of Integrinαvβ3 protein.Conclusions1.3-D multicellular spheroid was more compact than 2-D cultured cells and the sensitivity to chemotherapy of the 3-D multicellular spheroid was lower than 2-D cultured cells.2.The mRNA and protein expression levels of Integrinαv and Integrinβ3 in 3-D multicellular spheroid were higher than 2-D cultured cells.Treatment with Integrinαvβ3 antibody can sensitize 3-D multicellular spheroid to chemotherapy.3.Enhancement of Sema3F protein expression in Sema3F-transfected cells could increase the HT29 cell sensitivity to chemotherapy through downregulation of the Integrinαvβ3 protein.