结直肠癌3D细胞肿瘤球的构建及天马颗粒抗肿瘤作用的初探OA

Objective To establish a 3D cell tumorsphere model incorporating colorectal cancer cells(HCT-116)and endothelial cells(HUVEC),and validate the effects of Tianma Granules on the growth and angiogenesis of the 3D cell tumorspheres within this model.Methods HCT-116 and HUVEC were mixed in varying ratios and cultured in ultra-low attachment 96-well plates.The optimal culture ratio was determined by measuring the volume,cell count,and ATP content of the 3D cell tumorspheres.HCT-116 and HUVEC were mixed at a 2:1 ratio and divided into five groups:blank control group(15%blank serum),doxorubicin hydrochloride(15%blank serum+100 ng/mL doxorubicin),and low-,medium-,and high-dose Tianma Granules-containing serum(TGCS)groups(5%,10%,and 15%drug-containing serum respectively,supplemented with blank serum to a total of 15%).The volume,cell count,and ATP content of the 3D cell tumorspheres were then measured.Additionally,2D cells and 3D cell tumorspheres were divided into six groups:2D control(15%blank serum),3D control(15%blank serum),doxorubicin group(15%blank serum+100 ng/mL doxorubicin hydrochloride),and low-,medium-,and high-dose TGCS groups(5%,10%,and 15%drug-containing serum respectively,supplemented with blank serum to a total of 15%).RNA sequencing analysis was performed,and RT-qPCR was used to measure the mRNA expressions of pyruvate kinase M1(PKM1),pyruvate kinase M2(PKM2),and glucose transporter 1(Glut1).Western blot was conducted to check the protein expressions of CD31,P21 and P53.TSA multiple immunofluorescence was employed to examine the expressions of CD31 and proliferating cellnuclear antigen(PCNA).Results On the 9th day of culture,when the cell ratio was 2:1,the 3D cell tumorspheres exhibited the maximum volume,the highest cell count,and the highest ATP content(P＜0.05,P＜0.01).Compared with the blank control group,the 3D cell tumorspheres in all drug-administered groups showed reductions in the volume,cell count,and ATP content(P＜0.05).Compared with the doxorubicin hydrochloride group,the low-dose TGCS group showed increases in the volume,cell count,and ATP content of 3D cell tumorspheres(P＜0.05,P＜0.01);the medium-dose TGCS group showed reductions in the volume of 3D cell tumorspheres(P＜0.01);the high-dose TGCS group exhibited reductions in the volume,cell count,and ATP content of the 3D cell tumorspheres(P＜0.01).Compared with the low-dose TGCS group,the medium-and high-dose TGCS groups exhibited reductions in the volume,cell count,and ATP content of the 3D cell tumorspheres(P＜0.05,P＜0.01).Compared with the medium-dose TGCS group,the high-dose TGCS group exhibited reductions in the volume,cell count,and ATP content of the 3D cell tumorspheres(P＜0.05,P＜0.01).Compared with the 2D group,the glycolytic pathway was upregulated in the 3D group;conversely,compared with the 3D group,the glycolytic pathway was downregulated in the high-dose TGCS group.Compared with the 2D group,the mRNA expression levels of PKM1,PKM2 and Glut1,as well as the protein expression level of CD31,increased in the 3D group(P＜0.01),while the protein expression levels of P53 and P21 decreased(P＜0.05,P＜0.01).Compared with the 3D group,the mRNA expression levels of PKM1 and Glut1 reduced in all drug-administered groups(P＜0.05,P＜0.01),while the protein expression levels of P53 and P21 increased(P＜0.05,P＜0.01).The mRNA expression level of PKM2 and the protein expression level of CD31 decreased in the doxorubicin hydrochloride group,as well as the medium-and high-dose TGCS groups(P＜0.05,P＜0.01).Compared with the doxorubicin hydrochloride group,the low-dose TGCS group exhibited elevated mRNA expression levels of PKM1 and PKM2(P＜0.05,P＜0.01)and reduced protein expression level of P21(P＜0.05).The medium-dose TGCS group showed decreased mRNA expression level of PKM2 and protein expression levels of P21 and CD31(P＜0.05,P＜0.01).The high-dose TGCS group demonstrated reduced mRNA expression levels of PKM1,PKM2 and Glut1,as well as protein expression levels of P53,P21 and CD31(P＜0.05,P＜0.01).Compared with the low-dose TGCS group,the medium-dose group exhibited decreased mRNA expression levels of PKM1 and PKM2,as well as protein expression level of CD31(P＜0.05,P＜0.01);while the high-dose group showed reduced mRNA expression levels of PKM1,PKM2 and Glut1,as well as protein expression levels of P53,P21 and CD31(P＜0.05,P＜0.01).Compared with the medium-dose TGCS group,the high-dose group exhibited reduced mRNA expression level of Glut1 and protein expression levels of P53 and P21(P＜0.05,P＜0.01).Compared with the blank control group,both the doxorubicin hydrochloride group and the high-dose TGCS group showed a decrease in the number of PCNA-and CD31-positive cells(P＜0.05,P＜0.01).Compared with the doxorubicin hydrochloride group,the high-dose TGCS group demonstrated a reduction in the number of CD31-positive cells(P＜0.01).Conclusion A relatively stable 3D cell tumorsphere model can be established when the ratio of colorectal cancer cells to endothelial cells is 2:1.TGCS can inhibit tumorsphere growth and angiogenesis.