The Effects Of CT26.WT Colon Cancer Cells On RAW264.7 Macrophage Proliferation And Differentiation

Objective:In this study,different concentrations of CT26.WT colon cancer cell culture supernatant or CT26.WT colon cancer cell whole cell antistoste were used to simulate the in vitro colon cancer tumor microenvironment and cultured RAW264.7 macrophages.By observing the changes of macrophage morphology,activity,cytokine expression and migration ability,we further investigated the effects of CT26.WT colon cancer cells on the proliferation and differentiation of RAW264.7 macrophages.Methods:1.The experiment was divided into three groups.In control group,RAW264.7 macrophages were cultured with DMEM complete culture medium.In supernatant group,RAW264.7 macrophages were cultured in DMEM medium with different concentrations of CT26.WT colon cancer cell culture supernatant（25%,50%）for 3 and 5 days,respectively.In antigen group,RAW264.7 macrophages were cultured in DMEM medium with different concentrations of CT26.WT colon cancer cell whole cell antistoves（10%,20%）for 3 and 5 days,respectively.2.We used inverted microscope to observe the morphology of RAW264.7 macrophages and the morphological changes of RAW264.7macrophages in CT26.WT colon cancer cell culture supernatant or CT26.WT colon cancer cell culture with whole cell antistost.3.Complete the MTS experiment.We stimulated RAW264.7macrophages with different concentrations of CT26.WT colon cancer cell culture supernatant or CT26.WT colon cancer cell whole cell antistant for 3and 5 days,respectively.The OD of each group were detected at the wavelength of 490nm by MTS method.To investigate the effects of CT26.WT colon cancer cells on the proliferation of RAW264.7 macrophages.OD was positively correlated with the number of viable cells.4.Real-time Quantitative PCR.RAW264.7 macrophages were stimulated with different concentrations of CT26.WT colon cancer cell culture supernatant or CT26.WT colon cancer cell whole cell antigen for 3and 5 days respectively,withβ-Action as an internal parameter,the Ct of IL-6,IL-10,IL12 and TNF-αin RAW264.7 macrophages in each group were detected by Real-time Quantitative PCR,2^-△△Ctwas used to calculate the relative expression of m RNA of each target gene.5.Cell scratch assay.RAW264.7 macrophages were stimulated with different concentrations of CT26.WT colon cancer cell culture supernatant or CT26.WT colon cancer cell whole cell antigen for 3 and 5 days respectively,the change of scratch width was recorded to detect the migration ability of RAW264.7 macrophages.6.SPSS 26.0,graphpad prism 8 and Microsoft Excel were used to process and statistically analyze the experimental data,the measurement data were expressed as the mean±standard deviation（x±s）,one-way ANOVA was used for the comparison between different concentrations of the three groups,and the two pairs between the three groups were compared with post-hoc multiple comparisons,and the difference was statistically significant with P<0.05.Results:1.Morphological changes of macrophages:RAW264.7 macrophages of normal control are mostly round or oval adherent cells.Compared with the control group,macrophages exhibited long fusiform changes with pseudopodia extending out after stimulation with different concentrations of CT26.WT colon cancer cell culture supernatant respectively,and the morphological changes of cells were more obvious with the increase of stimulation concentration;after stimulation of CT26.WT colon cancer cell whole cell antigen solution at different concentrations,the macrophage volume increased slightly and became flattened and round.2.MTS results showed that compared with the control group,macrophages exhibited long fusiform changes with pseudopodia extending out after stimulation with different concentrations of CT26.WT colon cancer cell culture supernatant for 3 days respectively,the OD of 50%concentration of the supernatant group were reduced（P<0.05）as detected at the wavelength of 490 nm;the OD of each group was reduced（P<0.05）after 5days of stimulation,showing concentration-dependence;indicating that the proliferation of macrophages was inhibited to various degrees.After stimulation with different concentrations of CT26.WT colon cancer cell whole cell antigen for 3 days respectively,the OD of 10%concentration of antigen solution group were elevated（P<0.05）,indicating that the proliferation of macrophages was promoted to various degrees.3.Real-time Quantitative PCR results showed that,compared with the control group,macrophages exhibited long fusiform changes with pseudopodia extending out after stimulation with different concentrations of CT26.WT colon cancer cell culture supernatant for 3 days respectively,the expression levels of IL-6,IL-12 and TNF-αof RAW264.7 macrophages decreased to varying degrees（P<0.05）,the expression level of IL-10increased（P<0.05）;after 5 days of stimulation,the expression levels of IL-6,IL-10 and IL-12 were increased to varying degrees（P<0.05）,the expression levels of TNF-αdecreased（P<0.05）.After stimulation with different concentrations of CT26.WT colon cancer cell whole cell antigen for 3 and 5days respectively,the expression levels of IL-6,IL-12 and TNF-αof RAW264.7 macrophages increased to varying degrees（P<0.05）;the expression levels of IL-10 decreased to varying degrees（P<0.05）.4.The results of cell scratch experiments showed that after 72 hours of scratching,the scratch width of the control group narrowed more significantly compared to the group stimulated by different concentrations of CT26.WT colon cancer cell culture supernatant,indicating that the migration ability of RAW264.7 macrophages was weakened;the scratch width of the colon cancer cell whole cell antigen stimulated group with different concentrations of CT26.WT narrowgved more significantly compared to the control group,indicating the enhanced migration ability of RAW264.7macrophages.Conclusions:1.Supernatant from CT26.WT tumor cell culture has a tendency to inhibit the proliferation of RAW264.7 macrophages,induce the differentiation of RAW264.7 macrophages to M2-like phenotypes in the early stage,and induce the differentiation of RAW264.7 macrophages to M1/M2 mixed with the extension of culture time.2.CT26.WT whole-cell antigen solution of colon cancer cell has a tendency to promote the proliferation of RAW264.7 macrophages and induce the differentiation of RAW264.7 macrophages to M1-like phenotypes.