Induction Of Cancer Stem Cells In Vitro And Screening Specific Binding Peptide

Although cancer treatment technology has made great progress,the survival rate of cancer patients is still low,mainly due to the Cancer stem cells(CSCs).CSCs are cells in tumor characterized with stem cell properties.They have the potential of self-renewal and differentiation and can lead to strong chemotherapy resistance,tumor recurrence and metastasis.Only by eliminating CSCs can we really cure cancer.Cancer stem cell theory provides new ideas of tumor development and cancer treatment.Therefore,establishing effective methods to study on CSCs is of great significance.This article took breast cancer and lung cancer as objectives to induce CSCs.In serum-free medium,added with bFGF,EGF,insulin and B27,tumor cells formed tumor spheres.Alkaline phosphatase staining,immunofluorescence,flow cytometry,real-time PCR and western blot were applied to verify the stem cell properties of tumor spheres.Results showed high expression of CD133,CD44,Sox2,Oct4 and Nanog in tumor spheres,indicating that cancer stem cells had been induced by suspension culture.Drug endocytosis experiment,toxicity study and ABCG2 expression detection of breast cancer stem cells showed that drug concentration of MCF-7 CSCs was much lower than that of MCF-7,and MCF-7 CSCs’ relative survival rate was higher than that of MCF-7.Moreover,high expression of ABCG2 in MCF-7 CSCs was showed by western-blotting.All the evidence above verified CSC’s drug resistance.Phage display technology was used to get specific targeting peptides sequence of lung CSCs.After sorting out the side population cells from tumor spheres by flow cytometry,researchers employed two rounds of “negative/positive” phage library screening and the last round of affinity screening to screen out the specific targeting phages.Phages bound to CSCs were further enriched after each round of screening,.40 clones were randomly selected from the third round microtiter plates,some of which with the same nucleotide sequence were merged after sequencing and translated into amino acid.Peptides were synthesis with FAM fluorescence,and their specificity were tested by immunofluorescence.Results showed that the two peptides SPM and SP28 had specific affinity for A549 CSCs.