| BackgroundCancer drug resistance is a major obstacle for chemotherapy.Recent data now implicate intratumoural heterogeneity as a major driver of drug resistance.Single cell sequencing studies identified the genetically diversity in the multiple space within human tumours.The major factors determining the intratumoural heterogeneity are(ⅰ)genetic makeup,(ⅱ)stochastic processes,(ⅲ)tumor microenvironment and(ⅳ)cell and tissue plasticity.Precise medicine is based on tumor heterogeneity,mainly gene expression differences given to individualized treatment.And,this kind of heterogeneity should be true heterogeneity,that is,genomic differences,but the differences in gene expression are not due only to differences in the genome,but also caused by other reasons such as epigenetics or stochastic processes.Whether precision medicine in such cases can also bring benefits to patients need to be calmly thinked about.Similarly,one cancer cell line is believed to be composed of numerous clones with heterogeneity.However,how big the difference of drug-response pattern can exist in clones from a cell line or from a single cell has not been reported now.Here,we choose the cell lines as the models to demonstrate the drug-response diversity in clones from a cell line or from a single cell.MethodsWe studied three cell lines as follows:4T1,Bcap37 and K562.Firstly,using limited dilution method,the single cell clones from the three cell lines were isolated and expanded.The IC50 of the drugs were determined by MTT assay.The mRNA expressions of the drug-resistance related genes of the clones were detected by Real-time polymerase chain reaction.Secondly,one single cell clone was chosen in each cell line and the daughter clones(subclones)were isolated and expanded using the same method.The IC50 of the drugs and the mRNA expressions of the subclones were determined.Then,cluster analysis was carried out to test the differences among the clones or subclones in drug-response pattern and gene expression pattern.It is worth noting that stem cell like cells were sorted by three-dimensional culture and immunofluorescence assay of CD24/CD44 expression in 4T1 cell line.Results1.Clones and daughter clones from 4T1 cell line show heterogeneous phenotypes.The clones which could differentiate the acinar-like structure using the three-dimensional culture were CD24+CD44+ cells,and we call these clones S.The clones which could not differentiate the acinar-like structure were CD24+CD44-cells,and they were called N.The 11 S clones or 11 N clones derived from 4T1 cells were drastically different from each other.Each clone exhibited a distinct drug-response pattern against 11 anticancer drugs;they showed drastic difference in drug sensitivity toward anticancer drugs;accordingly,each clone had a distinct expression profile of 19 genes associated with drug sensitivity.Note that these clones were cultured under the same condition without drug pretreatment.It is surprising that similar results were obtained using daughter clones derived from a single 4T1 cell,i.e.,each daughter clone showed distinct drug-response pattern and gene expression profile,and they differed dramatically in drug sensitivity.Taking the IC50s or gene.expression as a variable to carry out a cluster analysis for the clones or the subclones,we found a number of categories do exist.2.Clones and daughter clones from Bcap37 cell line show heterogeneous phenotypes.The drug-response pattern diversity against 9 anticancer drugs existed among the clones and daughter clones from Bcap37 cell line.Unlike clones derived from 4T1,the difference of the fold change between clones in drug sensitivity was much smaller,e.g.,the biggest fold change was about 8(HCPT),whereas in 4T1 clones(S group)the biggest fold change was 273(MTX).It was noted that the absolute number of IC50s of Bcap37 and its clones were substantially higher than those of 4T1 and its clones,indicating the specific feature of individual cell lines.Accordingly,the gene expression profiles among the clones or daughter clones differed dramatically.The cluster analyses were also carried out for the clones or the subclones,and we found a number of categories do exist.3.Clones and daughter clones from K562 cell line show heterogeneous phenotypes.The clones from K562 cell line showed differences in the response to the targeted drug Imatinib,while the daughter clones did not.But,the drug-response pattern against the traditional anticancer drugs showed drastic differences either among the clones or daughter clones.The expression of protein c-Abl also differed significantly either among the clones or subclones.In addition,the expression profile of fusion gene BCR/ABL and other drug-resistance related 18 genes were also drastically different associated with drug sensitivity.Taking the IC50s or gene expression as a variable to carry out a cluster analysis for the clones or the subclones,we found a number of categories do exist.And the one-way analysis of variance of the expression of protein c-Abl showed the differences were statistical significant among the clones and subclones(P<0.05).ConclusionWe propose that a single cell can rapidly reconstitute a population of cells with high heterogeneity of drug response whose genetic makeups are the same.The acquisition of diverse drug-response phenotype does not require drug pretreatment,but entirely randomly.This is the first time to propose a heterogeneous drug response between progeny clones derived from a single cancer cell. |
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