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In-vivo Single Cell Transcript Analyses for Systems Modelling

To investigate the importance of minimal residual disease of BCR-ABL expressing cells chronic myelogenous leukemia (CML) was assessed using single cell molecular analytical approaches. CML is believed to occur as a consequence of the clonal expansion of leukemic stem cells and to be maintained by an expanding population of hematopoietic stem cells that have acquired a BCR-ABL fusion gene. Recent studies indicate that primitive CML cells are less responsive to tyrosine kinase inhibitors and are a reservoir for the emergence of tyrosine kinase resistant subclones. It also has been reported that BCR-ABL mediated cell adhesion may be involved in post-therapy residual disease of CML. The natural heterogeneity seen across both attached and unattached BCR-ABL expressing populations has been investigated. The study developed a means to utilize flow-assisted cell sorting of cell lines expressing BCR-ABL to derive individual attached and unattached cell sub-populations. Using a homogenous extraction procedure, cells individually flow sorted into microtitre plates were subjected to combinations of ABL and BCR-ABL RT qPCR, and also phosphorylated BCR-ABL protein was assessed using the proximity ligation assay and revealed the existence of lowly and highly BCR-ABL expressing cell line populations. The implications of this study will be discussed along with newly derived systems models and in-vivo sample procedures employing AFM.

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