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Midtveisevaluering

Midtveisevaluering - Stein Erik Gullaksen

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Abstract: Single cell signal transduction in myeloid leukaemia

The characterization of changes in intracellular signalling when Chronic Myeloid Leukaemia (CML) cells are challenged with therapeutic molecules or mechanical stimuli may shed new light on both disease biology and mechanism. Mass cytometry allows for the probing of more than 40 parameters per single cell, dramatically increasing the depth of data extracted from perturbed cells if compared to conventional flow cytometry. This permits not only a deep immunophenotyping when applicable, but also importantly probing a wide range of key intracellular phosphorylation targets.

In chronic myeloid leukaemia, we believe that an early and real-time evaluation of tyrosine kinase inhibition by measuring changes in signal transduction is needed to personalize treatment for patients. We have developed a panel for mass cytometry to investigate longitudinal peripheral blood samples of patients subjected to nilotinib in the ENEST1st trial (NCT01061177). We were able to reliably and consistently identify the major leukocyte subsets in healthy and diseased samples, and characterize changes in intracellular signalling. As a function of time and TKI treatment (patient sampling at diagnosis, after 3 hrs, 7 days, and 28 days), we saw a normalisation of the peripheral blood picture, indicating a complete haematological remission. In the hematopoietic progenitor cell compartment, statistically significant changes in phosphorylation of pSTAT3 Y705 were detected with treatment.

To investigate the physical force milieu of CML cells in vivo, we have successfully designed a high-throughput microfluidic chip device able to exert a uniform mechanical force on single cells, mimicking the passing of cells though narrow microvasculature. Here, single cells are forced to traverse an array of micro-pillars spaced by a distance less than the diameter of the CML cell line KCL-22. This pertains to cml as the uncontrolled proliferation of malignant cells during blast crisis increases the pressure in the bone marrow, which is in turn mechanically communicated to cells by mechanotransduction. Also, a common complication during blast crisis in the clinic is leukostasis, where cells mechanically obstruct vasculature during circulation. Currently, we are in the process of developing a mass cytometry panel able to measure a wide range of key signal transduction targets in both mechanotransduction and cell stress response.