No abstract

The migratory properties of leukemic cells are commonly associated with their pathological potential and can significantly affect the disease progression. While the research in immunopathology mostly employed powerful indirect methods such as flow cytometry, these cells were rarely observed directly using live imaging microscopy. This is especially true for the malignant cells of the B-cell lineage, such as those originating from chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In this study, we employed open-source image analysis tools to automatically and quantitatively describe the amoeboid migration of four B-cell leukemic and lymphoma cell lines and primary CLL cells. To avoid the effect of the shear stress of the medium on these usually non-adherent cells, we have confined the cells using a modified under-agarose assay. Surprisingly, the behavior of tested cell lines differed substantially in terms of basal motility or response to chemokines and VCAM1 stimulation. Since casein kinase 1 (CK1) was reported as a regulator of B-cell migration and a promoter of CLL, we looked at the effects of CK1 inhibition in more detail. Migration analysis revealed that CK1 inhibition induced rapid negative effects on the migratory polarity of these cells, which was quantitatively and morphologically distinct from the effect of ROCK inhibition. We have set up an assay that visualizes endocytic vesicles in the uropod and facilitates morphological analysis. This assay hints that the effect of CK1 inhibition might be connected to defects in polarized intracellular transport. In summary, 1) we introduce and validate a pipeline for the imaging and quantitative assessment of the amoeboid migration of CLL/MCL cells, 2) we provide evidence that the assay is sensitive enough to mechanistically study migration defects identified by the transwell assay, and 3) we describe the polarity defects induced by inhibition or deletion of CK1ε.

The cyclic adenosine monophosphate (cAMP) signaling pathway is involved in various physiological and pathophysiological processes. Forskolin (FSK), a labdane diterpene well known as an activator of cAMP production, is suggested to possess significant immunomodulatory potential. However, the specific effects of elevated cAMP levels caused by the FSK-mediated activation of adenylate cyclase (AC) on T helper (Th) cell differentiation and functions are still unclear. We speculated that the increased levels of cAMP in Th cells affect the differentiation program of distinct Th populations differently, in particular the development of Th1, Th2, and Th17 subsets. Only minor changes in the expressions of isoforms of ACs and phosphodiesterases (PDE), enzymes responsible for the degradation of cAMP, were observed in differentiating human Th cells with prevailing ADCY1, ADCY3, ADCY7, and ADCY9 and PDE3B, PDE4A/B/D, PDE7A/B, and PDE8A isoforms. FSK mediated elevation in Th1-specific markers reinforcing the Th1 cell phenotype. The differentiation of Th2 was not altered by FSK, though cell metabolism was affected. In contrast, the Th17 immunophenotype was severely suppressed leading to the highly specific upregulation of the level of CXCL13. The causality between FSK-elicited cAMP production and the observed modulation of Th2 differentiation was proven by using cAMP inhibitor 2,5-dideoxyadenosine that reverted the FSK effects. Overall, an FSK-mediated cAMP increase has an effect on Th1, Th2 and Th17 differentiation and can contribute to the identification of novel therapeutic targets for the treatment of Th cell-related pathological processes.