Recent computational studies indicate that the molecular noise of a cellular process may be a rich source of information about process dynamics and parameters. However, accessing this source requires stochastic models that are usually difficult to analyze. Therefore, parameter estimation for stochastic systems using distribution measurements, as provided for instance by flow cytometry, currently remains limited to very small and simple systems. Here we propose a new method that makes use of low-order moments of the measured distribution and thereby keeps the essential parts of the provided information, while still staying applicable to systems of realistic size. We demonstrate how cell-to-cell variability can be incorporated into the analysis obviating the need for the ubiquitous assumption that the measurements stem from a homogeneous cell population. We demonstrate the method for a simple example of gene expression using synthetic data generated by stochastic simulation. Subsequently, we use time-lapsed flow cytometry data for the osmo-stress induced transcriptional response in budding yeast to calibrate a stochastic model, which is then used as a basis for predictions. Our results show that measurements of the mean and the variance can be enough to determine the model parameters, even if the measured distributions are not well-characterized by low-order moments only-e.g., if they are bimodal.extrinsic variability | high-osmolarity glycerol pathway | moment dynamics | parameter inference | stochastic kinetic models B uilding predictive computational models of intracellular reaction kinetics is still a dauntingly ill-posed task (1), characterized by low-dimensional experimental readouts of the hypothesized high-dimensional process. Single-cell technologies hold promise to partly alleviate this ill-posedness by exploiting the observed variability for the calibration of stochastic kinetic models (2, 3). The same technologies, however, also reveal that isogenic cells in a single population exhibit large cell-to-cell variability (4, 5). The variation can be shown to be a convolution of two sources, namely the intrinsic molecular noise and extrinsic factors that render single cells different even in the absence of molecular noise; in many cases, the latter was reported to dominate the former (4, 5). Extrinsic factors comprise difference in cell size, cell-cycle stage, expression capacity, local growth conditionsto name but a few (6, 7). Thus, although single-cell technology offers a way out of the predicament of ill-posedness, it requires new methods to deal properly with intrinsic and extrinsic variability. The effect of extrinsic variability on the dynamics of stochastic models is studied in refs. 7 and 8, whereas first attempts have been made to address the inverse problem of quantifying the extrinsic (9) and any additional intrinsic (10) components from measurements. Because the latter is based on path sampling, its applicability remains limited to small systems. Naturally, extrinsic variability is bypassed when...

Key Points Motility of resting CLL cells requires chemokine-mediated RhoA activation but is independent of Tiam1/Rac signals. Tiam1/Rac signals are indispensible for CLL cell proliferation and chemoresistance.

Anaplastic large cell lymphoma (ALCL) is a rare, aggressive, non-Hodgkin's lymphoma that is characterized by CD30 expression and disease onset in young patients. About half of ALCL patients bear the t(2;5)(p23;q35) translocation, which results in the formation of the nucleophosmin-anaplastic lymphoma tyrosine kinase (NPM–ALK) fusion protein (ALCL ALK+). However, little is known about the molecular features and tumour drivers in ALK-negative ALCL (ALCL ALK−), which is characterized by a worse prognosis. We found that ALCL ALK−, in contrast to ALCL ALK+, lymphomas display high miR-155 expression. Consistent with this, we observed an inverse correlation between miR-155 promoter methylation and miR-155 expression in ALCL. However, no direct effect of the ALK kinase on miR-155 levels was observed. Ago2 immunoprecipitation revealed miR-155 as the most abundant miRNA, and enrichment of target mRNAs C/EBPβ and SOCS1. To investigate its function, we over-expressed miR-155 in ALCL ALK+ cell lines and demonstrated reduced levels of C/EBPβ and SOCS1. In murine engraftment models of ALCL ALK−, we showed that anti-miR-155 mimics are able to reduce tumour growth. This goes hand-in-hand with increased levels of cleaved caspase-3 and high SOCS1 in these tumours, which leads to suppression of STAT3 signalling. Moreover, miR-155 induces IL-22 expression and suppresses the C/EBPβ target IL-8. These data suggest that miR-155 can act as a tumour driver in ALCL ALK− and blocking miR-155 could be therapeutically relevant. Original miRNA array data are to be found in the supplementary material (Table S1). © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Homing to distinct lymphoid organs enables chronic lymphocytic leukemia (CLL) cells to receive pro-survival and proliferative signals. Cytogenetic aberrations can significantly affect CLL cell compartmentalization. Trisomy 12 (tri12) defines a CLL subgroup with specific clinical features and increased levels of the negative prognostic marker CD49d, the α4-subunit of the integrin VLA-4, which is a key regulator of CLL cell homing to bone marrow (BM). Chemokine-induced inside-out VLA-4 activation, particularly via the CXCL12-CXCR4 axis, increases the arrest of various cell types on VCAM-1 presenting endothelium. Here, we demonstrate that high CD49d expression in tri12 CLL is accompanied by decreased CXCR4 expression. Dissecting functional consequences of these alterations, we observed that tri12 CLL cell homing to murine BM is not affected by CXCR4-CXCL12 blockage using AMD3100 or olaptesed pegol/NOX-A12. In line, CCL21-CCR7 rather than CXCL12-CXCR4 interactions triggered VLA-4-mediated arrests of tri12 CLL cells to VCAM-1 under blood flow conditions. Concordantly, in real-time kinetic analyses we found CCL21 but not CXCL12 being capable to induce inside-out VLA-4 conformational changes in this CLL subgroup. Our results provide novel insights into the peculiar clinico-biological behaviour of tri12 CLL and emphasize its specific chemokine and integrin utilization during pathophysiologically and therapeutically relevant interactions with the microenvironment.

Internal organs heal injuries with new connective tissue, but the cellular and molecular events of this process remain obscure. By tagging extracellular matrix around the mesothelium lining in mouse peritoneum, liver and cecum, here we show that preexisting matrix was transferred across organs into wounds in various injury models. Using proteomics, genetic lineage-tracing and selective injury in juxtaposed organs, we found that the tissue of origin for the transferred matrix likely dictated the scarring or regeneration of the healing tissue. Single-cell RNA sequencing and genetic and chemical screens indicated that the preexisting matrix was transferred by neutrophils dependent on the HSF–integrin AM/B2-kindlin3 cascade. Pharmacologic inhibition of this axis prevented matrix transfer and the formation of peritoneal adhesions. Matrix transfer was thus an early event of wound repair and provides a therapeutic window to dampen scaring across a range of conditions.