Dataset Information

A Semi-automated and Scalable 3D Spheroid Assay to Study Neuroblast Migration.

ABSTRACT: The subventricular zone of the mammalian brain is the major source of adult born neurons. These neuroblasts normally migrate long distances to the olfactory bulbs but can be re-routed to locations of injury and promote neuroregeneration. Mechanistic understanding and pharmacological targets regulating neuroblast migration is sparse. Furthermore, lack of migration assays limits development of pharmaceutical interventions targeting neuroblast recruitment. We therefore developed a physiologically relevant 3D neuroblast spheroid migration assay that permits the investigation of large numbers of interventions. To verify the assay, 1,012 kinase inhibitors were screened for their effects on migration. Several induced significant increases or decreases in migration. MuSK and PIK3CB were selected as putative targets and their knockdown validated increased neuroblast migration. Thus, compounds identified through this assay system could be explored for their potential in augmenting neuroblast recruitment to sites of injury for neuroregeneration, or for decreasing malignant invasion.

SUBMITTER: Ducker M

PROVIDER: S-EPMC7486343 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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A Semi-automated and Scalable 3D Spheroid Assay to Study Neuroblast Migration.

Ducker Martin M Millar Valerie V Ebner Daniel D Szele Francis G FG

Stem cell reports 20200806 3

The subventricular zone of the mammalian brain is the major source of adult born neurons. These neuroblasts normally migrate long distances to the olfactory bulbs but can be re-routed to locations of injury and promote neuroregeneration. Mechanistic understanding and pharmacological targets regulating neuroblast migration is sparse. Furthermore, lack of migration assays limits development of pharmaceutical interventions targeting neuroblast recruitment. We therefore developed a physiologically r ...[more]

PMID: 32763162

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Project description:While it is known that cells with differential adhesion tend to segregate and preferentially sort, the physical forces governing sorting and invasion in heterogeneous tumors remain poorly understood. To investigate this, we tune matrix confinement, mimicking changes in the stiffness and confinement of the tumor microenvironment, to explore how physical confinement influences individual and collective cell migration in 3D spheroids. High levels of confinement lead to cell sorting while reducing matrix confinement triggers the collective fluidization of cell motion. Cell sorting, which depends on cell-cell adhesion, is crucial to this phenomenon. Burst-like migration does not occur for spheroids that have not undergone sorting, regardless of the degree of matrix confinement. Using computational Self-Propelled Voronoi modeling, we show that spheroid sorting and invasion into the matrix depend on the balance between cell-generated forces and matrix resistance. The findings support a model where matrix confinement modulates 3D spheroid sorting and unjamming in an adhesion-dependent manner, providing insights into the mechanisms of cell sorting and migration in the primary tumor and toward distant metastatic sites. STATEMENT OF SIGNIFICANCE: The mechanical properties of the tumor microenvironment significantly influence cancer cell migration within the primary tumor, yet how these properties affect intercellular interactions in heterogeneous tumors is not well understood. By utilizing calcium and calcium chelators, we dynamically alter collagen-alginate hydrogel stiffness and investigate tumor cell behavior within co-culture spheroids in response to varying degrees of matrix confinement. High confinement is found to trigger cell sorting while reducing confinement for sorted spheroids facilitates collective cell invasion. Notably, without prior sorting, spheroids do not exhibit burst-like migration, regardless of confinement levels. This work establishes that matrix confinement and intercellular adhesion regulate 3D spheroid dynamics, offering insights into cellular organization and migration within the primary tumor.

Project description:BackgroundThe haemostatic balance is an equilibrium of pro- and anticoagulant factors that work synergistically to prevent bleeding and thrombosis. As thrombin is the central enzyme in the coagulation pathway, it is desirable to measure thrombin generation (TG) in order to detect possible bleeding or thrombotic phenotypes, as well as to investigate the capacity of drugs affecting the formation of thrombin. By investigating the underlying processes of TG (i.e., prothrombin conversion and inactivation), additional information is collected about the dynamics of thrombin formation.ObjectivesTo obtain reference values for thrombin dynamics (TD) analysis in 112 healthy donors using an automated system for TG.MethodsTG was measured on the ST Genesia, fibrinogen on the Start, anti-thrombin (AT) on the STA R Max and α2Macroglobulin (α2M) with an in-house chromogenic assay.ResultsTG was measured using STG-BleedScreen, STG-ThromboScreen and STG-DrugScreen. The TG data was used as an input for TD analysis, in combination with plasma levels of AT, α2M and fibrinogen that were 113% (108-118%), 2.6 μM (2.2 μM-3.1 μM) and 2.9 g/L (2.6-3.2 g/L), respectively. The maximum rate of the prothrombinase complex (PCmax) and the total amount of prothrombin converted (PCtot) increased with increasing tissue factor (TF) concentration. PCtot increased from 902 to 988 nM, whereas PCmax increased from 172 to 508 nM/min. Thrombin (T)-AT and T-α2M complexes also increased with increasing TF concentration (i.e., from 860 to 955 nM and from 28 to 33 nm, respectively). PCtot, T-AT and T-α2M complex formation were strongly inhibited by addition of thrombomodulin (-44%, -43%, and -48%, respectively), whereas PCmax was affected less (-24%). PCtot, PCmax, T-AT, and T-α2M were higher in women using oral contraceptives (OC) compared to men/women without OC, and inhibition by thrombomodulin was also significantly less in women on OC (p < 0.05).ConclusionsTG measured on the ST Genesia can be used as an input for TD analysis. The data obtained can be used as reference values for future clinical studies as the balance between prothrombin conversion and thrombin inactivation has shown to be useful in several clinical settings.

Dataset Information

A Semi-automated and Scalable 3D Spheroid Assay to Study Neuroblast Migration.

Publications

A Semi-automated and Scalable 3D Spheroid Assay to Study Neuroblast Migration.

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