Biology subjects

Otto, O.

Publications and source records attributed to Otto, O..

4 recordsLinked to original sources

bioRxiv2018

Spheroid culture of mesenchymal stromal cells results in morpho-rheological properties appropriate for improved microcirculation

Human bone marrow mesenchymal stromal cells (MSCs) have been used in clinical trials for the treatment of systemic inflammatory diseases due to their regenerative and immunomodulatory properties. However, intravenous administration of MSCs is hampered by cell trapping within the pulmonary capillary networks. Here, we hypothesize that traditional twodimensional (2D) plastic-adherent cell expansion fails to result in appropriate morphorheological properties required for cell-circulation. To address this issue, we adapted a novel method to culture MSCs in non-adherent three-dimensional (3D) spheroids (mesenspheres). The biological properties of mesensphere-cultured MSCs remained identical to conventional 2D cultures. Morpho-rheological analyses revealed a smaller size and lower cell stiffness of mesensphere-derived MSCs compared to plastic-adherent MSCs, measured using real-time deformability cytometry (RT-DC) and atomic force microscopy, resulting in an increased ability to pass through micro-constrictions in an ex vivo microcirculation assay. This ability was confirmed in vivo by analysis of cell accumulation in various organ capillary networks after intravenous injection of mesensphere-derived MSCs in mouse. Our findings generally identify cellular morpho-rheological properties as attractive targets to improve microcirculation and specifically suggest mesensphere cultures as a promising approach for optimized MSC-based therapies.

biophysics↗

bioRxiv2017

Harnessing the adaptive potential of mechanoresponsive proteins to overwhelm pancreatic cancer dissemination and invasion

Metastatic disease is often characterized by altered cellular contractility and deformability, lending cells and groups of cells the flexibility to navigate through different microenvironments. This ability to change cell shape is driven in large part by the structural elements of the mechanobiome, which includes cytoskeletal proteins that sense and respond to mechanical stimuli. Here, we demonstrate that key mechanoresponsive proteins (those which accumulate in response to mechanical stress), specifically nonmuscle myosin IIA and IIC, -actinin 4, and filamin B, are highly upregulated in pancreatic ductal adenocarcinoma cancer (PDAC) and in patient-derived pancreatic cancer cell lines. Their less responsive sister paralogs (myosin IIB, -actinin 1, and filamin A) show a smaller dynamic range or disappear with PDAC progression. We demonstrate that these mechanoresponsive proteins directly impact cell mechanics using knock-down and overexpression cell lines. We further quantify the nonmuscle myosin II family members in patient-derived cell lines and identify a role for myosin IIC in the formation of transverse actin arcs in single cells and cortical actin belts in tissue spheroids. We harness the upregulation of myosin IIC and its impact of cytoskeletal architecture through the use of the mechanical modulator 4-hydroxyacetophenone (4-HAP), which increases myosin IIC assembly and stiffens cells. Here, 4-HAP decreases dissemination, induces cortical actin belts, and slows retrograde actin flow in spheroids. Finally, mice having undergone hemi-splenectomies with PDAC cells and then treated with 4-HAP have a reduction in liver metastases. Thus, increasing the activity of these mechanoresponsive proteins (in this case, by increasing myosin IIC assembly) to overwhelm the ability of cells to polarize and invade may be an effective strategy to improve the five-year survival rate of pancreatic cancer patients, currently hovering around 6%.

cancer biology↗

bioRxiv2017

Real-time fluorescence and deformability cytometry - flow cytometry goes mechanics

Cell mechanical characterization has recently approached the throughput of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we combine real-time 1D-imaging fluorescence and deformability cytometry (RT-FDC) to merge the two worlds in one instrument -- opening many new research avenues. We demonstrate its utility using sub-cellular fluorescence localization to identify mitotic cells and test for their mechanical changes in an RNAi screen.

biophysics↗

bioRxiv2017

Detection Of Human Disease Conditions By Single-Cell Morpho-Rheological Phenotyping Of Whole Blood

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of whole blood, without labeling, enrichment or separation, at rates of 1,000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in whole blood and adds a functional dimension to conventional blood analysis.

biophysics↗