Spatio-temporal modeling and live-cell imaging of proteolysis in the 4D microenvironment of breast cancer

With the increasing number of spaceflights, it is crucial to understand the changes occurring in human cells exposed to real microgravity (r-µg) conditions. We tested the effect of r-µg on MCF-7 breast cancer cells with the objective to investigate cytoskeletal alterations and early changes in the gene expression of factors belonging to the cytoskeleton, extracellular matrix, focal adhesion, and cytokines. In the Technische Experimente unter Schwerelosigkeit (TEXUS) 54 rocket mission, we had the opportunity to conduct our experiment during 6 min of r-µg and focused on cytoskeletal alterations of MCF-7 breast cancer cells expressing the Lifeact-GFP marker protein for the visualization of F-actin as well as the mCherry-tubulin fusion protein using the Fluorescence Microscopy Analysis System (FLUMIAS) for fast live-cell imaging under r-µg. Moreover, in a second mission we investigated changes in RNA transcription and morphology in breast cancer cells exposed to parabolic flight (PF) maneuvers (31st Deutsches Zentrum für Luft- und Raumfahrt (DLR) PF campaign). The MCF-7 cells showed a rearrangement of the F-actin and tubulin with holes, accumulations in the tubulin network, and the appearance of filopodia- and lamellipodia-like structures in the F-actin cytoskeleton shortly after the beginning of the r-µg period. PF maneuvers induced an early up-regulation of KRT8, RDX, TIMP1, CXCL8 mRNAs, and a down-regulation of VCL after the first parabola. E-cadherin protein was significantly reduced and is involved in cell adhesion processes, and plays a significant role in tumorigenesis. Changes in the E-cadherin protein synthesis can lead to tumor progression. Pathway analyses indicate that VCL protein has an activating effect on CDH1. In conclusion, live-cell imaging visualized similar changes as those occurring in thyroid cancer cells in r-µg. This result indicates the presence of a common mechanism of gravity perception and sensation.

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Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001334 ◽

2021 ◽

Vol 9 (1) ◽

pp. e001334

Author(s):

Jiri Eitler ◽

Natalie Wotschel ◽

Nicole Miller ◽

Laurent Boissel ◽

Hans G Klingemann ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Cell ◽

Nk Cells ◽

Cancer Cells ◽

Live Cell Imaging ◽

Cell Imaging ◽

Nk Cell ◽

Live Cell ◽

Cell Resistance ◽

Tumor Cell Resistance

BackgroundOn encountering a susceptible target, natural killer (NK) cells mediate cytotoxicity through highly regulated steps of directed degranulation. Cytotoxic granules converge at the microtubule organizing center and are polarized toward the immunological synapse (IS), followed by granule exocytosis. NK cell retargeting by chimeric antigen receptors (CARs) or mAbs represents a promising strategy for overcoming tumor cell resistance. However, little is known about the lytic granule dynamics of such retargeted NK cells toward NK-cell-resistant tumors.MethodsHere, we used spinning disk confocal microscopy for live-cell imaging to analyze granule-mediated NK cell cytotoxicity in ErbB2-targeted CAR-expressing NK-92 cells (NK-92/5.28.z) and high-affinity FcR transgenic NK-92 cells plus Herceptin toward ErbB2-positive breast cancer cells (MDA-MB-453), which are resistant to parental NK-92.ResultsUnmodified NK-92 cells cocultured with resistant cancer cells showed stable conjugate formation and granule clustering, but failed to polarize granules to the IS. In contrast, retargeting by CAR or FcR+Herceptin toward the MDA-MB-453 cells enabled granule polarization to the IS, resulting in highly effective cytotoxicity. We found that in NK-92 the phosphoinositide 3-kinase pathway was activated after contact with resistant MDA-MB-453, while phospholipase C-γ (PLCγ) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) were not activated. In contrast, retargeting by CAR or antibody-dependent cell-mediated cytotoxicity (ADCC) provided the missing PLCγ and MEK/ERK signals.ConclusionsThese observations suggest that NK cells can create conjugates with resistant cancer cells and respond by granule clustering, but the activation signals are insufficient to induce granule polarization and consequent release of lytic enzymes. Retargeting by CAR and/or the FcR/mAb (ADCC) axis provide the necessary signals, leading to granule polarization and thereby overcoming tumor cell resistance.Keywords: NK cells, NK-92, haNK, ADCC, Chimeric Antigen Receptor (CAR), breast cancer, cancer immunotherapy, live-cell imaging, granule polarization

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A live-cell imaging system for visualizing the transport of Marburg virus nucleocapsid-like structures

10.1101/658419 ◽

2019 ◽

Author(s):

Yuki Takamatsu ◽

Takeshi Noda ◽

Stephan Becker

Keyword(s):

Live Cell Imaging ◽

Ebola Virus ◽

Cell Imaging ◽

Temporal Dynamics ◽

Imaging System ◽

Marburg Virus ◽

Live Cell ◽

Infected Cells ◽

Spatio Temporal ◽

Living Organisms

AbstractLive-cell imaging is a powerful tool for visualization of the spatio-temporal dynamics of living organisms. Although this technique is utilized to visualize nucleocapsid transport in Marburg virus (MARV)- or Ebola virus-infected cells, the experiments require biosafety level-4 (BSL-4) laboratories, which are restricted to trained and authorized individuals. To overcome this limitation, we developed a live-cell imaging system to visualize MARV nucleocapsid-like structures using fluorescence-conjugated viral proteins, which can be conducted outside BSL-4 laboratories. Our experiments revealed that nucleocapsid-like structures have similar transport characteristics to nucleocapsids observed in MARV-infected cells. This system provides a safe platform to evaluate antiviral drugs that inhibit MARV nucleocapsid transport.

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Alterations of the Cytoskeleton in Breast Cancer Cells during Microgravity visualised by FLUMIAS Live-Cell Imaging

Frontiers in Physiology ◽

10.3389/conf.fphys.2018.26.00008 ◽

2018 ◽

Vol 9 ◽

Author(s):

Mohamed Nassef ◽

Sascha Kopp ◽

Daniela Melnik ◽

Marcus Krüger ◽

Markus Wehland ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Live Cell Imaging ◽

Breast Cancer Cells ◽

Cell Imaging ◽

Live Cell

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Anion selective chromogenic and fluorogenic chemosensor and its application in breast cancer live cell imaging

RSC Advances ◽

10.1039/c4ra04453c ◽

2014 ◽

Vol 4 (78) ◽

pp. 41446-41452 ◽

Cited By ~ 26

Author(s):

Darshna Sharma ◽

Anuradha Moirangthem ◽

Suban K. Sahoo ◽

Anupam Basu ◽

Sutapa M. Roy ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Live Cell Imaging ◽

Cell Imaging ◽

Breast Tumour ◽

Live Cell ◽

Cancer Cell Imaging ◽

Tumour Diagnosis

A novel anion selective chromogenic and fluorogenic chemosensor (L) bearing catechol and phenol groups was developed. The sensorLwas tested for live breast cancer cell imaging and can be applied to the breast tumour diagnosis.

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Long-term single-cell imaging and simulations of microtubules reveal principles behind wall patterning during proto-xylem development

Nature Communications ◽

10.1038/s41467-021-20894-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

René Schneider ◽

Kris van’t Klooster ◽

Kelsey L. Picard ◽

Jasper van der Gucht ◽

Taku Demura ◽

...

Keyword(s):

Cell Walls ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Quantitative Microscopy ◽

Microtubule Severing ◽

Single Cell Imaging ◽

Spatio Temporal

AbstractPlants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. Here, we establish long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to understand microtubule re-organization. We find that the re-organization requires local microtubule de-stabilization in band-interspersing gaps. Using microtubule simulations, we recapitulate the process in silico and predict that spatio-temporal control of microtubule nucleation is critical for pattern formation, which we confirm in vivo. By combining simulations and live-cell imaging we further explain how the xylem wall-deficient and microtubule-severing KATANIN contributes to microtubule and wall patterning. Hence, by combining quantitative microscopy and modelling we devise a framework to understand how microtubule re-organization supports wall patterning.

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Long-Term Live Cell Imaging of Breast Cancer Stem Cell Biomarkers Using Nanoparticle Labels

Optics in the Life Sciences ◽

10.1364/boda.2015.bt4a.5 ◽

2015 ◽

Author(s):

Maria Navas-Moreno ◽

Tatyana Chernenko ◽

Majid Mehrpouyen ◽

Ming Yan ◽

Demet Candas ◽

...

Keyword(s):

Breast Cancer ◽

Stem Cell ◽

Cancer Stem Cell ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Breast Cancer Stem Cell

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Live cell imaging of lytic granule motility in anti-ErbB2 CAR NK cells and FcR NK cells plus Herceptin towards ErbB2+ breast cancer cells

Cytotherapy ◽

10.1016/j.jcyt.2020.04.068 ◽

2020 ◽

Vol 22 (5) ◽

pp. S199

Author(s):

J. Eitler ◽

N. Wotschel ◽

H. Klingemann ◽

W.S. Wels ◽

T. Tonn

Keyword(s):

Breast Cancer ◽

Nk Cells ◽

Cancer Cells ◽

Live Cell Imaging ◽

Breast Cancer Cells ◽

Cell Imaging ◽

Live Cell ◽

Lytic Granule

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Fast, volumetric live-cell imaging using high-resolution light-field microscopy

10.1101/439315 ◽

2018 ◽

Author(s):

Haoyu Li ◽

Changliang Guo ◽

Deborah Kim-Holzapfel ◽

Weiyi Li ◽

Yelena Altshuller ◽

...

Keyword(s):

High Resolution ◽

Live Cell Imaging ◽

Light Field ◽

Cell Imaging ◽

Spatial Scales ◽

Functional Brain Imaging ◽

Live Cell ◽

Three Dimensions ◽

Acquisition Time ◽

Spatio Temporal

AbstractVisualizing diverse anatomical and functional traits that span many spatial scales with high spatio-temporal resolution provides insights into the fundamentals of living organisms. Light-field microscopy (LFM) has recently emerged as a scanning-free, scalable method that allows for high-speed, volumetric functional brain imaging. Given those promising applications at the tissue level, at its other extreme, this highly-scalable approach holds great potential for observing structures and dynamics in single-cell specimens. However, the challenge remains for current LFM to achieve subcellular level, near-diffraction-limited 3D spatial resolution. Here, we report high-resolution LFM (HR-LFM) for live-cell imaging with a resolution of 300-700 nm in all three dimensions, an imaging depth of several micrometers, and a volume acquisition time of milliseconds. We demonstrate the technique by imaging various cellular dynamics and structures and tracking single particles. The method may advance LFM as a particularly useful tool for understanding biological systems at multiple spatio-temporal levels.

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