Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures

Suman, Rakesh; Smith, Gabrielle; Hazel, Kathryn E.A.; Kasprowicz, Richard; Coles, Mark; O’Toole, Peter; Chawla, Sangeeta

doi:10.1038/srep22032

Cited by 37 publications

(33 citation statements)

References 21 publications

(33 reference statements)

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“…Intravital imaging of microglial morphology enables the study of cellular changes in the native microenvironment in real-time and thus is a critical approach to study behavioural changes in response to injury or infection [14,36]. Although it is possible to culture microglia in vitro, when removed from the brain, their behaviour changes [50] and so intravital imaging provides a useful means to study microglial arborisation in living cells in situ. The microglial behaviour captured here using intravital imaging broadly agrees with previous reports [14,36], together with morphological data recorded from brain tissue sections using immunohistochemistry [45].…”

Section: Discussionmentioning

confidence: 99%

Metastatic breast cancer cells induce altered microglial morphology and electrical excitability in vivo

Simon

Yang

Marrison

et al. 2020

J Neuroinflammation

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Background: An emerging problem in the treatment of breast cancer is the increasing incidence of metastases to the brain. Metastatic brain tumours are incurable and can cause epileptic seizures and cognitive impairment, so better understanding of this niche, and the cellular mechanisms, is urgently required. Microglia are the resident brain macrophage population, becoming "activated" by neuronal injury, eliciting an inflammatory response. Microglia promote proliferation, angiogenesis and invasion in brain tumours and metastases. However, the mechanisms underlying microglial involvement appear complex and better models are required to improve understanding of function. Methods: Here, we sought to address this need by developing a model to study metastatic breast cancer cellmicroglial interactions using intravital imaging combined with ex vivo electrophysiology. We implanted an optical window on the parietal bone to facilitate observation of cellular behaviour in situ in the outer cortex of heterozygous Cx3cr1 GFP/+ mice. Results: We detected GFP-expressing microglia in Cx3cr1 GFP/+ mice up to 350 μm below the window without significant loss of resolution. When DsRed-expressing metastatic MDA-MB-231 breast cancer cells were implanted in Matrigel under the optical window, significant accumulation of activated microglia around invading tumour cells could be observed. This inflammatory response resulted in significant cortical disorganisation and aberrant spontaneously-occurring local field potential spike events around the metastatic site. Conclusions: These data suggest that peritumoral microglial activation and accumulation may play a critical role in local tissue changes underpinning aberrant cortical activity, which offers a possible mechanism for the disrupted cognitive performance and seizures seen in patients with metastatic breast cancer.

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Section: Discussionmentioning

confidence: 99%

Metastatic breast cancer cells induce altered microglial morphology and electrical excitability in vivo

Simon

Yang

Marrison

et al. 2020

J Neuroinflammation

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“…Cell migration was measured using wound healing assays (Yang et al, 2012). Label-free ptychographic microscopy was used to monitor motility in real time (Marrison, Raty, Marriott, & O'Toole, 2013;Suman et al, 2016). Images were acquired over 16 h at 9 min intervals using a Phasefocus VL-21 microscope with an 10X (0.25 NA) lens using 640 nm illumination and equipped with an environmental chamber maintaining the cells at 37 °C in 5% CO2.…”

Section: Cell Migration Assaymentioning

confidence: 99%

Voltage-dependent activation of Rac1 by Na_v1.5 channels promotes cell migration

Yang

James

Suman³

et al. 2019

Preprint

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Ion channels can regulate the plasma membrane potential (Vm) and cell migration as a result of altered ion flux. However, the mechanism by which Vm regulates motility remains unclear.Here, we show that the Nav1.5 sodium channel carries persistent inward Na + current which depolarizes the resting Vm at the timescale of minutes. This Nav1.5-dependent Vm depolarization increases Rac1 colocalization with phosphatidylserine, to which it is anchored at the leading edge of migrating cells, promoting Rac1 activation. A genetically-encoded FRET biosensor of Rac1 activation shows that depolarization-induced Rac1 activation results in acquisition of a motile phenotype. By identifying Nav1.5-mediated Vm depolarization as a regulator of Rac1 activation, we link ionic and electrical signaling at the plasma membrane to small GTPase-dependent cytoskeletal reorganization and cellular migration. We uncover a novel and unexpected mechanism for Rac1 activation, which fine tunes cell migration in response to ionic and/or electric field changes in the local microenvironment.

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“…The high contrast images generated by the system are label-free and exempt from focal drift, allowing extended time-lapse imaging [17][18][19] . The high-contrast nature of the images facilitates automated individual cell segmentation and tracking with the Cell Analysis Toolbox ® software, which outputs extensive and specific feature measurements for each cell.…”

Section: Image Capture Using Ptychography and Image Analysis Using Cementioning

confidence: 99%

Tumor heterogeneity and therapy resistance - implications for future treatments of prostate cancer

Frame¹,

Noble²,

Klein³

et al. 2017

JCMT

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Aim:To develop new therapies for prostate cancer, disease heterogeneity must be addressed. This includes patient variation, multi-focal disease, cellular heterogeneity, genomic changes and epigenetic modification. This requires more representative models to be used in more innovative ways. Methods: This study used a panel of cell lines and primary prostate epithelial cell cultures derived from patient tissue. Several assays were used; alamar blue, colony forming assays, γH2AX and Ki67 immunofluorescence and comet assays. Ptychographic quantitative phase imaging (QPI), a label-free imaging technique, combined with Cell Analysis Toolbox software, was implemented to carry out real-time analysis of cells and to retrieve morphological, kinetic and population data. Results: A combination of radiation and Vorinostat may be more effective than radiation alone. Primary prostate cancer stem-like cells are more resistant to etoposide than more differentiated cells. Analysis of QPI images showed that cell lines and primary cells differ in their size, motility and proliferation rate. A QPI signature was developed in order to identify two subpopulations of cells within a heterogeneous primary culture. Conclusion: Use of primary prostate epithelial cultures allows assessment of therapies whilst taking into account cellular heterogeneity. Analysis of rare cell populations and embracing novel techniques may ultimately lead to identifying and overcoming treatment resistance.

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Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures

Cited by 37 publications

References 21 publications

Metastatic breast cancer cells induce altered microglial morphology and electrical excitability in vivo

Metastatic breast cancer cells induce altered microglial morphology and electrical excitability in vivo

Voltage-dependent activation of Rac1 by Na_v1.5 channels promotes cell migration

Tumor heterogeneity and therapy resistance - implications for future treatments of prostate cancer

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Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures

Cited by 37 publications

References 21 publications

Metastatic breast cancer cells induce altered microglial morphology and electrical excitability in vivo

Metastatic breast cancer cells induce altered microglial morphology and electrical excitability in vivo

Voltage-dependent activation of Rac1 by Nav1.5 channels promotes cell migration

Tumor heterogeneity and therapy resistance - implications for future treatments of prostate cancer

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Product

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Voltage-dependent activation of Rac1 by Na_v1.5 channels promotes cell migration