PhagoKinetic Track Assay: Imaging and Analysis of Single Cell Migration

Fokkelman, Michiel; Roosmalen, Wies van; Rogkoti, Vasiliki-Maria; Dévédec, Sylvia E. Le; Geiger, Benjamin; Water, Bob van de

doi:10.21769/bioprotoc.1699

Cited by 4 publications

(5 citation statements)

References 3 publications

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“…1). Image analysis of single cell tracks was performed using PhagoTracker (32,33) and quantitative output data was normalized (robust Z-score) to mock transfected control cells using KNIME. High and low Zscores of individual parameters already showed the effect of siRNA knockdown on cell motility, i.e.…”

Section: A Systematic High Throughput Signaling Rnai Screen For Tnbc mentioning

confidence: 99%

“…PKT assays were performed as described before (32,33). Briefly, black 96-well µClear plates (Greiner Bio-One, Frickenhausen, Germany) were coated with 10 µg/ml fibronectin (Sigma-Aldrich, Zwijndrecht, The Netherlands) for 1 h at 37°C.…”

Section: Phagokinetic Track (Pkt) Assaymentioning

confidence: 99%

“…Procedures for transfection, medium refreshment and PKT assay were optimized for laboratory automation by a liquid-handling robot (BioMek FX, Beckman Coulter). (32). Migratory tracks without cells or with more than 1 cell were excluded during image analysis.…”

Section: Phagokinetic Track (Pkt) Assaymentioning

confidence: 99%

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Uncovering the signaling landscape controlling breast cancer cell migration identifies splicing factor PRPF4B as a metastasis driver

Koedoot

Fokkelman

Rogkoti

et al. 2018

Preprint

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Metastasis is the major cause of death in cancer patients and migration of cancer cells from the primary tumor to distant sites is the prerequisite of metastasis formation. Here we applied an imaging-based RNAi phenotypic cell migration screen using two highly migratory basal breast cancer cell lines (Hs578T and MDA-MB-231) to provide a repository for signaling determinants that functionally drive cancer cell migration. We screened ~4,200 individual target genes covering most cell signaling components and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, of which 43 genes were common denominators of cell migration. Interaction networks of candidate migratory modulators were in common with networks of different clinical breast cancer prognostic and metastasis classifiers. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF were amplified in human primary breast tumors and the expression was associated with metastasisfree survival. Depletion of PRPF4B, BUD31 and BPTF caused primarily down-regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B was essential for triple negative breast cancer cell migration and critical for breast cancer metastasis formation in vivo, making PRPF4B a candidate for further drug development. Our systematic phenotypic screen provides an important repository of candidate metastasis drug targets.

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Section: A Systematic High Throughput Signaling Rnai Screen For Tnbc mentioning

confidence: 99%

Section: Phagokinetic Track (Pkt) Assaymentioning

confidence: 99%

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Uncovering the signaling landscape controlling breast cancer cell migration identifies splicing factor PRPF4B as a metastasis driver

Koedoot

Fokkelman

Rogkoti

et al. 2018

Preprint

Self Cite

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“…An advanced microfluidic version of the scratch wound assay, in which the artificial wound is precisely created using a laminar flow of trypsin solution, was introduced to study the effect of shear stress on cell migration. Moreover, the phagokinetic track method is implanted to determine the direct motility of the cells and directional effects on cell movement [ 38 ].…”

Section: Angiogenesis Assaysmentioning

confidence: 99%

An Overview of In Vitro, In Vivo, and Computational Techniques for Cancer-Associated Angiogenesis Studies

Rahman

Tan

Othman

et al. 2020

BioMed Research International

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Angiogenesis is a crucial area in scientific research because it involves many important physiological and pathological processes. Indeed, angiogenesis is critical for normal physiological processes, including wound healing and embryonic development, as well as being a component of many disorders, such as rheumatoid arthritis, obesity, and diabetic retinopathies. Investigations of angiogenic mechanisms require assays that can activate the critical steps of angiogenesis as well as provide a tool for assessing the efficacy of therapeutic agents. Thus, angiogenesis assays are key tools for studying the mechanisms of angiogenesis and identifying the potential therapeutic strategies to modulate neovascularization. However, the regulation of angiogenesis is highly complex and not fully understood. Difficulties in assessing the regulators of angiogenic response have necessitated the development of an alternative approach. In this paper, we review the standard models for the study of tumor angiogenesis on the macroscopic scale that include in vitro, in vivo, and computational models. We also highlight the differences in several modeling approaches and describe key advances in understanding the computational models that contributed to the knowledge base of the field.

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“…However, there are still many AS events that have been associated with cancer progression which cannot be attributed to specific splice factors yet. The introduction of the RNAi libraries and more recently the CRISPR Cas9 technology together with the development of high-throughput screening technologies [105, 163–165] would allow systematic evaluation of spliceosomal components in multiple aspects of breast cancer progression, such as proliferation and migration. Future studies should apply these technologies to uncover the complete signaling landscape of splice factors in breast cancer progression that then can be used to develop specific splice factor inhibitors preventing metastasis formation and patient deaths.…”

Section: Future Perspectives and Conclusionmentioning

confidence: 99%

Splicing regulatory factors in breast cancer hallmarks and disease progression

et al. 2019

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By regulating transcript isoform expression levels, alternative splicing provides an additional layer of protein control. Recent studies show evidence that cancer cells use different splicing events to fulfill their requirements in order to develop, progress and metastasize. However, there has been less attention for the role of the complex catalyzing the complicated multistep splicing reaction: the spliceosome. The spliceosome consists of multiple sub-complexes in total comprising 244 proteins or splice factors and 5 associated RNA molecules. Here we discuss the role of splice factors in the oncogenic processes tumors cells need to fulfill their oncogenic properties (the so-called the hallmarks of cancer). Despite the fact that splice factors have been investigated only recently, they seem to play a prominent role in already five hallmarks of cancer: angiogenesis, resisting cell death, sustaining proliferation, deregulating cellular energetics and invasion and metastasis formation by affecting major signaling pathways such as epithelial-to-mesenchymal transition, the Warburg effect, DNA damage response and hormone receptor dependent proliferation. Moreover, we could relate expression of representative genes of four other hallmarks (enabling replicative mortality, genomic instability, avoiding immune destruction and evading growth suppression) to splice factor levels in human breast cancer tumors, suggesting that also these hallmarks could be regulated by splice factors. Since many splice factors are involved in multiple hallmarks of cancer, inhibiting splice factors might provide a new layer of oncogenic control and a powerful method to combat breast cancer progression.

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PhagoKinetic Track Assay: Imaging and Analysis of Single Cell Migration

Cited by 4 publications

References 3 publications

Uncovering the signaling landscape controlling breast cancer cell migration identifies splicing factor PRPF4B as a metastasis driver

Uncovering the signaling landscape controlling breast cancer cell migration identifies splicing factor PRPF4B as a metastasis driver

An Overview of In Vitro, In Vivo, and Computational Techniques for Cancer-Associated Angiogenesis Studies

Splicing regulatory factors in breast cancer hallmarks and disease progression

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