scispace - formally typeset
Search or ask a question
Journal Article•DOI•

In vitro cell migration and invasion assays.

Nina Kramer1, Angelika Walzl1, Christine Unger1, Margit Rosner1, Georg Krupitza1, Markus Hengstschläger1, Helmut Dolznig1 •
Medical University of Vienna1
01 Jan 2013-Mutation Research (Mutat Res)-Vol. 752, Iss: 1, pp 10-24
TL;DR: A concise summary of established migration/invasion assays described in the literature is provided, list advantages, limitations and drawbacks, give a tabular overview for convenience and depict the basic principles of the assays graphically.
Abstract: Determining the migratory and invasive capacity of tumor and stromal cells and clarifying the underlying mechanisms is most relevant for novel strategies in cancer diagnosis, prognosis, drug development and treatment. Here we shortly summarize the different modes of cell travelling and review in vitro methods, which can be used to evaluate migration and invasion. We provide a concise summary of established migration/invasion assays described in the literature, list advantages, limitations and drawbacks, give a tabular overview for convenience and depict the basic principles of the assays graphically. In many cases particular research problems and specific cell types do not leave a choice for a broad variety of usable assays. However, for most standard applications using adherent cells, based on our experience we suggest to use exclusion zone assays to evaluate migration/invasion. We substantiate our choice by demonstrating that the advantages outbalance the drawbacks e.g. the simple setup, the easy readout, the kinetic analysis, the evaluation of cell morphology and the feasibility to perform the assay with standard laboratory equipment. Finally, innovative 3D migration and invasion models including heterotypic cell interactions are discussed. These methods recapitulate the in vivo situation most closely. Results obtained with these assays have already shed new light on cancer cell spreading and potentially will uncover unknown mechanisms.
Citations
More filters
Journal Article•DOI•
U1 snRNP regulates cancer cell migration and invasion in vitro

[...]

Jung-Min Oh1, Christopher C. Venters1, Chao Di1, Anna Maria Pinto1, Lili Wan1, Ihab Younis1, Zhiqiang Cai1, Chie Arai1, Byung Ran So1, Jingqi Duan1, Gideon Dreyfuss1 •
University of Pennsylvania1
07 Jan 2020-Nature Communications
TL;DR: An unexpected role for U1 homeostasis (available U1 relative to transcription) in oncogenic and activated cell states is revealed, and U1 is suggested as a potential target for their modulation.
Abstract: Stimulated cells and cancer cells have widespread shortening of mRNA 3'-untranslated regions (3'UTRs) and switches to shorter mRNA isoforms due to usage of more proximal polyadenylation signals (PASs) in introns and last exons. U1 snRNP (U1), vertebrates' most abundant non-coding (spliceosomal) small nuclear RNA, silences proximal PASs and its inhibition with antisense morpholino oligonucleotides (U1 AMO) triggers widespread premature transcription termination and mRNA shortening. Here we show that low U1 AMO doses increase cancer cells' migration and invasion in vitro by up to 500%, whereas U1 over-expression has the opposite effect. In addition to 3'UTR length, numerous transcriptome changes that could contribute to this phenotype are observed, including alternative splicing, and mRNA expression levels of proto-oncogenes and tumor suppressors. These findings reveal an unexpected role for U1 homeostasis (available U1 relative to transcription) in oncogenic and activated cell states, and suggest U1 as a potential target for their modulation.

3,432 citations

Posted Content•DOI•
U1 snRNP regulates cancer cell migration and invasion

[...]

Jung-Min Oh1, Christopher C. Venters1, Chao Di1, Anna Maria Pinto1, Lili Wan1, Ihab Younis1, Zhiqiang Cai1, Chie Arai1, Byung Ran So1, Gideon Dreyfuss1 •
University of Pennsylvania1
09 Aug 2019-bioRxiv
TL;DR: It is shown that U1 AMO also modulates cancer cells’ phenotype, dose-dependently increasing migration and invasion in vitro by up to 500%, whereas U1 over-expression has the opposite effect.
Abstract: Stimulated cells and cancer cells have widespread shortening of mRNA 3’-utranslated regions (3’UTRs) and switches to shorter mRNA isoforms due to usage of more proximal polyadenylation signals (PASs) in the last exon and in introns. U1 snRNA (U1), vertebrates’ most abundant non-coding (spliceosomal) small nuclear RNA, silences proximal PASs and its inhibition with antisense morpholino oligonucleotides (U1 AMO) triggers widespread mRNA shortening. Here we show that U1 AMO also modulates cancer cells’ phenotype, dose-dependently increasing migration and invasion in vitro by up to 500%, whereas U1 over-expression has the opposite effect. In addition to 3’UTR length, numerous transcriptome changes that could contribute to this phenotype are observed, including alternative splicing, and mRNA expression levels of proto-oncogenes and tumor suppressors. These findings reveal an unexpected link between U1 regulation and oncogenic and activated cell states, and suggest U1 as a potential target for their modulation.

1,660 citations

Journal Article•DOI•
In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform

[...]

Moriah E. Katt1, Amanda L. Placone1, Andrew D. Wong1, Zinnia S. Xu1, Peter C. Searson1 •
Johns Hopkins University1
12 Feb 2016-Frontiers in Bioengineering and Biotechnology
TL;DR: A wide range of current in vitro tumor models are reviewed and their advantages, disadvantages, and suitability in modeling specific aspects of the metastatic cascade and drug treatment are summarized.
Abstract: In vitro tumor models have provided important tools for cancer research and serve as low-cost screening platforms for drug therapies; however, cancer recurrence remains largely unchecked due to metastasis, which is the cause of the majority of cancer related deaths. The need for an improved understanding of the progression and treatment of cancer has pushed for increased accuracy and physiological relevance of in vitro tumor models. As a result, in vitro tumor models have concurrently increased in complexity and their output parameters further diversified, since these models have progressed beyond simple proliferation, invasion, and cytotoxicity screens and have begun recapitulating critical steps in the metastatic cascade, such as intravasation, extravasation, angiogenesis, matrix remodeling, and tumor cell dormancy. Advances in tumor cell biology, 3D cell culture, tissue engineering, biomaterials, microfabrication, and microfluidics have enabled rapid development of new in vitro tumor models that often incorporate multiple cell types, extracellular matrix materials, and spatial and temporal introduction of soluble factors. Other innovations include the incorporation of perfusable microvessels to simulate the tumor vasculature and model intravasation and extravasation. The drive towards precision medicine has increased interest in adapting in vitro tumor models for patient-specific therapies, clinical management, and assessment of metastatic potential. Here, we review the wide range of current in vitro tumor models and summarize their advantages, disadvantages, and suitability in modeling specific aspects of the metastatic cascade and drug treatment.

548 citations

Cites background or methods from "In vitro cell migration and invasio..."

  • ...ECM layers can range in thickness up to 1 mm (Kramer et al., 2013)....

    [...]

  • ...Migration, the simplest variation, involves seeding cancer cells directly on a porous membrane, while invasion assays involve seeding cells on a layer of ECM material on top of the porous membrane (Kramer et al., 2013)....

    [...]

  • ...Transwell-based assays are usually combined with a chemoattractant gradient, typically media with 10% FBS in the bottom chamber and media with ≤1% FBS in the upper chamber (Marshall, 2011; Kramer et al., 2013)....

    [...]

  • ...introduction Transwell-based assays are widely used to assess cancer cell migration and invasion (Figure 1) (Hulkower and Herber, 2011; Marshall, 2011; Kramer et al., 2013)....

    [...]

Journal Article•DOI•
Mutant KRAS is a druggable target for pancreatic cancer

[...]

Elina Zorde Khvalevsky, Racheli Gabai, Itzhak Haim Rachmut, Elad Horwitz, Zivia Brunschwig, Ariel Orbach, Adva Shemi, Talia Golan1, Abraham J. Domb2, Eylon Yavin2, Hilla Giladi2, Ludmila Rivkin2, Alina Simerzin2, Rami Eliakim1, Abed Khalaileh, Ayala Hubert, Maor Lahav1, Yael Kopelman, Eran Goldin3, A Dancour3, Yael Hants2, Sagit Arbel-Alon2, Rinat Abramovitch2, Amotz Shemi, Eithan Galun2 •
Sheba Medical Center1, Hebrew University of Jerusalem2, Shaare Zedek Medical Center3
17 Dec 2013-Proceedings of the National Academy of Sciences of the United States of America
TL;DR: A reproducible and safe delivery platform based on a miniature biodegradable polymeric matrix, for the controlled and prolonged delivery of siRNA that overcomes the major obstacles of toxicity and organ accessibility is reported.
Abstract: Pancreatic ductal adenocarcinoma (PDA) represents an unmet therapeutic challenge PDA is addicted to the activity of the mutated KRAS oncogene which is considered so far an undruggable therapeutic target We propose an approach to target KRAS effectively in patients using RNA interference To meet this challenge, we have developed a local prolonged siRNA delivery system (Local Drug EluteR, LODER) shedding siRNA against the mutated KRAS (siG12D LODER) The siG12D LODER was assessed for its structural, release, and delivery properties in vitro and in vivo The effect of the siG12D LODER on tumor growth was assessed in sc and orthotopic mouse models KRAS silencing effect was further assessed on the KRAS downstream signaling pathway The LODER-encapsulated siRNA was stable and active in vivo for 155 d Treatment of PDA cells with siG12D LODER resulted in a significant decrease in KRAS levels, leading to inhibition of proliferation and epithelial–mesenchymal transition In vivo, siG12D LODER impeded the growth of human pancreatic tumor cells and prolonged mouse survival We report a reproducible and safe delivery platform based on a miniature biodegradable polymeric matrix, for the controlled and prolonged delivery of siRNA This technology provides the following advantages: (i) siRNA is protected from degradation; (ii) the siRNA is slowly released locally within the tumor for prolonged periods; and (iii) the siG12D LODER elicits a therapeutic effect, thereby demonstrating that mutated KRAS is indeed a druggable target

250 citations

Cites methods from "In vitro cell migration and invasio..."

  • ...We analyzed the migration characteristics of Panc1 cells following transfection with siG12D or scrambled siRNA, using the scratch and Transwell-migration assays (17, 18)....

    [...]

Journal Article•DOI•
In vitro Cell Migration, Invasion, and Adhesion Assays: From Cell Imaging to Data Analysis.

[...]

Jordi Pijuan1, Carla Barceló1, David Moreno2, Oscar Maiques1, Pol Sisó1, Rosa M. Martí3, Anna Macià1, Anaïs Panosa1 •
University of Lleida1, Spanish National Research Council2, Hospital Universitari Arnau de Vilanova3
14 Jun 2019-Frontiers in Cell and Developmental Biology
TL;DR: Four in vitro protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification are presented, providing orthogonal information that can be used either individually or collectively in many different experimental setups.
Abstract: Cell migration is a key procedure involved in many biological processes including embryological development, tissue formation, immune defense or inflammation, and cancer progression. How physical, chemical, and molecular aspects can affect cell motility is a challenge to understand migratory cells behavior. In vitro assays are excellent approaches to extrapolate to in vivo situations and study live cells behavior. Here we present four in vitro protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on two-dimensional wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and three-dimensional spreading and transwell assays. All together, they cover different phenotypes and hallmarks of cell motility and adhesion, providing orthogonal information that can be used either individually or collectively in many different experimental setups. These optimized protocols will facilitate physiological and cellular characterization of these processes, which may be used for fast screening of specific therapeutic cancer drugs for migratory function, novel strategies in cancer diagnosis, and for assaying new molecules involved in adhesion and invasion metastatic properties of cancer cells.

246 citations

Cites background from "In vitro cell migration and invasio..."

  • ...Metastasis is the main cause of cancer lethality, 90% of deaths from solid tumors can be ascribed to metastatic dissemination (Kramer et al., 2013), and understanding the multi-step migration, adhesion and invasion progress, represents an enormous challenge in cancer treatment (Anderson et al....

    [...]

References
More filters
Journal Article•DOI•
The hallmarks of cancer.

[...]

Douglas Hanahan1, Robert A. Weinberg2•
University of California, San Francisco1, Massachusetts Institute of Technology2
07 Jan 2000-Cell
TL;DR: This work has been supported by the Department of the Army and the National Institutes of Health, and the author acknowledges the support and encouragement of the National Cancer Institute.

28,811 citations

"In vitro cell migration and invasio..." refers background in this paper

  • ...These are for example based on multi-step progression [6], ‘‘intrinsic’’ metastasis [7] or metastatic dissemination [8] although no clear mechanisms were defined....

    [...]

Journal Article•DOI•
The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells

[...]

Sendurai A. Mani1, Wenjun Guo1, Mai Jing Liao1, Elinor Ng Eaton1, Ayyakkannu Ayyanan2, Alicia Y. Zhou1, Mary W. Brooks1, Ferenc Reinhard1, Cheng Cheng Zhang1, Michail Shipitsin3, Lauren L. Campbell3, Kornelia Polyak3, Cathrin Brisken2, Jing Yang4, Robert A. Weinberg1 •
Massachusetts Institute of Technology1, École Polytechnique Fédérale de Lausanne2, Harvard University3, University of California, San Diego4
16 May 2008-Cell
TL;DR: It is reported that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers, and it is shown that those cells have an increased ability to form mammospheres, a property associated with mammARY epithelial stem cells.

8,052 citations

"In vitro cell migration and invasio..." refers background in this paper

  • ...also discussed to increase the proportion of cancer stem cells [23,24]....

    [...]

Journal Article•DOI•
Epithelial–mesenchymal transitions in tumour progression

[...]

Jean Paul Thiery1•
Curie Institute1
01 Jun 2002-Nature Reviews Cancer
TL;DR: Epithelial–mesenchymal transition provides a new basis for understanding the progression of carcinoma towards dedifferentiated and more malignant states.
Abstract: Without epithelial–mesenchymal transitions, in which polarized epithelial cells are converted into motile cells, multicellular organisms would be incapable of getting past the blastula stage of embryonic development. However, this important developmental programme has a more sinister role in tumour progression. Epithelial–mesenchymal transition provides a new basis for understanding the progression of carcinoma towards dedifferentiated and more malignant states.

6,362 citations

"In vitro cell migration and invasio..." refers background in this paper

  • ...Fibroblasts, sarcoma cells and highly dedifferentiated tumor cells, which have undergone epithelial to mesenchymal transition (EMT) [22], use this mode of motility....

    [...]

  • ...We employed this model to analyze EMT in hepatocellular carcinoma [136] and to clarify the function of hVps37A in ovarian carcinoma [137]....

    [...]

  • ...Contents lists available at SciVerse ScienceDirect Mutation Research/Reviews in Mutation Research jo u rn al h om epag e: ww w.els evier .c o m/lo cat e/ rev iew sm r Co mm un i ty ad dr es s : w ww.els evier . co m/lo c ate /mu t r es Abbreviations: ECM, extracellular matrix; BME, basal membrane extract; EMT, epithelial mesenchymal transition; PET, polyethylenterephthalat; PC, polycarbonat; ECIS, electric cell–substrate impedance sensing; EHS, Engelbreth–Holm–Swarm tumor; HTS, high throughput screening; MMP, matrix metalloprotease; CAF, cancer associated fibroblast; LMAT, leukocyte migration agarose technique; FFPE, formalin fixed paraffin embedded; EC, endothelial cell; SEM, scanning electron microscopy....

    [...]

  • ...Epithelial cells could therefore dedifferentiate via EMT to cancer stem cells....

    [...]

  • ...EMT has not only been proposed to play a key role in the acquisition of a migratory or invasive phenotype of many carcinoma cells, but is also discussed to increase the proportion of cancer stem cells [23,24]....

    [...]

Journal Article•DOI•
Cell migration: integrating signals from front to back.

[...]

Anne J. Ridley1, Martin A. Schwartz2, Keith Burridge3, Richard A. Firtel4, Mark H. Ginsberg5, Gary G. Borisy6, J. Thomas Parsons2, Alan Rick Horwitz2 •
Ludwig Institute for Cancer Research1, University of Virginia2, University of North Carolina at Chapel Hill3, University of California, San Diego4, Scripps Research Institute5, Northwestern University6
05 Dec 2003-Science
TL;DR: The mechanisms underlying the major steps of migration and the signaling pathways that regulate them are described, and recent advances investigating the nature of polarity in migrating cells and the pathways that establish it are outlined.
Abstract: Cell migration is a highly integrated multistep process that orchestrates embryonic morphogenesis; contributes to tissue repair and regeneration; and drives disease progression in cancer, mental retardation, atherosclerosis, and arthritis. The migrating cell is highly polarized with complex regulatory pathways that spatially and temporally integrate its component processes. This review describes the mechanisms underlying the major steps of migration and the signaling pathways that regulate them, and outlines recent advances investigating the nature of polarity in migrating cells and the pathways that establish it.

4,839 citations

"In vitro cell migration and invasio..." refers background in this paper

  • ...These cells display directional polarity, with a leading edge at the front and a lagging edge at the back of the cell body [14]....

    [...]

Journal Article•DOI•
Cancer Metastasis: Building a Framework

[...]

Gaorav P. Gupta1, Joan Massagué1•
Memorial Sloan Kettering Cancer Center1
17 Nov 2006-Cell
TL;DR: Understanding of the origins and nature of cancer metastasis and the selection of traits that are advantageous to cancer cells is promoted.

3,863 citations

"In vitro cell migration and invasio..." refers background in this paper

  • ...Metastasis involves multiple processes such as infiltrative growth through the extracellular matrix (ECM), cell migration through blood or lymph vessels and rise of distant colonies [9]....

    [...]

Related Papers (5)
In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro

[...]

01 Feb 2007-Nature Protocols
Chun Chi Liang, Ann Y. Park, Jun-Lin Guan
Hallmarks of cancer: the next generation.

[...]

04 Mar 2011-Cell
Douglas Hanahan, Robert A. Weinberg
Tumour-cell invasion and migration: diversity and escape mechanisms

[...]

01 May 2003-Nature Reviews Cancer
Peter Friedl, Katarina Wolf
The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.

[...]

01 Mar 1962-Journal of Experimental Medicine
Stephen Boyden
Collective cell migration in morphogenesis, regeneration and cancer

[...]

01 Jul 2009-Nature Reviews Molecular Cell Biology
Peter Friedl, Darren Gilmour
Trending Questions (1)
What are the investigation methods in cancer research in vitro and what are the main questions so far?

The paper discusses various in vitro methods for investigating cell migration and invasion in cancer research, but it does not specifically mention the main questions addressed in this field.

See answers from 5 other papers