Name: using microarrays to interrogate microenvironmental impact on cellular phenotypes in cancer
Uploaded: 2019-05-21T12:30:00
Description: Watch this Scientific Journal Video about Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer at JoVE.com

The work in this manuscript was supported by the NIH Common Fund Library of Network Cellular Signatures (LINCS) U54 grant HG008100 (J.W.G., L.M.H., and J.E.K) and NCI Cancer Systems Biology Consortium (CSBC) U54 CA209988grant (J.W.G., L.M.H., and J.E.K).

NOTE: An overview of the entire MEMA process, including estimated time, is outlined in the flow diagram shown in Figure 1. This protocol details the fabrication of MEMAs in 8-well plates. The protocol may be adapted for other plates or slides.
1. Preparation of Protein, Diluent, and Staining Buffers
<ol>
	<li>Equilibrate vials of ECMs, ligands, and cytokines to room temperature (RT) and briefly centrifuge. Add the appropriate volume of the appro...

<ol>
	<li>Hanahan, D., Coussens, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessories+to+the+crime:+functions+of+cells+recruited+to+the+tumor+microenvironment.">Accessories to the crime: functions of cells recruited to the tumor microenvironment.</a> Cancer Cell. 21 (3), 309-322 (2012).</li><li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumor+progression+and+metastasis.">Microenvironmental regulation of tumor progression and metastasis.</a> Nature Medicine. 19 (11), 1423-1437 (2013).</li><li>Hughes, C. S., Postovit, L. M., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+a+complex+protein+mixture+required+for+optimal+growth+of+cell+culture.">Matrigel: a complex protein mixture required for optimal growth of cell culture.</a> Proteomics. 10 (9), 1886-1890 (2010).</li><li>LaBarge, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mammary+progenitor+cell+fate+decisions+are+products+of+interactions+with+combinatorial+microenvironments.">Human mammary progenitor cell fate decisions are products of interactions with combinatorial microenvironments.</a> Integrative Biology (Cambridge). 1 (1), 70-79 (2009).</li><li>Watson, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironment-Mediated+Mechanisms+of+Resistance+to+HER2+Inhibitors+Differ+between+HER2++Breast+Cancer+Subtypes.">Microenvironment-Mediated Mechanisms of Resistance to HER2 Inhibitors Differ between HER2+ Breast Cancer Subtypes.</a> Cell Systems. 6 (3), 329-342 (2018).</li><li>Ranga, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+niche+microarrays+for+systems-level+analyses+of+cell+fate.">3D niche microarrays for systems-level analyses of cell fate.</a> Nature Communications. 5, 4324 (2014).</li><li>Malta, D. F. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+microarrays+to+study+inductive+signaling+for+endoderm+specification.">Extracellular matrix microarrays to study inductive signaling for endoderm specification.</a> Acta Biomater. 34, 30-40 (2016).</li><li>Kamentsky, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+structure,+function+and+compatibility+for+CellProfiler:+modular+high-throughput+image+analysis+software.">Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software.</a> Bioinformatics. 27 (8), 1179-1180 (2011).</li><li>Gagnon-Bartsch, J. A., Jacob, L., Speed, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removing+Unwanted+Variation+from+High+Dimensional+Data+with+Negative+Controls.">Removing Unwanted Variation from High Dimensional Data with Negative Controls.</a> University of California, Berkeley, Department of Statistics, University of California, Berkeley. , (2013).</li><li>Allan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OMERO:+flexible,+model-driven+data+management+for+experimental+biology.">OMERO: flexible, model-driven data management for experimental biology.</a> Nature Methods. 9 (3), 245-253 (2012).</li><li>Simian, M., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids:+A+historical+perspective+of+thinking+in+three+dimensions.">Organoids: A historical perspective of thinking in three dimensions.</a> Journal of Cell Biology. 216 (1), 31-40 (2017).</li><li>Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+differentiated+state+of+normal+and+malignant+cells+or+how+to+define+a+"normal"+cell+in+culture.">The differentiated state of normal and malignant cells or how to define a "normal" cell in culture.</a> International Review of Cytology. 70, 27-100 (1981).</li><li>Serban, M. A., Prestwich, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+extracellular+matrices:+solutions+for+the+puzzle.">Modular extracellular matrices: solutions for the puzzle.</a> Methods. 45 (1), 93-98 (2008).</li><li>Kaylan, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+lung+tumor+cell+drug+responses+as+a+function+of+matrix+context+and+genotype+using+cell+microarrays.">Mapping lung tumor cell drug responses as a function of matrix context and genotype using cell microarrays.</a> Integrative Biology (Cambridge). 8 (12), 1221-1231 (2016).</li><li>Lin, C. H., Jokela, T., Gray, J., LaBarge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+Microenvironments+Impose+a+Continuum+of+Cellular+Responses+to+a+Single+Pathway-Targeted+Anti-cancer+Compound.">Combinatorial Microenvironments Impose a Continuum of Cellular Responses to a Single Pathway-Targeted Anti-cancer Compound.</a> Cell Reports. 21 (2), 533-545 (2017).</li><li>Gjorevski, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designer+matrices+for+intestinal+stem+cell+and+organoid+culture.">Designer matrices for intestinal stem cell and organoid culture.</a> Nature. 539 (7630), 560-564 (2016).</li></ol>

The importance of &#34;dimensionality&#34;&#160;and context has been a motivating factor in the development of in vitro culture systems as tools in the characterization of cancer cells through their interaction with the microenvironment11, and the ability of in vitro culture systems to mimic the in vivo environment is a driving force behind the quest to improve those culture systems. In vitro systems, however, remain significant tools of cancer research precisely because of their ability to distil...

Culturing of cancer cell lines on plastic in two-dimensional (2D) monolayers remains one of the major workhorses for cancer researchers. However, the microenvironment is increasingly being recognized for its ability to impact cellular phenotypes. In cancer, the tumor microenvironment is known to influence multiple cellular behaviors, including growth, survival, invasion, and response to therapy1,2. Traditional monolayer cell cultures typically lack microenvironment influences, which has led to the development of more complex three-dimensional (3D) assays to grow cells, including commercially available purified basement membrane extracts. However, these purified matrices are typically complicated to use and suffer from technical problems such as batch variability3 and complex compositions3. As a result, it can be difficult to assign function to specific proteins that may be impacting cellular phenotypes3.
To address these limitations, we have developed the microenvironment microarray (MEMA) technology, which reduces the microenvironment down to simple combinations of extracellular matrix (ECM) and soluble growth factor proteins4,5. The MEMA platform enables identification of dominant microenvironmental factors that impact the behavior of cells. By using an array format, thousands of combinations of microenvironment factors can be assayed in a single experiment. The MEMA described here interrogates ~2,500 different unique microenvironment conditions. ECM proteins printed into well plates form growth pads upon which cells can be cultured. Soluble ligands are added to individual wells, creating unique combinatorial microenvironments (ECM + ligand) on each different spot to which the cells are exposed. Cells are cultured for several days, then fixed, stained, and imaged to assess cellular phenotypes as a result of exposure to these specific microenvironment combinations. Since the microenvironments are simple combinations, it is straightforward to identify proteins that drive major phenotypic changes in cells. MEMAs have been used successfully to identify factors that influence multiple cellular phenotypes, including those that drive cell fate decisions and response to therapy4,5,6,7. These responses can be validated in simple 2D experiments and can then be assessed under conditions that more fully recapitulate the complexity of the tumor microenvironment. The MEMA platform is highly adaptable to a variety of cell types and endpoints, provided that good phenotypic biomarkers are available.

using microarrays to interrogate microenvironmental impact on cellular phenotypes in cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

To simplify microenvironmental impacts on cell growth and proliferation and to identify conditions that promoted or inhibited cell growth and proliferation, the breast cancer cell line MCF7 was seeded on a set of eight 8-well MEMAs as described in the protocol. This assay exposed the cells to 48 different ECMs and 57 different ligands, for a total of 2736 combinatorial microenvironmental conditions. After 71 h in culture, cells were pulsed with EdU, fixed, permeablized, and stained with DAPI, the reaction for EdU detecti...

Watch this Scientific Journal Video about Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer at JoVE.com

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

Research

JoVE Journal

Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...