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
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	<li>Equilibrate vials of ECMs, ligands, and cytokines to room temperature (RT) and briefly centrifuge. Add the appropriate volume of the appro...

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The authors have nothing to disclose.

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...

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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 ...

Studying the tumor microenvironment is difficult due to its complexity. We've developed microenvironment microarrays consisting of simple combinatorial microenvironments that allow the identification of major drivers of tumor cell phenotypes. The major advantage of the MEMA platform is that because the microenvironment conditions are all defined, it is straightforward to identify microenvironment factors that drive phenotypes of interest.The MEMA platform is widely applicable in other non-cancer systems, including primary cell strains and stem cells. There are a number of engineering tricks and steps that we have developed to speed the wet bench work and simplify the protocol. To begin, use a touch pin printer to print extracellular matrix print mixture in fiducial spots into eight-well plates.Use 350-micrometer diameter pins arranged in a four-times-seven print head configuration to print the extracellular matrix for the microenvironment microarrays. The collagen block is printed onto MEMAs as a grid. The PBS-filled wells provide extra humidity to prevent drying out of the extracellular matrix print mixture source plate.Print the arrays in the eight-well plates as 20 columns by 35 rows, for a total of 700 spots. After printing, store plates in a desiccator for a minimum of three days prior to use. First, design a 96-well plate layout with spacing that allows for the use of a multi-channel pipette with four spaced tips, so that the treatment of many MEMA plates can be done at once.Then retrieve the ligands from the freezer, and thaw in ice in a laminar flow hood. Briefly flick and spin down each tube. Use manufacturer's recommended buffer to dilute the ligands to a 200-times working stock.Pipette 10 microliters of each 200-times ligand stock into the corresponding well within the 96-well plate. Use sealing film to seal the plates and store them at minus-20 degrees Celsius. Make ligand treatment plates in batches, and capture all metadata for downstream analysis.Before culturing cells, block the MEMAs with two milliliters per well of nonfouling blocking buffer for 20 minutes. Use a Y-splitter with two Pasteur pipettes to aspirate blocking buffer in multiple wells at once, and then triple wash the wells with PBS. To prevent desiccation, leave the final volume of PBS in the wells until ready for cell plating.Prior to a full MEMA experiment, optimize MCF7 cell seeding concentration by performing a cell titration experiment. An ideal spot, as shown here, has sufficient cell numbers to extract data, but not so many that the spot is overly confluent. In the MEMA, aspirate the remaining PBS and seed MCF7 cells at 30, 000 to 35, 000 cells per well in two milliliters of seeding medium.Place the MEMA in an incubator at 37 degrees Celsius. After adhesion overnight in the incubator, aspirate the medium and replace with two milliliters of reduced growth medium to isolate the stimulatory impact of specific ligands. Then thaw a previously-frozen ligand treatment plate on ice.Centrifuge thawed plate at 200 times g for one minute. Transfer 200 microliters of medium from each well in the MEMA culture plate to the appropriate well in the ligand treatment plate. Pipette up and down to mix the ligand with the medium, and transfer this mixture back to the appropriate well in the MEMA culture plate.Lightly rock by hand and return the MEMA plates to the incubator to culture the ligand-ECM combination at 37 degrees Celsius and 5%carbon dioxide. After 71 hours, add 100 times EdU to the MEMA wells for a final concentration of 10 micromolar. Incubate it for another one hour at 37 degrees Celsius and 5%carbon dioxide.After the final incubation, aspirate the wells. Fix MEMAs in two milliliters per well of 2%PFA for 15 minutes at room temperature. Then aspirate the PFA from the wells.Permeabilize with two milliliters per well of 0.1%nonionic surfactant for 15 minutes. Aspirate the surfactant, and then wash the wells with two milliliters of PBS and PBS-T, sequentially. After that, add 1.5 milliliters of EdU detection reaction reagents to each well.Incubate for one hour at room temperature, rocking and protected from light. Then quench the reaction with 1.5 milliliter commercial quench buffer per well. Aspirate again, and wash with PBS-T prior to incubating with optimized concentrations of stains or antibodies.Incubate MEMA wells with primary antibodies in staining buffer, rocking at four degrees Celsius overnight. Following primary antibody incubation, wash wells with PBS followed by PBS-T. Add secondary antibodies in 0.5 micrograms per milliliter DAPI, diluted in staining buffer.Incubate, rocking for one hour at room temperature in the dark. Wash wells two times with two milliliters per well of PBS, and leave them in the final two milliliters of PBS. To image stained MEMA, place it on the microscope stage of an automated imaging system with appropriate fluorescent detection channels.Output resulting image data to an image management system. Segment cells and calculate intensity levels using CellProfiler. In this protocol, cell cycle profiles of binned DAPI intensity values versus cell counts from one eight-well plate indicate cells in G1 versus G2 cell cycle phase.This corresponds to two nDNA content in cells in early growth phase, and four nDNA content in cells about to undergo mitosis. The impact of the microenvironment of ligands and ECM on both cell number and EdU incorporation is shown. Red indicates higher cell number, and blue is lower cell number.Many of the effects are ligand-driven, as the ECM condition did not strongly impact cell number or EdU positivity. Natagen 1 is a clear exception, as the presence of this ECM molecule inhibits cell binding and growth of MCF7. Ligands such as FGF6 and neuregulin-1 alpha enhance cell number and have high rates of EdU incorporation, while ligands such as amphiregulin and neuregulin-1 SMDF inhibit cell binding and growth of cells.An example of MCF7 cells growing on a MEMA spot treated with neuregulin-1 alpha shows high rates of EdU incorporation, indicated by pink nuclei. In this image, green stain is cell mask, and blue is DAPI. Since a full MEMA experiment is expensive, it is very important to perform optimization steps, such as cell and serum titrations, prior to performing the full experiment.Once microenvironmental conditions of interest are identified using the MEMA platform, the specific ligands and extracellular matrix proteins can be studied in more depth using traditional 2D and 3D cell culture systems.

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5.5K Views.  Oregon Health and Science University. Studying the tumor microenvironment is difficult due to its complexity. We've developed microenvironment microarrays consisting of simple combinatorial microenvironments that allow the identification of major drivers of tumor cell phenotypes. The major advantage of the MEMA platform is that because the microenvironment conditions are all defined, it is straightforward to identify microenvironment factors that drive phenotypes of interest.The MEMA platform is widely applicable in other ...