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 appropriate RT buffer as indicated on the product data sheet. Follow manufacturer&#8217;s recommendation for stock concentrations. 
	NOTE: A full list of the ligands and ECMs with their stock and final concentrations are provided in Table 1 and Table 2. Both ligands and ECMs are typically used at the highest concentration of the range recommended by the manufacturer that elicits a biological effect in standard 2 day&#160;culture assays. Handle proteins gently and in biosafety cabinets under laminar flow to avoid contamination.</li>
	<li>Incubate vials with gentle rocking at RT for 1 h. Do not vortex proteins as this can cause them to denature.</li>
	<li>Aliquot proteins for long term storage so that all aliquots are single use only to avoid degradation with repeated freeze/thaw cycles. Store lyophilized proteins at -80 &#176;C (unless otherwise specified) until needed. Take care to collect all metadata for future reference, such as: (i) protein name, (ii) date prepared, (iii) lot/batch number, (iv) supplier, (v) catalog number, (vi) concentration, (vii) volume, and (viii) preparer.</li>
	<li>Prepare diluent buffer containing 20% (v/v) glycerol, 10 mM EDTA, 200 mM Tris-HCl, pH 7.2, and filter sterilize. Keep this buffer sterile and store at RT.</li>
	<li>Prepare staining buffer containing 2% (w/v) BSA, 1 mM MgCl2, and 0.02% NaN3 in phosphate-buffered saline (PBS). Filter and store at 4 &#176;C.</li>
</ol>
2. Preparation of an ECM Source Plate
<ol>
	<li>Remove aliquoted stocks of ECM proteins to be printed and thaw on ice. Record all lot numbers for metadata tracking.</li>
	<li>Flick tubes of thawed proteins gently to ensure proper resuspension and spin down in a centrifuge.</li>
	<li>Make ECM print mixtures (EPMs) and a fluorescent fiducial to be used by a liquid handling robot that will create the randomized 384-well source plates. 
	NOTE: The 384-well source plates will be used by a touch pin array printer to create the printed arrays in 8 well plates.
	<ol>
		<li>Label 1.5 mL microcentrifuge tubes for each EPM and the fiducial.</li>
		<li>Prepare each EPM by combining 125 &#181;L of diluent buffer (see step 1.4) with the appropriate volume of ECM stock and bring the mixture up to a total volume of 250 &#181;L with PBS. The final concentrations in each EPM tube will be 1x ECM protein, 5 mM EDTA, 10% glycerol, and 100 mM Tris.</li>
		<li>Prepare a fluorescent fiducial by dissolving it in the appropriate buffer specified by the manufacturer and transfer 250 &#181;L to a labelled fiducial tube.</li>
	</ol>
	</li>
</ol>
3. Creation of the Source Plate Using a Liquid Handler
<ol>
	<li>Design a 384-well plate layout that randomizes the positions of the ECMs and is optimized for the array printer pin head being used. Design the placement of the fiducial so that it will be printed in the row 1, column 1 position of each well to assist in array orientation. 
	NOTE: A total of 14&#8722;15 replicates of each ECM are used to ensure robust data. Include additional replicates of collagen or another ECM that yields robust attachment for assessment of uniformity of binding. The layout may need to utilize multiple 384-well plates depending on the number of ECMs of interest.</li>
	<li>Transfer EPM tubes to a liquid handler, keeping tubes at 4 &#176;C either with a cooled tube rack or by using a liquid handling robot located in a cold room.</li>
	<li>Using the liquid handler&#8217;s software, run a program to transfer 15 &#181;L of each EPM and the fiducial to the predesignated wells within the 384-well source plate(s).</li>
	<li>Pipet PBS into any unused wells to increase humidity and guard against desiccation during the printing process. 
	NOTE: See Figure 2 for an example of a 384-well source plate set that is optimized for a 4 x 7 pin head and includes a collagen I block and PBS.</li>
	<li>Seal plate(s) and keep at 4 &#176;C until ready to print.</li>
</ol>
4. Printing MEMAs Using an Array Printing Robot
NOTE: The following part of the protocol specifically describes the preparation and use of MEMA to investigate the impact of different microenvironment proteins on the growth and proliferation of MCF7 cells. However, the protocol can easily be adapted to use different ligands, ECMs, and cells to study other cell lines and endpoints of interest.
<ol>
	<li>Using a touch pin printer, print EPMs and fiducial spots into 8 well plates. Print multiple replicates of each ECM condition to ensure reproducibility. 
	NOTE: Other plate formats or slides can be used for printing, but buffer optimization may be required to achieve optimal spot formation.
	<ol>
		<li>Print the ECMs for the MEMA using 350 &#181;m diameter pins arranged in a 4 x 7 print head configuration. Print the arrays in the 8-well plates as 20 columns by 35 rows, for a total of ~700 spots. Larger arrays are possible in these plates but come with a trade-off of increased edge effects in both cell binding and staining.</li>
	</ol>
	</li>
	<li>After printing, store plates in a desiccator for a minimum of 3 days prior to use.</li>
</ol>
5. Creation of Ligand Treatment Plates
<ol>
	<li>Design a 96-well plate layout including ligands of interest. To facilitate treatment of many MEMA plates at once, design this plate with spacing that allows for the use of a multi-channel pipet with 4 spaced tips to transfer liquids between the wells of 8-well MEMAs and a 96-well plate. 
	NOTE: In this protocol, the full set of ligands listed in Table 2 are utilized.</li>
	<li>Thaw ligands on ice. Briefly flick and spin down each tube.</li>
	<li>Dilute ligands to a 200x working stock using the manufacturer&#8217;s recommended buffer (typically PBS).</li>
	<li>Pipet 10 &#181;L of each 200x ligand stock into the corresponding well within the 96-well plate.</li>
	<li>Seal and store plates at -20 &#176;C. 
	NOTE: Make ligand treatment plates in batches, capturing all metadata for downstream analysis.</li>
</ol>
6. Culturing Cells on MEMAs
<ol>
	<li>Block MEMAs for 20 min with 2 mL per well of non-fouling blocking buffer containing 1% non-fouling blocking agent (Table of Materials) in double-distilled water (ddH2O).</li>
	<li>Aspirate blocking buffer and triple rinse wells with PBS. To prevent desiccation, leave final volume of PBS in wells until ready for cell plating. 
	NOTE: It is extremely helpful to have two bench workers for cell culture steps on MEMAs. One bench worker can perform aspiration steps, while the second performs addition steps. It is recommended to use a 1 mL multichannel pipet with tips spaced to match the 8-well plate for pipetting and a Y-splitter with two Pasteur pipettes to aspirate multiple wells at once.</li>
	<li>Seed 2 x 105 MCF7 cells per well in 2 mL of Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) medium containing 10% fetal bovine serum (FBS). 
	NOTE: Prior to a full MEMA experiment, perform a cell titration experiment to optimize cell numbers such that MEMA spots have high cell numbers (but are not confluent) at the end of the desired experimental duration.</li>
	<li>After 2&#8722;18 h of adhesion, aspirate medium and replace with 2 mL of reduced-growth medium (DMEM with 0.1% FBS). 
	NOTE: Reduced serum (e.g., 0.1% FBS) or growth factor-depleted conditions can be used at this time to isolate the stimulatory impact of specific ligands.</li>
	<li>Thaw a ligand treatment plate on ice. Centrifuge thawed plate at 200 x g for 1 min.</li>
	<li>Transfer 200 &#181;L of medium from each well in the culture plate to the appropriate well in the treatment plate. Pipet up and down to mix ligand volume with medium and transfer this mixture back to the appropriate well in the MEMA plate.</li>
	<li>Lightly rock by hand and return MEMA plates to the incubator. Culture for the duration of the experiment in the presence of the ligand/ECM combination at 37 &#176;C and 5% CO2. 
	NOTE: A typical MEMA experiment runs for 72 h; longer duration experiments may require replacement of medium and re-treatment with ligand.</li>
	<li>Pulse MEMA wells at 71 h with 100x 5-ethynyl-2&#8217;-deoxyuridine (EdU) for a final concentration of 10 &#181;M. Incubate in experimental conditions with EdU for 1 h at 37 &#176;C and 5% CO2. 
	NOTE: Other live cell treatments may also be used at this time.</li>
</ol>
7. Fixing and Staining MEMAs
<ol>
	<li>After 72 h and any live cell treatments, aspirate wells. Fix MEMAs in 2 mL per well of 2% paraformaldehyde (PFA) for 15 min at RT.</li>
	<li>Aspirate PFA. Permeabilize with 2 mL per well of 0.1% nonionic surfactant&#160;for 15 min.</li>
	<li>Aspirate the nonionic surfactant and wash with 2 mL per well of PBS. Aspirate PBS. Wash with 2 mL of PBS with 0.05% polysorbate 20 (PBS-T). 
	NOTE: The MEMA surface is hydrophobic, and failure to wash with PBS-T before stain and antibody incubation will result in the formation of voids in wells during incubation steps and give rise to staining artifacts.</li>
	<li>Aspirate PBS-T. Add EdU detection reaction reagents. Incubate for 1 h at RT, rocking and protected from light. After 1 h incubation, quench reaction with the provided commercial quench buffer. 
	NOTE: EdU detection and staining/antibody steps may be performed in 1.5 mL per well to reduce cost.</li>
	<li>Aspirate the quench buffer and wash with PBS-T prior to incubating with stains or antibodies.</li>
	<li>Incubate MEMA wells with antibodies against histone H3K9me3 (1:1,000) and fibrillarin (1:400) in staining buffer containing 2% (w/v) bovine serum albumin (BSA), 1 mM MgCl2 and 0.02% NaN3 overnight at 4 &#176;C. 
	NOTE: Perform antibody titrations to determine optimal concentrations prior to using them on a full MEMA set.</li>
	<li>Following primary antibody or stain incubation, wash wells 2x with PBS and once with PBS-T.</li>
	<li>Add secondary antibodies (donkey anti-mouse IgG and donkey anti-rabbit IgG, both 1:300) and 0.5 &#181;g/mL 4&#8242; 6&#8208;diamidino&#8208;2&#8208;phenylindole (DAPI). Incubate for 1 h at RT in the dark.</li>
	<li>Wash wells 2x with 2 mL per well of PBS, leaving them in the final 2 mL PBS.</li>
	<li>Proceed to imaging or store stained MEMAs for later imaging in PBS at 4 &#176;C protected from light.</li>
</ol>
8. Imaging of MEMAs
<ol>
	<li>Image MEMA on an automated imaging system with appropriate fluorescent detection channels.</li>
	<li>Output resulting image data to an image management system. Segment cells and calculate intensity levels using CellProfiler8.</li>
</ol>
9. Data Analysis
NOTE: Data analysis consists of normalization, variation correction, and summarization of the raw CellProfiler derived data. In this instance, the R-environment with custom code is used to perform all the steps. However, any statistical environment or software program can be utilized to perform the equivalent actions. An example of the open source custom code for the R environment for analysis is available at: https://www.synapse.org/#!Synapse:syn2862345/wiki/72486.
<ol>
	<li>Preprocess and normalize the segmented image data.</li>
	<li>Determine spot cell count using the DAPI stained nuclei.</li>
	<li>Auto-gate EdU intensity to label cells as EdU+. Measure proliferation using the proportion of EdU+ cells in each spot.</li>
	<li>Median summarize cytoplasmic stains and nuclear morphology measurements on the spot level.</li>
	<li>Perform removal of unwanted variation (RUV) normalization on the data to improve data quality9. 
	NOTE: This approach is applied to each intensity and morphology signal independently as a matrix with arrays using the rows and spots as the columns as described previously9.</li>
	<li>Apply bivariate loess normalization to the RUV normalized residuals using the array row and array column as the independent variables to correct for spatial or intensity related effects.</li>
	<li>Once normalization is completed, median summarize the replicates for each microenvironment condition for reporting and further analysis.</li>
</ol>

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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...

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 ...

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5.5K Views.  Oregon Health and Science University. 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.

Video: Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

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 context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

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

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

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

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 result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

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 within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

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 (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

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 cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

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 eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

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

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor cells into neighboring tissue. Thus, invasion is a crucial step in metastasis, making the invasion process research and development of anti-metastatic drugs, highly significant. To address this demand, there is a need to develop 3D in vitro models which imitate the architecture of solid tumors and their microenvironment most closely to in vivo state on one hand, but at the same time be reproducible, robust and suitable for high yield and high content measurements. Currently, most invasion assays lean on sophisticated microfluidic technologies which are adequate for research but not for high volume drug screening. Other assays using plate-based devices with isolated individual spheroids in each well are material consuming and have low sample size per condition. The goal of the current protocol is to provide a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. We developed a 3D model for invasion assay based on HMCA imaging plate for the research of tumor invasion and anti-metastatic drug discovery. This device enables the production of numerous uniform spheroids per well (high sample size per condition) surrounded by ECM components, while continuously and simultaneously observing and measuring the spheroids at single-element resolution for medium throughput screening of anti-metastatic drugs. This platform is presented here by the production of HeLa and MCF7 spheroids for exemplifying single cell and collective invasion. We compare the influence of the ECM component hyaluronic acid (HA) on the invasive capacity of collagen surrounding HeLa spheroids. Finally, we introduce Fisetin (invasion inhibitor) to HeLa spheroids and nitric oxide (NO) (invasion activator) to MCF7 spheroids. The results are analyzed by in-house software which enables semi-automatic, simple and fast analysis which facilitates multi-parameter examination.

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

A HMCA-based imaging plate is presented for invasion assay performance. This plate facilitates the formation of three-dimensional (3D) tumor spheroids and the measurement of cancer cell invasion into the extracellular matrix (ECM). The invasion assay quantification is achieved by semi-automatic analysis.

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 disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

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 anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

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

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

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Chapters in this video

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

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Using the Dot Assay to Analyze Migration of Cell Sheets

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

Acellular and Cellular Lung Model to Study Tumor Metastasis

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

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

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

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

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions