Funding was provided by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust (C-076) (L.M.S.); University of Illinois at Chicago Startup Funds (L.M.S.); Grant 543296 from the Ovarian Cancer Research Fund Alliance (M.D.); and UG3 ES029073 (J.E.B.) and by the National Center for Advancing Translational Sciences, National Institute of Health, through grant UL1TR002003 (JEB &#38; LMS).

All animal procedures were in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Institutional Animal Use and Care (IACUC) committee at the University of Illinois at Chicago.
1. Preparation of Reagents
<ol>
	<li>Maintain murine oviductal epithelium (MOE) cells at 37&#176;C with 5% CO2 in a humidified incubator in alpha Minimum Essential Medium (&#945;MEM) media supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 mg/mL insulin, transferrin, selenium (ITS), 1.8 ng/mL epidermal growth factor (EGF), 100 U/mL penicillin-streptomycin, 1 mg/mL gentamycin, and 18.2 ng/mL estradiol-17&#946;. Pass the cells every 3&#8211;4 days, ensuring there are enough cells on the same day the mice will be sacrificed.</li>
	<li>Prepare 2% agarose by mixing 1 g of low-melting agarose with 50 mL of distilled water. Autoclave the agarose, allow to cool, and then aliquot (1 mL) in 2 mL tubes (see Table of Materials) before agarose solidifies. Agarose can be stored at -20 &#176;C indefinitely.</li>
	<li>Prepare at least 2 mL of 1x Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) media.</li>
	<li>For the matrix for MALDI-TOF MS, prepare 10 mL of 5 mg/mL 1:1 alpha-cyano-4-hyroxycinnamic acid (CHCA):dihydroxybenzoic acid (DHB) (see Table of Materials) in 90:10 ACN:H2O + 0.1% trifluoroacetic acid (TFA). Sonicate solution until matrix is dissolved.</li>
</ol>
2. Mouse Colony and Ovary Removal
<ol>
	<li>Maintain breeding pairs of CD-1 mice using standard housing procedures. Near the day of parturition, check the mice every day so that the age of the pups is known.</li>
	<li>Euthanize pups at 16&#8211;18 days of age by CO2 inhalation per NIH guidelines. Deliver CO2 (100%) at a flow rate of 10%&#8211;20% chamber volume per minute and continue for two minutes after respiration had stopped. Confirm euthanasia by cervical dislocation.</li>
	<li>Disinfect all surgical equipment by submerging in 70% ethanol. Warm Leibovitz&#8217;s L-15 media with 1x penicillin-streptomycin to 37 &#176;C.</li>
	<li>Wet the abdominal surface with 70% ethanol to disinfect the area and minimize contamination with hair. Grasp the skin covering the abdominal wall using blunt forceps and use surgical scissors to make a large V-shape cut through the skin and abdominal wall, exposing the internal organs.</li>
	<li>Move the internal organs to the side using forceps. Individually grab each uterine horn with fine forceps and lift slightly.</li>
	<li>Locate the ovary, which will be at the end of the uterine horn, immediately below the kidney. Use surgical scissors to dissect the ovary free of connective tissue. Then, cut the ovarian horn in half.</li>
	<li>Move the ovary, oviduct, and approximately one half of each uterine horn to pre-warmed Leibovitz&#8217;s L-15 media.</li>
	<li>Under a dissecting microscope carefully move each ovary from the surrounding bursa, freeing it from the oviduct, bursa, and any adipose tissue. Move each ovary to a new dish of Leibovitz&#8217;s L-15 media.</li>
	<li>Cut each ovary in half axially. Keep the ovarian pieces (now called explants) kept at 37 &#176;C until plating of agarose.</li>
</ol>
3. Setting Up and Incubating the ITO-treated Slide for Cocultures
<ol>
	<li>Undivided cocultures
	<ol>
		<li>Liquify the agarose at 70 &#176;C on a hot plate.</li>
		<li>Place the 8-well divider on top of the indium tinoxide (ITO)-treated slide (Figure 1A). The rubber bottom on the divider aids in adhesion to the slide, but make sure to apply continuous gentle downward pressure during agarose plating to ensure no leaking or mixing between wells.</li>
		<li>Collect cells in a 15 mL conical tube, centrifuge (5 min at 800 rpm), and resuspend to 50k cells per 150 &#181;L in 1x DMEM media. If a different cell density is optimal, make sure that at this step the cell suspension is 2x the final density desired (e.g., for a final concentration of 50,000 cells in 300 &#181;L, cell density at this step is 50,000 cells in 150 &#181;L).</li>
		<li>Before plating cell culture, add ovarian explant to center of well (Figure 1B).</li>
		<li>Add agarose to each cell culture in individual 2 mL tubes just before plating. For each well, combine 200 &#181;L of cell suspension and 200 &#181;L of liquified agarose in a 2 mL tube. For example, for four wells, combine 800 &#181;L of cell suspension and 800 &#181;L of 2% agarose. Some mixture will be left over, but making slightly more than necessary avoids air bubbles during pipetting.
		<ol>
			<li>Add agarose to individual cell cultures immediately before plating that cell culture. The agarose will cool in under a minute, so be prepared to plate quickly.</li>
		</ol>
		</li>
		<li>Immediately add 300 &#181;L of the cell/agarose mixture to each well (Figure 1C). Figure 1C shows three cell conditions and one media condition, each plated with and without an ovary.</li>
		<li>Incubate slide at 37 &#176;C and 5% CO2 in a humidified incubator.</li>
	</ol>
	</li>
	<li>Divided Cocultures. 
	<ol>
		<li>Cut dividers from thin, smooth plastic (Figure 2A). 
		NOTE: This experiment uses the sides of a sterile disposable media basin because they are flat and thin. Cut them just wide enough to fit snugly into the hypotenuse of the well (~13 mm).</li>
		<li>Liquify the agarose at 70 &#176;C on a hot plate.</li>
		<li>Place the 8-well divider on top of the ITO-treated slide (Figure 2B). The rubber bottom on the divider aids in adhesion to the slide, but make sure to apply continuous gentle downward pressure during agarose plating to ensure no leaking or mixing between wells.</li>
		<li>Collect cells in a 15 mL conical tube, centrifuge (5 min at 800 rpm), and resuspend to 50k cells per 150 &#181;L in 1x DMEM media. If a different cell density is optimal, make sure that at this step the cell suspension is 2x the final density desired (e.g. for a final concentration of 50,000 cells in 300 &#181;L, cell density at this step is 50,000 cells in 150 &#181;L).</li>
		<li>Insert plastic dividers diagonally into wells (Figure 2C).</li>
		<li>Add agarose to each cell suspension one at a time just before plating. For each well, combine 100 &#181;L of cell suspension and 100 &#181;L of liquified agarose in a 2 mL tube. For example, for four wells, combine 400 &#181;L of cell culture and 400 &#181;L of 2% agarose. Some cell/agarose mixture will be left over but making slightly more than necessary avoids air bubbles during pipetting.
		<ol>
			<li>Add agarose to individual cell cultures immediately before plating that cell culture. The agarose will cool in under a minute, so be prepared to plate quickly.</li>
		</ol>
		</li>
		<li>On one side of the divider, plate 150 &#181;L of cell/agarose mixture. Allow agarose to cool and solidify (approximately one min) and then remove the divider (Figure 2D).</li>
		<li>Place ovary explant in the center of the empty half of the well. Place 150 &#181;L media/agarose mixture over top of the ovary, and only in the correct side of the well (Figure 2E).</li>
		<li>Incubate slide at 37 &#176;C and 5% CO2 in a humidified incubator.</li>
	</ol>
	</li>
</ol>
4. Drying Slide and Preparing for MALDI-TOF MS
<ol>
	<li>After four days (or any preferred time point), remove the chamber divider from the agarose plugs and the slide (Figure 1D). Gently detach the sides of the agarose from the chamber with a flat spatula and gently pull the chamber upward, being careful not to move any agarose plugs. If they do move, gently reposition them so that they are not touching one another.</li>
	<li>Place the slide in a 37 &#176;C oven for approximately 4 h, rotating 90&#176; every h. 
	NOTE: The rotation of the slide is important to ensure even heat distribution throughout the sample.</li>
	<li>Once dry, remove the slide from the oven (Figure 1E).</li>
	<li>Apply matrix solution using the sprayer (Figure 1F), with the following parameters: temperature = 30 &#176;C, flow rate = 0.2 mL/min, number of passes = 8, direction = CC, and nozzle distance = 40 mm. 
	NOTE: In lieu of a matrix sprayer, an artistic airbrush can be used to apply liquid matrix. The same matrix solution can be used to spray, but approximately twice as much solution is required. With the slide clamped so that it hangs vertically, spray the slide from a 90&#176; angle from approximately one foot away (Adapted from&#160;Hoffmann15) until the matrix layer is visible.</li>
	<li>Add 1 &#181;L of calibrant (Phosphorus Red for targets &#60;500 Da, a peptide mixture (see Table of Materials) for targets &#60;5,000 Da) to clear spot on slide. Phosphorus Red requires no mixing with matrix, but the peptide mixture requires 1:1 mixture with matrix to aid ionization. Wait for calibrant to dry.</li>
	<li>Draw an X using a permanent marker in each corner of the slide and take an optical image using a camera or a scanner at 1,200 dpi.</li>
</ol>
5. Imaging Mass Spectrometry Data Acquisition
<ol>
	<li>Open a new sequence on the IMS data analysis software (see Table of Materials). Set the raster width to desired spatial resolution, at least 50 &#181;m.</li>
	<li>Upload the optical image of the slide to the analysis software and set three teach points using the intersections in the X&#8217;s drawn in each corner.</li>
	<li>Designate regions of interest for imaging in the acquisition software and name them accordingly.</li>
	<li>Calibrate the instrument using the data acquisition software (see Table of Materials) within 5 ppm error.</li>
	<li>Save the optimized method and begin run. This experiment optimized the following parameters: Polarity = Positive, Detector = Reflectron, Laser size = 2, Laser power = 50%, Reflector mode detector gain = 3x.</li>
</ol>
6. Processing IMS Data
<ol>
	<li>Import .mis file into statistical software (see Table of Materials) for analysis of significance.</li>
</ol>

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The authors have nothing to disclose

There is a growing body of evidence that implicates norepinephrine&#8217;s role in HGSOC12,17,18, and this technique has contributed more mechanistic information. With at least eight biological conditions present on the same slide, the method can account for biological controls such as gene and cell specificity as well as media controls in a single IMS run. While the method was optimized to evaluate exchanged small molecules in ...

Imaging mass spectrometry (IMS) has been optimized to characterize the spatial distribution of molecular features in three widely used applications: tissue slices, spheroids, and microbial colonies1,2,3. Tissue slices can be used to evaluate the localization of metabolites in the context of biological conditions in a host, either targeted or untargeted within a specific mass range. However, differences between molecular features are the most significant and obvious when a healthy tissue is compared to a diseased condition, for example, a tumor. This IMS approach is particularly adapted to detection of disease biomarkers, however, acquiring tissue samples at discrete stages in disease progression (such as tumor grades) precludes the identification of signals that could be important for initiation of the disease. The exchange of information through space is a ubiquitous feature of many biological systems, and tissue slices cannot capture this dynamic chemical relay. One technique that is capable of visualizing chemical exchange and diffusion is IMS of microbial colonies grown on agar plates; small molecules are able to diffuse through and across the agar and can be captured via matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry4. This growth setup can be used to identify molecules exchanged between discrete biological entities (colonies) and can also determine directionality of metabolite production. The platform originally designed for microbial colony growth was adapted to explore the primary metabolism of tissue explants grown with mammalian cells, and IMS was used to evaluate the dynamic chemical exchange in an in vitro mammalian system.
In the past several years, it has become clear that high grade serous ovarian cancer (HGSOC) often originates in the fallopian tube epithelium (FTE) and then metastasizes to the ovary during early disease development5,6,7,8. The reason that tumorigenic FTE cells spread to the ovary, where large tumors eventually form and metastasize further, is currently unclear. Previous research has focused on the role of ovarian proteins in primary metastasis to the ovary; however, it has recently been demonstrated that the transition from a healthy to a tumorigenic tissue results in massive disruption of cellular metabolism and alters production of small molecules9,10,11. Therefore, we hypothesized that small molecules exchanged between the FTE and the ovary may be partly responsible for primary metastasis of HGSOC.
Using our newly developed IMS procedure, we have determined that coculture of tumorigenic FTE and healthy ovarian tissue induces the production of norepinephrine from the ovary. However, other cell types or normal FTE cells did not elicit this effect. An extraordinary benefit of this method is that the molecular production and exchange of signals that represent real molecules can be visualized, so even in a coculture it is possible to determine the source of a signal. This is an advantage over analysis of homogenized samples, where all spatial information is lost. In our model system, we were able to clearly assign the production of norepinephrine to the ovary. Norepinephrine has been linked to the metastasis and chemoresistance of ovarian cancers, and our detection of this molecule has validated that the novel IMS method can uncover biologically relevant molecules12,13,14. This validation lets us propose that this new application of IMS can be particularly helpful to research groups that are attempting to identify small molecules in coculture environments and to understand early events that influence cell transformation and metastasis. The overall goal of this method is to elucidate the identity and spatial distribution of small molecules during exchange between tissues and organs, represented either by in vitro 3D cell cultures or ex vivo tissue.

<table>
	<tbody>
		<tr>
			<td>15 mL Falcon tubes</td>
			<td>Denville</td>
			<td>C1017-O</td>
			<td>To collect cells</td>
		</tr>
		<tr>
			<td>8-well chamber (Millipore EZ-slide chamber)</td>
			<td>Millipore</td>
			<td>PEZGS0816</td>
			<td>Repurposed from Millipore Millicell EZ-slide chamber slide</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34998-4L</td>
			<td>Solvent for sprayed matrix</td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential Medium (&#945;MEM)</td>
			<td>Fisher</td>
			<td>10-022-CV</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>Autoflex speed MALDI-TOF LRF</td>
			<td>Bruker</td>
			<td></td>
			<td>For IMS data analysis</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td>To collect cells and remove supernatant</td>
		</tr>
		<tr>
			<td>CHCA Matrix</td>
			<td>Bruker Daltonic</td>
			<td>8201344</td>
			<td>Matrix sprayed onto dried slide</td>
		</tr>
		<tr>
			<td>DHB Matrix</td>
			<td>Bruker Daltonic</td>
			<td>8201346</td>
			<td>Matrix sprayed onto dried slide</td>
		</tr>
		<tr>
			<td>Disposable Scalpels</td>
			<td>Fisher</td>
			<td>22-079-707</td>
			<td>For removal of the ovaries</td>
		</tr>
		<tr>
			<td>Dissecting Scissors</td>
			<td>Fisher</td>
			<td>13-804-6</td>
			<td>For removal of the ovaries</td>
		</tr>
		<tr>
			<td>DMEM Media</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td>Media mixed with agarose</td>
		</tr>
		<tr>
			<td>epidermal growth factor</td>
			<td>Peprotech Inc.</td>
			<td>100-15</td>
			<td>Cell culture media supplement</td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Genesee Scientific</td>
			<td>22-282</td>
			<td>For agarose aliquots</td>
		</tr>
		<tr>
			<td>Estradiol-17&#946;</td>
			<td>Simga-Aldrich</td>
			<td>E2758</td>
			<td>Cell culture media supplement</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Denville</td>
			<td>fb5001</td>
			<td>Cell culture media supplement</td>
		</tr>
		<tr>
			<td>FlexControl 3.4</td>
			<td>Bruker Daltonic</td>
			<td></td>
			<td>IMS data acquisition software</td>
		</tr>
		<tr>
			<td>FlexImaging 4.1</td>
			<td>Bruker Daltonic</td>
			<td></td>
			<td>IMS data analysis software</td>
		</tr>
		<tr>
			<td>Forceps (fine)</td>
			<td>Fsiher</td>
			<td>22-327379</td>
			<td>For removal of the ovaries</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Cellgro</td>
			<td>30-005-CR</td>
			<td>Cell culture media supplement</td>
		</tr>
		<tr>
			<td>Insulin, Transferrin, Selenium (ITS)</td>
			<td>Sigma-Aldrich</td>
			<td>11074547001</td>
			<td>Cell culture media supplement</td>
		</tr>
		<tr>
			<td>ITO-coated slide</td>
			<td>Bruker</td>
			<td>8237001</td>
			<td>Platform for co-culture incubation</td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>Gibco</td>
			<td>11415064</td>
			<td>Media used during tissue dissection</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>Cell culture media supplement</td>
		</tr>
		<tr>
			<td>Low-melting agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9414-10G</td>
			<td>Mixed with media for plating</td>
		</tr>
		<tr>
			<td>Media basin</td>
			<td>Corning</td>
			<td>4870</td>
			<td>Used to cut plastic dividers for divided chambers</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Cell culture media supplement</td>
		</tr>
		<tr>
			<td>Peptide Calibration Standard</td>
			<td>Bruker Daltonic</td>
			<td>8206195</td>
			<td>Calibrant for medium mass range</td>
		</tr>
		<tr>
			<td>Phophorus red</td>
			<td>Sigma-Aldrich</td>
			<td>343-242-5G</td>
			<td>Calibrant for low mass range</td>
		</tr>
		<tr>
			<td>SCiLS Lab 2015</td>
			<td>Bruker Daltonic</td>
			<td></td>
			<td>IMS data statistical analysis</td>
		</tr>
		<tr>
			<td>Surgical Forceps (blunt)</td>
			<td>Fisher</td>
			<td>08-875-8B</td>
			<td>For removal of the ovaries</td>
		</tr>
		<tr>
			<td>TFA</td>
			<td>Fisher Technologies</td>
			<td>A116-50</td>
			<td>Added to matrix solution</td>
		</tr>
		<tr>
			<td>TM Sprayer</td>
			<td>HTX Technologies</td>
			<td></td>
			<td>For applying matrix</td>
		</tr>
	</tbody>
</table>

capturing small molecule communication between tissues and cells using imaging mass spectrometry

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

An optimally dried ITO slide will result in a flat desiccated sample with minimal to no wrinkles across the surface of the agarose and agarose pieces that maintain spatial separation on the slide (Figure 3). Figure 3A shows optimal drying, while Figure 3B shows the wrinkles that should be avoided. This optimization requires careful monitoring of the slide in the oven, as exact times can vary based on...

Watch this Scientific Journal Video about Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry at JoVE.com

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

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Research

JoVE Journal

Cancer Research

6.6K Views.  University of Illinois at Chicago. A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

Video: Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Morphology-Based Distinction Between Healthy and Pathological Cells Utilizing Fourier Transforms and Self-Organizing Maps

The appearance and the movements of immune cells are driven by their environment. As a reaction to a pathogen invasion, the immune cells are recruited to the site of inflammation and are activated to prevent a further spreading of the invasion. This is also reflected by changes in the behavior and the morphological appearance of the immune cells. In cancerous tissue, similar morphokinetic changes have been observed in the behavior of microglial cells: intra-tumoral microglia have less complex 3-dimensional shapes, having less-branched cellular processes, and move more rapidly than those in healthy tissue. The examination of such morphokinetic properties requires complex 3D microscopy techniques, which can be extremely challenging when executed longitudinally. Therefore, the recording of a static 3D shape of a cell is much simpler, because this does not require intravital measurements and can be performed on excised tissue as well. However, it is essential to possess analysis tools that allow the fast and precise description of the 3D shapes and allows the diagnostic classification of healthy and pathogenic tissue samples based solely on static, shape-related information. Here, we present a toolkit that analyzes the discrete Fourier components of the outline of a set of 2D projections of the 3D cell surfaces via Self-Organizing Maps. The application of artificial intelligence methods allows our framework to learn about various cell shapes as it is applied to more and more tissue samples, whilst the workflow remains simple.

Here, we provide a workflow that allows the identification of healthy and pathological cells based on their 3-dimensional shape. We describe the process of using 2D projection outlines based on the 3D surfaces to train a Self-Organizing Map that will provide objective clustering of the investigated cell populations.

The appearance and the movements of immune cells are driven by their environment. As a reaction to a pathogen invasion, the immune cells are recruited to ...

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced stage, which seriously hampers therapy. Mitochondrial changes are a hallmark of human ovarian cancers, and mitochondria are the centers of energy metabolism, cell signaling, and oxidative stress. In-depth insights into the changes of the mitochondrial proteome in ovarian cancers compared to control ovarian tissue will benefit in-depth understanding of the molecular mechanisms of ovarian cancer, and the discovery of effective and reliable biomarkers and therapeutic targets. An effective mitochondrial preparation method coupled with an isobaric tag for relative and absolute quantification (iTRAQ) quantitative proteomics are presented here to analyze human ovarian cancer and control mitochondrial proteomes, including differential-speed centrifugation, density gradient centrifugation, quality assessment of mitochondrial samples, protein digestion with trypsin, iTRAQ labeling, strong cation exchange fractionation (SCX), liquid chromatography (LC), tandem mass spectrometry (MS/MS), database analysis, and quantitative analysis of mitochondrial proteins. Many proteins have been successfully identified to maximize the coverage of the human ovarian cancer mitochondrial proteome and to achieve the differentially expressed mitochondrial protein profile in human ovarian cancers.

This article presents a protocol of differential-speed centrifugation in combination with density gradient centrifugation to separate mitochondria from human ovarian cancer tissues and control ovarian tissues for quantitative proteomics analysis, resulting in a high-quality mitochondrial sample and high-throughput and high-reproducibility quantitative proteomics analysis of a human ovarian cancer mitochondrial proteome.

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced ...