Name: capturing small molecule communication between tissues and cells using imaging mass spectrometry
Uploaded: 2019-04-03T15:00:00
Description: Watch this Scientific Journal Video about Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry at JoVE.com

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), ...

<ol>
	<li>Paine, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Reproductive+System+Non-Negative+Matrix+Factorization+Mass+Spectrometry+Imaging+of+an+Early-Stage+Ovarian+Cancer+Mouse+Model.">Whole Reproductive System Non-Negative Matrix Factorization Mass Spectrometry Imaging of an Early-Stage Ovarian Cancer Mouse Model.</a> PLoS One. 11 (5), e0154837 (2016).</li><li>Yang, Y. L., Xu, Y., Straight, P., Dorrestein, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+Metabolic+Exchange+with+Imaging+Mass+Spectrometry.">Translating Metabolic Exchange with Imaging Mass Spectrometry.</a> Nature Chemical Biology. 5 (12), 885-887 (2009).</li><li>Li, H., Hummon, A. 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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Grade+Serous+Ovarian+Carcinomas+Originate+in+the+Fallopian+Tube.">High Grade Serous Ovarian Carcinomas Originate in the Fallopian Tube.</a> Nature Communications. 8 (1), (2018).</li><li>Klinkebiel, D., Zhang, W., Akers, S. N., Odunsi, K., Karpf, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Methylome+Analyses+Implicate+Fallopian+Tube+Epithelia+as+the+Origin+for+High-Grade+Serous+Ovarian+Cancer.">DNA Methylome Analyses Implicate Fallopian Tube Epithelia as the Origin for High-Grade Serous Ovarian Cancer.</a> Molecular Cancer Research. 14 (9), 787-794 (2016).</li><li>Falconer, H., Yin, L., Gronberg, H., Altman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+Cancer+Risk+After+Salpingectomy:+A+Nationwide+Population-Based+Study.">Ovarian Cancer Risk After Salpingectomy: A Nationwide Population-Based Study.</a> JNCI Journal of the National Cancer Institute. 107 (2), dju410 (2015).</li><li>Dean, M., Davis, D. A., Burdette, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activin+A+Stimulates+Migration+of+the+Fallopian+Tube+Epithelium,+an+Origin+of+High-Grade+Serous+Ovarian+Cancer,+through+Non-Canonical+Signaling.">Activin A Stimulates Migration of the Fallopian Tube Epithelium, an Origin of High-Grade Serous Ovarian Cancer, through Non-Canonical Signaling.</a> Cancer Letters. 391, 114-124 (2017).</li><li>King, S. M., Burdette, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+Progenitor+Cells+of+Ovarian+Cancer:+Analysis+of+Current+Animal+Models.">Evaluating the Progenitor Cells of Ovarian Cancer: Analysis of Current Animal Models.</a> BMB Reports. 44 (7), 435 (2011).</li><li>Reznik, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Landscape+of+Metabolic+Variation+across+Tumor+Types.">Landscape of Metabolic Variation across Tumor Types.</a> Cell Systems. 6 (3), 301-313 (2018).</li><li>Watkins, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Impact+of+Selective+and+Nonselective+Beta-Blockers+on+Survival+in+Patients+with+Ovarian+Cancer:+Beta-Blockers+and+Ovarian+Cancer+Survival.">Clinical Impact of Selective and Nonselective Beta-Blockers on Survival in Patients with Ovarian Cancer: Beta-Blockers and Ovarian Cancer Survival.</a> Cancer. 121 (19), 3444-3451 (2015).</li><li>Lutgendorf, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress-Related+Mediators+Stimulate+Vascular+Endothelial+Growth+Factor+Secretion+by+Two+Ovarian+Cancer+Cell+Lines.">Stress-Related Mediators Stimulate Vascular Endothelial Growth Factor Secretion by Two Ovarian Cancer Cell Lines.</a> Clinical Cancer Research. 9 (12), 4514-4521 (2003).</li><li>Sood, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+Hormone-Mediated+Invasion+of+Ovarian+Cancer+Cells.">Stress Hormone-Mediated Invasion of Ovarian Cancer Cells.</a> Clinical Cancer Research. 12 (2), 369-375 (2006).</li><li>Hoffmann, T., Dorrestein, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneous+Matrix+Deposition+on+Dried+Agar+for+MALDI+Imaging+Mass+Spectrometry+of+Microbial+Cultures.">Homogeneous Matrix Deposition on Dried Agar for MALDI Imaging Mass Spectrometry of Microbial Cultures.</a> Journal of The American Society for Mass Spectrometry. 26 (11), 1959-1962 (2015).</li><li>Zink, K. E., Dean, M., Burdette, J. E., Sanchez, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Mass+Spectrometry+Reveals+Crosstalk+between+the+Fallopian+Tube+and+the+Ovary+That+Drives+Primary+Metastasis+of+Ovarian+Cancer.">Imaging Mass Spectrometry Reveals Crosstalk between the Fallopian Tube and the Ovary That Drives Primary Metastasis of Ovarian Cancer.</a> ACS Central Science. 4 (10), 1360-1370 (2018).</li><li>Armaiz-Pena, G. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Src+Activation+by+&#946;-Adrenoreceptors+Is+a+Key+Switch+for+Tumour+Metastasis.">Src Activation by &#946;-Adrenoreceptors Is a Key Switch for Tumour Metastasis.</a> Nature Communications. 4, 1403 (2013).</li><li>Choi, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTERT+Mediates+Norepinephrine-Induced+Slug+Expression+and+Ovarian+Cancer+Aggressiveness.">HTERT Mediates Norepinephrine-Induced Slug Expression and Ovarian Cancer Aggressiveness.</a> Oncogene. 34 (26), 3402 (2015).</li></ol>

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 ...

Our protocol can map small molecule chemical communication between cells and tissues, and we have specifically applied this to explore small molecules in ovarian cancer metathesis. The main advantage of our technique is the ability to measure small molecules in a label-free manner while maintaining the spatial integrity of cells and tissues in 2D. This technique has allowed us to interrogate the contribution of small molecules in ovarian cancer metastases in an untargeted manner.This could unveil new therapeutic targets and pathways that have previously been overlooked. This methodology could easily be extended to other cancers that can be grown in agarose and any other disease in which a spatial component might be important for the resulting cellular phenotype. Ensuring that all the cells and components are in hand and patience with the drying process is essential to this protocol.A rushed desiccation step can often lead to poor quality mass spectrometry data. Visual demonstration of this method is helpful in understanding our sample preparation and desiccation steps, as these deviate greatly from typical imaging mass spectrometry experiments that use tissues or microbes on agar. To begin, liquefy the agarose at 70 degrees Celsius on a hot plate.Place the eight-well divider on top of the ITO-treated slide. Via standard trypsin treatment, collect MOE cells in a 15-milliliter conical tube, centrifuge, and add one times DMEM media to resuspend to obtain a concentration of 50, 000 cells per 150 microliters, which is two times the final density desired. For undivided cocultures, use forceps to add ovarian explant to the center of the four wells.Just before plating, combine 200 microliters of cell suspension and 200 microliters of liquified agarose in individual two-milliliter tubes. Avoid air bubbles during pipetting. Immediately, add 300 microliters of the cell-agarose mixture to each well, and apply continuous gentle downward pressure on the divider to ensure no leaking or mixing between wells.Make sure there are no air bubbles and the ovary remains in the center of the well. If the pipetted agarose disturbed the ovary, gently use the pipette tip to center it before the agarose cools. Then, incubate the slide at 37 degrees Celsius and 5%carbon dioxide in a humidified incubator.For divided cocultures, cut dividers from thin, smooth plastic. The sides of a sterile, disposable media basin are used. Cut them just wide enough to fit snugly into the hypotenuse of the well.Place the eight-well divider on top of the ITO-treated slide, and insert plastic dividers diagonally into wells. Prepare cell cultures as done previously, and combine 100 microliters of cell suspension and 100 microliters of liquified agarose in a two-milliliter tube. Immediately, plate 150 microliters of cell-agarose mixture on one side of the divider while keeping downward pressure on the divider.Allow agarose to cool and solidify for approximately one minute, and then remove the divider. Use forceps to place ovary explant in the center of the empty half of the well. Add 150 microliters of cell-agarose mixture over the top of the ovary.Incubate the slide at 37 degrees Celsius and 5%carbon dioxide in a humidified incubator. After four days, remove the chamber divider from the agarose plugs. With a flat spatula, detach the sides of the agarose from the chamber, and gently pull the chamber upward.If any agarose plugs are moved, gently reposition them so that they are not touching one another. Place the slide in a 37-degree Celsius oven for approximately four hours, and rotate 90 degrees every hour to ensure even heat distribution throughout the sample. The slide must be completely dried.Otherwise, it can lead to an explosion of the sample in the high vacuum environment of the MALDI-TOF mass spectrometer. Drying the slide and monitoring the progress is the most critical step to achieve high spatial resolution and quality mass spectra. If flaking or excessive wrinkling occur, we do not recommend collecting the mass spectrometry data.With the parameters set upon the matrix sprayer, apply matrix solution to the slide. To use Phosphorus Red as calibrant, add one microliter of Phosphorus Red to a clear spot on the slide. To use the peptide mixture as calibrant, mix it with matrix on Parafilm in a one-to-one ratio to aid ionization, and spot 0.5 to one microliter onto the slide.Wait for calibrant to dry before imaging mass spectrometry data acquisition. 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 1200 dpi. In this experiment, careful monitoring of the slide in the oven is essential to ensure an optimal dried ITO slide, which results in a flat desiccated sample with minimal to no wrinkles across the surface of the agarose.This figure shows the wrinkles that should be avoided. Wrinkles can slightly affect the height and therefore the mass accuracy of the imaging mass spectrometry signals. Slight wrinkles will not impact the quality of the data.An optimally located tissue should be as close to the center as possible. The tissue used should be in the center of the well if it is undivided or in the center of the half triangle if it is divided so that acquisition of IMS data is not contaminated with edge effects. Badly positioned tissue, when imaged, may result in false mass signals from the edge effects.The dried slide image is used to teach the MALDI-TOF mass spectrometer which regions to image. Small regions around the ovarian tissue were selected for IMS at 50 micrometers. A representative image of an m-over-z signal is shown for undivided cocultures, as well as for divided chamber setup.The most important thing to remember is that the resulting data are only as good as the sample preparation. Pay special attention to the drying process. The most exciting aspect is validating the small molecules discovered in this way using other experiments, such as invasion assays or colonization assays, to inform on the effective concentration and translate to human biology.We expect that this will open the door to discover new pathways of potential therapeutic targets for ovarian cancer by better understanding the processes in the primary metathesis of ovarian cancer.

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Cancer Research

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 ...

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 ...

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results ...