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>
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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. 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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. 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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. 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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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