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. B. <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+of+Three-Dimensional+Cell+Culture+Systems.">Imaging Mass Spectrometry of Three-Dimensional Cell Culture Systems.</a> Analytical Chemistry. 83 (22), 8794-8801 (2011).</li><li>Yang, J. Y., 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=Primer+on+Agar-Based+Microbial+Imaging+Mass+Spectrometry.">Primer on Agar-Based Microbial Imaging Mass Spectrometry.</a> Journal of Bacteriology. 194 (22), 6023-6028 (2012).</li><li>Coscia, F., 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=Integrative+Proteomic+Profiling+of+Ovarian+Cancer+Cell+Lines+Reveals+Precursor+Cell+Associated+Proteins+and+Functional+Status.">Integrative Proteomic Profiling of Ovarian Cancer Cell Lines Reveals Precursor Cell Associated Proteins and Functional Status.</a> Nature Communications. 7, 12645 (2016).</li><li>Labidi-Galy, S. 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

6.5K visualizações.  University of Illinois at Chicago. Nosso protocolo pode mapear a comunicação química de pequenas moléculas entre células e tecidos, e nós especificamente aplicamos isso para explorar pequenas moléculas na metatese do câncer de ovário. A principal vantagem de nossa técnica é a capacidade de medir pequenas moléculas de forma livre de rótulos, mantendo a integridade espacial das células e tecidos em 2D. Esta técnica nos permitiu interrogar a contribuição de pequenas moléculas em metástases de câncer de ovário...