Name: Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry (Video) | JoVE Journal
Uploaded: 2019-04-03T15:00:00.000+00:00
Duration: 7 min 58 s
Description: Learn imaging mass spectrometry for mapping small molecule communication in ovarian cancer research. This label-free method preserves spatial integrity while revealing therapeutic targets and pathways.

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5 minutes

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>

<ol><li>Paine, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+Reproductive+System+Non-Negative+Matrix+Factorization+Mass+Spectrometry+Imaging+of+an+Early-Stage+Ovarian+Cancer+Mouse+Model%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Translating+Metabolic+Exchange+with+Imaging+Mass+Spectrometry%5BTitle%5D)+AND+%22Nature+Chemical+Biology%22%5BJournal%5D)">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/pubmed?term=((Imaging+Mass+Spectrometry+of+Three-Dimensional+Cell+Culture+Systems%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Primer+on+Agar-Based+Microbial+Imaging+Mass+Spectrometry%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">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/pubmed?term=((Integrative+Proteomic+Profiling+of+Ovarian+Cancer+Cell+Lines+Reveals+Precursor+Cell+Associated+Proteins+and+Functional+Status%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((High+Grade+Serous+Ovarian+Carcinomas+Originate+in+the+Fallopian+Tube%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((DNA+Methylome+Analyses+Implicate+Fallopian+Tube+Epithelia+as+the+Origin+for+High-Grade+Serous+Ovarian+Cancer%5BTitle%5D)+AND+%22Molecular+Cancer+Research%22%5BJournal%5D)">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/pubmed?term=((Ovarian+Cancer+Risk+After+Salpingectomy%3A+A+Nationwide+Population-Based+Study%5BTitle%5D)+AND+%22JNCI+Journal+of+the+National+Cancer+Institute%22%5BJournal%5D)">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/pubmed?term=((Activin+A+Stimulates+Migration+of+the+Fallopian+Tube+Epithelium%2C+an+Origin+of+High-Grade+Serous+Ovarian+Cancer%2C+through+Non-Canonical+Signaling%5BTitle%5D)+AND+%22Cancer+Letters%22%5BJournal%5D)">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/pubmed?term=((Evaluating+the+Progenitor+Cells+of+Ovarian+Cancer%3A+Analysis+of+Current+Animal+Models%5BTitle%5D)+AND+%22BMB+Reports%22%5BJournal%5D)">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/pubmed?term=((Landscape+of+Metabolic+Variation+across+Tumor+Types%5BTitle%5D)+AND+%22Cell+Systems%22%5BJournal%5D)">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/pubmed?term=((Clinical+Impact+of+Selective+and+Nonselective+Beta-Blockers+on+Survival+in+Patients+with+Ovarian+Cancer%3A+Beta-Blockers+and+Ovarian+Cancer+Survival%5BTitle%5D)+AND+%22Cancer%22%5BJournal%5D)">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/pubmed?term=((Stress-Related+Mediators+Stimulate+Vascular+Endothelial+Growth+Factor+Secretion+by+Two+Ovarian+Cancer+Cell+Lines%5BTitle%5D)+AND+%22Clinical+Cancer+Research%22%5BJournal%5D)">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/pubmed?term=((Stress+Hormone-Mediated+Invasion+of+Ovarian+Cancer+Cells%5BTitle%5D)+AND+%22Clinical+Cancer+Research%22%5BJournal%5D)">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/pubmed?term=((Homogeneous+Matrix+Deposition+on+Dried+Agar+for+MALDI+Imaging+Mass+Spectrometry+of+Microbial+Cultures%5BTitle%5D)+AND+%22Journal+of+The+American+Society+for+Mass+Spectrometry%22%5BJournal%5D)">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/pubmed?term=((Imaging+Mass+Spectrometry+Reveals+Crosstalk+between+the+Fallopian+Tube+and+the+Ovary+That+Drives+Primary+Metastasis+of+Ovarian+Cancer%5BTitle%5D)+AND+%22ACS+Central+Science%22%5BJournal%5D)">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/pubmed?term=((Src+Activation+by+%CE%B2-Adrenoreceptors+Is+a+Key+Switch+for+Tumour+Metastasis%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Src Activation by β-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/pubmed?term=((HTERT+Mediates+Norepinephrine-Induced+Slug+Expression+and+Ovarian+Cancer+Aggressiveness%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">HTERT Mediates Norepinephrine-Induced Slug Expression and Ovarian Cancer Aggressiveness.</a> Oncogene. 34 (26), 3402(2015).</li>
</ol>

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 (&amp;alpha;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&amp;beta;</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&#039;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

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...

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

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

An advanced method was developed for mass spectrometry imaging (MSI) of brain organoids that allows mapping metabolite distributions within these models. This technology offers insights into brain metabolic pathways and metabolite signatures during early development and in disease, promising a deeper understanding of the human brain function.

Molecular Imaging of Human Brain Organoids Using Mass Spectrometry

This protocol outlines the use of microMS for fluorescence-guided, single-cell MALDI-2 mass spectrometry, enabling enhanced molecular profiling of primary rat neuronal cells.

Fluorescence-Guided Matrix-assisted Laser Desorption/Ionization with Laser-Induced Postionization Mass Spectrometry of Individual Rat Neural Cells

This protocol aims to preserve cell metabolites for single-cell mass spectrometry (SCMS) by combining volatile salt solution washing, rapid liquid nitrogen quenching, freeze-drying, and −8 °C storage. It demonstrates that liquid nitrogen quenching is essential for maintaining metabolite profiles, while prolonged cold storage should be minimized to prevent metabolic alterations.

Sample Preparation for Single Cell Mass Spectrometry Metabolomics Studies: Combined Cell Washing, Quenching, Drying, and Storage

This protocol describes single-cell mass spectrometry imaging of OVCAR-8 cells using desorption electrospray ionization coupled to Orbitrap mass spectrometers for high-spatial-resolution metabolomic analysis.

Single-Cell Mass Spectrometry Imaging Using Desorption Electrospray Ionization Coupled To Orbitrap Mass Spectrometers

Learn imaging mass spectrometry for mapping small molecule communication in ovarian cancer research. This label-free method preserves spatial integrity while revealing therapeutic targets and pathways.

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

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