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

Summary

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

Abstract

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.

Introduction

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 colonies^1,2,3. 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 development^5,6,7,8. 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 molecules^9,10,11. 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 molecules^12,13,14. 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.

Protocol

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

1. Maintain murine oviductal epithelium (MOE) cells at 37°C with 5% CO₂ in a humidified incubator in alpha Minimum Essential Medium (αMEM) 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-17β. Pass the cells every 3–4 days, ensuring there are enough cells on the same day the mice will be sacrificed.
2. 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 °C indefinitely.
3. Prepare at least 2 mL of 1x Dulbecco’s Modified Eagle’s Medium (DMEM) media.
4. 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.

2. Mouse Colony and Ovary Removal

1. 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.
2. Euthanize pups at 16–18 days of age by CO₂ inhalation per NIH guidelines. Deliver CO₂ (100%) at a flow rate of 10%–20% chamber volume per minute and continue for two minutes after respiration had stopped. Confirm euthanasia by cervical dislocation.
3. Disinfect all surgical equipment by submerging in 70% ethanol. Warm Leibovitz’s L-15 media with 1x penicillin-streptomycin to 37 °C.
4. 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.
5. Move the internal organs to the side using forceps. Individually grab each uterine horn with fine forceps and lift slightly.
6. 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.
7. Move the ovary, oviduct, and approximately one half of each uterine horn to pre-warmed Leibovitz’s L-15 media.
8. 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’s L-15 media.
9. Cut each ovary in half axially. Keep the ovarian pieces (now called explants) kept at 37 °C until plating of agarose.

3. Setting Up and Incubating the ITO-treated Slide for Cocultures

1. Undivided cocultures
   1. Liquify the agarose at 70 °C on a hot plate.
   2. 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.
   3. Collect cells in a 15 mL conical tube, centrifuge (5 min at 800 rpm), and resuspend to 50k cells per 150 µL 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 µL, cell density at this step is 50,000 cells in 150 µL).
   4. Before plating cell culture, add ovarian explant to center of well (Figure 1B).
   5. Add agarose to each cell culture in individual 2 mL tubes just before plating. For each well, combine 200 µL of cell suspension and 200 µL of liquified agarose in a 2 mL tube. For example, for four wells, combine 800 µL of cell suspension and 800 µL of 2% agarose. Some mixture will be left over, but making slightly more than necessary avoids air bubbles during pipetting.
      1. 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.
   6. Immediately add 300 µL 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.
   7. Incubate slide at 37 °C and 5% CO₂ in a humidified incubator.
2. Divided Cocultures.
   1. 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).
   2. Liquify the agarose at 70 °C on a hot plate.
   3. 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.
   4. Collect cells in a 15 mL conical tube, centrifuge (5 min at 800 rpm), and resuspend to 50k cells per 150 µL 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 µL, cell density at this step is 50,000 cells in 150 µL).
   5. Insert plastic dividers diagonally into wells (Figure 2C).
   6. Add agarose to each cell suspension one at a time just before plating. For each well, combine 100 µL of cell suspension and 100 µL of liquified agarose in a 2 mL tube. For example, for four wells, combine 400 µL of cell culture and 400 µL of 2% agarose. Some cell/agarose mixture will be left over but making slightly more than necessary avoids air bubbles during pipetting.
      1. 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.
   7. On one side of the divider, plate 150 µL of cell/agarose mixture. Allow agarose to cool and solidify (approximately one min) and then remove the divider (Figure 2D).
   8. Place ovary explant in the center of the empty half of the well. Place 150 µL media/agarose mixture over top of the ovary, and only in the correct side of the well (Figure 2E).
   9. Incubate slide at 37 °C and 5% CO₂ in a humidified incubator.

4. Drying Slide and Preparing for MALDI-TOF MS

1. 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.
2. Place the slide in a 37 °C oven for approximately 4 h, rotating 90° every h.
   NOTE: The rotation of the slide is important to ensure even heat distribution throughout the sample.
3. Once dry, remove the slide from the oven (Figure 1E).
4. Apply matrix solution using the sprayer (Figure 1F), with the following parameters: temperature = 30 °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° angle from approximately one foot away (Adapted from Hoffmann¹⁵) until the matrix layer is visible.
5. Add 1 µL of calibrant (Phosphorus Red for targets <500 Da, a peptide mixture (see Table of Materials) for targets <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.
6. 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.

5. Imaging Mass Spectrometry Data Acquisition

1. 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 µm.
2. Upload the optical image of the slide to the analysis software and set three teach points using the intersections in the X’s drawn in each corner.
3. Designate regions of interest for imaging in the acquisition software and name them accordingly.
4. Calibrate the instrument using the data acquisition software (see Table of Materials) within 5 ppm error.
5. 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.

6. Processing IMS Data

1. Import .mis file into statistical software (see Table of Materials) for analysis of significance.

Results

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...

Discussion

There is a growing body of evidence that implicates norepinephrine’s role in HGSOC^12,17,18, 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 ...

Materials

List of materials used in this article

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