Modeling Ovarian Cancer Communication for Imaging Mass Spectrometry Analysis: A Method to Identify Small Molecule Chemical Communicators in Metastatic Ovarian Tumor Development

Overview

This protocol describes a technique for sample preparation by co-culturing ovarian explants with the fallopian tube cancer cells. These samples are visualized using imaging mass spectroscopy, or IMS,  to detect small molecule exchange between the cells and tissues leading to tumor development.

Protocol

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. 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 the center of the 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.

 2. 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 hours, rotating 90° every hour.
   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 approximately one foot away 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 spots on the slide. Phosphorus Red requires no mixing with the matrix, but the peptide mixture requires 1:1 mixture with the matrix to aid ionization. Wait for the calibrant to dry.
6. Draw an X using a permanent marker on each corner of the slide and take an optical image using a camera or a scanner at 1,200 dpi.

Representative Results

Figure 1: Workflow for sample preparation of undivided coculture. (A) Adhere 8-well chamber to conductive side of ITO-coated slide. (B) Place halved ovaries in the center of wells for coculture conditions. (C) Add 300 μL of agarose/cell suspension directly into wells. Make sure there are no air bubbles and that the ovary remains in the center of the well. If the pipetted agarose disturbed the ovary, gently use the pipet tip to center it before the agarose cools. (D) After four days of incubation (or otherwise optimized time) remove the 8-well chamber from the slide. If agarose remains attached to the chamber, gently detach the agarose plug from the chamber using a spatula and reposition it on the slide. The agarose will not adhere to the slide, so it will be easy to move. Draw an ‘X’ on each corner of the slide and take a photo. (E) Dry the slide in a 37 °C oven for 4h, rotating 90° each hour. Agarose should be fully desiccated and should lie flat on the slide. (F) Apply matrix of choice via a sprayer or airbrush to slide. Matrix layer should be visible as yellow. Scan the slide on a scanner at 1,200 dpi and add calibrants or standards for MALDI-TOF MS analysis.

Figure 2: Workflow for sample preparation of divided coculture. (A) Cut tabs out of the media basin (~13 mm) and ensure that the sides are straight. (B) Attach an 8-well chamber to the conductive side of the ITO-coated slide. (C) Insert dividers diagonally into wells. (D) Add 150 μL cell culture in agarose to one side of the divider, allow the agarose to cool, and remove the divider. (E) Add ovary to the center of the empty well half and cover with 150 μL of media and agarose suspension. This figure is reproduced with permission from Zink et al. 2018.

Materials

Name	Company	Catalog Number	Comments
Denville	C1017-O	To collect cells
Millipore	PEZGS0816	Repurposed from Millipore Millicell EZ-slide chamber slide
Sigma-Aldrich	34998-4L	Solvent for sprayed matrix
Eppendorf	5810 R	To collect cells and remove supernatant
Bruker Daltonic	8201344	Matrix sprayed onto dried slide
Bruker Daltonic	8201346	Matrix sprayed onto dried slide
Gibco	11995-065	Media mixed with agarose
Genesee Scientific	22-282	For agarose aliquots
Bruker	8237001	Platform for co-culture incubation
Sigma-Aldrich	A9414-10G	Mixed with media for plating
Corning	4870	Used to cut plastic dividers for divided chambers
Bruker Daltonic	8206195	Calibrant for medium mass range
Sigma-Aldrich	343-242-5G	Calibrant for low mass range
Simga-Aldrich	E2758	Cell culture media supplement
Peprotech Inc.	100-15	Cell culture media supplement
Cellgro	30-005-CR	Cell culture media supplement
Sigma-Aldrich	11074547001	Cell culture media supplement
Gibco	25030-081	Cell culture media supplement
Gibco	15140-122	Cell culture media supplement
Fisher Technologies	A116-50	Added to matrix solution
HTX Technologies		For applying matrix