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.
