This study investigates the interaction between tumorigenic cells from the fallopian tube and healthy ovarian cells, focusing on the production of chemical communicators that facilitate tumor metastasis. The methodology involves co-culturing these cells in a 3D agarose matrix to visualize the small molecules produced.
Tumorigenic cells from the fallopian tube stimulate healthy ovary cells to produce chemical communicators or small molecules that promote tumor metastasis. To visualize these chemical communicators, begin by assembling a well-divider on top of a suitable glass slide. Position a plastic divider diagonally in the well to separate the well into two equal-sized compartments. Dispense a suspension of fallopian tube tumor cells in liquid agarose into one compartment of the well. Upon cooling, the agarose solidifies and forms a 3D network enabling tumor cells' entrapment.
Remove the divider and place a healthy ovarian tissue centrally in the empty compartment. Cover it with a liquid agarose mixture. Allow the agarose to solidify and embed the ovarian tissue in proximity to the cancer cells. Incubate for the desired duration. Both the tumor cells and healthy ovarian cells produce small molecules that diffuse through the agarose network towards their neighboring cells.
Remove the well-divider. Dry the co-culture to desiccate and flatten the agar, and capture the released small molecules within the matrix. Visualize the distribution of small molecules using imaging mass spectroscopy or IMS.
Place the 8-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 liquefied agarose in a 2-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 1 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 could 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 1 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 1 to 1 ratio to aid ionization and spot 0.5 to 1 microliter onto the slide. Wait for calibrant to dry before imaging mass spectrometry data acquisition.
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