Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

This research demonstrates a novel approach to the study of appendiceal cancer therapeutics and pathobiology in a preclinical setting. These methods may be more broadly applicable to the study of other malignancies. Appendiceal cancer is difficult to study given its low incidence in the absence of an appendix in mice for disease modeling.

This technique allows the study of cell interactions within the tumor microenvironment. This method can be applied to study the biology of multiple types of cancers that have the propensity to metastasize to the omentum, such as colorectal and ovarian cancers. Begin by preparing the transport and culture media.

Upon tissue arrival, transfer the pseudomyxoma peritonei tumor tissues into 35 complete DMEM containing six centimeter diameter dishes. In addition to removing any liquified mucin, or highly mucinous tissue regions, remove any tissue that is difficult to cut with a scalpel. Cut tumor tissue nodules roughly into one cube centimeter smaller pieces.

for agarose preparation and tissue embedding, prepare a 5%solution of low-melt agarose in PBS without fetal bovine serum or FBS on the same day if tissues arrival. Add the liquid 5%agarose solution to the six centimeter dishes containing two to three smaller tumor specimens until it covers the specimen. Place the embedded tissues in a four degree Celsius refrigerator for 30 minutes.

Demount the tissue specimens on the vibratome, remove the solidified auger from the refrigerator, and cut the agarose cubes slightly larger than the width of the tissue using a scalpel. Apply super glue to the vibratome stage and gently place the agarose cube onto the super glue for one to two minutes for fixing. Fix the blade to the vibratome, and set the thickness of the tissue section to roughly 150 to 250 microns for optimal downstream imaging.

To culture the tissue slices, prepare a permeable insert plate by adding two milliliters of complete DMEM media above the permeable membrane and three milliliters below the permeable membrane. Next, gently lift the tissue slices out of the cutting chamber of the vibratome using a thin paintbrush, and place two to four slices into permeable culture dishes containing complete DMEM. Culture the slices for up to seven days at 37 degrees Celsius in 5%carbon dioxide.

During media replacement, every 24 hours, add bortezomib as controls for cell killing, and testable chemotherapies 5-Fluorouracil, or 5-FU, to the culture media to perform pharmacological intervention. Place tumor slices for confocal imaging analysis in a single well of a 12-well dish containing PBS with 1%FBS. For calcium imaging experiments, instead of PBS, incubate the slices in an extra cellular calcium solution.

Stain samples with imaging dyes to determine the viability of tissue slices. Incubate for 10 minutes to one hour after adding the viability dye. After incubation, transfer the slices to an optically clear glass bottom Petri dish containing PBS with 1%FBS to be used for imaging on a confocal imaging device.

Cut a small piece from the end of a one milliliter pipette tip to widen the opening, allowing vigorous mechanical dissociation by pipetting. Incubate slices at 37 degrees Celsius with rotation for five to 15 minutes in one milliliter of digestion buffer, with vigorous tissue disruption two to three times during incubation using mechanical dissociation. Once the tissue is digested, transfer the disrupted tissue with digestion media on a 70 micrometer filter placed on top of a 50 milliliter conical tube.

Pick larger pieces using sterile forceps or a pipette. Using the blunt backend of a plastic five milliliter syringe, mash any larger undissociated slices and wash them with four milliliters of PBS containing 2%FBS. Take the dissociated cell supernatant and centrifuge at 300G for five to 10 minutes.

Remove the supernatant and wash the pellet with one milliliter of PBS containing 2%FBS. To prepare the sample for flow cytometry, add the blocking buffer containing 50 microliters of human FC block, and incubate at room temperature for 15 minutes. Perform extra cellular staining using the respective antibodies in 50 microliters of PBS containing 2%FBS.

Aliquot five milliliters of complete media for control and treatment conditions. On the two to four slices plated onto the permeable dishes, add the media from the control treatment aliquots and the additional combination therapies to be used for downstream viability, live dead analysis. Leave tissue slices in a culture containing complete DMEM for two to five days, changing media as well bortezomib and 5-FU every other day.

For luminescent viability analysis, add 500 microliters of PBS into a 12-well dish, and transfer the sequentially matched tissue slices to the PBS solution using a paintbrush or a one milliliter pipette. Remove the PBS from the slices. Add 500 microliters of the luminescent viability solution per condition and incubate with a slow rotation on the shaker at room temperature for 30 minutes before reading the luminescence using a luminescence plate reader.

The mucin staining in tissue slices was visualized using periodic acid shift staining and mucin-specific antibodies. Incubation in calcein AM and propidium iodide determined the health and viability. The nuclei overlapped the cells proliferated during culture.

These results were quantified to determine the number of proliferating cells within the tumor slice. The slices incubated in CD11b PE conjugated antibody and Fluo-4 AM, labeled the local immune cells in CTU. Pseudocolor scaled images showing intracellular calcium levels allowed visualizing differential levels of intracellular calcium.

Spontaneous activity tracked and time lapsed for before, during and after intercellular calcium response within the highlighted CD11b positive immune cell is shown here. The raw traces of calcium responses in the CD11b immune cell, along with other responsive and non-responsive cells were quantified. Representative results of immunotyping of a patient tumor sample with pseudomyxoma peritonei are shown here.

Quantification of the tumor immuno profile showed that the donor specimen 337 has high levels of M2 macrophages, indicating that utilizing pseudomyxoma peritonei-derived tissue slices allowed the interrogation of the unique donor cellular landscape of the patient tumor samples. Pharmacotyping and downstream analysis of tissue slices from pseudomyxoma peritonei human tissues showed the killing of cells in tumor slices as confirmed by cytotoxicity analysis and tunnel confocal imaging. In response to the cytotoxic drug, bortezomib and 5-fluorouracil.

Luminescent viability analysis was performed using sequentially matched slices. Successful production of slices from mucinous tumors requires meticulous dissection and removal of cellular and acellular tissue components like fat and dense abdominal wall tissue. This technique can allow for modeling interactions between cancer cells and multiple cell types in the tumor microenvironment, such as adipose, vascular and immune cell interactions.