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    2020-03-17

    r> In this study we describe a novel murine model of the pathophysiologic process that leads to port site metastasis in women with ovarian cancer. We were able to predictably induce a metastatic deposit within the abdominal wall in immune competent and immunocompromised mice using the syngeneic murine ID8 EOC cell line [15]. This metastatic model allows for study of a clinically-relevant metastatic KRN 7000 in an immunocompetent mouse and can be used as a secondary outcome for pre-clinical drug studies in mice.
    Methods 
    luciferase activity 3–4 weeks following injection, and thereafter as indicated. Mice with radiographic evidence of intraperitoneal tumor were treated with puncture of the right inferior abdominal wall just medial to the nipple with an 18 gauge hollow bore needle. Control sites were identified in the midline of the upper abdomen remote from the ID8 injection site or the puncture site. Mice were sacrificed 3–4 weeks following abdominal wall puncture using CO2 gas per institutional protocols.
    Mouse Imaging
    Imaging was performed as a modification of a previously described protocol [19,20]. Briefly, mice were injected with 200 μL of a suspension of 15 mg/mL D-Luciferin Potassium Salt (Gold Biotech-nology, St. Louis, MI) in 9% sodium chloride (Baxter, Deerfield, IL) into the peritoneum via the left lower quadrant. Mice were then anesthetized with isoflurane gas. Images were obtained 10 min after Luciferin injection with the Xenogen VivoVision IVIS Bioluminescent and Fluorescent Imager (PerkinElmer, Waltham, MA).
    Tissue Processing and Pathology
    Biopsies of the abdominal wall were obtained immediately upon mouse sacrifice. Abdominal wall hair was removed with Nair™. If a palpable nodule or scar was identified in the right lower quadrant in the expected area of the needle puncture (just medial to the nipple), this was marked with a skin pen. If there was no scar or nodule, the area just medial to the nipple was marked. The anterior abdominal wall including the marked site was then excised using a 5 mm Keyes punch biopsy. Abdominal wall biopsies were taken in the same manner remote for the ID8 injection and contralateral to the puncture site and used as paired control sites. Specimens were placed in 4% paraformaldehyde within marked cassettes. Blocks were processed by the Dartmouth Pathology Core Resource. Specimens were embedded into a paraffin block and oriented such that a skin edge is visible on the slide. Slides were cut at 4 microns, air dried, and loaded onto Akura Tissue-Tek Prisma Autostrainer (Leica Biosys-tems, Buffalo Grove, IL). Slides were dried for 25 minutes, deparaffinized in Xylene, and hydrated through graduated alcohols to water. Cells were stained with Hematoxylin 2 for five minutes and washed in water. Cells were then washed in bluing agent for one minute then washed in water and then 95% alcohol for 30 seconds. Cells were then stained with Eosin-Y for 30 seconds. Slides were dehydrated in 100% alcohol and cleared with xylene. Slides were then mounted with Tissue Tek mounting medium. Staining and dehydrating materials were obtained from Richard Allen Scientific (Grand Island, NY). Pathology slides were interpreted by LJT, our collaborator within the pathology department, who was blind to specimen origin or treatment group.
    Mice and Cells
    C57BL/6 mice were purchased from Charles River (Wilmington, MA). NOD SCID gamma (NSG) mice were purchased from the Dartmouth Mouse Modeling Shared Resource (Lebanon, NH). All animal experiments were approved by the Institutional Animal Care and Use Committee. ID8 murine ovarian cancer cells transduced with pFB-neo-Luciferase (ID8-luc cells) were previously described and selected with 0.8 mg/ml G418 [15,16].