5×106 OVCAR-8-RFP cells suspended in PBS were injected intraperitoneally into NCr nu/nu athymic female mice (N=5) aged 10 to 12 weeks (Taconic, Rensselaer, NY). Tumor burden was monitored on a weekly basis using a Xenogen IVIS® Spectrum In Vivo Imaging System (PerkinElmer, Waltham, MA). Mice were also given weekly vaginal lavages using 200 μL of sterile PBS throughout the study to collect cells from the local microenvironment of the reproductive organs. Cells sourced from lavages were counted and spun down to remove PBS as it has been shown to suppress MALDI-TOF ionization.21 (link) This initial centrifugation step also removes mucous and mucous-associated proteins, such as mucin. Cells are then normalized to 10,000 cells/μL using DI water and stored at −80°C following collection. Upon addition of DI water, cells undergo osmotic stress and lyse, which removes the need for a wash step as all remaining proteins will be in solution. This lysing step prior to analysis also allows for proteins or other similarly sized molecules to be detected via MALDI-TOF MS. After two months of tumor progression, all animals were humanely sacrificed followed by collection of tumors and reproductive organs.