Tumor Conditioned Media in Nude Mice

This animal study was performed in accordance with the Cornell University animal care guidelines and was approved by Cornell University’s Institutional Animal Care and Use Committee. Three-week-old, female athymic nude-Foxn1^nu mice (n = 5 per condition) were purchased from Envigo and housed five animals per cage with ad libitum access to food and water. For 3 weeks, mice received daily intraperitoneal injections of TCM or blank media control. To generate TCM for injections, MDA-MB-231 breast cancer cells (American Type Culture Collection) at 90% confluency were incubated in serum-free Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penicillin/streptomycin (Invitrogen) for 24 hours. Conditioned medium was collected, normalized to cell number, concentrated 10-fold in an Amicon centrifugal filter unit (molecular weight cutoff, 3 kDa; EMD Millipore), and subsequently diluted fivefold with serum-free DMEM. Each injection (300 μl in volume) delivered conditioned medium that was produced by an equivalent of 280,000 tumor cells [or a multicellular tumor spheroid with a diameter of ~2 to 3 mm (67 (link))]. Blank low-serum DMEM was processed similarly (control) for mice receiving control injections. TCM and control treatments were prepared in one batch, frozen in aliquots, and thawed immediately before injection to ensure consistency between injections.