Tumor Xenograft Murine Model.

Athymic nude, outbred, female mice (5.5 to 6.5 wk old at the time of tumor cell injection) were provided by The OSUCCC Target Validation Shared Resource (TVSR); the original breeders (strains 553 and 554) were purchased either from the National Cancer Institute (NCI) Frederick facility or Charles River. All animal studies were performed in compliance with the Guide for the Care and Use of the Laboratory Animals as outlined by The Ohio State University Institutional Animal Care and Use Committee.

Parental 143.98.2 cell line and a 143.98.2 clone stably expressing tdTomato-DTA_N-I_N-p66α were expanded to obtain ∼90% viable (by Trypan Blue exclusion) subconfluent cell suspension of 50 × 10⁶ cells/mL in ice-cold phosphate-buffered saline (PBS). The cells were mixed with Matrigel (Corning) at 2:1 volume ratio (cells to Matrigel) and injected s.c. in murine flanks 2.5 × 10⁶ cells per site. Tumor size was measured with a caliper and the murine body weights were examined every 3 d, starting 1 wk post-cell inoculation. Tumor volumes were calculated using an equation [tumor volume (mm³) = (tumor length) × (tumor width)² × 0.5]. Isolation of cells from tumor xenografts is described in SI Appendix, Methods.

For split-toxin treatment, 1-wk post-tumor cell inoculation, when tumors had reached ∼100 to 200 mm³ in size, animals were randomized into two groups and injected s.c. at three to four sites adjacent to the tumor (100 μL total volume per mouse): one group with sterile PBS only and another group with PBS containing 8 µg LF_N-MBD2-I_C-DTA_C (3.4 µM final concentration) and 24 µg PA_WT (5.8 µM final concentrations). The injections were administered every 2 to 3 d for a total of five times. Three-days post-last injection, all animals were killed; tumors were then weighed and processed for downstream applications. Endotoxin was removed from LF_N-MBD2-I_C-DTA_C and PA_WT protein preps used for the injections by means of High-Capacity Endotoxin Removal resin (Pierce/Thermo Fisher Scientific); endotoxin levels were evaluated using the Chromogenic Endotoxin Quant kit (Pierce/Thermo Fisher Scientific). Stability of LF_N-MBD2-I_C-DTA_C in mouse serum was tested as described in SI Appendix, Methods.