D luciferin

Subcutaneous injection model: One million luciferase-expressing PDA^T cells were injected subcutaneously into the flanks of mice. 12 days after transplantation, animals were randomly assigned to the vehicle control group (0.5% carboxymethylcellulose, 0.1% Tween80 in ddH₂O) or TEPP46 treatment group (50 mg kg⁻¹). Animals were dosed twice a day by oral gavage for 7 days. Tumor volume was monitored by caliper measurements using the following formula: tumor volume [mm³] = (length [mm]) × (width [mm]) × (height [mm]) × (π/6). To monitor for potential metastasis, mice were subject to bioluminescent imaging using the IVIS spectrum (PerkinElmer Cat# 124262) after injection with 150 mg kg⁻¹ D-Luciferin (Goldbio Cat# LUCK).
Intrasplenic injection model: 50,000 luciferase-expressing PDA^T cells were injected into the spleen as described above, after which animals were randomly assigned to the vehicle control group (0.5% carboxymethylcellulose, 0.1% Tween80 in ddH₂O) or TEPP46 treatment group (50 mg kg⁻¹). Animals were dosed twice a day by oral gavage. Body weight and tumor burden were measured at enrollment and once a week throughout the course of the study. To monitor tumor burden in the liver, mice were subject to bioluminescent imaging using the IVIS spectrum (PerkinElmer Cat# 124262) after injection with 150 mg kg⁻¹ D-Luciferin (Goldbio Cat# LUCK).

All animal studies were performed according to the guidelines established by the Institutional Animal Care and Use Committee (IACUC) at the Penn State College of Medicine. An orthotopic xenograft breast cancer model was generated by injecting 2.0×10⁶ MDA-MB-231 cells into the inguinal mammary fat pad of female NOD SCID Gamma (NSG) mice, aged 6–8 weeks. Cells were injected in a 50:50 mixture of PBS and matrigel basement membrane matrix (Fisher Scientific; cat#CB-40234). Mice were imaged for luciferase expression on a weekly basis for 7 weeks via a Xenogen IVIS bioluminescent imager. Mice were injected with 5ul/gram body weight of 30mg/ml Luciferin-D (Gold Biotechnology, cat# LUCK-1G) in PBS, 5 minutes prior to imaging. Photon flux was calculated using region of interest (ROI) measurements of either the primary tumor site, or the ventral thoracic area for lung metastasis. Tumor volume was also measured using calipers and calculated as πLW²/6. At the experiment end point, mice were euthanized and tumor, lung, and liver tissue were harvested for ex vivo analysis and subsequent histology.