Xenogen ivis lumina bioluminescence imaging system

Prior to bioluminescence imaging the region where tumors were located was shaved. Mice were then anesthetized with 2.5% isoflurane in O₂, then administered the d-luciferin substrate (150 mg/kg in PBS) by IP injection. Following the injection of the d-luciferin substrate mice, were imaged using the Xenogen IVIS Lumina Bioluminescence Imaging System (PerkinElmer). The peak of luciferase photon flux was recorded 6 minutes after injection of the d-luciferin substrate. The total photon flux was analyzed and restricted to tumor region of interest using Living Image v2.60.1 software (Imaging Systems).
More information regarding human TYMS and DHFR preparation, tritium-based TYMS catalytic activity assay, competitive drug displacement assay, DHFR activity assay, initial computational screening for potential inhibitors, and molecular modeling of the proposed inhibition modes is provided in Supplemental Methods.

Luc-PANC-1 (5 × 10⁶) and Luc-CM (0.5 × 10⁶) cells were resuspended in 200 μL of PBS and injected subcutaneously or IP, respectively, into 6- to 8-week-old NSG mice. Tumor-bearing mice were imaged using Xenogen IVIS Lumina Bioluminescence Imaging System (PerkinElmer) every week after cell injection. Luc-PANC-1– and Luc-CM–injected mice were randomized based on slope of luciferase signal at 28 days and 24 days, respectively, and treatment was initiated. Mice received daily 19-S or 19-S7 at the indicated doses by IP injection or by oral gavage (PO), and vehicle control mice received corn oil. For all animals, body weight was recorded weekly, and Luc-PANC-1 tumor volume was measured weekly with a caliper. For scheduled sacrifice experiments, animals were euthanized after 4 weeks of treatment. For survival studies, animals were sacrificed when tumors reached 1,500 mm³ or when they showed any signs of ulceration. Harvested tumors were excised and weighed, then fixed in alcoholic formalin (67.5% ethanol, 10% formaldehyde 37%, 22.5% water) for pathology analysis. Tumor volume was calculated as volume = 0.52(length × width²).