Ivis lumina 3 imager

The Renca cell line (syngeneic to BALB/c mice) was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), engineered to express firefly luciferase, and cultured as described [4 (link),29 (link),32 (link)]. Cells were confirmed negative for mycoplasma, passaged, and used at the same passage number to limit experimental variation. Intrarenal tumor challenges and bioluminescent imaging (BLI) using an IVIS Lumina III Imager (Perkin Elmer; Waltham, MA, USA; University of Alabama at Birmingham (UAB) Small Animal Imaging Facility) were performed as described [4 (link),29 (link),32 (link)]. At day six post-tumor challenge, BLI was used to assess tumor burdens in live mice prior to therapy randomization. At day seven post-tumor challenge, mice were re-injected in the tumor-bearing kidney with either sterile saline or 10⁹ pfu of replication-deficient adenovirus (Ad) encoding a membrane-bound version of full-length murine TRAIL protein (University of Iowa Viral Vector Core) plus 100 µg CpG1826 (Integrated DNA Technologies) (AdT/CpG treatment) and further randomized to receive no therapy, InVivoMAb polyclonal Armenian hamster IgG control (BioXCell; Lebanon, NH, USA), or InVivoMAb anti (α)-mouse CTLA-4 (clone UC10-4F10-11; BioXCell) i.p. at a dose of 100 μg/mouse on days 10, 13, and 16 post-tumor challenge.

In vivo fluorescence imaging of P144 was performed as previously described.¹³ Briefly, mice were intravenously injected with 1 mg/kg of Alexa Fluor 647‐conjugated P144 (dye‐to‐protein molar ratio of 4.78:1). Alexa Fluor 647 (Thermo Fisher Scientific) alone served as the control. Mice were anesthetized with isoflurane (Hebei Yipin Pharmaceutical Co. Ltd) and imaged using an IVIS Lumina III imager (PerkinElmer chemagen Technologie GmbH) using identical settings (2‐s exposure, f/stop ¼ 2).

The Renca renal carcinoma cell line (derived from and syngeneic to BALB/c mice) was purchased from ATCC, engineered to express firefly-luciferase through lentiviral transduction, and cultured as previously described [32 (link),34 (link),50 (link),51 (link)]. Cells were not authenticated after purchase. Cells were confirmed negative for mycoplasma, passaged, and used at the same passage number (passage 13) in all tumor studies described here to limit experimental variation. Intrarenal tumor challenges were performed where indicated as previously described [32 (link),50 (link),51 (link)]. BALB/c mice were orthotopically injected with 5 × 10⁴ live Renca cells in a volume of 100 μL/mouse. Tumor burdens were assessed from live anesthetized mice or from excised lungs by bioluminescent imaging (BLI) using intraperitoneal injections with 1 mg of luciferin and an IVIS Lumina III imager from Perkin Elmer, provided by the Small Animal Imaging Facility at UAB. In the rare event that BLI yielded negative radiance values when imaging lungs (n = 3 lungs), the negative value was replaced with the background BLI radiance value for tumor-free lungs (1.5 × 10⁴ photons/s/cm²/sr). Primary renal tumors were excised and weighed.