Lumina xr imaging system

To study cellular uptake of various nanoparticles, we performed near infrared imaging on cells incubated with the DiR formulations. Briefly, six cell lines including 293 T, HT29, HepG2, MDA-MB-486, MCF-7, 4T1 were seeded into a 24-well plate at the density of 10⁵ cells/well. After 80–90% confluence, the cells were incubated with various fluorescent formulations (free DiR, PDN and the PDNM) containing equal levels of DiR for 2 h. Treatment with PBS was used as a negative control. Subsequently, the cells in each well were washed with precooled PBS for three times and imaged by Lumina XR imaging system (PerkinElmer, IVIS Lumina, Waltham, MA).

C57BL/6 mice were anesthetized and injected with 1×10⁵ GL261-luc tumor cells in the right cerebral hemisphere of the brain using a standard stereotaxic instrument (RWD Life Science, USA). Established GL261-bearing S15^KO and S15^WT model mice were randomized into different groups, receiving craniocerebral radiation (4 Gy) on Day 7 using an RS-2000 Biological Irradiator (RadSource, Canada) or intraperitoneal injection of an anti-PD-1 mAb (clone G4, in-house) 200μg/mouse on Day 10. Tumor growth was monitored by bioluminescence imaging every 3-7 days using a Lumina XR Imaging System (PerkinElmer, USA). Tumor tissues were isolated on the indicated days for further analysis. The survival of the mice was monitored over a 28-day observation period.

Human breast cancer cell lines (MCF-7, SKBR-3 and MDA-MB-468), mouse breast cancer cell lines (4T1, E0771), mouse glioma cell (GL261) and OVA transfection the EL4 thymoma cell (EG7) were cultured in RPMI-1640 or DMEM containing 10% FBS (Sigma-Aldrich). B7H4-overexpressing tumor cells (GL261-B7H4 and EG7-B7H4) were prepared by transfection of the pcDNA-mB7H4 plasmid encoding full-length mouse B7H4. C57BL/6 mice were anesthetized and 5 × 10⁵ GL261 or GL261-B7H4 tumor cells were injected into the right cerebral hemisphere of the brain at 4 mm depth below the surface of the scull using a Hamilton PB-600-1 Repeatable Dispenser. Tumor growth was monitored by bioluminescent imaging every 3–7 days using a Lumina XR imaging system (PerkinElmer), and the survival of mice was monitored daily. A total of 5 × 10⁵ EG7 or EG7-B7H4 cells were s.c. injected in the right flank of C57BL/6 mice or NSG mice. Tumor sizes were measured with digital calipers every 3 days and calculated using the equation (l + w)/2, where l and w refer to the larger and smaller dimensions, respectively. The mice were sacrificed as death for humane treatment after tumors reached a size of 2.0 cm in each dimension.