Combination therapy of nano-FdUMP and nano-Folox with PD-L1 blockade for CRC liver metastasis mouse model

The CRC liver metastasis mouse model was established as previously described [7]. In brief, mice were anesthetized using 2.5% isoflurane, and the spleen was exteriorized, tied and sectioned. Afterwards, ~ 2 × 10⁵ CT26-FL3 cells were injected to the distal section of the spleen. The hemi-spleen injected by CT26-FL3 cells was removed, and the other half was placed back into the cavity. Following tumor inoculation at Day 0, tumor growth was monitored using the IVIS® Kinetics Optical System. When tumor growth was reached at ~ 0.5 to 1 × 10⁸ p/sec/cm²/sr, mice were i.v. administrated with Nano-Folox containing 1.5 mg/kg of Pt (~ 4.5 mg/kg of FnA) as described in fig. 7, which were followed by i.v. administration of Nano-FdUMP (10 mg/kg of fluorine drug) at 8 h post-injection. After this, mice were i.p. injected with or without anti-PD-L1 mAb (Bioxcell, clone 10F.9G2, 100 μg per mouse). The tumor growth was observed using the IVIS® Kinetics Optical System (n = 6). Separately, one day following two injections, tumors were obtained on Day 12 for TUNEL analysis (n = 4), flow cytometry (n = 4) and RT-PCR experiment (n = 4), as described above.

The combination therapy of Nano-FdUMP/Nano-Folox and anti-PD-L1 antibody for CRC liver metastasis mouse model. a) Treatment schedule and IVIS images. b) The liver metastases over a 16-day period (n = 6, * p < 0.05 and ** p < 0.01). c) Animal survival (median survival: PBS = 20 days, anti-PD-L1 antibody = 21 days, and Nano-FdUMP/Nano-Folox = 48 days) (n = 6, *** p < 0.01). d) Immunofluorescent staining of tumors on Day 12 (DNA fragments = green; nuclei = blue) to determine apoptosis (n = 4, ** p < 0.01, relative to Nano-FdUMP/Nano-Folox). e) Level of CD8⁺ T cells, CD4⁺ T cells, memory CD8⁺ T cells, memory CD4⁺ T cells, and activated DCs in tumors on Day 12, analyzed by flow cytometry (n = 4, * p < 0.05 and NS = no significance). f) The mRNA expression of IFN-γ, IL-12, IL-4, IL-6 and IL-10 in tumors on Day 12 (n = 4, * p < 0.05 and NS = no significance)