Fig. 4: In vivo NIR fluorescence imaging and nano-immunocomplex-mediated activatable sono-metabolic checkpoint trimodal cancer therapy.

a Pharmacokinetic analysis of blood concentration of HP in BALB/c mice at t = 1, 2, 4, 8, 12, or 24 h post-injection of HNP or HPNP (n = 3). b NIR fluorescence imaging of 4T1 tumor-bearing BALB/c mice at t = 0, 2, 4, 6, 8, 12, or 24 h post-injection of HNP or HPNP (injection dose: 200 μL, [HP] = 1 mmol/L, or [ADA] = 40 U/mL). c Biodistribution of HNP or HPNP in 4T1 tumor-bearing mice at 24 h after systemic administration (n = 3). HPNP versus HNP in tumors: p = 0.0035. d Quantitative analysis of SOSG MFIs in tumor tissues from HNP-, PNP-, or HPNP-injected mice with or without sono-irradiation (1.0 MHz, 1.2 W/cm², 50% duty cycle) for 6 min (n = 5). HNP + US versus HNP: p < 0.0001; HPNP + US versus HPNP: p < 0.0001. e Schematic illustration of the schedule for bilateral tumor model implantation and nano-immunocomplex-mediated activatable sono-metabolic checkpoint trimodal cancer therapy. Growth curves of primary tumors (f) and distant tumors (g) after different treatments (n = 5). 6 versus 1 in f: p < 0.0001; 8 versus 1 in f: p < 0.0001; 8 versus 1 in g: p < 0.0001. h Survival curves for the mice after different treatments using the Kaplan–Meier method (n = 5). p < 0.0001. i Quantification of caspase-3 expression (n = 5). 6 versus 1 in primary tumors: p < 0.0001; 8 versus 1 in primary tumors: p < 0.0001; 8 versus 1 in distant tumors: p < 0.0001. j Histological H&E staining of lung in 4T1 tumor-bearing mice. Images are representative of three biologically independent mice. k Schematic illustration of the schedule for tumor rechallenge study. l Growth curves of the reinoculated tumors (n = 5). HPNP versus HNP: p = 0.0014. m Survival curves for the mice with reinoculated tumors using the Kaplan–Meier method (n = 5). p = 0.0003. Cell viability of 3T3 and 4T1 cells as target cells (T) after incubation with effector T cells (E) isolated from spleen of the rechallenged and HNP- (n) or HPNP-injected (o) mice as a function of the E/T ratios (n = 4). 4T1 versus 3T3 in n: p = 0.0027; 4T1 versus 3T3 in o: p < 0.0001. Statistical significance in c, d, i, n, and o was calculated via one-way ANOVA with a Tukey post-hoc test. Statistical significance in f, g, and l was calculated via two-tailed Student’s t-test. Statistical significance in h and m was calculated via the log-rank test. **p < 0.01, ***p < 0.001, and ****p < 0.0001. The mean values and SD are presented. Source data are provided as a Source Data file.