Article Figures & Data
Figures
- FIGURE 1.
Radiochemistry and target binding of huPAR and muPAR immuno-PET antibodies. (A) Bead binding assay with muPAR- and huPAR-coated magnetic beads via His tag shows high binding of [89Zr]Zr-DFO-anti-muPAR to all conditions; still, significant increase in bound activity was found for muPAR antigen beads. Binding was significantly below that of control beads with huPAR antigen, and there was no significant difference from control with blocked beads. (B) Identical bead binding with [89Zr]Zr-DFO-anti-huPAR shows better specificity to control beads and significant difference for both muPAR and huPAR antigens, confirming mouse–human cross reactivity of [89Zr]Zr-DFO-anti-huPAR antibody. (C) Increased β-gal staining alongside morphologic changes shows that TP induces senescence in KPC and MiaPaCa2 cells. (D) Murine pancreatic cancer line KPC was treated with TP for 1, 6, or 12 d, showing significant increase in [89Zr]Zr-DFO-anti-muPAR binding, which can be blocked. (E) Uptake of [89Zr]Zr-DFO-anti-huPAR in MiaPaCa2, despite lower activity uptake overall, was significantly increased as well on TP treatment. There were 4 technical replicates in A and B and 5 technical replicates in D and E. Scale bar in C represents 100 μm. *P < 0.05. **P < 0.005. ***P < 0.0005. ****P < 0.0001. Ab = antibody.
- FIGURE 2.
Background imaging of muPAR in healthy CD1 mice shows decrease in antibody distribution and increase in soluble uPAR with age. (A) [89Zr]Zr-DFO-anti-muPAR distribution by PET in 1.5-mo-old mice was found to be much more diffuse by 96 h than in geriatric mice at age of 21 mo. (B) Terminal biodistribution confirmed immuno-PET imaging, with significant decrease in radiotracer distribution in geriatric mouse blood, lungs, and bone. (C) Blood from littermates of immuno-PET study at 1.5, 4, and 21 mo of age were assayed by enzyme-linked immunosorbent assay for soluble uPAR, showing significant increase in soluble uPAR between 1.5- and 21-mo-old mice. There were 5 mice per cohort, aged in Memorial Sloan Kettering Cancer Center vivarium, in A and B, and 4 mice per age group with 3 technical replicates per mouse in C. One-way ANOVA statistical analysis was used in C. *P < 0.05. ***P < 0.0005. ****P < 0.0001. %IA = percentage injected activity; %ID = percentage injected dose; bars = median; Cor. = coronal projection; error = SEM; MIP = maximum-intensity projection; suPAR = soluble uPAR.
- FIGURE 3.
With greater specificity than [18F]FDG, immuno-PET of muPAR and huPAR showed that human and murine pancreatic cancer increased with senescence-inducing therapy. (A) [89Zr]Zr-DFO-anti-muPAR PET imaging at 24–144 h in KPC-bearing mice showed clear uptake in tumor with and without TP treatment, though with reduced distribution in liver and increased uptake in blood after TP treatment. Terminal biodistribution can be found in Supplemental Figure 5A. (B) [89Zr]Zr-DFO-anti-huPAR PET imaging at 24–144 h in MiaPaCa2 showed lower tumor targeting in untreated mice than did [89Zr]Zr-DFO-anti-muPAR imaging. On TP treatment, tumor uptake increased significantly with TP, as seen in terminal biodistribution (Supplemental Fig. 5B), and was more specific to tumor than with [18F]FDG imaging (Supplemental Fig. 5D). There were 5 mice per group. Region-of-interest analysis of heart, liver, and tumor can be found for KPC and MiaPaCa2 models in Supplemental Figure 6. %IA = percentage injected activity.
- FIGURE 4.
[89Zr]Zr-DFO-anti-muPAR targeting of 10- and 52-wk-old mice bearing KPC flank tumors on or off TP therapy. (A) Ten-week-old mice bearing flank KPC tumors showed rapid accumulation of [89Zr]Zr-DFO-anti-muPAR in tumor after 24 h and through 72 h, with no discernable difference between untreated and TP-treated tumors. (B) Mice aged 52 wk that were implanted and imaged at same time as mice in A showed less uptake of [89Zr]Zr-DFO-anti-muPAR in untreated group with increase in TP treatment. (C) In 52-wk-old untreated mice, larger tumors were seen than in 10-wk-old mice, as expected for mice with advanced age and diminished immune capacity. No other differences in gross organ findings between 10 and 52 wk old were observed other than enlargement of liver in aged mice. (D) Terminal biodistribution at 72 h shows decrease in [89Zr]Zr-DFO-anti-muPAR in blood as seen in Figure 2C for aged mice, and significant decrease in tumor targeting was also seen for untreated mice. TP-treated mice had slightly higher average uptake values, but differences were not statistically significant. Although [89Zr]Zr-DFO-anti-muPAR rapidly identified KPC flank tumors, TP treatment did not yield significant increase in tumor uptake in either TP group; tumor size was same as that of mice 10 wk old. %IA = percentage injected activity; %ID = percentage injected dose.
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