EGFR-Targeted ImmunoPET of UMUC3 Orthotopic Bladder Tumors

Abstract

Purpose: Immuno-positron emission tomography (immunoPET) combines the specificity of an antibody with the sensitivity of PET to image dysregulated pathways in cancer. This study examines the performance of immunoPET using the radioimmunoconjugate [⁸⁹Zr]Zr-DFO-Panitumumab to detect epidermal growth factor receptor (EGFR) expression in an orthotopic model of bladder cancer (BCa).

Procedures: Expression and quantification of EGFR receptors were confirmed in four different BCa cell lines. Binding assays validated [⁸⁹Zr]Zr-DFO-Panitumumab specificity for EGFR-expressing UMUC3 BCa cells. Subcutaneous and orthotopic UMUC3 xenografts were then used for PET imaging and ex vivo biodistribution of the radioimmunoconjugate. Control cohorts included non-tumor mice, ⁸⁹Zr-labeled non-specific IgG, and blocking experiments.

Results: [⁸⁹Zr]Zr-DFO-Panitumumab binds specifically to EGFR-expressing UMUC3 cells with a B_max value of 5.9 × 10⁴ EGFRs/cell in vitro. ImmunoPET/CT images show localization of the antibody in subcutaneous UMUC3 xenografts and murine bladder tumors. In the orthotopic model, the immunoPET signal correlates with the respective tumor volume. Ex vivo biodistribution analysis further confirmed imaging results.

Conclusion: The preclinical data presents a proof of concept for utilizing EGFR-targeted immunoPET to image BCa with altered EGFR protein levels.

Keywords: Bladder Cancer; EGFR; ImmunoPET.

Figure 1.

(A) Western blot analysis of EGFR protein levels in RT112, T24, UMUC3, and UMUC14 bladder cancer cell lines. (B, C) ⁸⁹Zr-labeled Panitumumab binding to EGFR-expressing UMUC3 bladder cancer cells in the absence (B) and presence (C) of excess Panitumumab. UMUC3 bladder cancer cells (1, 2, 4, or 10 million) were incubated with 37 KBq of [⁸⁹Zr]Zr-DFO-Panitumumab (0.33–0.36 μg) for 1 h at 4°C. For blocking of ⁸⁹Zr-labeled Panitumumab binding to cancer cells, cells were incubated with ⁸⁹Zr-labeled Panitumumab in the presence of 33–36 μg DFO-Panitumumab. Data are presented as mean ± S.E.M, n = 6 (2 independent experiments). (D) UMUC3 cells were incubated with [⁸⁹Zr]Zr-DFO-Panitumumab (0–125 nM) for 3 h at 4°C. Specific binding of [⁸⁹Zr]Zr-DFO-Panitumumab and non-linear regression curve fit are represented in black spheres and dotted lines. Data are presented as mean ± S.E.M, n = 3.

Figure 2.

(A) Representative coronal PET images and (B) biodistribution data of [⁸⁹Zr]Zr-DFO-Panitumumab in athymic nude mice bearing subcutaneous UMUC3 tumors. PET images and biodistribution were performed at 72 h after tail vein injection of [⁸⁹Zr]Zr-DFO-Panitumumab (8.25–9.18 MBq, 58–65 μg protein). Bars, n = 4 mice per group, mean ± S.E.M. (C) ⁸⁹Zr-labeled IgG or ⁸⁹Zr-labeled Panitumumab accumulation in UMUC3 subcutaneous tumors. Tumors were collected at 72 h after tail vein injection of [⁸⁹Zr]Zr-DFO-Panitumumab or [⁸⁹Zr]Zr-DFO-IgG (8.25–9.18 MBq, 58–65 μg protein). Blocking experiments were performed by administering [⁸⁹Zr]Zr-DFO-Panitumumab in the presence of 2.32 mg DFO-Panitumumab. Data are presented as mean ± S.E.M, n = 4 mice per group. ^**P < 0.01, based on a Student’s t-test. %ID/g, percentage of injected dose per gram.

Figure 3.

(A) Schematic of PET imaging and biodistribution studies of [⁸⁹Zr]Zr-DFO-Panitumumab in orthotopic UMUC3 tumors. Right panel shows representative ultrasound images of murine bladders at 11 days after UMUC3 cells’ implantation in the bladder. (B) Representative coronal and MIP PET images of [⁸⁹Zr]Zr-DFO-Panitumumab in athymic nude mice bearing orthotopic UMUC3 tumors or mice without tumors (M1, M2, M5, and M6 are mouse identifications of 2 mice included in the respective cohorts). PET images were collected at 72 h after tail vein injection of [⁸⁹Zr]Zr-DFO-Panitumumab (8.25–9.18 Mq, 58–65 μg protein). %ID/g, percentage of injected dose per gram.

Figure 4.

(A) Biodistribution data of [⁸⁹Zr]Zr-DFO-Panitumumab in athymic nude mice without tumors (top panel, labeled as M5-M8 where each bar represents an individual mouse) or with UMUC3 orthotopic tumors (bottom panel, labeled as M1-M4 where each bar represents an individual mouse). Biodistribution was performed at 72 h after tail vein injection of [⁸⁹Zr]Zr-DFO-Panitumumab (8.25–9.18 MBq, 58–65 μg protein). Each bar represents data collected for one mouse of each cohort. (B) Bladder tumors at 14 days after UMUC3 cells’ implantation in the bladder. (C) ⁸⁹Zr-labeled Panitumumab accumulation in UMUC3 orthotopic tumors or non-tumor bladders at 14 days after UMUC3 cells’ implantation in the bladder. Bladders were collected at 72 h after tail vein injection of [⁸⁹Zr]Zr-DFO-Panitumumab (8.25–9.18 MBq, 58–65 μg protein). Data are presented as mean ± S.E.M, n = 4 mice per group. ^*P < 0.05, based on a Student’s t-test. (D) EGFR protein levels in UMUC3 orthotopic bladder tumors. Tumors were collected and EGFR expression analyzed by western blot for mice labeled as M1, M2, M3, and M4. β-actin is a loading control.