Performance of PSMA-targeted radiotheranostics in an experimental model of renal cell carcinoma

Abstract

Introduction: Renal cell carcinoma (RCC) represents cancer originating from the renal epithelium and accounts for > 90% of cancers in the kidney. Prostate-specific membrane antigen (PSMA) is overexpressed in tumor-associated neovascular endothelial cells of many solid tumors, including metastatic RCC. Although studied in several small clinical studies, PSMA-based imaging and therapy have not been pursued rigorously in preclinical RCC. This study aimed to evaluate the preclinical performance of PSMA-based radiotheranostic agents in a relevant murine model.

Methods: A PSMA-overexpressing murine cell line, PSMA+ RENCA, was developed by lentiviral transduction. PSMA-based theranostic agents, ⁶⁸Ga-L1/¹⁷⁷Lu-L1/²²⁵Ac-L1, were synthesized in high radiochemical yield and purity following our reported methods. Immunocompetent BALB/c mice were used for flank and orthotopic tumor inoculation. ⁶⁸Ga-L1 was evaluated in small animal PET/CT imaging in flank and PET/MR imaging in orthotopic models. Cell viability studies were conducted for ¹⁷⁷Lu-L1 and ²²⁵Ac-L1. Proof-of-concept treatment studies were performed using ²²⁵Ac-L1 (0, 37 kBq, 2 kBq × 37 kBq, 1 week apart) using PSMA+ RENCA in the flank model.

Results: Cellular uptake of ⁶⁸Ga-L1, ¹⁷⁷Lu-L1, and ²²⁵Ac-L1 confirmed the specificity of the agents to PSMA+ RENCA cells rather than to RENCA (wt) cells, which are low in PSMA expression. The uptake in PSMA+ RENCA cells at 1 h for ⁶⁸Ga-L1 (49.0% incubated dose [ID] ± 3.6%ID/million cells), ¹⁷⁷Lu-L1 (22.1%ID ± 0.5%ID)/million cells), and ²²⁵Ac-L1 (4.1% ± 0.2% ID)/million cells), respectively, were higher than the RENCA (wt) cells (~ 1%ID-2%ID/million cells). PET/CT images displayed > 7-fold higher accumulation of ⁶⁸Ga-L1 in PSMA+ RENCA compared to RENCA (wt) in flank implantation at 1 h. A twofold higher accumulation of ⁶⁸Ga-L1 was observed in orthotopic tumors than in normal kidneys during 1-3 h postinjection. High lung uptake was observed with ⁶⁸Ga-L1 PET/MR imaging 3 weeks after orthotopic implantation of PSMA+ RENCA due to spontaneous lung metastases. The imaging data were further confirmed by immunohistochemical characterization. ²²⁵Ac-L1 (0-37 kBq) displayed a dose-dependent reduction of cell proliferation in the PSMA+ RENCA cells after 48 h incubation; ~ 40% reduction in the cells with treated 37 kBq compared to vehicle (p < 0.001); however, no effect was observed with ¹⁷⁷Lu-L1 (0-3700 kBq) up to 144 h postinoculation, suggesting lower efficacy of β-particle-emitting radiations in cellular studies compared to α-particle-emitting ²²⁵Ac-L1. Animals treated with ²²⁵Ac-L1 at 1 week posttumor inoculation in flank models displayed significant tumor growth delay (p < 0.03) and longer median survival of 21 days and 24 days for the treatment groups 37 kBq and 2 kBq × 37 kBq, respectively, compared to the vehicle group (12 days).

Conclusion: The results suggest that a theranostic strategy targeting PSMA, employing PET and α-emitting radiopharmaceuticals, enabled tumor growth control and enhanced survival in a relevant immunocompetent murine model of RCC. These studies provide the rationale for clinical studies of PSMA-targeted theranostic agents in patients with RCC.

Keywords: actinium-225; alpha-particle emitting radionuclide; gallium-68; lutetium-177; positron-emission tomography; prostate-specific membrane antigen; targeted radiopharmaceutical therapy; β-particle emitting radionuclide.

Figure 1

Characterization of PSMA+ RENCA and RENCA (wt) cell lines. (A) Flow cytometry of PSMA surface expression. Quantitation of mean fluorescence intensity shows that PSMA+ RENCA cells have higher levels of PSMA surface expression than RENCA (wt) cells. (B) PSMA total protein levels in PSMA+ RENCA and RENCA (wt) cells by Western blot. (C) Relative fold change in PSMA total protein levels compared to GAPDH. (D) PSMA mRNA levels in PSMA+ RENCA and RENCA (wt) cells by RT-qPCR. (E) Chemical structure of PSMA-based radiopharmaceutical agents used in this study. (F–H) Cell uptake (average ± SD, n = 3) of (F) ⁶⁸Ga-L1, (G) ¹⁷⁷Lu-L1, and (H) ²²⁵Ac-L1 in PSMA+ RENCA without or with blockade and RENCA (wt) cells at 37°C.

Figure 2

(A) PSMA PET-CT imaging in murine renal cell carcinoma, PSMA+ RENCA (right flank), and RENCA (left flank) with ⁶⁸Ga-L1. Male BALB/c tumor-bearing mice were injected with 7.4 MBq of ⁶⁸Ga-L1 through the tail vein, and PET-CT images were acquired 0.5 h, 1 h, and 2 h postinjection. Red arrow: PSMA+ RENCA; dotted black arrow: RENCA (wt). (B) Tissue biodistribution data for ²²⁵Ac-L1 in flank models bearing PSMA+ RENCA (right) and RENCA (wt) (left). (C) Tissue biodistribution data for ²²⁵Ac-L1 in orthotopic models bearing PSMA+ RENCA in the right kidney. Data, mean ± SEM of three mice.

Figure 3

(A) Whole-body PET/MR images of PSMA+ RENCA orthotopic tumor-bearing mice: M2 (32 days post-tumor inoculation; top panel) and M2 + blocker (34 days post-tumor inoculation; coinjection with PSMA-targeted ZJ43; middle panel) in prone (left side) and supine view (right side) at 3 h postinjection of 68Ga-L1. K, kidney (black dotted circle, white arrow); T, orthotopic tumor (yellow dotted circle, yellow arrow); L, lung (red dotted circle, red arrow); B, bladder (yellow solid circle, white dotted arrow); R, right; L, left. All images are decay-corrected and adjusted to the maximum value. (B) Axial PET (left panel), fused PET/MR (middle panel), and T2-weighed MR (right panel) images of Ctrl (tumor-free; top panel) and RENCA-orthotopic tumor-bearing mice (M1, 21 days posttumor inoculation; M2, 32 days posttumor inoculation; and M2 with blocker, 34 days posttumor inoculation) at 3 h postinjection of ⁶⁸Ga-L1.

Figure 4

(A) Representative photographs of kidneys (left panel) and H&E staining of kidney sections (middle panel, scale bar, 5 mm) in orthotopic RENCA tumor-bearing mice (M1 and M2) harvested at the times indicated. Histopathological analysis of tumor-bearing kidneys by H&E and PSMA staining (scale bar, 100 µM; ×20). Areas characterized by intense purple staining (H&E) indicate the presence of renal tumors and demonstrate lower PSMA staining. (B) Representative images (scale bar, 100 µM; ×20) of H&E (middle panel) and PSMA (bottom panel) staining observed in nonmetastatic and micrometastatic regions of the lungs (represented by dense purple staining in H&E) were indicated by b and a, respectively, in the top panel. High PSMA expression was observed in the micrometastatic sites. (C) Coronal view PET/MRI imaging of the lungs of M2 showing high soft tissue contrast in MR imaging and high sensitivity of PET imaging.

Figure 5

(A) Cell viability was assessed with ²²⁵Ac-L1 in a dose-dependent manner (0–37 kBq) against PSMA+ (R+) and RENCA (wt) (R-) cells by a cell Titer-Glo luminescent cell viability assay. (B) Study design to evaluate the treatment effect in PSMA+ RENCA flank tumors following administration of ²²⁵Ac-L1 via tail-vein injection. (C) Relative tumor volume (v_t /v_o ); tumor growth curves relative to tumor volume at day 7 (set to 1). (D) Body weights of control and treatment groups. (E) The Kaplan–Meier curve revealed significant tumor growth control in the RENCA xenograft model for the treatment groups (p < 0.03) compared to the untreated control group. (F) Representative photographs were collected on day 18 after the tumor inoculation from Ctrl (0 kBq) and treatment groups (²²⁵Ac-L1; 37 kBq and 2 × 37 kBq). (G) The average weight of tumors collected from Ctrl (0 kBq) and treatment groups (²²⁵Ac-L1; 37 kBq and 2 × 37 kBq). (H) Representative H&E-stained PSMA+ RENCA tumor sections (left first panel; scale bar, 5 mm; ×0.2) from control and treatment groups. Representative images (scale bar, 100 µM; ×20) of H&E (left second panel), IHC of PSMA (middle panel), CD31 (right third panel), and γ-H2AX (right last panel) staining, respectively, observed in PSMA+ RENCA tumors indicated by a yellow shaded triangle in the left panel from the control and treatment groups.