Establishing 177Lu-PSMA-617 Radioligand Therapy in a Syngeneic Model of Murine Prostate Cancer

Abstract

Clinical ¹⁷⁷Lu-PSMA-617 radioligand therapy (RLT) is applied in advanced-stage prostate cancer. However, to the best of our knowledge murine models to study the biologic effects of various activity levels have not been established. The aim of this study was to optimize specific and total activity for ¹⁷⁷Lu-PSMA-617 RLT in a syngeneic model of murine prostate cancer. Methods: Murine-reconstituted, oncogene-driven prostate cancer cells (0.1 × 10⁶) (RM1), transduced to express human prostate-specific membrane antigen (PSMA), were injected into the left flank of C57Bl6 immunocompetent mice. RLT was performed by administering a single tail vein injection of ¹⁷⁷Lu-PSMA-617 at different formulations for specific (60 MBq at high, 62 MBq/nmol; intermediate, 31 MBq/nmol; or low 15 MBq/nmol specific activity) or total activity (30, 60, or 120 MBq). Organ distribution was determined by ex vivo γ-counter measurement. DNA double-strand breaks were measured using anti-gamma-H2A.X (phospho S139) immunohistochemistry. Efficacy was assessed by serial CT tumor volumetry and ¹⁸F-FDG PET metabolic volume. Toxicity was evaluated 4 wk after the start of RLT. Results: Mean tumor-to-kidney ratios ± SEM were 19 ± 5, 10 ± 5, and 2 ± 0 for high, intermediate, and low (each n = 3) specific activity, respectively. Four of 6 (67%) mice treated with intermediate or high specific activity and none of 6 (0%) mice treated with low specific activity or formulation demonstrated significant DNA double-strand breaks (≥5% γ-H2A.X-positive cells). High when compared with intermediate or low specific activity resulted in a lower mean ± SEM tumor load by histopathology (vital tissue, 4 ± 2 vs. 8 ± 3 mm²; n = 3 vs. 6), day-4 ¹⁸F-FDG PET (metabolic volume, 87 ± 23 vs. 118 ± 14 mm³; n = 6 vs. 12), and day-7 CT (volume, 323 ± 122 vs. 590 ± 46 mm³; n = 3 vs. 6; P = 0.039). ¹⁷⁷Lu-PSMA-617 (120 MBq) with high specific activity induced superior tumor growth inhibition (P = 0.021, n = 5/group) without subacute hematologic toxicity (n = 3/group). Conclusion:¹⁷⁷Lu-PSMA-617 (120 MBq) and high specific activity resulted in the highest efficacy in a syngeneic model of murine prostate cancer. The model will be useful for studying the effects of PSMA-directed RLT combined with potentially synergistic pharmacologic approaches.

Keywords: 177Lu; PSMA; prostate cancer; specific activity; syngeneic.

FIGURE 1.

Biodistribution of ¹⁷⁷Lu-PSMA-617 at different levels of specific activity (experiment 1). (A) Mice received 60 MBq of ¹⁷⁷Lu-PSMA-617 at high (62 MBq/nmol), intermediate (31 MBq/nmol), or low (15 MBq/nmol) specific activity. Mice were sacrificed on day 4 to determine ¹⁷⁷Lu-PSMA-617 biodistribution (B) and tumor–to–organ uptake ratios (C). Data are mean %IA/g tissue + SEM (n = 3/group). Difference between high- versus intermediate- or low-specific-activity groups was assessed by Mann–Whitney test. *P < 0.05. GI = gastrointestinal tract.

FIGURE 2.

Response after ¹⁷⁷Lu-PSMA-617 RLT using high, intermediate, or low specific activity (experiment 1). (A) Mice received formulation (NT group) or 60 MBq of ¹⁷⁷Lu-PSMA-617 at high, intermediate, or low specific activity. CT tumor volume is shown until first animal per group was required to be sacrificed. Higher tumor uptake translates into more effective tumor growth inhibition (B, *P = 0.039), prolonged survival (C), and metabolic response (D). One mouse in high group had complete response and remained tumor free for more than 60 d (C). Difference between high- versus intermediate- to low-specific-activity groups was assessed by Mann–Whitney test. Data are presented as mean + SEM of n = 3 (B and C) or n = 6 (D) per group. *P < 0.05.

FIGURE 3.

Tumor growth inhibition (A) and survival (B) after RLT using 30, 60, or 120 MBq of ¹⁷⁷Lu-PSMA-617 (experiment 2). (A) Mice received formulation (NT group) or 120, 60, or 30 MBq of ¹⁷⁷Lu-PSMA-617 at high specific activity. One hundred twenty MBq of ¹⁷⁷Lu-PSMA-617 induced most effective tumor growth inhibition (B, *P = 0.021) with improved survival (C). Difference between 120-MBq versus other groups was assessed by Mann–Whitney test on day 12. Data are mean + SEM of n = 5 per group. *P < 0.05; ^‡1 mouse each died during PET/CT.

FIGURE 4.

PSMA expression of mouse RM1 versus human C4-2 prostate cancer cell lines. (A) PSMA expression was determined in vitro by flow cytometry for RM1-hPSMA (green), RM1-PGLS (blue), and C4-2 (red). RM1-PGLS cells exhibit heterogeneous PSMA expression, with PSMA-low (light blue) and PSMA-high (dark blue) subpopulations. (B) Mean absolute numbers of PSMA on cell surface were quantified on basis of mean fluorescence intensity values using antihuman IgG quantification beads. (C) Representative ⁶⁸Ga-PSMA-11 PET/CT maximum-intensity projection of mice bearing equally sized subcutaneous mouse allografts or human xenografts. Mean %IA/g was 22.7, 17.4, and 15.5 for RM1-hPSMA, RM1-PGLS, and C4-2, respectively (n = 2 each).

FIGURE 5.

DNA damage induced by ¹⁷⁷Lu-PSMA-617 RLT using high, intermediate, or low specific activity (experiment 1). (A) Representative areas of tumor tissue stained for γ-H2A.X are shown at 10-fold magnification (conditions in separate rows). Proportion of γ-H2A.X–positive cells was evaluated by a masked reader visually (A) and by ImageJ software (B). + = 5%–10%; ++ = 10%–20%; +++ = >20% γ-H2A.X–positive cells. Dots represent single values; bars are mean values (n = 3 per group).

FIGURE 6.

Hemoglobin and blood count level 4 wk after RLT using 30, 60, or 120 MBq of ¹⁷⁷Lu-PSMA-617 (experiment 3). ¹⁷⁷Lu-PSMA-617 RLT was not associated with relevant hematotoxicity. Data are mean ± SEM of n = 3/group. LLN = lower limit of reference range.