Study design

¹⁷⁷Lu-PSMA617 RLT efficacy as function of PSMA expression/cell (Figure 1A). RM1-YFP (PSMA-, YFP-only), RM1-PSMA low (PSMA⁺; ~0.5×10⁴ PSMA/cell), RM1-PSMA medium (PSMA⁺⁺; ~1.3×10⁴ PSMA/cell),) and RM1-PSMA high (PSMA⁺⁺⁺; ~4.9×10⁴ PSMA/cell; Supplementary Figure S1A), respectively, were injected into Nod Scid gamma (NSG) mice (12 mice/group; day 0) to generate xenografts.

The impact of the fraction of PSMA-positive vs. PSMA-negative RM1 tumor cells on ¹⁷⁷Lu-PSMA617 RLT efficacy (Figure 2A). PSMA- and PSMA⁺⁺⁺ cells were mixed at different ratios (PSMA-: PSMA⁺⁺⁺ = 100:0, 75:25, 50:50, 25:75, 0:100) and injected into NSG mice (10 mice/group; day 0).

To confirm the data obtained in the RM1 model, we tested the relevance of PSMA expression for RLT outcome in a human-derived PC model (study 3; Figure 3A). NSG mice were injected with PC-3-PIP (PSMA⁺⁺⁺) and PC-3 (PSMA-) at different ratios (PSMA-: PSMA⁺⁺⁺ = 100:0, 66:33, 66:33, 0:100; 12 mice/group). The ratios of PSMA-positive to -negative cells was changed to 0-33-66-100% based on the results of study 2 (Figure 2B, Supplementary Figure S4A).

Lastly, to further elucidate the consequences of PSMA heterogeneity, PSMA- and PSMA⁺⁺⁺ RM1 cells were mixed at different ratios (PSMA-: PSMA⁺⁺⁺ = 100:0, 66:33, 66:33, 0:100) and injected into NSG mice (10 mice/group; day 0) for treatment with ²²⁵Ac-PSMA617 (Figure 4A).

Baseline tumor volume was determined on day 4 (RM1 model) or day 19 (PC-3 model). One to two days later, mice were treated with ¹⁷⁷Lu-PSMA617 (studies 1, 2, 3), or ²²⁵Ac-PSMA617 (study 4). Tumor volume was measured 3x/week (RM1 model) or 1–2x/week (PC-3 model) by CT volumetry. In the RM1 model, tumor growth studies were terminated already at day 14 post RLT because of tumor size and mouse condition (beginning ulceration) as per veterinarian instruction. Absolute tumor volumes for all studies are shown in Supplementary Figure S2A–D.

In a subset of mice (3 mice/group; randomized group allocation), tumors were resected 2 days post RLT. We chose this time point based on our preliminary biodistribution data (data not shown) which suggested a higher ¹⁷⁷Lu-PSMA617 uptake at 48h vs. 4h following injection of 30 MBq ¹⁷⁷Lu-PSMA617 into mice with C4–2 tumors. The percent injected ¹⁷⁷Lu-/²²⁵Ac-PSMA617 activity incorporated into the tumors was determined by ex vivo gamma-counting. DNA damage and PSMA expression in the tumor samples were analyzed by anti-53BP1 and anti-PSMA immunohistochemistry (IHC), respectively.

To ensure comparable tumor growth characteristics across groups and for comparison to tumor volumes in RLT-treated mice tumor volumes of untreated mice were measured by CT (5 mice/group).