Preparation of [177Lu]Lu-DOTA-TOC

[177Lu]Lu-DOTA-TOC (“PRRT”) was readily prepared by ITM according to clinical production standards with an average molar activity of 223 MBq/nmol. Radiochemical purity (rcp) was assayed via radio-TLC (AR-2000, Bioscan) to ensure > 97% rcp for in vivo use. [177Lu]Lu-DOTA-TOC was diluted in RPMI-1640 medium (Gibco, #61870036) for in vitro and in 0.9% NaCl (B. Braun) for in vivo use. Chemicals and solvents were sourced from ITW Reagents, Sigma Aldrich and Avantor.

Preparation of PARPi

For in vitro assays, the PARPi (Olaparib, Rucaparib; MedChemExpress, #HY-10162, #HY-10617A) were prepared as 20 mM or 100 mM stock solutions in dimethyl sulfoxide (DMSO) and subsequently diluted in medium. All treatment and control (“untreated/medium”) conditions were adjusted to contain the same final DMSO (max. 0.5% (v/v)) concentration to ensure that observed treatment effects were not DMSO-related. For in vivo use, stock solutions of Olaparib and Rucaparib in DMSO were prepared at a concentration of 75 mg/mL, which were diluted with PEG300 (MedChemExpress, #HY-Y0873) and PBS to yield a final concentration of 1.5 mg/mL (2% DMSO, 30% PEG300 in PBS) directly before use.

Synthesis of a fluorescent sst-analog

A fluorescent sst-antagonist containing SulfoCy5 (Jena Bioscience; CLK-A130-5) was synthesized using strain-promoted azide-alkyne cycloaddition (SPAAC) as described previously [21]. Briefly, azide functionalized peptide (0.5 mg, 0.27 µmol) was dissolved in 1 mL dry DMSO. DBCO-SulfoCy5 (0.5 mg, 1.07 µmol) was added to the reaction mixture, stirred at 37 °C for 3 h, then at room temperature overnight. The product “Fluo-Oct” was purified by HPLC with H2O/MeCN + 0.1% TFA as eluent, yielding 0.65 mg (0.23 µmol, 84%; Suppl. Figure 1). For flow cytometry, a 1 mM stock solution in DMSO was prepared.

Cell culture

The human SCLC cell lines H69, H446 and rat pancreatic cancer cells AR42J (all ATCC) were cultured in RPMI-1640 growth medium with 10% fetal bovine serum (FBS, Gibco, #A5256801) and 1% penicillin and streptomycin solution (Pen-strep, Gibco, #15140–122) using standard aseptic technique. Cells were maintained in an incubator at 37 °C in 5% atmospheric CO2 concentration and were passaged every 3–4 days. Cells were regularly subjected to mycoplasma testing, which was always reported negative.

Flow cytometry

To investigate the binding of the fluorescent octreotide analog (Fluo-Oct) to the SSTR2-positive cell lines AR42J, H69, and H446, cells were prepared in a concentration of 1 × 106 cells/mL in ice-cold FACS buffer (PBS with 5% FBS and 0.1% NaN3). Cells were incubated with 0.25 µM Fluo-Oct for 30 min at 4 °C in a dark environment. In the blocking group, cells were incubated with 25 µM octreotide (MedChemExpress, #HY-17365) for 15 min at 4 °C, followed 0.25 µM Fluo-Oct for 15 min in the dark. Then, cells were stained with 0.5 µg/mL DAPI (Biolegend, #422801) for 5 min before flow cytometry analysis on a BD FACSCanto instrument. Data were analyzed using FlowJo software.

Cell viability assessment

Cell viability was determined using the AlamarBlue high sensitivity (HS) assay (Thermo Fisher Scientific, #A50101). 5,000 cells per well were seeded in 96-well black clear F-bottom plates (H446) or 96-well black clear V-bottom plates (H69). H69 suspension cells were centrifuged and resuspended for each washing step. 24 h after seeding, PARPi (0.1 – 100 µM final concentration per well), [177Lu]Lu-DOTA-TOC (10 – 250 kBq final activity per well) or combinations thereof were added to the cells for 24 h ([177Lu]Lu-DOTA-TOC) and/or 72 h (PARPi). All wells, including untreated wells, contained 0.5% (v/v) DMSO throughout the assay. 96 h after seeding, medium was removed and resazurin (AlamarBlue HS) solution was added to each well at a final concentration of 10% (v/v) and incubated for 1 h at 37 °C before fluorescence was measured using a Biotek Synergy HT plate reader (Excitation: 540/35 nm, Emission: 590/20 nm). Relative viability was calculated in relation to “untreated” wells. We carried out three biological repeats with 3–6 technical replicates per plate. IC50-values were computed by applying the nonlinear regression function (Dose–response – Inhibition; Inhibitor vs. normalized response) in GraphPad Prism 9.

DNA damage determination (γH2AX)

30,000 H446 cells were seeded into 8-well glass bottom slides (Ibidi, #80807) 24 h before receiving the respective treatments for 1 h (Medium, 1 µM PARPi, 25 kBq [177Lu]Lu-DOTA-TOC, 1 µM PARPi + 25 kBq [177Lu]Lu-DOTA-TOC). After treatment, cells were either stained immediately for γH2AX foci or subjected to a 23 h post-incubation resting period in medium. For immunofluorescence staining, cells were fixed with 4% PFA and permeabilized with 0.1% TritonX-100/PBS for 10 min before blocking with 3% BSA/PBS for 1 h. Then, cells were incubated with the primary Phospho-Histone H2A.X antibody (1:500; MA5-27753, ThermoFisher) for 1 h, followed by the secondary AlexaFluor-488-conjugated goat-anti-mouse IgG antibody (1:2000; A110001, ThermoFisher) for 1 h. Nuclei were counterstained with 10 µg/mL Hoechst 33,342 (Invitrogen, #H3570) for 5 min. Cells were imaged on an EVOS M7000 microscope (Thermo Fisher Scientific). Quantification of foci per cell was performed using the Software CellProfiler Version 4.2.1 and the “Speckle counting” pipeline.

Clonogenic assay

H446 cells were seeded in 6-well plates 48 h prior to the experiment. Seeding numbers were adjusted in each treatment group (Medium: 300, 400; PARPi: 400, 600; [177Lu]Lu-DOTA-TOC: 600, 1200; combination therapy: 1200, 3000). Cells were treated with medium, 0.5 µM PARPi for 72 h, 12.5 kBq [177Lu]Lu-DOTA-TOC for 24 h, or the combination therapy of 0.5 µM PARPi for 72 h and 12.5 kBq [177Lu]Lu-DOTA-TOC during the first 24 h. Following treatment, compounds were removed, and fresh medium was added. After 7 days, colonies were fixed with cold methanol, stained with 5% crystal violet in PBS and subsequently counted. Survival fractions were calculated by dividing the plating efficiency as counted colonies divided by seeded cells of the respective treatment group through the plating efficiency of the untreated control group.

Animal experiments

Authorization for all animal experiments was obtained from the Regierung von Oberbayern. Mice were housed under specific-pathogen free conditions with access to mouse chow and water ad libitum. Female 7–8 weeks old athymic nude mice (Crl:NU/NCr-Foxn1nu, Charles River Laboratories, Sulzfeld) were subcutaneously injected with 1–4 × 106 H69 or H446 cells in 1:1 RPMI-1640 medium:Matrigel (Corning Inc., #11543550). Animals were regularly monitored as defined in the license.

Biodistribution

After the subcutaneous tumors reached a size of approximately 100–200 mm3 animals were injected intravenously with approximately 40 MBq [177Lu]Lu-DOTA-TOC with or without prior injection of 1 mg Octreotide for blocking (n = 3/model). After 4 or 72 h, the mice were euthanized and dissected. Organ weights were determined using an analytical balance (Sartorius). A Wizard2 gamma counter (PerkinElmer) was used to determine radioactivity in each organ after calibration with dilutions of [177Lu]Lu-DOTA-TOC solution.

SPECT imaging

SPECT imaging was performed for 60 min per animal on a nanoScan SPECT/CT (Mediso) at different timepoints (1 h, 24 h, 72 h) after injection of approx. 25 MBq [177Lu]Lu-DOTA-TOC per animal (n = 3/model). Reconstruction, image analysis and quantification of SPECT data and image analysis were performed using Nucline and Interview fusion software (both Mediso).

In vivo treatment study – single PRRT dose

10–15 days after xenografting, mice were randomized into 4 (H446) or 6 (H69) groups after stratification for tumor size to ensure a uniform initial tumor size distribution (H446: ~ 150 mm3; H69: ~ 60 mm3; Supplementary Fig. 2A, B). Mice in the PARPi and combination therapy groups received daily i.p. injections of PARPi on days 1–5 and 8–12 at a dose of 10 mg/kg. Mice in the [177Lu]Lu-DOTA-TOC and the combination therapy groups received a single dose of approx. 40 MBq [177Lu]Lu-DOTA-TOC on day 3 of PARPi treatment. Length, width and depth of the subcutaneous tumors were measured daily using a caliper and tumor volume was calculated using the ellipsoid formula. Mice were weighed regularly after start of the treatment. Mice were euthanized when they reached pre-defined criteria. During the study, only the criteria of tumor growth (single dimension ≤ 15 mm) or maximum observation time came into effect. In the H69 cohort, one animal per group was euthanized 3 days after PRRT (or equivalent time) and tumor, bone and kidney tissues were processed for histopathology.

In vivo treatment study – fractionated PRRT dose

In a second treatment study using the H69 model, the single 40 MBq dose of [177Lu]Lu-DOTA-TOC PRRT was fractionated into multiple (2 or 3) lower doses of 20 MBq and combined with Olaparib (daily i.p. doses of 10 mg/kg). Prior to the beginning of treatment, mice were randomly assigned to 7 treatment groups (n = 5–6 animals/group) with an initial mean tumor volume of approx. 50 mm3 per group (Supplementary Fig. 2C). The following cohorts were investigated: i) untreated, ii) Olaparib with 2 × dosing cycles on days 1–5 and 7–11, iii) Olaparib with 3 × dosing cycles on days 1–5, 7–11 and 13–17, iv) 2 × doses [177Lu]Lu-DOTA-TOC on days 3 and 9, v) 3 × doses of [177Lu]Lu-DOTA-TOC on days 3, 9 and 15, vi) combination group (2x) receiving Olaparib on days 1–5 and 7–11 and [177Lu]Lu-DOTA-TOC on days 3 and 9 of PARPi treatment, and vii) combination group (3x) receiving Olaparib on days 1–5, 7–11 and 13–17 and [177Lu]Lu-DOTA-TOC on days 3, 9 and 15. Body weight and tumor volumes were determined every 2–3 days. Animals were sacrificed when pre-defined endpoint criteria or humane endpoint was reached. In this study, only the pre-defined endpoint, a tumor volume of 1000 mm3 with a maximal size of ≤ 15 mm in a single dimension, was reached. After a maximum observation period of 4 weeks after the last treatment, the study terminated in accordance with the animal protocol.

Histology

All samples were fixated in 4% PFA for 24 h and subsequently stored in 70% ethanol at 4 °C until the radioactivity had fully decayed. Samples containing bone for bone marrow analysis were decalcified using OSTEOSOFT® (Merck) solution. Formalin-fixed paraffin embedded (FFPE) tissues were cut into 2 µm sections using a microtome. After deparaffinization and rehydration, tissue sections were subjected to standard H&E staining. Tumor sections were additionally subjected to IHC. IHC staining was performed on a Bond RXm (Leica, Nussloch) autostainer using the following primary antibodies: Cleaved Caspase 3 (Clone 5A1E, Cell signaling #9664L; 1:150 dilution), PARP1 (Proteintech #13,372–1-AP, 1:250 dilution), SSTR2 (Clone umb1, abcam #ab134152. 1:150 dilution), γH2AX (pSER139, Novus #NB100-2280, 1:500 dilution) with optimized protocols of the Comparative Experimental Pathology Core Facility. Stained tissue sections were digitalized, and the Aperio ImageScope Software was used for data analysis.

Statistical analysis

Results are given as mean ± SD, mean ± SEM or percent of control, as stated in the figure caption, respectively. Animal survival of in vivo therapy studies was depicted as Kaplan Meier plots and statistical significance analyzed by log-rank (Mantel-Cox) test. p-values < 0.05 were considered as significant. Data visualization and statistical analysis was performed using GraphPad Prism (Version 9.5.1).