PARP targeted Auger emitter therapy with [125I]PARPi-01 for triple-negative breast cancer

Radiosynthesis of [123/125I]PARPi-01

Radiolabelling was conducted as previously described [17] (Additional file 1: Fig. S1). Briefly, the volume of the [123/125I]Iodide (PerkinElmer Inc.) was reduced if necessary, by evaporation followed by redissolution in water (< 15 µL). 3 µL of a PARPi-01 precursor solution (Tributyl stannylated derivative of Olaparib, 15 mM in DCM) were evaporated to dryness, and re-dissolved in 20µL HOAc. 15 µL of the concentrated radio iodide solution were added to the acid precursor solution and labelling was started by adding 20 µL of a fresh chloramine-T solution (1 mM in MeCN). After 10 min the crude reaction mixture was purified using gradient RP HPLC with a classical C18 column (LiChrospher 100 RP18 EC 5µ, 250 × 4 mm, Merck, Germany). The product fraction was diluted with 15 mL water and was solid phase extracted by a preconditioned SepPak light C18 cartridge (WAT023501, Waters). The [123/125I]PARPi-01 was eluted using 1 mL CH3CN and evaporated to dryness. The residue was taken up in little EtOH and diluted by isotonic saline to keep the EtOH concentration below 8%. The resulting overall radiochemical yield was 78 ± 12% with purities of > 95%. Molar activity of obtained [123I]PARPi-01 and [125I]PARPi-01 were 279 ± 216 GBq/µmol (n = 3) and 30.5 ± 6.4 GBq/µmol (n = 4), respectively.

Cell-line culture conditions

All cell lines were obtained from the Clinic for gynaecology and obstetrics, University Hospital RWTH Aachen and cultured in DMEM supplemented with 5% Fetal bovine serum and 1% Penicillin/Streptomycin. at 37 °C with 5% CO2. Cells in culture were tested for absence of mycoplasma every two weeks.

Phosphorimager analysis of [125I]PARPi-01 nuclear uptake

Breast cancer cells were seeded (0.5 × 106) in a 12-well plate and treated with [125I]PARPi-01 (1 MBq/106 cells) for 24 h. Subcellular fractionation was performed using a Subcellular Fractionation kit (ThermoFischer inc., #78840. Protein concertation was assessed using BCA assay. SDS-PAGE was performed using the fractions (30 µg/lane) and Any KD Mini-Protean TGX gels (90 V for 1.5 h). The separated proteins were transferred to PVDF membranes using a wet blotting (60 V, 2 h) technique. The blots were then blocked with 1% Milk/TBST and incubated with a primary antibody (anti-PARP1 (1:1000); Abcam ab227244; anti-GAPDH (1:4000); cell signalling 14C10, or anti-Histon H3 1:1000) Abcam ab1791). For the phosphorimager analysis, after blotting, the blots were sandwiched with a Fujifilm Imaging plate for Bioanalyzer (BAS-MS 2040), and incubated overnight, and the protein bound activity was imaged using a phosphorimager (Typhoon FLA 7000 version 1.2). To assess the tracer uptake, both fractions were measured using a gamma counter (Wizard2, Perkin Elmer, USA).

Tumour model, study design and animal care

Female NOD/SCID mice at 5–6 weeks of age (Janvier, France) was used for developing subcutaneous TNBC tumour. Animals were housed in 20–24 °C in a 12 h day light cycle. For each mouse, 3 × 106 MDA-MB-231 cells were suspended in a 1:1 (volume) mixture of Culture media: Matrigel® matrix (Corning®, Product No: 356237). Mice were inoculated with this cell suspension (max 200 µl) subcutaneously in the right flank. Tumour growth was monitored daily using calliper measurements. Tumour volume was calculated daily according to the formula

$$}\;} = }\;} \times \left( }\;}} \right)^} \times \, 0.$$

Upon reaching of a tumour volume of 500 mm3, animals were proceeded for experiments. For biodistribution study animals were administered with [123I]PARPi-01 (n = 3). For therapy studies animals were randomized for control (n = 10) or [125I]PARPi-01 administration (n = 10). Randomisation was done based on equivalent distribution of tumour volumes. In addition to the daily calliper measurements image analysis of CT based tumour volumes was blinded to avoid bias. Animals were sacrificed upon completion of biodistribution study or upon reaching a humane end point for the therapy study. Humane end point is determined as reaching of either of the following conditions: 15 mm tumour diameter, 1500 mm3 tumour volume, or development of ulcers/ascites.

Small animal SPECT/CT imaging

Imaging was performed with a small animal SPECT/CT system (X-Cube and γ-Cube, Molecubes, Gent, Belgium). Mice bearing subcutaneous MDA-MB-231 tumours upon reaching tumour volume of 500 mm3 were intravenously administered with [123I]PARPi-01 (9.3 ± 2.7 MBq) via lateral tail-vein using a catheter under 1.5 to 2.5% isoflurane anaesthesia in oxygen at 0.8 L/min. [123I]PARPi-01 was prepared by diluting the tracer in 0.9% NaCl to a total volume of 100 μL. Administered dose was calculated by subtraction of decay-corrected syringe activity post-injection from pre-injection activity. The mice were scanned either at 4 h or at 24 h post injection (p.i.). The CT acquisition settings were as follows: default high resolution protocol, 440 uA, 50 kVp, 32 ms exposure time, and 1080° rotation in a spiral mode with 960 exposures/360°; the duration of each CT scan was 5 min. A SPECT scan was initiated at the end of each CT scan. The SPECT acquisition settings were as follows: 7-headed camera equipped with a general-purpose collimator with 28 pinholes (i.e., 0.75 mm apertures and a radius of rotation of 20 mm), helical scan with 82 bed positions; the total duration of each SPECT scan was 30 min. Both CT and SPECT axial fields of view were selected at 105 mm. During the scans, the isoflurane concentration was adapted to achieve a respiratory rate between 75 and 50 breaths per minute. Images were exported and post-processed on PMOD software, version 3.8 (PMOD technologies, Switzerland).

In vivo Biodistribution study

For biodistribution, animals imaged at 4 h p.i. were killed 24 h p.i., while the ones scanned 24 h p.i. were killed 5 days p.i.. Subsequently, organs were excised, weighed, and assayed for radioactivity in a gamma counter. Mean tumour and organ uptake was determined from decay-corrected tissue radioactivity normalized to injected dose and tissue sample weight (%ID/g tissue wet weight).

Endogenous therapy study

Mice were administered intravenously via lateral tail vain with Saline (0.9% NaCl) or [125I]PARPi-01 (8.15 ± 2.9 MBq/dose) in four doses at 10-day intervals. The tumour volume was measured using [18F]FDG based PET/CT (Trifoil, USA) once per week. [18F]FDG (2.3 ± 0.4 MBq, AAA, Düsseldorf, Germany) was intraperitoneally administered and scanned 30 min later. Upon reaching 520 mm3 tumour volume, one additional CT imaging per week (Trifoil, USA) apart from the PET/CT was performed for monitoring tumour growth.

Small animal PET/CT imaging

After injection with [18F]FDG, the mice were placed on the scanner bed and the CT scan was initiated. The exposure settings used were as follows: 130 uA, 75 kVp, 230 ms exposure time, and 360° rotation with 512 views; the duration of the CT scans was ~ 5 min. A dynamic 30 min PET scan was initiated at the end of the CT scan. The CT had an axial field of view of 91.1 mm and a PET of 112 mm. During the scans, the isoflurane concentration was adapted to achieve a respiratory rate between 75–50 breaths per minute. Mice were maintained at 1% Isoflurane and heated at 37 °C from anaesthesia induction to the end of imaging. The CT images were reconstructed using a Feldkamp filtered back projection reconstruction process to a voxel size of 0.154 × 0.154 × 0.154 mm in a 592 × 592 × 560 matrix. Using vendor software, the CT values were converted into Hounsfield units (HU) using the following formula

$$} = 000 \times \left( } - \, \mu }} \right) \, \div \, \mu }} \right)$$

where µw is the linear attenuation coefficient of the water and µt is the linear attenuation coefficient of the tissue. The PET data were reconstructed using a 3D ordered-subset expectation maximization (i.e., OSEM-3D with three iterations and eight subsets) with a maximum a posteriori probability algorithm (30 iterations) into a 240 × 240 × 192 image matrix (resulting in final voxel dimensions of 0.25 × 0.25 × 0.597 mm). PET normalization, CT attenuation correction, and CT scatter correction were applied to all PET reconstructions. The PET images were automatically aligned to the CT using a custom-made transformation in PMOD software package from a capillary phantom.

Apoptosis (TUNEL) staining

Organs excised and retrieved from control (n = 3) and therapy mice (n = 3) were fixed with 4% PFA overnight at 4 °C. For cryopreservation, the organs were kept for 2–3 days at 4 °C in 30% sucrose Cryosections (6 µm) of excised and preserved organs were made using a cryostat (Leica, CM3050S). For TUNEL fluorescence quantification, therapy mice (n = 3) and control mice (n = 3) slices were stained simultaneously. The TUNEL staining kit (Roche # 11684795910) was used and protocol was followed as mentioned in the staining kit. Tissues were washed with PBS and permeabilised with 0.1 % Triton-X in 0.1 % Sodium nitrate on ice for 2 min. Following permeabilization, sections were incubated with TUNEL reaction mixture (Fluorescence labelled Terminal deoxynucleotidyl transferase enzyme dissolved in nucleotide buffer) for 1 h at 37 °C. After PBS wash, the cells were mounted with RotiMount® Vectashield containing DAPI and analysed using fluorescence microscopy (Zen Lite, Carl Zeiss). The images were processed and quantified using CellProfiler 3.0 software.

Anti-PARP1 staining

For anti-PARP1 staining, cryosections were washed with PBS and then antigen-retrieval was performed by heating the sections in citrate buffer (pH 6) at 600 W for 20 min. The sections were washed with PBS (2 min) and then incubated with blocking buffer (1 % BSA) for 1 h. The tissues were then incubated overnight with a 1:3000 dilution of the primary antibody (Mouse Anti-PARP1; Sigma #AMAB90959). Tissues were then briefly washed with PBS and then incubated with secondary antibody (Goat Anti-Mouse DyLight 488, Invitrogen #35502) in a dilution of 1:500 for 1 h. After PBS wash, the cells were mounted with RotiMount® Vectashield containing DAPI and analysed using fluorescence microscopy (Zen Lite, Carl Zeiss).

H&E staining

For Haematoxylin–Eosin staining the (6 µm) cryosections were washed with PBS thrice 10 min each followed by Haematoxylin (10 min) and washed with water. Subsequently tissues were incubated with Eosin (1 min) followed by wash twice in PBS for 5 min. Dehydration steps (70% Ethanol, 96% Ethanol and 100% Ethanol, Xylol) were then performed 2 min in each solvent. The tissues were then mounted and proceeded for bright light microscopy (Zen Lite, Carl Zeiss).

Statistical analyses

All statistical analyses were performed using the Graph-Pad Prism software (Version 9.1.1). Biodistribution data is expressed as mean ± standard deviation. Kaplan–Meier Survival curve was analysed using survival analysis (Log rank Mantel-Cox test). The data points of the normalized tumour volume as a function of the time of the groups (Control, 125I-PARPi, Control—excluded ulcers, 125I-PARPi—excluded ulcers) were subjected to nonlinear regression analysis using the exponential growth formula Y = Y0*exp(k*x) (Malthusian). Accordingly, the doubling time is equal to ln(2)/k. The Goodness-of-Fit was evaluated determining R2 for these groups, which was 0.79, 0.81, 0.94 and 0.87, respectively, demonstrating that the selection of the model is appropriate. During regression analysis, the best-fit value k was compared between two datasets each using the extra sum-of-squares F test, testing the null hypothesis that k is the same for both datasets. Time activity analysis was performed using nonlinear exponential decay curve fitting and area under the curve was computed using the Graph-Pad Prism software. The area under the curve is expressed as total peak area ± standard error. For statistical analysis of fluorescent signals, unpaired t-test was used.

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