Cell culture

Human prostate cancer cell line C4-2 (ATCC CRL-3314, Manassas, VA) was subcultured in RPMI-1640 medium (Sigma-Aldrich (Merck), Oslo, Norway) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Cytiva, Marlborough, MA), 100 units/mL penicillin and 10 µg/mL streptomycin (Sigma-Aldrich) and maintained at 5% CO2, 37 °C in a humidified incubator. Routine testing for mycoplasma was performed using MycoAlert Mycoplasma Detection kit (Lonza, Basel, Switzerland).

BET inhibitors

The BET inhibitors AZD5153 (Cayman Chemical, Ann Arbor, MI) and JQ1 (MedChemExpress, NJ) were prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at a stock concentration of 25 mM and stored at −20 °C.

Preparation of [212Pb]Pb-AB001

High purity 212Pb (≥ 99.99%) was generated by 228Th-based gas-diffusion generator and extracted in 0.1 M HCl, as described by Li et al. [26]. Prior to radiolabelling, the 212PbCl2 solution was pH-adjusted to 5–6 using 5 M sodium acetate. The AB001 ligand (previously referred to as NG001, MedKoo Biosciences Inc., Morrisville, NC, USA) was then added to achieve a specific activity of 2 MBq/µg (3.32 MBq/nmol), following the procedure described by Stenberg et al. [27]. The radioactivity of 212Pb in equilibrium with its progenies was measured using Cobra II Autogamma Counter (Packard Instrument Company, Downer Grove, IL) with a counting window 50–120 keV and Capintec CRC-127R radioisotope dose calibrator (Capintec Inc., Ramsey, NJ) with calibration number 667 [28]. Radiochemical purity was assessed using instant thin-layer chromatography (iTLC) with Tec-Control chromatography strips (Biodex Medical Systems, Shirley, NY, USA). Only radioligand preparations demonstrating a purity greater than 95% were used in the experiments.

Treatments and assessment of metabolic activity in 2D cell monolayer

Cells were seeded in 96-well plates one day prior to initiating treatment. For single therapy treatments, the culture medium was replaced with either medium containing BET inhibitors for the entire treatment duration or medium containing [212Pb]Pb-AB001 for a 4-h period, followed by replacement of the radioligand with fresh medium. For combination therapy treatments, medium containing BET inhibitors was added 3–4 h prior to a 4-h incubation with [212Pb]Pb-AB001, after which, radioligand-containing medium was replaced with medium containing BET inhibitors.

For the C4-2 cell line, which expresses PSMA with a dissociation constant of approximately 22 nM for the ligand [29], the maximum radioactivity concentration of 50 kBq/mL was chosen. Given a specific activity of 2 MBq/µg, this corresponds to a ligand concentration of ~ 25 ng/mL, or ~ 15 nM — remaining below 22 nM ensures that the majority of PSMA receptors remain unoccupied by cold ligand, thereby allowing the observed cytotoxic effects to be attributed primarily to the radiolabelled compound rather than competitive binding by excess unlabelled AB001.

Metabolic cell activity, based on ATP quantification, was assessed using the CellTiter-Glo™ Assay (Promega, Madison, WI, USA) following the manufacturer’s protocol. Luminescence, proportional to intracellular ATP levels, was measured in clear-bottomed white plates with an integration time of 1000 ms using a Tecan Spark multimode microplate reader (Tecan, Mannedorf, Switzerland).

Assessment of cytotoxicity in 3D spheroid model

C4-2 spheroids were generated by the liquid-overlay technique as described by Stenberg et al. [30]. Briefly, 96-well flat-bottomed plates were coated with a layer of 1.5% agarose in PBS with Mg2+ & Ca2+. C4-2 cells (500 cells/100 µL) were then added, and plates were centrifuged at 470 × g for 15 min. Spheroids formed over 4–5 days at 5% CO2, 37˚C in a humidified incubator. Spheroids were treated with drugs as described above.

Cell viability in spheroids was assessed using fluorescein diacetate (FDA; 16 µg/mL in PBS with Mg2+ & Ca2+) for live cells and propidium iodide (PI; 40 µg/mL in PBS with Mg2+ & Ca2+) for dead cells.

Spheroid growth and viability were monitored for up to 21 days using Axiovert 200 M microscope (Carl Zeiss AG, Oberkochen, Baden-Wurttemberg, Germany) and AxioVision Rel. 4.8 software (Carl Zeiss AG), with medium changes 2–3 times a week.

Spheroid size was assessed by measuring the cross-sectional area of each spheroid from brightfield images acquired at defined time points. Growth kinetics were assessed based on area changes over time. Doubling time (Td) was calculated by fitting the exponential growth phase using SigmaPlot15 to obtain the growth rate constant (a), with Td = ln(2)/a.

Evaluation of cell viability, DNA damage and cell cycle changes

C4-2 cells (0.5–2 × 106 cells/flask) were seeded in T25 or T75 cell culture flasks one day before treatment. Cells were treated with drugs as described above. Cell death, DNA damage, cell cycle changes and the percentage of mitotic cells were assessed by harvesting cells at 1 h (only groups treated with [212Pb]Pb-AB001), 1, 3 and 6 days post-treatment.

Cell viability was assessed immediately post harvesting by staining with Annexin V FITC (ImmunoTools, Friesoythe, Germany) and PI (Sigma-Aldrich Norway AS) in Annexin V binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl2) for 15 min at room temperature.

DNA damage and cell cycle changes were assessed by staining cells with FVD-eFluor 450 (cat. no. 65–0863-14, Invitrogen, Thermo Fisher Scientific, Oslo, Norway) for 1 h on ice, followed by fixation with 100% methanol (Sigma-Aldrich). Washes were performed with 0.2% v/v Tween-20 in PBS without Mg2+ & Ca2+ (PBST). Cells were stained with 1 µg/mL rabbit anti-PS10H3 (cat. no. 06–570, Merck Millipore, Darmstadt, Germany) and 1 µg/mL mouse anti-pγH2AX (cat. no. 05–636, Merck Millipore) primary antibodies in PBST with 2% v/v FBS and 0.4 mg/mL PureLink RNase A (cat. no. 12091–021, Invitrogen, Thermo Fisher Scientific) at room temperature, in the dark for 1 h. After removing the primary antibodies, cells were incubated with 2 µg/mL donkey anti-rabbit AlexaFluor647 (cat. no. A31573, Invitrogen, Thermo Fisher Scientific) and 2 µg/mL goat anti-mouse FITC (cat. no. F0479, Dako, Agilent Technologies, Santa Clara, CA, USA) secondary antibodies in PBST with 2% v/v FBS at room temperature in the dark for 30 min. Finally, cells were incubated with 10 µg/mL PI in PBST. Samples were then measured using a CytoFlex S Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA) with C Flow Plus Software and the data analysed using FlowJo 10.7.1 software (FlowJo LLC, Ashland, OR, USA).

Statistical analysis

Data are presented as mean ± standard deviation. Statistical analyses were conducted using SigmaPlot 15.0 software (Systat Software, Inc., San Jose, CA, USA) and differences were deemed statistically significant at p-values < 0.05. The statistical difference between groups treated with [212Pb]Pb-AB001 versus combination treatment was evaluated using Student’s t-test when both normality (Shapiro–Wilk test) and equal variance assumptions were satisfied; otherwise, non-parametric alternatives (Mann–Whitney U test) were applied. For comparisons involving more than two groups, one-way ANOVA followed by Holm–Šídák post hoc correction for multiple comparisons was used, where applicable.

The Bliss independence test was performed to evaluate the presence of synergy between the two therapies [31]. It was assumed that both BET inhibitors and [212Pb]Pb-AB001 had independent effects, allowing for a comparison between the expected additive values and the observed values. The statistical significance of the difference between the calculated additive and observed values was assessed using the Student’s t-test, provided the normality and equal variance assumptions were met. Statistical significance was considered at p < 0.05. The combination index (CI) values were calculated from the mean values of two or three independent experiments using CompuSyn software (Combosyn, Inc., Paramus, NJ, USA). Interpretation of CI values followed the criteria: CI > 1 indicated antagonism, CI ≈ 1 indicated an additive effect, and CI < 1 indicated synergy [32].

All exact p-values are provided in Supplementary Tables S1–S5.