The murine mammary carcinoma cell line 4T1 was obtained from ATCC (American Type Culture Collection, USA). The 4T1 cell line was maintained at 37 °C in a humidified atmosphere (5% CO2, 95% air) using RPMI 1640 medium supplemented with 10 mM Hepes, 2.5 g/L glucose, 1 mM sodium pyruvate, and 10% fetal bovine serum. Cell number and viability were assessed by trypan blue exclusion assay using a 0.25% trypan blue solution.

All animal procedures conducted in this study (Ethical protocol: #266,110) were approved by the Institutional Animal Care and Use Committee in compliance with Greek authorities (Protocol number: 22,066). The animal facility where the experiments were conducted is accredited by the Greek National Committee for Laboratory Animals and under the License EL 25 BIOexp 045.

Mice were maintained under controlled environmental conditions, with temperatures set at 22 ± 2 °C, humidity maintained at 55 ± 10%, and subjected to a 12-hour light-dark cycle. Food and water were provided ad libitum throughout the study period.

One hundred and eighty healthy female BALB/c (BALB/cByJ) mice, aged 7–8 weeks, were obtained from Charles River and acclimatized for one week. Tumor inoculation was performed under anesthesia using isoflurane. The skin over the lateral thorax was incised to expose the mammary fat pad (MFP) before the injection of 1 × 105 4T1 breast cells, suspended in 50 µL RPMI 1640 medium, followed by suturing. Tumors were allowed to grow until the volume reached 1000 mm3 prior to the beginning of anti-cancer treatment.

Upon reaching palpable size (100 mm3), the mice were randomly allocated into 9 groups: (1) 177Lu-SN201 vehicle (Ringer’s buffer, Fresenius Kabi), also serving as the ICI vehicle control), (2) Paclitaxel vehicle (10% Cremophor and 10% ethanol), (3) Carboplatin vehicle (5% DMSO), (4) Niraparib vehicle (10% DMSO, 40% PEG 300, 5% Tween-80, and 45% saline), (5) 177Lu-SN201 (Spago Nanomedical), (6) ICI (InVivoMAb anti-mouse PD-1, BioXCell, # BE0146 and InVivoMAb anti-mouse CTLA-4, BioXCell, #BE0164), (7) Paclitaxel (Selleckchem, # S1150), (8) Carboplatin (MCE, HY-17,393), (9) Niraparib (Selleckchem, # S2741). The formulation and radiolabeling of 177Lu-SN201 were performed as described in a previous study [4]. A dose of 4 MBq was administered intravenously on Day 1 of the treatment schedule. ICI (anti-PD-1 200 µg/mouse and anti-CTLA-4 100 µg/mouse) were dissolved in PBS and administered intraperitoneally on days 1, 4, 7, and 10 [14]. Paclitaxel at a dose of 6 mg/kg was administered intraperitoneally on days 1, 3, 5, 7, and 9 [15]. Carboplatin was administered at a dose of 100 mg/kg via intraperitoneal injection on days 1 and 8 [16]. Niraparib at a dose of 50 mg/kg was administered orally once daily from day 1 to day 10 [17]. Vehicles were administered following the same schedule as their corresponding drugs.

Tumor volume and body weight were measured throughout the monitoring period. Mice were terminated when humane endpoints were reached. Upon termination, tumors and lungs were weighed, and plasma samples were collected for further analysis.

Total bilirubin, creatinine (Cre), blood urea nitrogen (BUN), alkaline phosphatase (ALP), and alanine aminotransferase (ALT) were quantified in terminal plasma samples using the DRI-CHEM NX600V Fujifilm analyzer.

Statistical analysis was conducted using GraphPad (Version 10.0, Prism). Specifically, a one-way ANOVA test (Kruskal-Wallis) was employed for multiple comparisons, and mixed-effect models were utilized to calculate statistical differences for tumor volume and body weight. Kaplan-Meier graphs with log-rank tests were employed to compare differences in survival.