Fig. 4: Breast tumor cells challenged with the secretome of PreNeu rely on PARP to survive.

a Quantification and representative images of western blots for PARP1, Ku70, Ku80, and DNA-PKcs levels in MCF-7 cells treated with or without conditioned media obtained from human cord blood-derived neutrophil precursors (cm-PreNeu; n = 4 for all the molecules), except n = 7 for PARP1). Data are reported as mean ± SEM. Statistical analyses (unpaired Student t test). b Quantification and representative image of western blot for PARP1 in MCF-7 cells silenced or not for the succinate receptor (SUCNR1) and treated or not with cm-PreNeu (n = 4 for each group). Data are reported as mean ± SEM. Statistical analysis (one-way ANOVA). c Quantification and representative image of western blot for PARP1 in MCF-7 cells silenced or not for the succinate receptor (SUCNR1) and treated or not with succinate (n = 3 for each group). Data are reported as mean ± SEM. Statistical analysis (unpaired t test). d Graph showing fold change in mRNA level of PARP1 in MCF-7 cells and LNCaP cells. Cells were previously irradiated at 10 Gy and then treated or control (Ø) with cm-PreNeu or succinate (2 mM) for 6 h (n = 3 for each group). Data are reported as mean ± SEM. Statistical analysis (unpaired t test). e Experimental scheme created in BioRender. Garda, c. (2025) https://BioRender.com/uueypah. PARP1-luciferase MCF-7 cells were transfected with pGL3 plasmid, treated or control (Ø) with cm-PreNeu in the presence or absence of αSLC13a5 for 6 hours. Cells were analyzed for luciferase activity using Dual-Glo Luciferase Assay System (Promega, E2920) following manufacturer’s instructions. Expression of PARP1 (Renilla) was normalized to the expression of pGL3 (Firefly) to calculate luciferase activity. The values were further normalized to the condition treated with cm-PreNeu. (n = 7 for each group, each dot is the mean of three independent technical replicates from two independent experiments). Data are reported as mean ± SEM. Statistical analysis (unpaired t test). f Cell proliferation of MCF-7 cells treated or control (Ø) with cm-PreNeu and challenge with PARPi (1 µM) after 24 h. g Annexin V-positive intensity in MCF-7 cells treated or control (Ø) with cm-PreNeu and challenge with PARPi (1 µM) after 24 h. f, g Aggregated data from three independent experiments are reported as mean ± SEM. Statistical analyses (two-way ANOVA and Ordinary one-way ANOVA multiple comparison test, respectively). h–j Tumor growth from the h E0771, i PyMT-N, and j PyMT-M models in female mice treated with PARPi or vehicle (Ø) when tumors were palpable as indicated in the graph (for the E0771 model n = 4 for each group, for the PyMT-N model n = 4 for each group, for the PyMT-M model n = 5 for Ø and n = 4 for PARPi groups). Data are reported as mean ± SEM. Statistical analysis (two-way ANOVA). k Representative histology images and their quantification. H&E and Cleaved-Caspase 3 immunohistochemical staining (Cleaved-Caspase 3 brown; nuclei, blue) of representative E0771, PyMT-N, and PyMT-M tumors from mice at the endpoint, treated with or without PARPi (n = 8 for E0771 + Ø, n = 9 for E0771 + PARPi, n = 30 for both PyMT-N and PyMT-M models; mean of three sections per mouse, ≥3 fields per section). Magnification ×20. Scale bar 200 µm. Quantification of Cleaved-Caspase 3 was reported as a percentage of the total cells within the tumor tissues. Data are represented as mean ± SEM. Statistical analyses (Unpaired Student t test). Source data are provided as a Source data file.