Kaluza analysis software

For apoptosis, each group of cells was collected and washed twice with PBS, using an apoptosis detection kit (BD Biosciences, Bedford, MA, United States), according to the manufacturer's protocol.
For cell cycle, each group of cells was collected and fixed with 70% precooled ethanol, using a cell cycle detection kit (Beyotime Institute of Biotechnology, China), according to the manufacturer's protocol.
Cell cycle and cell apoptosis were detected by CytoFLEX (Becton Dickinson, USA) and cell cycle was analyzed by Kaluza software (Kaluza® Analysis Software, Beckman Coulter).

For B-cell count and B-cell subpopulations determination, 200,000 PBMC were transferred into 96 U-well plate (Greiner Bio-One, Germany). After PBS was added up to volume of 200 µl, the plate was centrifuged (360 × g, 6 min, RT). Cells were stained with 50 µl of mixture of anti-human CD19-Pc7 (clone J3-119; Beckman Coulter, USA), anti-human CD27-BV421 (clone M-T271; Becton Dickinson, USA), anti-human IgM-PerCP Cy5.5 (clone MHM-88; BioLegend, USA) and PBS with 0.5% FCS for 30 min, 4 °C, dark. Afterwards 150 µl PBS with 0.5% FCS was added and the samples were centrifuged (360;× g, 6 min, RT), supernatant discarded. The cells were resuspend in 100 µl of PBS, followed by adding 100 μl of 4% PFA (paraformaldehyde) (Sigma-Aldrich Chemie GmbH, Germany) and the suspension was well mixed. The samples were kept in fridge (4 °C) overnight. Next day, the samples were washed before the measurement and cells were resuspended in PBS with 0.5% FCS. Samples were measured on a BD LSRII SORP flow cytometer with BD FACS Diva software version 8.0.1 (Becton Dickinson, USA) and analyzed with Kaluza Analysis Software (Beckman Coulter, USA). The gating strategy is shown in Supplementary Fig. 8.

2 × 10⁵ cells were stained with 100 μl of cell death staining solution (1 ml of Ringer’s solution (Fresenius Kabi, Bad Homburg, Germany), 0.75 μl/ml of AnnexinV-FITC (AxV) (1 mg/ml) (GeneArt, Regensburg, Germany), and 1.0 μl/ml of Propidium iodide (Pi) (1 mg/ml) (Sigma-Aldrich, Munich, Germany)). After incubation for 30 minutes, the measurement was performed on a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed with the Kaluza Analysis software (Beckman Coulter, Brea, CA, USA).
To determine the clonogenic potential of the breast cancer cells, 250 tumor cells/well were seeded in a 6-well plate 6h before irradiation. Afterwards they were irradiated with the indicated doses and cultured for two weeks, fixed and stained with 1% crystal violet in ethanol (Sigma-Aldrich, St. Louis, MO). Colonies with more than 50 cells were counted and normalized to mock-treated samples. The survival curves were calculated by adding a curve fit (dek(hx)). All calculations were performed with GraphPad Prism.

Cell count and -viability were assessed using flow cytometry. Before the analysis, cells were resuspended in PBS + 1% bovine serum albumin (BSA) solution. Propidium iodide (PI) (Sigma-Aldrich) was added to measure cell viability. Flow count beads (Analis) were added to acquire absolute cell counts. The samples were analyzed using Beckman Coulter Cytomics FC500 and CXP analysis software. A minimum of 10.000 events was acquired for each sample. Data analysis was done using the Kaluza analysis software (Beckman Coulter Life Sciences). The different gates are depicted in Supplementary Fig. S1.

Exosomes isolated from serum were incubated with aldehyde/sulfate latex beads (Invitrogen, Carlsbad, CA, USA) for 15 min at room temperature (RT). PBS supplemented with 3% bovine serum albumin was then added and the samples were incubated overnight on a rotating mixer. The bead-coupled exosomes were pelleted by centrifugation at 3000× g for 10 min and washed with PBS. The samples were then pelleted by further centrifugation at 3000× g for 10 min. Subsequently, the supernatants were removed from the samples and the pellets were resuspended in PBS containing anti-CD9 and anti-CD63 antibodies (BioLegend, San Diego, CA, USA) for 1 h at RT. Afterwards, the samples were again pelleted by centrifugation at 3000× g for 10 min before the pellets were resuspended in PBS. Exosome markers were detected using flow cytometry (Galios, Beckman Coulter, Brea, CA, USA). Following detection, analysis was performed with Kaluza analysis software (Beckman Coulter, Brea, CA, USA).