Kaluza 1.3 flow analysis software

To estimate apoptosis, HeLa cells were plated in 35-mm dishes (1.5 × 10⁵ cells/dish), for 24 h, and treated with 5 Gy or 15 Gy of γ-radiation, or with 60 J/m² ultraviolet C (UVC; positive control for apoptosis induction). Then, adhered and suspended cells were harvested by successive rounds of PBS washing-trypsinization-centrifugation, 48 h or 72 h after γ-radiation. Harvested cells were resuspended in Annexin-V binding buffer (50 mM HEPES, pH 7.4, containing 0.7 M NaCl and 12.5 mM CaCl₂) for a final density of 1 × 10⁶ cells/mL, and 5 μL Annexin-V-FITC (BD Biosciences, Franklin Lakes, NJ, USA) and 1.5 μL propidium iodide (1 mg/mL) were added to 100-μL aliquots of cell suspension (1 × 10⁵ cells). Samples were incubated for 15 min at room temperature (and protected from light), and then 400 μL of Annexin-V binding buffer was added to each sample, and cells were analyzed by flow cytometry in a FACSVerse (BD Biosciences, Franklin Lakes, NJ, USA). Flow cytometry data were analyzed on the Kaluza 1.3 Flow Analysis software (Beckman Coulter, Brea, CA, USA).

TIL in fresh breast tissues from untreated primary BC were analyzed by FACS as previously published (39 (link)). Multi-color FACS was performed using antibodies to well-defined immune cell markers, including CD3, CD4, CD8, CD19 and CD45 and a panel of immune checkpoint molecules: PD-1, PD-L1, PD-L2, CTLA-4, LAG3, and TIM3 (details of the antibodies are provided in Table S2A in Supplementary Material). Intracellular CTLA-4 (iCTLA-4) was labeled by fixing and permeabilizing membrane-labeled cells with the BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD 554714) following the manufacturer’s instructions. Sample data were acquired on a GALLIOS 10/3 cytometer and analyzed using Kaluza^® 1.3 Flow Analysis Software (Beckman Coulter, Brea, CA, USA). N = X in the data plots indicate the number of analyzable tumors for a given TIL marker (tumors with insufficient FACS data acquisition were excluded). The amount of material for experimental analysis was limited by the routine requirements of the pathology laboratory, which explains why it was not always possible to analyze all markers for each sample.

To estimate different apoptosis phases (early and late) and necrosis, HeLa and RhoA-N19 cells were treated with UV-radiation and collected (including the supernatants possibly containing cells) 48h and 72h after stress. Cells were then resuspended in Annexin-V binding buffer (50mM HEPES, pH 7.4, containing 0.7M NaCl and 12.5mM CaCl₂) in a final density of 1 × 10⁶ cells/mL. In aliquots of 100 μL of cell suspension (containing 1 × 10⁵ cells) were added 5μL of Annexin-V-FITC (BD Biosciences) and 1.5μL of 1 mg/mL propidium iodide. Samples were incubated for 15min at room temperature in a dark chamber and 400μL of Annexin-V binding buffer was added to each sample. Cells were analyzed by flow cytometry in a FACS Verse (BD Biosciences) and the data were analyzed on the Kaluza^® 1.3 Flow Analysis software (Beckman Coulter). The results were presented as percentage of cells in early apoptosis (positive for Annexin V and negative for PI), late apoptosis (positive for both Annexin V an PI) and necrosis (negative for Annexin V and positive for PI).