Emr8 5

The expression of HLA-I complex and its subunits were confirmed by IHC using the OPTIVIEW universal DAB kit (Ventana), the Ventana Bench mark XT autostainer (Ventana) and antibodies against HLA-ABC (EMR8-5, 1:8000, Abcam, Cambridge, UK), HLA-A (EP1395Y, 1:5000, Abcam), HLA-B (1:700, Abcam), HLA-C (1:1000, Abcam) and B2M (B2M/961, 1:2000, Abcam). Immunostaining of TILs was performed with antibodies specific to CD3 (1:100; Dako, Glostrup, Denmark) and CD8 (1:100; Dako) using the Bond polymer kit (Leica Microsystems) and Leica BOND-MAX autostainer (Leica Microsystems). IHC of PD-L1 was performed on the Autostainer Link 48 with EnVision DAB Detection System (Agilent Technologies, Santa Clara, CA) and 22C3 pharmDx antibody (prediluted; Dako), according to the manufacturer’s instructions.

Formalin-fixed, paraffin-embedded CM tissue biopsies were studied. Histopathological features were determined according to AJCC-UICC staging (40 (link)) (Table S1 in Supplementary Material). Proliferative index (PI) was determined by Ki-67⁺ staining (41 (link)) (%): Ki-67⁺ tumor cells/(Ki-67⁺ tumor cells + Ki-67⁻ tumor cells) × 100. PI was determined in 1 mm² tumor hot spot zone (clone MIB-1, Dako). HLA-I expression (%) was determined in tumor tissues with clone EMR8-5 (Abcam). The avidin–biotin–peroxidase (ABC) system (Vectastain, Vector Labs) was afterward used. Sections were examined by optical microscopy (Olympus BX40 microscope, DP2-BSW software), and digitalized pictures were analyzed with ImageJ software (NIH).

Cells on coverslips were fixed in cold 2% paraformaldehyde in PBS, and then permeabilized in 0.2% Triton X-100 in PBS for 20 min. Cells were subsequently washed in PBS, blocked for 1 h at room temperature (RT) in PBS containing 2% bovine serum albumin (Sigma-Aldrich). Fixed cells were then incubated with the primary antibody in 2% BSA/PBS solution overnight at 4 °C. Cells were washed in PBS three times, and the secondary antibody was added in 2% BSA/PBS solution for 1 h at RT in the dark. Cells were washed three times in PBS and then incubated for 2 min in Hoechst 33342 solution, after which they were washed in PBS and mounted. Confocal images were obtained on an Olympus upright Fluoview 2000 confocal microscope (Center for Biologic Imaging, University of Pittsburgh, supported by NIH #1S10OD019973-01) using a 60x (UPlanApo NA = 1.42) or 20x (UPlanSApo NA = 0.85) objective. The primary antibodies used were rabbit anti-human cleaved caspase-3 (9661, Cell Signaling Technology), mouse anti-human E-cadherin (Clone 36, BD), mouse anti-human HLA Class 1 ABC antibody (EMR8-5, Abcam) and mouse anti-human PD-L1 (28-8, Abcam). Secondary antibodies used were goat anti-mouse Alexa Fluor® 488 or goat anti-rabbit Alexa Fluor® 647 (Life Technologies). Hoechst 33342 was applied for nuclei counterstaining.

To detect human breast cancer cells in the mouse organs, immunohistochemical analysis was performed using a human specific cytokeratin 7 antibody (Abcam, clone RCK105, 1:50 dilution) or HLA (Abcam, clone EMR8-5, 1:50 dilution). The paraffin-embedded tissue was deparaffinized in xylene and rehydrated through a series of different graded ethanol, followed by heat induced antigen retrieval in 1 mM EDTA (pH 8.0) or 10 mM sodium citrate buffer (pH 6.0) for 20 min in the 2100 retriever (Aptum) for cytokeratin 7 and HLA, respectively. The slides were allowed to cool to room temperature for 30 min. After the slides were washed, exogenous peroxidase activity was blocked by incubating the slides in 3% peroxidase for 20 min. The slides were then washed, blocked in 5% BSA for 30 min at room temperature, and incubated overnight at 4°C in primary antibody diluted in 5% BSA. Following primary antibody incubation and washing, the slides were incubated in secondary biotinylated anti-mouse IgG reagent (Dako) at room temperature for 1 hour, washed with PBS and then detected using Envision+ (Dako). Counterstaining with hematoxylin was performed and the slides were dehydrated through graded ethanol and then xylene and mounted with Entelen (Electron Microscopy, Inc.).