Tumor cell expression of CD163 is associated to postoperative radiotherapy and poor prognosis in patients with breast cancer treated with breast-conserving surgery

We collected data on all patients (n = 1164) with BC with isolated ipsilateral local recurrence (ILR) during the years of 1983–2008 from the breast cancer registry of the southeastern region of Sweden. For comparison, we selected an age-matched patient cohort (n = 1164), treated during the same period and without ILR. Only patients with radically removed tumors (R0), without lymph node metastases (N0) or distant metastases (M0) were included. All patients were treated with conventional breast-conserving surgery at surgical departments within the county of Östergötland, Sweden. Ethical approval from the Regional Ethics Committee in Linköping was obtained according to Swedish Biobank Law (Reference Number 2010/311–31). All data are presented in the main manuscript.

Tumor histology was reviewed by an experienced pathologist (SG), and formalin-fixed paraffin-embedded tissue blocks with invasive BC were chosen for tissue microarray, constructed using two tissue cores (diameter 0.6 mm). Eighty-three patient samples were included in total. Liver samples were used as a position control.

CD163 is considered as a macrophage-specific marker and is generally not expressed in cell types other than monocytes/macrophages. Based on the cell fusion theory, we used CD163-expression as a surrogate marker for macrophage phenotype in breast cancer cells. Five micrometer sections were obtained from formalin-fixed paraffin-embedded TMA tumor specimens. The sections were de-paraffinized in xylene and hydrated in a series of graded alcohols, pretreated with heat induced epitope retrieval and Tris-ethylenediamine tetraacetate acid buffer (1 mM, pH 9, 20/5/20 min; Decloaking Chamber NxGen, Biocare Medical), and stained for CD163 (anti-human, monoclonal antibody, clone 10D6, Novocastra, Leica). Staining for estrogen receptor (ER; clone SP1, Ventana Roche), progesterone receptor (PR; clone 1E2, Ventana Roche), Ki-67 (clone MIB-1, Dako Agilent), and human epidermal growth factor receptor 2 (HER2; clone 4B5, Ventana Roche) was done according to clinical laboratory standards. All slides were scanned to digital images using the Hamamtsu NanoZoomer XL (Visiopharm LRI AB). Image analysis and evaluation of immunostaining were performed by ImageScope viewing software (Leica Biosystems).

All immunostaining was evaluated by two experienced pathologists (SG and HO), blinded to patient characteristics and outcome. Macrophages and cancer cells were distinguished histomorphologically, the macrophages exhibiting small, regular nuclei and the cancer cells atypical nuclei with variations in size, shape, and chromatin staining. TAM-infiltration was evaluated semi-quantitatively, classified in three grades: no/low, moderate, or high (Fig. 1a–c). The fraction of CD163-positive cancer cells was calculated based on a count of 200 tumor cells in each TMA core. The tumors were considered CD163-positive if > 15% of the tumor cells expressed CD163. The expression of Ki-67, ER, PR, and HER-2 in cancer cells was evaluated according to ESMO guidelines (2015) (Senkus et al. 2015).

MCF-7/green fluorescent protein (GFP)-breast cancer cell line (AKR-211, Cell Biolabs, Inc., USA) was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 1% penicillin–streptomycin (PEST) (Thermo Fisher Scientific, USA), 10% Fetal Bovine Serum (FBS), and GlutaMax (Gibco^®, Life Technologies, USA) in T-75 tissue culture flasks (Sigma–Aldrich, USA) and incubated at 37 °C 5% CO₂ atmosphere. Cell medium was changed every 2–3 days, and the cells were passaged with 0.25% trypsin (Gibco, USA) at 95% confluence.

Monocytes were isolated from buffy coat obtained from healthy male blood donors at the department of transfusion medicine at Linköping University Hospital (Linköping, Sweden) and county hospital Ryhov (Jönköping, Sweden). All the blood donors gave informed consent according to the local guidelines and the Swedish National Law on ethical review of research involving humans (2003:460: 3–4§). The buffy coat was mixed with 70 ml 0.9% NaCl, layered onto 20 ml Lymphoprep (Thermo Fisher Scientific, USA) in 50 ml centrifuge tubes, and centrifuged at 480g in room temperature for 40 min. The buffy coat layer was transferred into new 50 ml tubes containing PBS-Heparin [500 ml PBS, pH 7.3, and 50 µl Heparin (0.01% Heparin 5000 IE/ml; Medicago Leo Pharma, Denmark)] and centrifuged at 300g for 10 min at 4 °C. The cell pellets were washed twice in PBS-Heparin (220 g, 5 min, 4 °C), followed by three washing procedures in Krebs–Ringer bicarbonate buffer (Sigma–Aldrich, USA) without Ca²⁺ (220 g, 5 min, 4 °C). White blood cells were re-suspended in 20 ml RPMI1640 medium supplemented with 1% PEST, seeded into T-75 tissue culture flasks, and incubated for 1–2 h at 37 °C with 5% CO₂ to allow monocyte adhesion. The non-adherent cells were eliminated by washing 2–3 times using PBS 37 °C and remaining attached cells incubated for 24 h at 37 °C with 5% CO₂ before differentiation to macrophages by incubation (at 37 °C in 5% CO₂) with 40 ng/ml of macrophage colony-stimulating factor, M-CSF (Nordic Biosite, Sweden), for 5–7 days and thereafter induced to M2 polarization with 20 ng/ml human interleukin-4 (Nordic Biosite, Sweden) for 18–24 h.

Spontaneous cell fusion occurred between macrophages and MCF-7/GFP-cancer cells upon co-culturing the cells at a ratio 3–5:1 (macrophage:MCF-7) in RPMI 1640 medium (supplemented with 10% FBS, 5% PEST, GlutaMax) at 37 °C for 2 days. The cells were harvested with a 0.05% trypsin–EDTA solution (Gibco, USA), centrifuged at 300g for 5 min at 4 °C, washed with 1 ml PBS 4 °C, and resuspended in 95 µl cell staining buffer (Nordic Biosite, Sweden) at a concentration of approximately 5 × 10⁶ cells/ml. The cell suspension was incubated on ice for 10 min with 5 µl TrueStainFcX solution (BioLegend, USA). Combinations of direct conjugated monoclonal anti-human CD163 (APC Anti-human CD163 (IgG1 k), clone GHI/61, 100 µg/ml) and anti-human CD45 (CF405M anti-human CD45 (IgG1 k), clone HI30, 50 µg/ml) antibodies or their respective isotype controls (APC and CF405M mouse IgG1 k, clone MOPC-21, 200 µg/ml; all antibodies from Biolegend, USA) were added to the cell suspension at concentrations recommended by the manufacturer and incubated at 4 °C for 30 min in darkness. The samples were centrifuged at 300g for 5 min at 4 °C and excess of antibodies was removed. The labelled cells were washed twice in 1 ml cell staining buffer, diluted in 1 ml PBS, and filtered in a pre-separation filter (30 µm, Miltenyi Biotech, Sweden) before they were sorted with BD FACSAria^™ III (BD Bioscience, USA; violet laser 405 nm, blue laser 488 nm, green laser 561 nm, red laser 632 nm). The cells were initially sorted by GFP-expression (positive selection of MCF-7/GFP origin) and subsequently by CD163-and CD45-expression. Macrophage/MCF-7-hybrids were defined as expressing both GFP and macrophage markers (CD163 and CD45). Cells positive for these markers were collected in tubes (BD FalconTM, Thermo Fisher Scientific) containing 0.5 ml FBS at 4 °C.

MCF-7/GFP-cells and M2-macrophage/MCF-7-hybrids (5 × 10⁵cells) were seeded in T-25 tissue culture flasks with RPMI 1640 medium and allowed to grow for 2 days (90–95% confluency). At day 3, the cell cultures were exposed to γ-radiation (Clinac 600C/D, Varian Medical Systems Incorporated, Herlev, Denmark, one AP field, linear accelerated 6MV Photons), at a dose-rate of 5 Gy/min and doses of 0 (control), 2.5 and 5.0 Gy at room temperature. The culture flasks were surrounded with 3 cm poly methyl methacrylate (PMMA) with a density comparable to that of human tissue.

After radiation procedure and storage at 4 °C, the cells were trypsinated and resuspended in RPMI medium. Cell counts were determined from two aliquots (TC10^™ Automated Cell Counter, Bio-Rad Laboratories AB, Sweden). Mean was used to prepare triplicates of100 cells per each 60 mm petri dishes (150288 Nunc^™, ThermoFischer Scientific, Denmark). The cultures were incubated with 4 ml RPMI medium (10% FBS, 5% PEST, GlutaMax) at 37 °C with 5% CO₂ for 6 days. After incubation, the cultures were washed with PBS (Medicago, Sweden) followed by incubation for 30 min in 6% glutaraldehyde (Fisher Scientific GTF) and 0.5% Crystal Violet staining solution (ServaElectrophoresis GmbH, Germany). The dishes were washed with water and allowed to dry at room temperature in darkness. Colonies (> 50 cells/colony) were counted using a visible light source (Olympus CH-2, Japan). Plating efficiency (PE) was defined as the proportion of colonies developed from the seeded cells and calculated according to the equation: PE = number of colonies/number of seeded cells. The survival fraction (SF) was estimated as SF = number of colonies formed after irradiation/(number of seeded cells × PE/100)(Franken et al. 2006).