Fetal calf serum (fcs)

In this study, we used the human monocyte leukemia cell line Thp-1 (ATCC TIB202 (47 (link), 48 (link)). Thp-1 cells are widely used as a model for monocyte-macrophage-like cells and can be differentiated into active macrophages, for instance using phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) treatment (47 (link)). For priming, we applied PMA (50 ng/well (53 (link)); for 2 days to Thp-1 cells, then gave them a one-day resting period, and then co-incubated the cells with the bacteria on the fourth day, for 4 h. In addition, we employed the NF-κB reporter cell line Thp1_luc (kindly provided by Karsten Tedin) for quantitating NF-κB-dependent responses, and the NLRP3-deficient Thp-1 cell line derivative Thp-1 dNLRP3def (Invivogen). All cell lines were cultured in RPMI1640 medium (buffered with 20 mM Hepes, Glutamax; Thermo Fisher Scientific - Gibco) supplemented with 10% FCS (PromoCell) and, in the case of Thp1_luc cells, were additionally supplied with 0.5 µg/ml puromycin (except when co-incubated with live bacteria or bacterial products). Cells were routinely cultured in a 5% CO₂ atmosphere incubator. Co-incubation times for each experimental setting are indicated in the figure captions and in the results.

N-Acetyl-d-penicillamine (NAP), sodium nitrate, vanadium (III) chloride (VCl₃), sulfanilamide, N-(1-naphthyl) ethylene diamine dihydrochloride (NEDD), PCL (Mn = 80,000), BioUltra grade PEG (Mn = 4000), dimethyl sulfoxide (DMSO), and thiazolyl blue tetrazolium were purchased from Sigma-Aldrich (St. Louis, MO). Poly caprolactone (PCL) filament (Mn ≈ 100,000 g/mol, white, diameter 1.75 mm) under the commercial name Resomer^® C was supplied by Evonik Corp. (USA). Hydroxyl (OH) functionalized multiwalled carbon nanotubes (MWCNTs, OD: 8–15 nm, L: 10–50 µm) were purchased from IoLiTec (Germany). Sulfuric acid (H₂SO₄), hydrochloric acid (HCl), ortho-phosphoric acid (H₃PO₄), methanol (MeOH), tetrahydrofuran (THF), toluene, tryptic soy broth (TSB), Luria broth (LB), and sodium nitrite (NaNO₂) were obtained from Merck (Darmstadt, Germany). Endothelial cell basal medium, FCS and supplement pack endothelial cell obtained from PromoCell (Heidelberg, Germany). Calcium and magnesium free Dulbecco’s phosphate buffered saline (PBS, pH 7.4), Dulbecco’s modified Eagle’s medium (DMED), fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco (Scotland, UK). Live/Dead viability/cytotoxicity kit, Alexa Fluor™ 594 Phalloidin, and DAPI were supplied by Thermo Fisher, Invitrogen™ and BD Pharmingen, respectively.

MARC‐145 cell layers were maintained in Dulbecco's Modified Eagle Medium (Sigma‐Aldrich) cell culture medium supplemented with 1% penicillin (500 U/ml; Sigma‐Aldrich), streptomycin (500 mg/ml; Sigma‐Aldrich) and 5% foetal calf serum (FCS; Gibco, part of Thermo Fisher Scientific, Dreieich, Germany) and incubated at 37°C and 5% CO₂ until confluency. Primary BUVEC were maintained in modified ECGM [endothelial cell growth medium (PromoCell, Heidelberg, Germany); 30% (v/v) ECMG and 70% (v/v) M199, supplemented with 1% penicillin and streptomycin and 5% FCS] at 37°C in 5% CO₂ atmosphere until confluency.
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PBMCs were obtained from 10 mL venous peripheral blood by centrifugation in a density gradient. The blood sample was harvested from the breast cancer patients before the surgical intervention in EDTA collection tubes. For PBMCs isolation, we used 10 mL Ficoll-Paque PLUS (Sigma-Aldrich), which was placed on the bottom of a 50 mL Falcon tube, while on top, we slowly pipetted the peripheral blood diluted with PBS (Gibco) at a ratio of 1:1. We collected the mononuclear cells ring after centrifugation at 500× g and deceleration 0 for 25 min. After washing the PBMCs twice with PBS (Gibco), the cells were cryopreserved in liquid nitrogen (−196 °C) in a medium containing FCS (PromoCell) and 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich), at a concentration of 10⁶ cells/mL, for further use.
The blood samples were submitted for HLA-typing in an independent study, and we selected only the PBMCs and TAF from patients with HLA type A*11 to be compatible with the SK-BR-3 tumor cell line.
All tissue and biological samples were obtained after signing the informed consent elaborated under a protocol approved by the Ethical Commission of the County Emergency Hospital “Pius Brinzeu” Timisoara, according to the World Medical Association Declaration of Helsinki.