Epirubicin

Viable CTCs were enriched from a cryo-conserved DLA product by using the Parsortix system and were cultured as previously reported [25 (link)]. Briefly, CTCs were cultured in low attachment plates (Corning, Corning, NY, USA) with RPMI 1640 medium supplemented with 1 × B27 (Thermo Fisher Scientific), 20 ng/mL human epidermal growth factor (Merck), 20 ng/mL fibroblast growth factor (Merck), and 1% penicillin-streptomycin (Thermo Fisher Scientific) in a humidified atmosphere with 5% CO2 and 4% O2.
For drug testing 100 CTCs were seeded per well of a 96-well plate after ten days of pre-culture. The cells were treated with capivasertib (MedChem Express, Monmouth Junction, NJ, USA), everolimus (Merck), epirubicin (Merck), and paclitaxel (Merck). Each drug concentration was tested in triplicates. After incubation for 6 days, cells were spun on glass slides, stained for cytokeratin, and numbers were determined by counting. As references, cell lines MDA-MB-231, SK-BR-3, T-47D, and MCF7 were used (ATCC, Manassas, VA, USA; catalog numbers: MDA-MB 231: HTB-26, SK-BR-3: HTB-30, T-47D: HTB133, and MCF7: HTB-22). Cells were authenticated via short tandem repeat analysis and regularly tested negative for Mycoplasma.

Cells (1x10⁶) were seeded in 6-well plates. After 12 hours in culture, fresh medium with 1 µg/ml doxorubicin or epirubicin (Sigma) was added for 24 hours. The conditioned medium was then harvested and measured for IFNβ using mouse IFNβ colorimetric ELISA kit (Thermo Scientific).

The anti-LC3B rabbit polyclonal antibody used in the western blot assay was from Cell Signaling Technology (#12741S; Beverly, MA, USA), the anti-p62/sequestosome 1 (SQSTM1) rabbit polyclonal antibody was obtained from Proteintech (#18420-1-AP; Chicago, IL, USA) and the anti-GAPDH mouse monoclonal antibody was from Beijing CoWin Biotech Co., Ltd. (cw0100A; Beijing, China). Goat anti-rabbit immunoglobulin G-horseradish peroxidase (IgG-HRP) and goat anti-mouse IgG-HRP (Pierce, Thermo Fisher Scientific, Rockford, IL, USA) were used as secondary antibodies, and enhanced chemiluminescence reagent (Pierce, Thermo Fisher Scientific) was used for developing the blots. The agents epirubicin, doxycycline, 3-methyladenine (3-MA; Sigma-Aldrich, St. Louis, MO, USA) and purimycin (Invitrogen Life Technologies, Carlsbad, CA, USA) were dissolved in phosphate-buffered saline (PBS) to form stock solutions and then added directly into the media to the required concentration.

Cells were cultivated in RPMI-1640 medium (Biochrom AG, Berlin, Germany) supplemented with 10% fetal bovine serum (FBS; Pan Biotech, Aidenbach, Germany) and 1% L-glutamine-penicillin-streptomycin solution (GPS; Sigma-Aldrich, St. Louis, Missouri, USA). Prior to further stimulation, THP-1 were differentiated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA; Abcam, Cambridge, UK) for 18 h.
Cells were treated with freshly prepared, sterile-filtered epirubicin (Sigma Aldrich) at concentrations between 0.01 to 5 µg/mL for 24 h, unless stated otherwise. After epirubicin treatment, PM and THP-1 were stimulated for 3 h with LPS from E.coli K12 (InvivoGen, San Diego, CA, USA) at concentrations of 10 or 100 ng/mL, respectively, followed by treatment with freshly prepared 1 mM ATP (InvivoGen, San Diego, CA, USA) for 1 h. For the inhibition of autophagy, 300 nM bafilomycin (Invivogen, San Diego, CA, USA) or 10 mM 3-methyl adenine (3-MA; Invivogen, San Diego, CA, USA) were added to the cells simultaneously with LPS, and maintained until the end of the experiment. For TLR2 ligation, cells were incubated either with 10⁸ cells/mL of heat-killed listeria monocytogenes (HKLM; Invivogen, San Diego, CA, USA) or 100 ng/mL Pam3CSK4 (Invivogen, San Diego, CA, USA) for 4 h.

The panel of drugs used were Daunorubicin (30450, Sigma-Aldrich), Doxorubicin (D1515, Sigma-Aldrich), Epirubicin (E9406, Sigma-Aldrich), Mitoxantrone (M6545, Sigma-Aldrich) and Trastuzumab (HYP9907, MedChem Express). All drugs were dissolved in molecular biology grade water to a concentration of 10 mM per stock. DOX, DNR, EPI, and MTX stocks were stored at -80°C and working stocks used at 4°C for up to one week. TRZ was stored at a 1 mM concentration at 4°C for up to one month.