Bsa 0.5

The cells were stimulated for 6, 12, 24, and 72 h with tumor necrosis factor-α (TNF-α) (10 ng/mL), lipopolysaccharide (LPS) (100 ng/mL), or both. HFLS Basal Medium with BSA 0.5% (Sigma) was used as a negative control. The samples were performed in triplicate. After the time points, the cell pellets were used for total RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.

Tumor tissue specimens derived either from CUP patients (AGN906 and AGN47), or PDX (AGN901, AGN43, AGN67, and AGN914), or a melanoma patient (mMEL321) were minced and digested with collagenase I (Gibco) at 2 mg/ml in culture medium for 40 min at 37 °C. After filtration and red cell lysis, single-cell suspensions were resuspended in culture medium, composed by: DMEM:F12 (Sigma), N2 supplement (Life Technologies-GIBCO), BSA 0.5% (Sigma), heparin 4 μg/ml (Sigma), 2 mM glutamine (Sigma), penicillin–streptomycin (EuroClone), and seeded in ultra-low-attachment flasks (Corning-Sigma). For AS914 and AS67, the culture medium was supplemented with a chemically defined Lipid Concentrate (Gibco). Culture medium was replaced the next day and used throughout propagation. Spheroids (agnospheres) appeared in culture few days after seeding, and stabilization occurred on average after 3–4 months. For dissociation, trypsin was required for all agnospheres except for AS906 and AS914. mCRC729 and mCRC0155, previously derived from colorectal cancer liver metastases^{17 (link)}, were kept in the same culture medium as above, supplemented with Lipid Concentrate (Gibco). Agnospheres and tumorspheres will be available upon institutional material transfer agreement approval.

AU565 (ERBB2+, positive control) and HCC38 (ERBB2−, negative control) breast cancer cell lines were seeded at 60–70% confluency on glass coverslips (EXACTA-OPTECH, area 10 × 10 mm², thickness 0.13–0.16 mm) in 24-well plate.
Following PBS 1× washing, cells were fixed with PFA 4% for 10 min, and then washed with PBS 1×. After fixation, cells were then blocked with blocking buffer, prepared with BSA 0.5% (Sigma-Aldrich) and NH₄Cl 50 mM (Sigma-Aldrich) in PBS 1×, for 1 h at room temperature (RT). Cells were incubated in humidified environment at RT in the dark with 1:100 primary antibody (BB700-conjugated mouse antihuman-ERBB2 Ab, BD Bioscience). Incubation time was set according to the experimental plan. Primary antibody was prepared in blocking buffer.
Following antibody incubation, cells were washed three times with PBS 1× and once more with ddH₂O. Then, coverslips were mounted in Fluoroshield^™ with DAPI (Sigma-Aldrich) and fluorescence images were acquired using a Nikon Ti Eclipse microscope equipped with a mercury lamp (Intensilight, Nikon), a EMCCD digital camera (iXon Ultra 897, Andor Technology Ltd) and a ×20 air objective (CFI Plan Fluor DLL ×20, 0.5 NA, Nikon Instruments).