Phg0313

GFP-tagged human CTC-derived Brx50 cells or patient-derived cancer cells were added to the capture chamber of the device via a syringe pump at a flow rate of 50 µL min⁻¹, and cells were cultured on-chip under hypoxic conditions (5% O₂). After the isolation of cancer cells from liquid biopsies, e.g., blood or ascites fluid, the chamber was washed with 1 mL 1X PBS. Subsequently, the wash solution was exchanged with CTC growth medium. CTC growth medium (RPMI 1640 Medium (Invitrogen, 52400-025) containing 20 ng mL⁻¹ recombinant human epidermal growth factor (Gibco, PHG0313), 20 ng mL⁻¹ recombinant human fibroblast growth factor (Gibco, 100-18B), 1X B27 supplement (Invitrogen, 17504-044) and 1X antibiotic-antimycotic (Invitrogen, 15240062)) was added every 48–72 h. A total volume of 100 µL CTC growth medium was added to a truncated 20–200 µL pipette tip, which was inserted into the capture chamber inlet, and an empty tip was inserted into the outlet, creating a steady flow of medium throughout the entire chamber. Generally, while for some patient-derived cancer cells proliferation is indefinite (i.e., we are able to maintain patient-derived cell lines), most proliferation is limited to a few days or weeks. This is sufficient for drug screen purposes but insufficient to derive permanently growing cell lines.

The CaP2 and CaP8 cell lines were established as described previously^{16 (link)} and cultured in DMEM media (Gibco 11995065) containing: FBS (10%, Applied StemCell B7227), Penicillin-Streptomycin (1%, Gibco 15070063), L-Glutamine (1%, Gibco A2916801), Insulin (5ug/ml, Gibco 12585-014), EGF (6 ng/ml, Gibco PHG0313), Bovine Pituitary Extract (25ug/ml, Gibco 13028-014). The PC3 and LNCaP cell lines were purchased from ATCC and the PC3 PTEN inducible cell line are generated as describe^{17 (link)} and cultured using Tet-free serum (Omega Scientific FB-15). All cell was cultured at 37 °C in 5% CO² condition. PTEN re-expression was induced by adding Doxcycline (1 ug/ml, sigma D9891) in culture media for 4 days. IFNγ (PEPROTECH, 315-05) was add at 10IU concentration for 72 h, PDL1 expression in CAP2/CAP8 cell was determined by FACS.

GFP- or RFP-tagged human CTC-derived cell lines were cultured under hypoxic conditions (5% O₂) in ultralow attachment (ULA) 6-well plates (Corning, 3471-COR). Every third day, CTC cultures were given CTC growth medium, which was made of RPMI 1640 medium (Invitrogen, 52400-025) containing 20 ng mL⁻¹ recombinant human epidermal growth factor (Gibco, PHG0313), 20 ng mL⁻¹ recombinant human fibroblast growth factor (Gibco, 100-18B), 1X B27 supplement (Invitrogen, 17504-044) and 1X antibiotic-antimycotic (Invitrogen, 15240062).

SZ95 cells are an immortalized human sebocyte cell line generated from the face of an 87-year-old female and transformed with Simian virus 40 (RRID:CVCL_9803). These cells were previously obtained from Cristos Zouboulis (Harris et al., 2019 (link)). SZ95 sebocytes were maintained in Sebomed Basal Medium (Fisher Scientific NC9711618) supplemented with 10% fetal bovine serum (GeminiBio 100-106), 5 ng/ml human epidermal growth factor (Thermo Fisher PHG0313), and 1% antibiotic–antimycotic (Gibco 15240062). Cells were cultured in 5% CO₂ incubator at 37°C. Cells were stimulated with 1 μg/ml LPS (Sigma L4524). Sixteen hours poststimulation cells were harvested and analyzed as described below. For TLR agonists treatment, reagents in human TLR1-9 Agonist kit (InvivoGen, tlrl-kit1hw) were diluted to working concentration in PBS. 100 ng/ml Pam3CSK4, 1 × 10⁸ cells/ml HKLM, 500 ng/ml Poly (I:C), 500 ng/ml Flagellin, 100 ng/ml FSL-1, and 1 μg/ml Imiquimod were used for stimulation of SZ95 cells. For heat-inactivated bacteria treatment, bacteria were grown to mid-logarithmic phase, spun down, washed, and resuspended in PBS. Bacteria were heat-inactivated by incubation at 95^oC for 20 min. Then 1 × 10⁸ cells/ml were used for each treatment.

HCC827P, PC9P, HCC827R and PC9R cells were subjected to flow cytometric analysis to obtain CD133⁺ABCG2⁺ CSCs (labelled HCC827P‐CSCs, PC9P‐CSCs, HCC827R‐CSCs and PC9R‐CSCs, respectively), with corresponding antibodies against CSC markers (anti‐CD133, ab216323, Abcam, Cambridge, UK; anti‐ABCG2, ab229193 Abcam) used in the analysis.
CSCs were cultured in serum‐free DMEM with 50‐mg/ml insulin (A1895602, Gibco), 100‐mg/ml transferrin (T2872, Gibco), 10‐mg/ml tetramethylenediamine (T21407, Sigma), .03‐mM sodium selenite (10102‐18‐8, Sigma), 2‐mM progesterone (15775‐74‐3, Sigma), .6% glucose (A2494001, Gibco), 5‐mM HEPES (15630080, Gibco), .1% sodium bicarbonate (144‐55‐8, Sigma), .4% bovine serum albumin (30063788, Gibco), 2‐mM l‐glutamine (G8540‐25G, Sigma), 50‐IU/ml penicillin (36945‐98‐9, Sigma) and 50‐mg/ml streptomycin (3810‐74‐0, Sigma), 20‐mg/ml EGF (PHG0313, Life Technologies, Foster City, CA) and 10‐mg/ml bFGF (PHG0261, Life Technologies) with 100% humidity and 5% CO₂ at 37°C. After the incubation, CSCs were trypsinized and harvested.
Detection of CD166⁺ (CD166‐PE, 559263, BD Biosciences, San Jose, CA) and CD44⁺ (CD44‐FITC, 555478, BD Biosciences) using flow cytometry and in vitro sphere‐forming assay were conducted for the identification of CSCs.