Enzyme r

All primary tumors were diagnosed at the Cross Cancer Institute (Edmonton, AB, Canada). The use of these tissues has been approved by our Institutional Ethics Committee. All archival tissues were formalin-fixed and paraffin-embedded. All fresh tumor samples were processed immediately after surgery to isolate primary BC cells using a protocol described previously [9 (link)]. Briefly, tumors were cut into small pieces of 2–4 mm in the greatest dimension, which were then incubated with RPMI supplemented with Enzyme H, Enzyme R and Enzyme A (Macs Miltenyi Biotec, Auburn, CA, USA) in MACS C-tubes. Tissue shearing was performed by providing three pulses every 30 min at 37 °C. Cells were collected after centrifugation at 300× g for 7 min. The subsequent steps of BC isolation were based on the manufacturer’s instructions (Cancer Cell Isolation kit, Panomics, Redwood, CA, USA). After culturing for 1–2 days, cells were infected with lentivirus containing either mCMV or the SRR2 reporter. Infection was repeated twice (24 h apart) and cells were sorted into RU or RR cells approximately 48 h later, based on the green fluorescence protein (GFP) expression [9 (link)].

Fresh samples isolated from patients in operation were preserved in MACS Tissue Storage Solution (Miltenyi Biotec) at 4 °C. Tumor tissues were cut into a range of 0.2–1.0 g small pieces and dissociated in 5 mL enzyme mix containing 4.7 ml RPMI 1640 (Gibco), 200 μL Enzyme H, 100 μL Enzyme R and 25 μL Enzyme A (Miltenyi Biotec, MACS Tumor Dissociation Kit, human). The samples were subsequently incubated in a 37 °C thermostatic shaker for 35 min. Then suspended samples were filtered through a 40-μm Cell-Strainer nylon mesh (BD) with 30 mL of RPMI 1640 and centrifuged at 300 × g for 7 min. After removing the supernatant, we used Red Blood Cell Lysis Solution (Miltenyi Biotec #130-094-183) and the Dead Cell Removal Kit (Miltenyi Biotec #130-090-101) to remove red blood cells and obtain live cells. Cell suspension was centrifugated at 300 × g for 7 min and the pellet was re-suspended in 1 mL PBS solution. Once the desired cell suspension was obtained, the sample was immediately placed on ice for subsequent GEMs preparation and reverse transcription. The single cell libraries were prepared according to the standard protocols and sequenced on Illumina NovaSeq 6000 Systems using paired-end sequencing (150 bp in length).

Gingival samples were processed to form a single-cell suspension, using a gentle MACS dissociator, a tissue dissociation kit and an optimized protocol. Briefly, gingival samples were kept in Dulbecco’s Phosphate-Buffered Saline (DPBS), washed twice with DPBS, and minced into small pieces. Each sample was then transferred into a c-tube which contained 2.35 mL of serum-free RPMI 1640 with 100 μL of Enzyme H, 50 μL of Enzyme R, and 12.5 μL of Enzyme A (Miltenyi Biotech, CA, United States). We used a Gentle mac-dissociator for mechanical and enzymatic disruption and kept the samples in a 37°C incubator for 30 min. The Gentle mac-dissociator was used three times and samples were placed in an incubator twice during the process. A 70-micrometer filter was used to achieve a single-cell suspension and each sample was washed with 15 ml RPMI solution and transferred into a centrifuge for 7 min under a 0.4 relative centrifugal field (RCF). The pellet was mixed with a freezing medium, which included 10% of dimethyl sulfoxide (DMSO) and 90% of fetal bovine serum (FBS). The samples were kept in an isopropanol chamber at −20°C for 24 h to preserve viability, then finally transferred into liquid nitrogen.