Dead cell removal microbeads

Tumor-bearing mice treated with vehicle or CB-1158 (100 mg/kg BID) were sacrificed for flow cytometry analysis on study day 9 (B16), day 10 (4T1), or day 14 (CT26 and LLC). Excised tumors were placed on ice in RPMI-1640 medium containing 5% FBS, minced with a razor blade, and dissociated in RPMI-1640 supplemented with mouse tumor dissociation enzymes (Miltenyi Biotec, Bergisch Gladbach, Germany) on a GentleMACS Octo Dissociator With Heat (Miltenyi Biotec) according to the manufacturer’s instructions. Dissociated tumors were strained through 70 μm nylon mesh, washed with cold PBS containing 2% FBS, blocked with anti-CD16/CD32 (Fc block antibody, eBioscience), and stained for cell surface antigens. For B16 and 4T1 tumors, washed dissociated tumor cells were incubated with Dead Cell Removal MicroBeads (Miltenyi Biotec) and applied to a magnetic column prior to staining. For intracellular staining, cells were fixed and permeabilized using buffers purchased from R&D Systems or eBioscience for cytoplasmic or nuclear antigens, respectively. All tumor flow experiments were acquired on an Attune NxT flow cytometer and analyzed with FlowJo software version 10 (Ashland, OR), using fluorescence-minus-one controls for gating and single-stained OneComp eBeads (eBioscience) to set compensation matrices.

The mice were anesthetized and perfused with 10 mL pre‐chilled PBS via the left heart ventricle. The kidney and spleen were removed and stored in cold 1640 medium (Gibco, USA), cut into 1 mm³ pieces with a small scissor on ice, then incubated in 5 mL of digestion buffer containing 0.25 mg mL⁻¹ Liberase TH (Thermolysin High) Research Grade enzyme (0.25 mg mL⁻¹, Roche, USA) and 50 µg mL⁻¹ DNase I (NEB, USA) at 37 °C for 30 min, shaking gently twice during the period. Then the digestion was stopped with 5% FBS. The digested tissue was then passed through a 70 µm cell strainer (Falcon, BD Biosciences, USA) into pre‐chilled PBS twice on ice and the cells were palleted by centrifugation at 400 g at 4 °C for 5 min. After centrifugation, the cell pellet was incubated with 3 mL of red blood cell (RBC) lysis buffer (Invitrogen, USA) on ice for 5 min and palleted by centrifugation on 400 g at 4°C for 5 min. Then, the cells were washed and resuspended in pre‐chilled PBS twice and filtered through a 40 µm cell strainer (Falcon, BD Biosciences, USA) to remove debris or cell aggregates. Finally, the cells were centrifuged at 400 g at 4 °C for 5 min and resuspended in 500 µL of pre‐chilled PBS with 0.04% BSA and the Dead Cell Removal Micro Beads (Miltenyi Biotec, Germany) were used to remove dead cells.

Three fresh samples were collected per group for scRNA-seq. In brief, the wound tissues were firstly digested by the Epidermis Dissociation Kit (Epidermis Dissociation Kit, mouse; Miltenyi Biotec) for enzymatic epidermal-dermal separation. The epidermis part was dissociated by a gentleMACS Dissociator (Miltenyi), then filtered (70-mm cell strainer, Corning, Durham), centrifuged (300 g, 10 min, 4 °C), and resuspended with phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA). The dermis part was cut into 1 mm width pieces and mixed with mixed enzyme solution containing type I collagenase (Gibco, Grand Island) and trypsin (Gibco, Canada), then dissociated by gentleMACS Dissociator (Miltenyi), and digested for 2.5 hours in a hybridization oven (Peqlab PerfectBlot). After being dissociated, filtered, centrifuged, and resuspended in red blood cell lysis buffer (Solarbio), the dermis cells were mixed with the epidermis part. Then the dead cells and debris were removed by Dead Cell Removal MicroBeads (Miltenyi).

Lymphoid cells were depleted from bone marrow and blood samples using magnetic separation. Briefly, 1 × 10⁷ cells were suspended in cold rinsing solution (Miltenyi Biotec, 130-091-222) supplemented with bovine serum albumin and incubated with anti-CD3 and anti-CD19 MicroBeads (Miltenyi Biotec, 130-050-101 and 130-050301, respectively) before passing through filters and columns. To remove dead cells, up to 1 × 10⁷ cells were suspended in Dead Cell Removal MicroBeads (Miltenyi Biotec, 130-090-101) and filtered through magnetic columns. Cells were recovered in primary media with cytokines for 2 h at 37 °C prior to performing experiments.

Centrifuge cell suspension at 300 × g for 10 min. Resuspend cells with Dead Cell Removal MicroBeads (Miltenyi Biotec, Germany). Mix well and incubate for 15 min at room temperature. Add the cell suspension to the column. Rinse with 1× binding buffer and collect effluent as living cells. In the cell suspension, early apoptosis, late apoptosis and necrotic cells are magnetically labelled and retained within the column, and the living cells in good condition are passed through the separation column.

Following a treatment that lasted 6 weeks in SC31 and 7 days in GS3 using E2 (1 mg) or a placebo pellet, tumors were harvested and digested into a single-cell suspension. Although results of longer E2/placebo treatments of GS3 showed larger differences in gene expression according to the bulk RNA-Seq analysis, the viability of single cells isolated from GS3 tumors decreased after the tumors shrank. Since the presence of a large number of dead cells would affect the quality of single-cell analysis, we decided to treat GS3-PDX for 7 days in order to keep single-cell viability over 80%. Single-cell samples were prepared for scRNA-Seq using a 10x Genomics platform. Single-cell preparations from two biological replicates from each treatment were combined and processed for scRNA-Seq (Figure S2). SC31 and GS3 tumors were cut into small, 2 mm thick strips and digested with 1.5 mg/mL DNAse I (#10104159001, Millipore Sigma, St. Louis, MO, USA), 0.4 mg/mL collagenase IV (CLS-4, Lot: 47E17528A, Worthington, Lakewood, NJ, USA), 5% FBS, and 10 mM HEPES in HBSS. The mixture was strained through a 70 μm cell strainer. Then, 1 mL of ACK lysis buffer was used to remove residual red blood cells from the sample. Dead cells were removed using Dead Cell Removal MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany).