Ficoll centrifugation

BM mononuclear cells (BMMNCs) were obtained by conducting equal-volume Ficoll centrifugation (2000 rpm, 40 minutes) (GE Healthcare, UK). The BMMNCs were then seeded in a flask at a cell density of 2×10⁶/cm². The basic growth medium consisted of α-MEM (GIBCO, CA, USA)+20% FBS (Corning, NY, USA)+4 ng/mL of bFGF (Millipore, MA, USA)+1xPS (GIBCO, CA, USA). The paired subcutaneous adipose tissues were dissected into small pieces and digested with collagenase IV (SERVA Electrophoresis GmbH, Heidelberg, Germany) and hyaluronidase (Sigma-Aldrich, MO, USA), and were cultured in the basic growth medium (α-MEM+20% FBS+4 ng/mL bFGF+1xPS) at 37°C for 50 minutes with intermittent shaking. The resulting suspensions were filtered using a 70-µm strainer (BD Bioscience, NJ, USA) to remove debris and then centrifuged at 2000 rpm for 10 minutes. The supernatants were discarded, and the cell pellets were resuspended in the basic growth medium and cultured at density of 1.3×10⁵/cm² in a tissue flask (37°C, 5% CO₂). The basic growth medium was changed every 3 days and the cells were maintained at subconfluent levels (Passage 0, P0). The attached cells (BMMSCs and ADMSCs) were then harvested using trypsin-EDTA (GIBCO, CA, USA), and were subcultured at a density of 2–3×10³ cells/cm² under the same conditions used in the primary culture (Passage 1, P1).

Peripheral blood mononuclear cells were isolated from whole blood via Ficoll centrifugation (GE Healthcare, IL, USA) and stained for CD14 and CD16. Afterwards, samples were washed once with PBS + 1% BSA, fixed for 30 min at 4 °C with PrimeFlow RNA Fixation Buffer (Affymetrix) and permeabilized with Permeabilization Buffer (Affymetrix) for another 30 min at 4 °C. In situ hybridization for IL-6 and IL-8 was performed using Prime FlowRNA assay kit and probe sets according to the manufacturer (Affymetrix)26 (link). In short, samples were incubated with the appropriate target probes for 2 h at 40 °C, washed twice with PrimeFlow RNA Wash Buffer, resuspended in Storage Buffer and acquired on a FACS Canto II (BD Biosciences). Gating strategy is displayed in Fig. 4B. In short, 100.000 events per sample were recorded and the monocyte population was gated using CD14 as a selection marker. Afterwards monocyte subsets were evaluated according to the respective expression of CD16. Compensation was achieved by using single stains of BD CompBead beads with the respective secondary labels.

Human peripheral blood mononuclear cells (PBMCs) were extracted by Ficoll centrifugation (Cat# 17-1440-02, GE Healthcare, USA) from peripheral blood provided by healthy donors. We then isolated CD8⁺ T cells from PBMCs using the human CD8⁺ T Cell Isolation kit (Cat# 130-096-495, Miltenyi Biotec, Germany). Isolated CD8⁺ T cells were activated by the addition of human anti-CD3/CD28 Dynabeads (Cat# 40203D, Thermo Fisher Scientific, USA) for 3 days and cultured in RPMI-1640 media containing 10% FBS, an antibiotic mixture, and 200 U/mL IL-2. After adherent overnight, control or IL-6-overexpressing A549 and H1703 cells were incubated with activated CD8⁺ T cells for 48 h, and exposed to either the isotype control or durvalumab (1500 μg/mL). In addition, after adherent overnight, A549 and H1703 cells were incubated with activated CD8⁺ T cells for 48 h with or without exposure to the IL-6 (20 ng/mL) isotype control or durvalumab (1,500 μg/mL). The ratio of CD8⁺ T cells to cancer cells was 5:1. After the removal of T cells and cellular debris, living cancer cells were then quantified using the Cell Counting Kit-8 (Cat# CK04, DOJINDO, Japan). The tumoricidal activity was calculated using the following formula: killing activity (%) = [1 − (optical density value of experimental group − optical density value of T cells group)/optical density value of tumor cells group] × 100%.

We isolated CD4⁺ T helper cells from PBMCs according to the manufacturer’s instructions (https://www.miltenyibiotec.com/_Resources/Persistent/3c804fa07b66b63215bbacbf43387804b151d77f/SP_CD4.pdf). Venous blood samples were collected from the patients with ASO or the healthy donors at the First Affiliated Hospital of Sun Yat-sen University, and PBMCs were isolated through Ficoll centrifugation (GE Healthcare, Catalog #17144002). CD14⁺ cells were isolated from PBMCs with CD14 magnetic bead kits (Miltenyi, Catalog# 130-050-201) and FITC-conjugated anti-human CD14 antibody (BD, Catalog# 555397) according to the manufacturer’s instructions. CD4⁺ T cells were isolated from the rest of the CD14⁻ cell suspensions by positive selection with a CD4 magnetic bead kit (Miltenyi, Catalog# 130-045-101) and PE-conjugated anti-human CD4 antibody (BD, Catalog# 555347) according to the manufacturer’s instructions. Isolated CD4⁺ T cells, CD14⁺ cells and CD4⁻CD14⁻ cells were validated by FACS and frozen in liquid nitrogen for future experiments.

Six de-identified neuroblastoma patient bone marrow samples were obtained from subjects enrolled on Children’s Oncology Group trial ADVL0912 (NCT00939770) (31 (link)). Written informed consent from parents or guardians was obtained, and the institutional review boards of participating institutions approved the protocol. Four additional de-identified and discarded bone marrow samples were obtained from the Children’s Hospital of Philadelphia; the use of these de-identified samples is not human subjects’ research according to our Institutional Review Board, thereby waiving the need for consent. Peripheral blood for spiking experiments was obtained from normal donors after obtaining written informed consent and using protocols approved by the Institutional Review Board of the Hospital at the University of Pennsylvania. Buffy coats from bone marrow were enriched by Ficoll centrifugation (GE Healthcare Life Sciences, Piscataway, NJ, USA). If cells were not immediately used for additional assays, fixation was performed using Miltenyi Inside Fix (Miltenyi Biotec Inc., Auburn, CA, USA).