Recombinant human sdf 1α

H460 (large cell carcinoma), A549 (adenocarcinoma), and H1299 (large cell carcinoma) NSCLC cell lines, authenticated by short tandem repeat (STR) profiling, and murine RAW 264.7, murine 3B11, and HUVECs (all purchased from ATCC) were cultured in RPMI 1640 + 10% fetal bovine serum (FBS) or in endothelial growth media (EGM-2, Lonza). The LT73 cell line was derived from a primary NSCLC adenocarcinoma and cultured in vitro in RPMI 1640 + 10% FBS.
CCR2⁺ cells were positively isolated from BM cells flushed from SCID mouse femurs using anti- CCR2⁻allophycocyanin (APC)⁺ anti-APC MicroBeads and an autoMACS Pro separator (Miltenyi Biotec). 1 × 10⁵ sorted CCR2⁺ IMs or CCR2⁻ myeloid cells were co-cultured at a 1:1 ratio with tumor cells or used to recover CM after 48 h.
Short-term NSCLC primary cultures were obtained from four early stage (I–IIb) NSCLC undergoing surgical resection using differential filtration of dissociated primary tumor tissues as described in Bertolini et al.³¹ (link)
For in vitro experiments, cells were treated with the following: peptide R (10 μM), recombinant human SDF-1α (25 ng/mL) (300-28A), cytoMCP-1 (CCL2) (25 ng/mL) (300-04), IL-8 (50 ng/mL) (200-08), GRO-α/MGSA (25 ng/mL) (300-11), and IL-6 (10 ng/mL) (200-06) (all from PeproTech); as well as neutralizing Ab anti-human/mouse SDF-1 (25 μg/mL) (MAB310) and mouse VEGF (150 ng/mL) (VEGF164) (both from R&D Systems).

For the migration assay, only samples containing more than 85% CLL cells were used. Moreover, we selected samples with CD49d-positive CLL cells. CLL cells were isolated by Ficoll gradient centrifugation and seeded in the upper chamber of a transwell (5 µm pore size; Sarstedt; Hildesheim, Germany) at 2x10⁶ in 100 mL of serumfree RPMI 1640. The transwells had been previously coated with human FN (10 mg/cm^{2 (link)}, Bio-Techne; Minneapolis, MN, USA), recombinant human VCAM1-Fc Chimera (1 µg/cm^{2 (link)}, Biolegend; San Diego, CA, USA), or bovine serum albumin (BSA, 1% [v/v] in PBS; Merck) as control. Lower chambers were filled with either 600 mL of serumfree RPMI 1640 medium or serum free RPMI 1640 medium supplemented with recombinant human SDF1α (200 ng/mL, Peprotech; London, UK). After 5 hours (h) at 37°C, migrated cells in the lower chambers were collected and mixed with 25 mL of CountBright absolute counting beads (ThermoFisher Scientific; Waltham, MA, USA) together with 5 mL of 7AAD to exclude dead cells. Samples were acquired on a BD FACS Canto I. Acquisition was stopped after collecting 2,000 events in the gate drawn around the beads. The cell number was estimated using the following equation: absolute count (cells/mL) = (cell count x counting bead volume) / (counting bead count x cell volume) x counting bead concentration

Gelatin (type A, from porcine skin) was purchased from Sigma-Aldrich, USA. Methacrylic anhydride, 2-mercapto-1-methylimidazole were purchased from Macklin, China. Dopamine hydrochloride and sodium hydroxide were purchased from Aladdin, China. Recombinant human SDF-1α was purchased from Peprotech, USA. Phosphate buffered saline (PBS, pH = 7.4) and fetal bovine serum were purchased from Gibco, USA. Cell counting kit-8 (CCK-8) was purchased from Beyotime Biotechnology, China. LIVE/DEAD cell imaging kit was purchased from Thermal Fisher, USA. Bone wax was purchased from Guangzhou Beogene Biotechnology Co., Ltd. Guangzhou, China. All the other reagents were analytical grade and used without further purification.