Cxcl5

Recombinant human (rh) CXCL1, CXCL5, CXCL6, M-CSF and RANKL were purchased from PeproTech and rh NDP from R&D Systems. Primary antibodies were produced in-house (ErbB4) or purchased from Santa Cruz Biotechnology (p-Tyr, NRG3). Trypan Blue solution 0.4% was delivered by Sigma-Aldrich, cell culture media and supplements by Gibco (Life Technologies), and epidermal growth factor (EGF) and Matrigel from BD Biosciences.

To analyze drug sensitivity, PDO fragments were plated into 4-well or 8-well chamber slides at 200 to 600 fragments/well and 500 nM fulvestrant and/or 1uM palbociclib was added the following day. PDOs of early passages (passage 5–8) were used to characterize drug sensitivity. To make conditioned media, CAFs were grown in empty advanced DMEM/F12 media, and conditioned media were harvested 48 h later and mixed 1:1 with completed PDO media. To assess the effect of CAFs on drug resistance, 2000 to 6000 CAFs were plated around a dome containing PDOs in BME, or PDOs were grown in nonconditioned and CAF-CM. For cytokine treatment, PDOs were stimulated with 100 ng/ml of recombinant human GROα, IL-8, CXCL5, HGF, CCL19 (all from PeproTech), or PBS and spiked at 48 h. Cell proliferation was assessed after 96 h of treatment by incubating PDOs with 10 μM -EdU for 4 h.

NH₂-SiO₂ NPs (500 µg/mL) were mixed with CXCL5 (4 µM) (Peprotech, Rocky Hill, NJ, USA) in water, then 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (Fluorochem Ltd., Hadfield, UK) 10 µM was added, and the reaction was stirred at room temperature for 4 h. The solution was then centrifugated twice (1100 rpm for 1.5 min) using 100 kDa Amicon tubes (Merck Millipore, Burlington, MA, USA), and the NPs were dispersed in water at the same initial concentration.

Ret melanoma cell line was established from skin melanomas isolated from RET transgenic mice [54 (link)]. Mouse lymphatic endothelial cell line SVEC4-10 was established from endothelial cells from axillary LN vessels [55 (link)]. Mouse mesothelioma AB12 cell line was purchased from the CellBank Australia. All cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (PAN-Biotech GmbH, Aidenbach, Germany) with 10% heat-inactivated FBS (Merck KGaA, Darmstadt, Germany) and 1% penicillin/streptomycin (Thermo Fisher, Waltham, MA, USA) at 37 °C and 5% CO₂. All cell lines were regularly tested for the absence of mycoplasma contamination. The CXCR2 inhibitor SB265610 (100 nM) (Research And Diagnostic Systems, Inc., Minneapolis, MN, USA) and the CXC chemokines CXCL1 (100 ng/mL) and CXCL5 (100 ng/mL) (both from PeproTech, Inc., Rocky Hill, NJ, USA) were used for the treatment in vitro.

For BM neutrophils isolation, BM cells were stained with anti-mouse Ly6G (1A8; Biolegend) followed by purification using anti-biotin magnetic beads and LS columns of MACS cell separation system (both Miltenyi Biotec). For in vitro infection, 10⁵ neutrophils were seeded per well and infected with Mtb HN878 at MOI 2 or 10. After 3 h of infection, cells were recovered for RNA analysis or reactive oxygen species detection with DHE and DHR probes. Cells were incubated with DHE or DHR at 37 °C for 10 or 30 min, respectively. Samples were then acquired on a BD FACS Canto II to detect red-fluorescence (DHE) or green fluorescence (DHR) due to probes oxidization. For the chemotaxis assay, 10⁵ neutrophils were seeded on the upper chamber of the 5 μm-pore transwells (96 well plate; Corning) and allowed to migrate for 3 h towards the lower chamber containing media with CXCL1, CXCL2 and CXCL5 (all from Peprotech) at 500, 50, 5 and 0.5 ng/ml or without cytokines. As control for the amount of cells seeded, cells were also seeded in the lower chamber. Cells were then recovered and stained with SAV PE-Cy7 (Biolegend). To quantify the number of migrated cells, counting beads (Biolegend) were added to the cells prior to acquisition in a BD FACS Canto II. All cytometry data were analysed using FlowJo version 10.1.r7.

Mouse BM cells were co-cultured with mouse brain microvascular ECs, CCL5 (PeproTech, 250-07), or CXCL5 (PeproTech, 300-22) under normoxia or hypoxia (1% O₂). ECs were seeded in 6-well plates (5 × 10³/well) and then cultured overnight with DMEM medium containing 5% FBS, followed by treatment with glioma-CM as described above. ECs were washed with PBS and co-cultured with freshly prepared BMDMs (10⁴ cells per well) for 5 days in RPMI-1640 media supplemented with 5% FBS. Cells were treated with specific neutralizing antibodies against CSF-1 (1 μg/ml, R&D Systems, AF416) and IL-6 (1 μg/ml, BioLegend, 504505), or control IgG (Sigma). Cells were fixed and subjected to immunofluorescence analysis. Single-cell suspension was prepared by using Versene solution (0.02% EDTA, Thermo) and then subjected to flow cytometry analysis.