Illumina HiSeq 4000 paired end sequencing; Single cell transcriptomics profiling of vessel co-option
Single cell transcriptomics profiling of vessel co-option
EC4_p
RENCA cells were injected into the tail vein of the mice using a 29G needle (2 × 105 cells in 0.1 mL phosphate buffered saline (PBS)). The animals were sacrificed on day 10, day 21 or day 36 of the experiment and lungs were immediately processed for metastatic cell or EC isolation. Mice were treated with Sunitinib (LC laboratories) (40 mg/kg i.p., every day), or with a control vehicle (0.5% carboxymethylcellulose sodium, 1.8% w/v NaCl, 0.4% w/v Tween 80, 0.9% w/v benzyl alcohol dissolved in reverse osmosis deionized water adjusted to pH 6) by daily gavage of 0.2 mL. Mice were randomly allocated to the treatment condition, balanced for body weight; treatment was started from day 10 after injection of RENCA cells and continued until day 20 (Bridgeman et al., 2017) or day 35. For metastatic cells and TECs, 5 mice per treatment group were anesthetized and killed via cervical dislocation, lungs were dissected and placed under a dissection microscope. Per mouse, at least 60 metastases were micro-dissected. For NECs, control (non-tumor bearing) mice were anesthetized, lungs were perfused with 5 mL PBS at 120 mL/h and both lungs were dissected. The dissected tissue was placed into a GentleMACS C Tube (Miltenyi Biotec) containing 5 mL of digestion buffer on ice (0.1% collagenase II (Thermo Fisher Scientific), 0.25% collagenase IV (Worthington Biochemical) and DNase I (15 M) in DMEM supplemented with 1x sodium pyruvate, 1x MEM NEAAs, ECGF/Heparin, antibiotic/antimycotic (2x) and 1% penicillin/streptomycin (Thermo Fisher Scientific)). The dissected tissues were dissociated using the gentleMACSTM Dissociator (Miltenyi Biotec; on m_lung_01 protocol) and incubated in a water bath at 37°C for 30 min with manual shaking every 5 min. At the end of the tissue digestion process, the samples were again placed in the gentleMACSTM Dissociator (Miltenyi Biotec; on m_lung_02 protocol) and the reaction was stopped by adding 10 mL of an isolation buffer containing PBS, 0.5% BSA and 40 mM EDTA. Subsequently, the cell suspension was filtered through a 40 μm cell strainer (Sigma-Aldrich) and cells were rinsed with isolation buffer. Next, for TECs and NECs, ECs were enriched by magnetic bead sorting using the MACS system (Miltenyi Biotec) at room temperature. First, immune cells and epithelial cells were depleted using selection with CD45 and CD326 MicroBeads (Miltenyi Biotec), followed by positive selection of ECs with CD31 MicroBeads (Miltenyi Biotec), using LS and MS columns (Miltenyi Biotec), according to the manufacturer's instructions. For further processing for scRNA-seq, the samples were resuspended in PBS containing 0.4% UltraPure BSA (50 mg/mL; Thermo Fisher Scientific) and filtered over 40 μm cell strainers on ice. Overall, it took between 3 to 4 h from obtaining metastases collection to obtaining single cell EC suspensions. For metastatic cells, no selection procedure was applied and it took between 1 to 2 h from collecting metastases to generating single cell metastatic cell suspensions. Nucleic acid extraction was done in single cells using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (v2, 10x Genomics) according to manufacturer's instructions. The single cell suspensions of freshly isolated metastatic cells and ECs (MACS-bead enriched as described above) were resuspended in PBS with 0.04% ultra-pure BSA and converted to barcoded scRNA-seq libraries using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (10x Genomics), aiming for 6,000 cells per library. Samples were processed using kits pertaining to V2 barcoding chemistry of 10x Genomics. Single samples were always processed in a single well of a PCR plate, allowing all cells from a sample to be treated with the same master mix and in the same reaction vessel.