70 m cell filter

Fresh tissues were washed three times with Hank's solution containing 1% fetal calf serum (FCS) and cut into small pieces. Specimens were collected in complete RPMI 1640 medium supplemented with 10% FCS (R‐10) containing collagenase IV (1 mg mL⁻¹) and deoxyribonuclease I (10 mg mL⁻¹) and mechanically separated using the gentle MACS Dissociator (Miltenyi Biotec). Dissociated cell suspensions were further incubated for 1 h under continuous rotation at 37 °C. The cell suspensions were then filtered by a 70 µm cell filter (BD Labware). Cell viability, as measured by trypan blue exclusion staining, was typically >95%.

PBMCs were isolated from whole blood anticoagulated with heparin (132 Units per 8 ml blood) (BD Biosciences, Oxford, UK) using standard methods. Of note is that the material used for density gradient centrifugation was adjusted dependent on the macaque species, with a Ficoll Histopaque gradient (GE Healthcare, USA) used with rhesus macaque blood and a Percoll gradient (GE Healthcare) used with cynomolgus macaques. Mononuclear cells (MNC) were isolated from spleen and lung tissue samples using an OctoMACS tissue dissociation device (Miltenyi Biotec). Lung tissue samples were dissected into approximately 5mm³ pieces and incubated for 1 h in a solution of 772.8 U/ml collagenase + 426 U/ml DNase (both from Sigma) diluted in Earle’s balanced salt solution supplemented with 200 mg/ml Calcium Chloride (Gibco, Life Technologies, Renfrew, UK), at 37 °C with continual gentle mixing of the tube. The homogenised solution was passed through a 70 µm cell filter (BD Biosciences) and the mononuclear cells separated by Ficoll Histopaque density gradient centrifugation. PBMCs and MNC isolated from tissues were stored at −180 °C until resuscitated for analysis.

Sphere-formation assay is the gold standard for evaluating cancer stem cells (CSCs). It judges the ability of a single cell to self-renew under suitable medium conditions. In general, it is necessary to test the self-renewal ability of cells after successive passage. Single-cell suspensions (1 × 10³ cancer cells/ well) were seeded in a poly-HEMA coated 6-well plate in MammoCult^TM medium (#05620, STEMCELL Technologies, Vancouver, Canada). After 10 days, tumor spheres were filtered by 70 µm cell filter (BD), then washed with PBS, and then centrifuged at 1000 rpm for 5 min to collect tumor spheres, and digested into single cells by 0.25% pancreatin. 1 × 10³ cells were collected for secondary pellet culture. Tumor spheres with a diameter larger than 75 µm were counted under an invert microscopy and sphere colony formation efficiency (SFE) was evaluated according to the following formula: (numbers of colonies/numbers of cells inoculated) × 100%.