Ammonium chloride

The #3 and/or #4 mammary glands (lymph node removed) from female virgin mice were manually minced for 1 min and then enzymatically dissociated for 1.5 h in DMEM/F12 (1:1) supplemented with 2 mg/ml collagenase (Roche) and 200 U/ml hyaluronidase (Sigma). Samples were briefly vortexed every 30 min. The mammary glands were then processed to single cells as previously described⁵² . Briefly, after dissociation red blood cells were lysed in ammonium chloride (Stem Cell Technologies) and processed to a single cell suspension by sequential digestion with 0.25% Trypsin (Stem Cell Technologies), 5 mg/ml dispase (Stem Cell Technologies) and 1 mg/ml DNase (Sigma) and filtered through a cell strainer (BD Biosciences).

11 primary endometrial and ovarian tumor cell cultures were established from tumors of patients undergoing surgery at the Division of Gynecologic Oncology, UZ Leuven (Belgium). Tissue was washed with PBS supplemented with penicillin/streptomycin and fungizone, digested with collagenases type IV (1 mg/ml; Roche, Vilvoorde, Belgium) and DNAse I (0.1 mg/ml; Roche) in RPMI+ medium. Single cell suspensions were prepared by filtration through a 70-µm filter. Red blood cells were lysed using ammonium chloride (Stem Cell Technologies, Grenoble, France). Single cells were plated into a 25-cm (Parsons et al., 2012 (link)) culture flask. After 1–3 weeks, when cells reached 60–70% confluency, fibroblasts were removed using mouse anti-human CD90 (Clone AS02; Dianova, Hamburg, Germany) bound to Mouse Pan IgG Dynabeads (Life Technologies, Erembodegem, Belgium). Cell cultures were subsequently passaged at 70–90% confluency and banked at −80°C. Primary tumor cell cultures were grown in RPMI Medium 1640 supplemented with 20% fetal bovine serum (FBS), 2 mM L-Glutamine, 100U/ml penicillin, 100 μg/ml streptomycin, 1 μg/ml fungizone, and 10 μg/ml gentamicin (Life Technologies) up to 25 passages.

Subsequently, 6–12 weeks old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and housed in the Animal Resource Centre at UHN. Mice were sublethally irradiated (2.5 Gy) one day prior to intravenous injection of 2–3 × 10⁶ AML blasts obtained from patients. On day 17 and day 20, mice were injected with PBS or 20 × 10⁶ Ide-DNT or ctrl-DNTs with 10,000 IU of IL-2 intravenously and followed by weekly 10,000 IU of IL-2 intraperitoneal injections. Mice body weight was measured twice a week. On day 24–100, mice were euthanized and the bone marrow (BM), spleen, liver, and lung were harvested. For the liver and lung, small tissue sections were fixed in 10% formalin and sent for hematoxylin and eosin (H&E) staining. The rest of tissues were digested with collagenase D at 37 ℃ for 30 min, filtered through 40 µm cell strainer, and underwent density gradient centrifugation at 1200× g for 20 min. The buffy coats were collected to check for the frequency and number of DNTs and AML using flow cytometry, as described previously. To longitudinally monitor the DNT level in mice periphery, 75–100 µL of blood was collected from lateral saphenous vein weekly, and red blood cells were lysed using ammonium chloride (StemCell Technologies). Subsequently, DNT and AML levels were assessed by flow cytometry.

Mice were analyzed between 4–12 weeks after birth, unless otherwise specified. RBCs were lysed with ammonium chloride (STEMCELL Technologies). Trypan blue (Amresco)–negative cells were counted as live cells.

MDA-MB-231Luc2+GFP+ were harvested from the hind limbs of tumour-positive mice. Tibia and femurs were dissected, centrifuged to harvest bone marrow and crushed using a pestle and mortar. The single cell suspension of bone and bone marrow underwent red blood lysis using ammonium chloride (STEM cell technologies, Vancouver, BC, Canada). Lysed samples were stained with TOPRO-3 (Thermo Fisher, Waltham, MA, USA) to determine live cells. GFP+ tumour cells were identified using the FACSMelody (BD).
Once GFP+ tumour cells were isolated from vehicle and treated animals, cells were lysed according to the Human MAPK phosphokinase array instructions (Abcam, Cambridge, UK). Protein concentrations were measured using a BCA assay and protein blots conducted as per supplier’s instructions (abcam- ab211061). Blots were imaged using the Bio-Rad Gel Doc system and software (Image Lab version 6.0.1) to determine band intensities.

Peripheral blood samples were collected from the tail vein or by heart puncture. For the isolation of haematopoietic organs, the mice were euthanized by CO₂ exposure. Unless otherwise indicated, the mice were killed two months post treatment. Bone marrow was isolated from the femur and tibia of the hind legs. All isolated organs were directly processed for analysis, frozen for cryopreservation or fixed in neutral buffered formalin (Sigma-Aldrich) for subsequent paraffin embedding. HPCs were isolated from BM using an EasySep mouse hematopoietic progenitor cell isolation kit (STEMCELL Technologies) according to the manufacturer’s instructions. If required, RBCs were lysed for 10 min in ammonium chloride (STEMCELL Technologies) and washed twice before downstream analysis.