All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Establishment of human PBMC-based model
- Myeloablation of NOD/SCID mice using cyclophosphamide: determination of optimal doses
- Purchase female nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice (6-8 weeks).
NOTE: All mice involved in this study were female.
- Prepare cyclophosphamide (CP) at different doses (50, 100 and 150 mg/kg) in saline. Prepare disulfiram (DS) in 0.8% Tween-80 in saline at 125 mg/kg.
NOTE: Different concentrations of CP were prepared to enable administration of equal volumes of drug solution to mice getting different doses of CP.
- Treat the animals with CP (intraperitoneal, i.p.) and DS (peroral, p.o.) once a day for 2 days. Give DS (p.o.) 2 h after each dose of CP.
NOTE: DS decreases the urotoxicity of CP in mice, and CP combined with DS has been suggested to have longer-lasting neutropenia than animals treated with CP alone. The dose regimen of CP might need to be pre-determined prior to actual studies and was found to vary slightly between different immunodeficient mouse strains.
- Collect blood samples from the orbital venous sinus and transfer to potassium ethylenediaminetetraacetic acid (EDTA-K) coated tubes on ice on day 0 (1 h before the 1st dose), day 2 (24 h after the 2nd dose) and day 4 (72 h after the 2nd dose).
- Examine the myeloablation effect after CP and DS treatment by fluorescence-activated cell sorting (FACS). Use rat anti-mouse CD11b (M1/70), rat anti-mouse Ly6C (HK1.4) and rat anti-mouse Ly6G (1A8) for gating CD11b+Ly6Chigh as neutrophils, CD11b+Ly6Chigh as monocytes.
- Record body weight and health conditions of the mice daily for one week. The optimal dose of CP and DS is determined as the regimen that results in maximum depletion of neutrophils and monocytes without causing severe toxicity to mice.
- Human PBMC transplantation and tumor engraftment: model set-up
- Isolate human PBMCs from healthy donors by density gradient centrifugation according to the manufacturer’s instructions.
- Pre-treat the mice with CP and DS as indicated by step 1.1.2 and 1.1.3 to increase transplantation efficiency.
- 20 to 24 h after the second dose of CP and DS, inject human tumor cell line such as A431 cells (ATCC, 2.5 x 106) and 5 x 106 isolated PBMCs (mixed in a total of 200 μL phosphate-buffered saline (PBS) containing 50% Matrigel), or tumor fragments (3 x 3 x 3 mm3, in a total volume of 200 μL PBS containing 50% Matrigel) and 200 μL of 5 x 106 PBMCs (100 μL each to the left and right side of engrafted tumor fragment) subcutaneously (s.c.) in the right flank of the animals.
- Measure primary tumor volume and record twice a week for 4-6 weeks.
NOTE: The mice will be euthanized once their body weights lose over 20% or their tumor volume reaches 2,000 mm3 or the tumor is ulcerated.
- Euthanize the mice in gas chambers with carbon dioxide. Collect the whole tumor tissues in sacrificed mice with ophthalmic scissors and process them for histology and immunohistochemistry (IHC) analysis. Examine the Human CD8, PD-1 and PD-L1 expressions in these tissues.
