A Technique to Generate a Human PBMC-Engrafted Humanized Xenograft Mouse Model

Abstract

Source: Li, Z., et al. A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research. J. Vis. Exp. (2019)

This video demonstrates an assay to generate a humanized xenograft mouse model. An immunocompromised mouse is injected with cyclophosphamide to cause immune cell depletion. Patient-derived tumor cells mixed with human peripheral blood mononuclear cells (PBMCs) in a hydrogel are subcutaneously injected into the mouse, forming a tumor with infiltrating T cells.

Protocol

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

1. Myeloablation of NOD/SCID mice using cyclophosphamide: determination of optimal doses​
   1. Purchase female nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice (6-8 weeks).
      NOTE: All mice involved in this study were female.
   2. 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.
   3. 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.
   4. 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 2^nd dose) and day 4 (72 h after the 2^nd dose).
   5. 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⁺Ly6C^high as neutrophils, CD11b⁺Ly6C^high as monocytes.
   6. 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.
2. Human PBMC transplantation and tumor engraftment: model set-up​
   1. Isolate human PBMCs from healthy donors by density gradient centrifugation according to the manufacturer’s instructions.
   2. Pre-treat the mice with CP and DS as indicated by step 1.1.2 and 1.1.3 to increase transplantation efficiency.
   3. 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.
   4. 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.
   5. 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.

Materials

PBMC separation /cell culture
Histopaque-1077	Sigma	10771	Cell isolation
DMEM	Corning	10-013-CVR	Cell culture
DPBS	Corning	21-031-CVR	Cell culture
FBS	Corning	35-076-CV	Cell culture
Penicillin-Streptomycin, Liquid	Gibco	15140-163	Cell culture
Trypsin-EDTA (0.25%), phenol red	Gibco	25200-114	Cell culture
Matrigel	Corning	356237	CDX inoculation
FACS analysis
Deoxyribonuclease I from bovine pancreas	Sigma	DN25	Sample preparation
Collagenase Type I	Sigma	C0130	Sample preparation
Anti-mouse/human CD11b (M1/70) antibody	BioLegend	101206	FACS
Anti-mouse Ly-6C (HK1.4) antibody	BioLegend	128008	FACS
Anti-mouse Ly-6G (1A8) antibody	BioLegend	127614	FACS
Anti-human CD8 (OKT8) antibody	Sungene Biotech	H10082-11H	FACS
Anti-human CD279 (MIH4) antibody	eBioscience	12-9969-42	FACS
Anti-human CD3 (HIT3a) antibody	4A Biotech	—	FACS
Guava easyCyte 8HT Benchtop Flow Cytometer	Millipore	0500-4008	FACS
Tumor/PDX implantation /dosing / measurement
Cyclophosphamide	J&K	Cat#419656, CAS#6055-19-2	In vivo efficacy
Disulfiram	J&K	Cat#591123, CAS#97-77-8	In vivo efficacy
Syringe	BD	300841	CDX inoculation
Hypodermic needles (14G)	Shanghai SA Mediciall & Plastic Instruments Co., Ltd.	0.7*32 TW SB	PDX inoculation
Vernier Caliper (MarCal)	Mahr	16ER	Tumor measurement
IVC individual ventilated cages	Lingyunboji Ltd.	IVC-128	Animal facility