A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

Podsumowanie

We describe a human peripheral blood mononuclear cell (PBMC) — based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

Streszczenie

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) — based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

Wprowadzenie

Immuno-oncology (I-O) is a rapidly expanding field of cancer treatment. Researchers have recently started to appreciate the therapeutic potential of modulating functions of the immune system to attack tumors. Immune checkpoint blockades have demonstrated encouraging activities in a variety of cancer types, including melanoma, renal cell carcinoma, head and neck, lung, bladder and prostate cancers^1,2. Contrary to targeted therapies that directly kill  cancer cells, I-O therapies potentiate the body’s immune system to attack tumors³.

To date, numerous relevant I-O animal models have been established. These include: 1) mouse tumor cell lines or tumor homograft in syngeneic mice; 2) spontaneous tumors derived from genetically engineered mouse (GEM) or carcinogen-induction; 3) chimeric GEMs with the knock-in of human drug target(s) in a functional murine immune system; and 4) mice with reconstituted human immunity transplanted with human cancer cells or patient-derived xenografts (PDXs). Each of these models have obvious advantages as well as limitations, which have been described and reviewed extensively elsewhere⁴.

Reconstitution of human immunity in immunodeficient mice have been growingly appreciated as a clinically relevant approach for translational I-O research. This is usually achieved through either 1) engraftment of adult immune cells (e.g., peripheral blood mononuclear cells (PMBC))^5,6, or 2) engraftment of hematopoietic stem cells (HSC) from, for example, umbilical cord blood or fetal liver^7,8. These humanized mice could support the growth of human tumors, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Despite the advantages, applications of humanized mice in I-O research were usually hindered by several concerns, such as long model development time and considerably high cost.

Here, we describe a human PBMC-based model that could be widely applied for translational I-O studies. This model is comparatively time- and cost-effective with high reproducibility in efficacy studies. It has been used in-house for the evaluations of several I-O therapeutics currently under preclinical and clinical development. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody⁹ , is used as the example to discuss model development, characterization, and possible applications for anti-tumor efficacy analyses.

Protokół

All procedures performed in studies involving human participants were in accordance with the ethical standards of BeiGene and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. All procedures performed in studies involving animals were approved by the Internal Review Board at BeiGene. This protocol has been specifically adjusted for the evaluation of BGB-A317 (Tislelizumab) in humanized NOD/SCID mice.

1. Establishment of human PBMC-based model

1. Myeloablation of NOD/SCID mice using cyclophosphamide: determination of optimal doses
   1. Purchase female 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 (i.p.) and DS (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 alone¹⁰ 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 EDTA-K coated tubes on ice on day 0 (1 h before the 1^st 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 FACS. Use rat anti-mouse CD11b (M1/70), rat anti-mouse Ly6C (HK1.4, ) and rat anti-mouse Ly6G (1A8)  for gating CD11b⁺ Ly6G^high as neutrophils, CD11b⁺Ly6C^high as monocytes^11,12.
   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) (s.c.) subcutaneously 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. See protocol step 4.

2. PBMC Donor Screen

1. Screen a panel of PBMC donors due to the anticipated variations resulted from PBMCs collected from individuals. Use A431 cells co-injecting with PBMCs from different donors according to the procedures as indicated by step 1.2.
   NOTE: Over 50 healthy PBMC donors were screened in the study in order to obtain enough number of suitable donors. Researchers who would like to adopt this protocol might decide on their own how many healthy PBMC donors to be screened, based on the design of the planned studies.
2. Monitor tumor volume twice a week by measuring with a caliper.
   NOTE: Tumor growth rate may vary with PBMC from different donors.
3. Collect the tumor tissues at an average volume of 200-500 mm³ and process them for histology and immunohistochemistry (IHC) analysis. Examine human CD8, PD-1 and PD-L1 expressions. See step 4 for detailed protocol.
4. Select PBMC donors that result in moderate tumor growth (tumor volume > 200 mm³ 14 days post inoculation) and relatively high PD-1, PD-L1 and CD8 expressions (mean IHC scores > 2). See step 4 for detailed IHC scoring protocol.

3. Human Cancer Cell Line and PDX Screen

1. Screen cell lines and PDXs according to the procedures stated in step 1.2, to evaluate tumor growth rate, human PD-L1 expression of the tumors and immune cell infiltrations.
   NOTE: Over 30 human cancer cell lines and over 20 PDXs of different cancer types were screened by the authors. Data of selected tumor models were shown in the results section.

4. Immunohistochemistry (IHC)

1. Harvest as indicated by step 1.2.5 and fix tumor tissues by immersing in formalin. Dehydrate and embed fixed tissues in paraffin. Section the fixed tissues at 3 μm and place them on polylysine-coated slides.
2. Deparaffinize in xylenes three times 7 min each. Hydrate the sections through graded alcohols: 100% ethanol twice for 3 min each, followed by 90%, 80% and 70% ethanol in turn for 3 min each. Rinse by deionized H₂O three times and remove excess liquid from the slides.
3. Perform antigen retrieval by placing the slides in a container and cover with 10 mM sodium citrate buffer (pH 6.0), or Tris-EDTA (pH 9.0). Heat the slides container by microwave for 3 min. Boil in a water bath at 95 °C for 30 min and then cool down to room temperature. Rinse by deionized H₂O three times and aspirate excess liquid from the slides.
4. Block the sections by 3% bovine serum albumin in PBS for 1 h and 0.3% H₂O₂ solution in PBS for 10 min. Stain by antibodies against human CD8 (EP334), PD-1 (NAT105,) and PD-L1 (E1L3N) at 4 °C overnight, and HRP conjugated 2^nd antibodies at RT for 1 h. Drop the substrate DAB (3,3'-diaminobenzidine) onto the slides and control the reaction time (seconds to minutes) by monitoring the brown color from microscope.
5. Cover the slides with neutral balsam after immersing the slides in 0.5% hydrochloric acid alcohol and 0.5% ammonia water in turn for 5 s each, then in 80%, 90% and 100% ethanol in sequence for 3 min each, and finally in xylenes using three changes for 5 min each. Detect the antibodies by observing the brown color of DAB using microscope.
   NOTE: Human CD8 and PD-1 expression on tumor-infiltrating leucocytes (TIL) were assessed by assigning an expression score on a 5-point scale (IHC score, range 0-4) at high objective magnification (20x, 40x). 0, absent; 1, weak intensity/ less than 20% cells; 2, weak-to-moderate intensity/ 20%-50% cells; 3, moderate-to-strong intensity/ 50%-80% cells; 4, strong intensity/ more than 80% cells. Human PD-L1 staining within tumor cells was scored using an adjusted scoring system on a 5-point scale (IHC score, range 0-4) because of its relatively diffused signal. 0, absent; 1, weak intensity/ less than 10% cells; 2, weak-to-moderate intensity/10%-30% cells; 3, moderate/ 30%-50% cells; 4, strong intensity/ more than 50% cells.

5. In Vivo efficacy and Pharmacodynamics Studies in Humanized PBMC-NOD/SCID Xenograft Models

1. Pre-treat NOD/SCID mice as indicated by step 1.1.3. In brief, treat the mice with 100 mg/kg CP (i.p.) and 125 mg/kg DS (p.o.) once a day for 2 days.
2. 20 to 24 h after the second dose, inject subcutaneously (s.c.) with indicated number of human cancer cells and 2.5-5 x 10⁶ PBMCs (a total of 200 μL cell mixture in 50% Matrigel) in the right front flank of animals.
   NOTE: The number of PBMC used for any individual mouse in one single study should be the same. However, due to variations in the availability of total isolated PBMC at the time of each study, the authors have chosen to use 2.5 x 10⁶, 4 x 10⁶, or 5 x 10⁶ PBMC at different studies. Although this 2-fold difference in the administered amount of PBMCs might affect the degree of humanization, the authors do not observe significant differences in evaluating anti-tumor efficacies of the tested immunotherapies.
3. For PDXs engraftment, inject subcutaneously tumor fragments (3 x 3 x 3 mm³) in the right front flank of animals. Inject subcutaneously 200 μL of 5 x 10⁶ PBMCs (100 μL each side) to the left and right of engrafted tumor fragment.
   NOTE: PDX tumor tissues were administered in a Matrigel solution, same as described for the cell line models.
4. On the day of cell inoculation, randomly group the animals and treat as the planned study protocol.  Assess the anti-tumor activity of candidate drugs, BGB-A317 (QW, i.p.) in this case, at the indicated doses in various tumor models.
   NOTE: The three human cancer cell lines (i.e., A431 (epidemoid carcinoma), SKOV3 (ovarian cancer) and SK-MES-1 (lung cancer)), as well as two PDX models (i.e., BCLU-054 (lung cancer) and BCCO-028 (colon cancer)), are considered good tumor models for I-O therapy evaluation in this humanized mouse model.
5. Measure primary tumor volume twice every week, using a caliper.
   NOTE: Gand body weights loss were observed around 4-6 weeks post PBMC engraftment in our studies, allowing a 1-2 months window for therapeutic efficacy evaluations.
6. For the pharmacodynamics analysis of tumor infiltrated immune cells, cut the tumor tissues into small pieces and digest them with collagenase type I (1 mg/mL) and DNase I (100 μg/mL) in RPMI1640 plus 5% fetal bovine serum (FBS) for 30 min at 37 °C. Pass the digested tissues through 40 μm cell strainers to obtain single cell suspensions.
7. Wash the cells and adjust cell number to a concentration of 1 x 10⁷ cells/mL in ice cold FACS Buffer (PBS, 1% FBS) in 96-well round bottom plates. Wash the cells by centrifuging and block them by adding 20 μg/mL human IgG for 30 min, followed by staining with anti-human CD3 (HIT3a), CD8 (OKT8) and PD-1 (MIH4) antibodies at 4 °C for 30 min. Then subject the stained samples to flow cytometry and analyze using guavaSoft 3.1.1. 

Wyniki

Following the procedures presented here, a PBMC-based humanized xenograft model was successfully established. In brief, CP myeloablation effects in NOD/SCID mice was determined by flow cytometry analysis of neutrophil and monocyte populations post CP and DS treatment (Figure 1). 100 mg/kg CP plus 125 mg/kg DS was determined as the optimal dose and used in later studies as the regimen results in maximum depletion of neutrophils and monocytes without causing severe toxicity to mice. Next, huma...

Dyskusje

Our knowledge of cancer development and progression has advanced significantly in recent years, with focus on a comprehensive understanding of both the tumor cells and its associated stroma. Harnessing the host immune mechanisms could induce a greater impact against cancer cells, representing a promising treatment strategy. Murine models with intact mouse immune systems, such as syngeneic and GEM models, have been widely used to study checkpoint-mediated immunity. Efficacy assessments using these models depend largely on...

Materiały

Name	Company	Catalog Number	Comments
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
IHC
Leica ASP200 Vacuum tissue processor	Leica	ASP200	IHC
Leica RM2235 Manual Rotary Microtome for Routine Sectioning	Leica	RM2235	IHC
Leica EG1150 H Heated Paraffin Embedding Module	Leica	EG1150 H	IHC
Ariol-Clinical IHC and FISH Scanner	Leica	Ariol	IHC
Anti-human CD8 (EP334) antibody	ZSGB-Bio	ZA-0508	IHC
Anti-human PD1 [NAT105] antibody	Abcam	ab52587	IHC
Anti-human PD-L1 (E1L3N) antibody	Cell Signaling Technology	13684S	IHC
Polink-2 plus Polymer HRP Detection System	ZSGB-Bio	PV-9001/9002	IHC