Name: a human peripheral blood mononuclear cell (pbmc) engrafted humanized xenograft model for translational immuno-oncology (i-o) research
Uploaded: 2019-08-15T14:00:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research at JoVE.com

We thank members of our laboratories for helpful discussions. This work was partially supported by the Biomedical and Life Science Innovation and Cultivation Research Program of the Beijing Municipal Science and Technology Commission under Grant Agreement No. Z151100003915070 (project &#34;Preclinical study on a novel immune oncology anti-tumor drug BGB-A317&#34;), and it was also partially supported by internal company funding for preclinical research.

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

<ol>
	<li>Myeloablation of NOD/SCID mice using cyclophosphamide: determination of optimal doses
	<ol>
		<li>Purchase female NOD/SCID mice (6-8 weeks). 
		NOTE: All mice involved in this study were female.</li>
		<li>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.</li>
		<li>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:&#160; 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&#160;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.</li>
		<li>Collect blood samples from the orbital venous sinus and transfer to 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).</li>
		<li>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) &#160;for gating CD11b+ Ly6Ghigh as neutrophils, CD11b+Ly6Chigh as monocytes11,12.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Human PBMC transplantation and tumor engraftment: model set-up
	<ol>
		<li>Isolate human PBMCs from healthy donors by density gradient centrifugation according to the manufacturer&#8217;s instructions.</li>
		<li>Pre-treat the mice with CP and DS as indicated by step 1.1.2 and 1.1.3 to increase transplantation efficiency.</li>
		<li>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 &#956;L phosphate-buffered saline (PBS) containing 50% Matrigel), or tumor fragments (3 x 3 x 3 mm3, in a total volume of 200 &#956;L PBS containing 50% Matrigel) and 200 &#956;L of 5 x 106 PBMCs (100 &#956;L each to the left and right side of engrafted tumor fragment) (s.c.) subcutaneously in the right flank of the animals.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

2. PBMC Donor Screen

<ol>
	<li>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.</li>
	<li>Monitor tumor volume twice a week by measuring with a caliper. 
	NOTE: Tumor growth rate may vary with PBMC from different donors.</li>
	<li>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.</li>
	<li>Select PBMC donors that result in moderate tumor growth (tumor volume &#62; 200 mm3 14 days post inoculation) and relatively high PD-1, PD-L1 and CD8 expressions (mean IHC scores &#62; 2). See step 4 for detailed IHC scoring protocol.</li>
</ol>

3. Human Cancer Cell Line and PDX Screen

<ol>
	<li>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.</li>
</ol>

4. Immunohistochemistry (IHC)

<ol>
	<li>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 &#956;m and place them on polylysine-coated slides.</li>
	<li>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.</li>
	<li>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 &#176;C for 30 min and then cool down to room temperature. Rinse by deionized H2O three times and aspirate excess liquid from the slides.</li>
	<li>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 &#176;C overnight, and HRP conjugated 2nd antibodies at RT for 1 h. Drop the substrate DAB (3,3&#39;-diaminobenzidine) onto the slides and control the reaction time (seconds to minutes) by monitoring the brown color from microscope.</li>
	<li>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.</li>
</ol>

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

<ol>
	<li>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.</li>
	<li>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 &#956;L 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.</li>
	<li>For PDXs engraftment, inject subcutaneously tumor fragments (3 x 3 x 3 mm3) in the right front flank of animals. Inject subcutaneously 200 &#956;L of 5 x 106 PBMCs (100 &#956;L 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.</li>
	<li>On the day of cell inoculation, randomly group the animals and treat as the planned study protocol. &#160;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.</li>
	<li>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.</li>
	<li>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 &#956;g/mL) in RPMI1640 plus 5% fetal bovine serum (FBS) for 30 min at 37 &#176;C. Pass the digested tissues through 40 &#956;m cell strainers to obtain single cell suspensions.</li>
	<li>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 &#956;g/mL human IgG for 30 min, followed by staining with anti-human CD3 (HIT3a), CD8 (OKT8) and PD-1 (MIH4) antibodies at 4 &#176;C for 30 min. Then subject the stained samples to flow cytometry and analyze using guavaSoft 3.1.1.&#160;</li>
</ol>

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All authors have ownership interest in BeiGene. Tong Zhang and Kang Li are inventors on a patent covering BGB-A317 described in this study.

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 surrogate anti-mouse target antibodies13,14. However, inherent differences between human and murine immune systems and the lack of some human targets in murine models limit preclinical studies of I-O anti-tumor effects15,16. Therefore, robust mouse models that include both human immune cells and human tumors are urgently desired, which will significantly improve the translation and development of novel I-O therapeutics.
Here, we describe a human PBMC-based xenograft mouse model that could potentially be widely used for translational I-O studies. PBMC donor as well as cancer cell line/PDX screens are critical to ensure robustness and reproducibility of in vivo efficacy studies. PBMC donors were screened in vivo to ensure successful human tumor and immune cell engraftment. Meanwhile, over 50 cell lines and PDXs of various human cancer origins were screened to evaluate tumor growth rate, human PD-L1 expression, and immune cell infiltrations. Our analyses suggest that about 20% of cancer cell lines and PDXs examined demonstrate acceptable tumor growth rate while at the same time having relatively high TILs and PD-L1 staining, which are considered good models for I-O efficacy evaluations.
HLA matching is routinely used in the clinic to match patients and donors for organ or marrow transplants17. The authors, however, have only performed limited characterization on HLA typing, and this remains an interesting topic to be investigated in future studies. The authors would like to note that a PBMC donor might be suitable for one cancer cell line/ PDX but not ideal for others. Therefore, PBMC donors might need to be screened for each cancer model to ensure optimal results.
Engraftment of human PBMC into NOD/SCID or NSG mice invariably leads to a xenogeneic graft-vs-host disease (xGvHD), a post-transplant disorder that results from immune-mediated attack of recipient tissue by donor T cells18,19. Clinical observations commonly associated with xGVHD have been observed in our humanized model, such as erythema, hunched posture, weight loss and mortality (data not shown). These phenotypes were usually observed towards the end of our studies, usually at 1-2 months post engraftment, indicating the propagation and infiltration of human T cells in xGVHD target organs. This allows a 1-2 months window for therapeutic I-O therapy evaluations. Several approaches have been utilized to decrease mouse innate immunity and enhance human immune cell engraftment20,21. For example, NSG mice defective in murine MHC Class I and Class II expression support engraftment of functional human T cells in the absence of acute xGvHD following injection of PBMC22.
NOD/SCID mice were used in this protocol. Mice homozygous for the SCID mutation have impaired T and B cell lymphocyte development and the NOD background additionally results in deficient natural killer (NK) cell function20. Other more highly immunodeficient mice, such as the NSG (The Jackson Laboratory), NCG (Charles River) and NOG (CIEA) strains, have been established. When engrafted with PBMCs, these mice have been shown develop human immune cells and form an environment that resembles the human immune system23,24. Alternatively, these mice could be engrafted with CD34+ human hematopoietic progenitor cells (HPCs) and display more sustained T cell differentiation and maturation25. In addition, next generation immunodeficient mouse models with further genetic modifications have been established to support better human myeloid lineage development and increased engraftment efficiency (refer to the variants portfolio webpage of The Jackson Laboratory and Taconic Biosciences).
More details of using these new strains for human immunity reconstitution are pending further investigations. Nevertheless, the protocol outlined in this article could serve as a general guideline of establishing and characterizing immunodeficient mouse models reconstituted with human PBMC. Three human cancer cell lines and two human patient-derived xenografts, covering a range of cancer types, are demonstrated in this article, suggesting the potential broad applications of our protocol in translational I-O studies. Most humanized PBMC models, to our knowledge, have chosen IV or IP as the route of injection26,27. Our models instead provide partially reconstituted human immunity in human tumor bearing mice through subcutaneously admixing of human PBMC with cancer xenografts. This approach provides a rapid and cost-effective, yet highly reproducible alternative to full stem cell reconstitution (e.g., CD34+ hematopoietic stem cell-engrafted humanized mice). Our model has been proved to be useful for evaluating T cell-engaging cancer immunotherapies, particularly when working on short timelines or to select agents before moving to a more complex multi-lineage immunity model.

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 cancers1,2. Contrary to targeted therapies that directly kill &#160;cancer cells, I-O therapies potentiate the body&#8217;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 liver7,8. 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.

<table><tbody><tr><td>&lt;strong&gt;PBMC separation /cell culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Histopaque-1077</td><td>Sigma</td><td>10771</td><td>Cell isolation</td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CVR</td><td>Cell culture</td></tr><tr><td>DPBS</td><td>Corning</td><td>21-031-CVR</td><td>Cell culture</td></tr><tr><td>FBS</td><td>Corning</td><td>35-076-CV</td><td>Cell culture</td></tr><tr><td>Penicillin-Streptomycin, Liquid</td><td>Gibco</td><td>15140-163</td><td>Cell culture</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Gibco</td><td>25200-114</td><td>Cell culture</td></tr><tr><td>Matrigel</td><td>Corning</td><td>356237</td><td>CDX inoculation</td></tr><tr><td>&lt;strong&gt;FACS analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma</td><td>DN25</td><td>Sample preparation</td></tr><tr><td>Collagenase Type I</td><td>Sigma</td><td>C0130</td><td>Sample preparation</td></tr><tr><td>Anti-mouse/human CD11b (M1/70) antibody</td><td>BioLegend</td><td>101206</td><td>FACS</td></tr><tr><td>Anti-mouse Ly-6C (HK1.4) antibody</td><td>BioLegend</td><td>128008</td><td>FACS</td></tr><tr><td>Anti-mouse Ly-6G (1A8) antibody</td><td>BioLegend</td><td>127614</td><td>FACS</td></tr><tr><td>Anti-human CD8 (OKT8) antibody</td><td>Sungene Biotech</td><td>H10082-11H</td><td>FACS</td></tr><tr><td>Anti-human CD279 (MIH4) antibody</td><td>eBioscience</td><td>12-9969-42</td><td>FACS</td></tr><tr><td>Anti-human CD3 (HIT3a) antibody</td><td>4A Biotech</td><td>--</td><td>FACS</td></tr><tr><td>Guava easyCyte 8HT Benchtop Flow Cytometer</td><td>Millipore</td><td>0500-4008</td><td>FACS</td></tr><tr><td>&lt;strong&gt;Tumor/PDX implantation /dosing / measurement&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cyclophosphamide</td><td>J&amp;amp;K</td><td>Cat#419656, CAS#6055-19-2</td><td>In vivo efficacy</td></tr><tr><td>Disulfiram</td><td>J&amp;amp;K</td><td>Cat#591123, CAS#97-77-8</td><td>In vivo efficacy</td></tr><tr><td>Syringe</td><td>BD</td><td>300841</td><td>CDX inoculation</td></tr><tr><td>Hypodermic needles (14 G)</td><td>Shanghai SA Mediciall &amp;amp; Plastic Instruments Co., Ltd.</td><td>0.7*32 TW SB</td><td>PDX inoculation</td></tr><tr><td>Vernier Caliper (MarCal)</td><td>Mahr</td><td>16ER</td><td>Tumor measurement</td></tr><tr><td>IVC individual ventilated cages</td><td>Lingyunboji Ltd.</td><td>IVC-128</td><td>Animal facility</td></tr><tr><td>&lt;strong&gt;IHC&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Leica ASP200 Vacuum tissue processor</td><td>Leica</td><td>ASP200</td><td>IHC</td></tr><tr><td>Leica RM2235 Manual Rotary Microtome for Routine Sectioning</td><td>Leica</td><td>RM2235</td><td>IHC</td></tr><tr><td>Leica EG1150 H Heated Paraffin Embedding Module</td><td>Leica</td><td>EG1150 H</td><td>IHC</td></tr><tr><td>Ariol-Clinical IHC and FISH Scanner</td><td>Leica</td><td>Ariol</td><td>IHC</td></tr><tr><td>Anti-human CD8 (EP334) antibody</td><td>ZSGB-Bio</td><td>ZA-0508</td><td>IHC</td></tr><tr><td>Anti-human PD1 [NAT105] antibody</td><td>Abcam</td><td>ab52587</td><td>IHC</td></tr><tr><td>Anti-human PD-L1 (E1L3N) antibody</td><td>Cell Signaling Technology</td><td>13684S</td><td>IHC</td></tr><tr><td>Polink-2 plus Polymer HRP Detection System</td><td>ZSGB-Bio</td><td>PV-9001/9002</td><td>IHC</td></tr></tbody></table>

a human peripheral blood mononuclear cell (pbmc) engrafted humanized xenograft model for translational immuno-oncology (i-o) research

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) &#8212; 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.

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, human PBMC and tumor transplantation was performed. Presence of human immune cell infiltrates in the tumor microenvironment was verified by IHC (Figure 2).
A panel of PBMC donors were screened in vivo to ensure relatively high immune cell infiltrations in the tumor microenvironment and acceptable tumor growth rate (section 2, Figure 3A). Meanwhile, over 30 human cancer cell lines as well as over 20 PDXs of different cancer types were screened to evaluate tumor growth rate, tumor PD-L1 expression and immune cell infiltrations (section 3). Representative results were shown in Figure 3B.
These PBMC-engrafted humanized mice were then used to examine the anti-tumor activity of BGB-A317. Human PBMCs from selected healthy donors were co-injected with human tumor cells (A431, SKOV3 and SK-MES-1) or primary tumor tissue fragments derived from cancer patients (BCCO-028 and BCLU-054) subcutaneously. The mice were treated, as indicated in Figure 4, with BGB-A317 or PBS intraperitoneally once a week from the day of tumor implantation. In all abovementioned models, BGB-A317 demonstrated significant anti-tumor activities (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59679/59679fig1.jpg" /> 
Figure 1: Myeloablation of NOD/SCID mice using cyclophosphamide (CP) and disulfiram (DS). (A) Gating strategy used to identify myeloid cell subsets including neutrophils and monocytes. (B) Representative results of myeloid cell (CD11b+), neutrophil (CD11b+Ly6G+) and monocyte (CD11b+Ly6C+) numbers upon different dosages of CP treatment. The boxes represent the 75th, 50th and 25th percentile of the values. The top and bottom lines represent maximum and minimal data points within the 1.5x IQ (inter quarter) range, respectively. n = 3 for the vehicle group and n = 6 for CP and/or DS treated groups. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59679/59679fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59679/59679fig2.jpg" /> 
Figure 2: Human PBMC transplantation and tumor engraftment. (A) Schematic diagram showing the general workflow of PBMC-based humanized xenograft model. (B) Tumor growth of A431 cells upon subcutaneous co-injection with donor PBMC with the indicated conditions (data represents mean tumor volume &#177; SEM, n = 6). (C) IHC analysis of tumors developed in mice treated with or without CP+DS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59679/59679fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59679/59679fig3.jpg" /> 
Figure 3: PBMC donor and human cancer cell line screen. (A) Representative summary data from PBMC donor screen. PBMC were mixed with A431 cells and inoculated subcutaneously in humanized NOD/SCID mice (see step 2). Each dot represents the mean data value of 3 mice engrafted with PBMCs from 1 donor. (B) Representative results from human cancer cell line screen. &#160;PBMC from selected donors were co-injected with A431, SK-MES-1 or SKOV3 cells. Data represents mean tumor volume &#177; SEM collected from 3 mice, 14 day post inoculation of the indicated cell lines. Mean IHC score &#177; SEM represents the average expression of human CD8, PD-1, and PD-L1 of all 3 mice. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59679/59679fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59679/59679fig4.jpg" /> 
Figure 4: Anti-tumor activities and pharmacodynamics analysis of BGB-A317 in PBMC-based humanized xenograft model. The anti-tumor activity of BGB-A317 at indicated doses (i.p., QW) was assessed using human cancer cell lines (A) A431 (with 5 x 106&#160;PBMC), (C) SKOV3 (with 5 x 106&#160;PBMC), (D) SK-MES-1 (with 5 x 106&#160;PBMC), and patient-derived xenografts (PDXs) (E) BCLU-054 (with 5 x 106&#160;PBMC) and (F) BCCO-028 (with 5 x 106&#160;PBMC). PBMC from selected healthy donors and corresponding tumor cells were co-injected subcutaneously into humanized NOD/SCID mice (n = 8 to 10). (B) Quantification of tumor-infiltrating hCD8+ and hCD8+hPD-1+ cells in BGB-A317 treated A431 tumors. n = 8-10 animals per group in A, and C to F, n = 4-6 animals per group in B; data represents mean &#177; SEM. The significance was evaluated using a two-tailed unpaired Student&#39;s t-test under the assumption of unequal variance. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59679/59679fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research at JoVE.com

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

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; 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.

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

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

a-human-peripheral-blood-mononuclear-cell-pbmc-engrafted-humanized

Nanyang Technological University

Research

JoVE Journal

Cancer Research

14.2K Views.  BeiGene (Beijing) Co., Ltd.. Ons protocol kan dienen als een algemene richtlijn voor het vaststellen van muismodellen gereconstitueerd met menselijke PBMC en tumoren voor immuno-oncologie onderzoek. Deze procedures bieden een kosteneffectieve en zeer reproduceerbare aanpak voor het gedeeltelijk reconstrueren van menselijke immuniteit bij menselijke tumordragende muizen door onderhuids en mengen van menselijke PBMC met kanker xenografts. Het demonstreren van de procedures met mij zal Xinxin Cui, Yanjuan Zhang, Huichen Bai...

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

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Immunology and Infection

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

Creation and Maintenance of a Living Biobank - How We Do It

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires a platform for preclinical research. Over recent years, we have established a biobank of colorectal and pancreatic cancers comprising of primary tumor tissue, normal tissue, sera, isolated peripheral blood lymphocytes (PBL), patient-derived xenografts (PDX), as well as primary and secondary cancer cell lines. Since original tumor tissue is limited and the establishment rate of primary cancer cell lines is still relatively low, PDX allow not only the preservation and extension of the biobank but also the generation of secondary cancer cell lines. Moreover, PDX-models have been proven to be the ideal in vivo model for preclinical drug testing. However, biobanking requires careful preparation, strict guidelines and a well attuned infrastructure. Colectomy, duodenopancreatectomy or resected metastases specimens are collected immediately after resection and transferred to the pathology department. Respecting priority of an unbiased histopathological report, at the discretion of the attending pathologist who carries out the dissections, small tumor pieces and non-tumor tissue are harvested. 
Necrotic parts are discarded and the remaining tumor tissue is cut into small, identical cubes and cryopreserved for later use. Additionally, a small portion of the tumor is minced and strained for primary cancer cell culture. Additionally, blood samples drawn from the patient pre- and postoperatively, are processed to obtain serum and PBLs. For PDX engraftment, the cryopreserved specimens are defrosted and implanted subcutaneously into the flanks of immunodeficient mice. The resulting PDX closely recapitulate the histology of the "donor" tumors and can be either used for subsequent xenografting or cryopreserved for later use. In the following work, we describe the individual steps of creation, maintenance and administration of a large biobank of colorectal and pancreatic cancer. Moreover, we highlight the crucial details and caveats associated with biobanking.

In the following work, we describe the consecutive steps necessary for the establishment of a large biobank of colorectal and pancreatic cancer.

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Medicine

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, &#34;humanized&#34;) mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2R&#947;C-/- NODSIRP&#945;) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; &#34;hot&#34; tumors exhibit large immune infiltration while &#34;cold&#34; tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the tumor.

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

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

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