eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;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
<ol>
	<li>Myeloablation of NOD/SCID mice using cyclophosphamide: determination of optimal doses&#8203;
	<ol>
		<li>Purchase female nonobese diabetic/severe combined immunodeficiency (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. 
		&#8203;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 (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: &#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 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.</li>
		<li>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).</li>
		<li>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.</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&#8203;
	<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) subcutaneously (s.c.) 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.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PBMC separation /cell culture</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>FACS analysis</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>Tumor/PDX implantation /dosing / measurement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclophosphamide</td>
			<td>J&#38;K</td>
			<td>Cat#419656, CAS#6055-19-2</td>
			<td>In vivo efficacy</td>
		</tr>
		<tr>
			<td>Disulfiram</td>
			<td>J&#38;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 (14G)</td>
			<td>Shanghai SA Mediciall &#38; 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>
	</tbody>
</table>

a technique to generate a human pbmc-engrafted humanized xenograft mouse model

Source: Li, Z., et al. <a href="https://app.jove.com/v/59679">A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research.</a> 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.

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

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. All procedures involving animal ...

Take an immunocompromised mouse possessing only neutrophils and monocytes as functional immune cells.
Intraperitoneally inject cyclophosphamide, a drug that targets immune progenitor cells.
The drug enters actively proliferating immune progenitor cells and crosslinks nucleic acids, causing cell death. The death of progenitor cells leads to immune cell depletion.
Orally administer disulfiram, a drug that forms an adduct with cyclophosphamide, reducing cyclophosphamide-induced side effects.
Subcutaneously inject a mixture of human tumor cells and peripheral blood mononuclear cells, or PBMCs, in a suitable gelatinous protein mixture.
The protein mixture forms a gel, mimicking the physiological extracellular matrix, and entraps the tumor cells.
Functional immune cell depletion allows the tumor cells to proliferate and form new blood vessels, transforming into a solid tumor xenograft.
Simultaneously, T cells from the injected PBMCs infiltrate the tumor.
The tumor xenograft exhibits growth and tumor-infiltrating T cells, establishing a humanized xenograft mouse model.

a-technique-to-generate-human-pbmc-engrafted-humanized-xenograft

Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

98 Views. 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, fo...

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

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 ...

JoVE Journal

Cancer Research

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 ...

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

Transcript

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

A Melanoma Patient-Derived Xenograft Model

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo