Name: tumor engraftment in a xenograft mouse model of human mantle cell lymphoma
Uploaded: 2018-03-30T12:30:00
Description: Watch this Scientific Journal Video about Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma at JoVE.com

This work was supported by the Ligue Genevoise contre le Cancer, Fondation Dr. Dubois Ferriere Dinu-Lipatti, Oncosuisse KPS-OCS, OCS-02260-08-2008 and 2914-02-2012, and Swiss National Science Foundation Grant 31003A_156760 and 310030-153456.

The human blood samples were used according to procedures approved by the local ethics and human experimentation committees of the Geneva University Hospital.
Animal procedures were performed in accordance with the Institutional Ethical Committee of Animal Care in Geneva, Switzerland and the Cantonal Veterinary Office (authorization number: GE/26/15).
1. Preparation of Primary Peripheral Blood Mononuclear Cells (PBMCs) by Density Gradient Separation
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Clinical trials are possible for drugs that are in an advanced stage of development but cannot be used for drug discovery. Efforts to develop animal recipients for human cell xenotransplantation in order to test early stage drugs have long been pursued. Here we present an animal model that avoids human graft rejection and can establish a model for human diseases, such as MCL. This is at present a state of the art xenograft model to study the mechanisms of human tumor engraftment and tumor growth. Here we use NSG mice, on...

Lymphocytes by nature play a major role in immune surveillance, and lymphocyte trafficking is a critical step in mounting antigen specific immunity1,2. This process includes migration of na&#239;ve T lymphocytes from the thymus to the blood stream, and from there to secondary lymphoid organs, including lymph nodes, Peyer&#39;s patches, or spleen, where they meet cognate antigens. The B lymphocytes differentiate in the bone marrow and migrate as na&#239;ve cells into follicles of secondary lymphoid organs3. Some of these B cells bind antigen with their receptor and are activated by specific T cells. Proliferation and differentiation of these B cells pushes the non-activated, naive B cells into the mantle zone of the follicle. Activated cells can then differentiate into memory B cells, which patrol the body, or mature into immunoglobulin secreting plasma cells that migrate to the bone marrow4.
MCL occurs when na&#239;ve B lymphocytes in the mantle zone transform into a tumor. These lymphoma cells reside in the microenvironment of the lymphoid organs and proliferate independently of specific T lymphocyte control. However, at a certain stage of density they escape from this niche and recirculate in the bloodstream in search for niches in other organs. Considering the complexity of adhesion molecules and the promiscuity of chemokines and their receptors, the mechanism of this cellular trafficking in vivo is poorly understood and therefore hampers therapy. Novel methods are needed to effectively block this migration process to prevent the lymphoma B cells from reaching new microenvironments.
MCL is one of the most difficult to treat B cell malignancies. The development of a neoplastic phenotype of MCL is the result of a multistep cascade, characterized by the acquisition of unique biologic properties. At the time of diagnosis, most patients (70%) already present with a disseminated disease, with a majority of cases exhibiting extranodal involvement in spleen, bone marrow, and/or the gastrointestinal tract5,6. In treated patients, relapse by resistant tumors within a few years is common, even though conventional chemotherapy induces high remission rates at short term7,8. Here we present a new disease model that can help understand MCL dissemination and its underlying biology: we established a human MCL xenograft mouse model that originated from primary tumor cells of patients. We hope that this model will help develop therapeutic strategies against MCL dissemination, and possibly provide new clinical perspectives for optimal diagnosis and treatment of relapsed patients.

<table>
	<tbody>
		<tr>
			<td>Ficoll-paque media</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>for separation of mononuclear cells</td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>Gibco-Life technologies</td>
			<td>61870-010</td>
			<td>for dilution of blood sample</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>washing of cells</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A8412</td>
			<td>for preparation of PBS with 1% BSA used in washing cells during isolation</td>
		</tr>
		<tr>
			<td>CD19-APC700</td>
			<td>Beckman Coulter</td>
			<td>B49212</td>
			<td>human pan-B cell marker</td>
		</tr>
		<tr>
			<td>CD20-APC</td>
			<td>Beckman Coulter</td>
			<td>A21693</td>
			<td>human pan-B cell marker</td>
		</tr>
		<tr>
			<td>CD20-ECD</td>
			<td>Beckman Coulter</td>
			<td>IM3607</td>
			<td>human pan-B cell marker</td>
		</tr>
		<tr>
			<td>CD5-PC 5.5</td>
			<td>Beckman Coulter</td>
			<td>PN A70203</td>
			<td>human T cell marker</td>
		</tr>
		<tr>
			<td>CD23-PE</td>
			<td>Pharmingen</td>
			<td>555711</td>
			<td>Cell surface protein typically absent in MCL</td>
		</tr>
		<tr>
			<td>CD45 KO</td>
			<td>Beckman Coulter</td>
			<td>B36294</td>
			<td>Pan-leucocyte marker</td>
		</tr>
		<tr>
			<td>CD200-PE</td>
			<td>Pharmingen</td>
			<td>552475</td>
			<td>Cell surface protein typically absent in MCL</td>
		</tr>
		<tr>
			<td>NOD scid gamma (NSG) mice</td>
			<td>Charles River Laboratories</td>
			<td>5557</td>
			<td>used to develop MCL xenografts in this study</td>
		</tr>
		<tr>
			<td>Easy sep Human B cell enrichment kit</td>
			<td>Stem cell technologies</td>
			<td>19054</td>
			<td>used to enrich B cells to obtain pure cells for injecting into mice</td>
		</tr>
		<tr>
			<td>FACS</td>
			<td>Beckman Coulter</td>
			<td>Navios</td>
			<td>used to characterize MCL sample and to study the organs for MCL engraftment</td>
		</tr>
		<tr>
			<td>1X ammonium chloride potassium buffer</td>
			<td></td>
			<td></td>
			<td>red blood lysis buffer (NH4Cl 8,024 mg/l; KHCO3 1,001 mg/l; EDTA.Na2&#183;2H2O 3.722 mg/l )</td>
		</tr>
	</tbody>
</table>

tumor engraftment in a xenograft mouse model of human mantle cell lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

The manuscript describes an optimized protocol for the successful development of a xenograft mouse model for engraftment of MCL cells. Preparation of a pure cell population (in this case MCL cells), is very critical to develop successful MCL xenografts. Figure 1 represents the preparative steps for mononuclear cell isolation from MCL patient&#39;s blood by density gradient separation. The mononuclear cells are further processed to obtain pure B cells using a ...

Watch this Scientific Journal Video about Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma at JoVE.com

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

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