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

Summary

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.

Abstract

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.

Introduction

Lymphocytes by nature play a major role in immune surveillance, and lymphocyte trafficking is a critical step in mounting antigen specific immunity^1,2. This process includes migration of naïve T lymphocytes from the thymus to the blood stream, and from there to secondary lymphoid organs, including lymph nodes, Peyer's patches, or spleen, where they meet cognate antigens. The B lymphocytes differentiate in the bone marrow and migrate as naïve cells into follicles of secondary lymphoid organs³. Some of these B cells bind antigen with their receptor and are activated by speci....

Protocol

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

NOTE: 3 - 5 mL of peripheral blood was obtained from patients presenting with MCL in a leukemic phase. The diagnosis was established accordi....

Results

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's blood by density gradient separation. The mononuclear cells are further processed to obtain pure B cells using a nega.......

Discussion

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.......

Materials

List of materials used in this article

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