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 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 naïve 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 tract^5,6. In treated patients, relapse by resistant tumors within a few years is common, even though conventional chemotherapy induces high remission rates at short term^7,8. 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.

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 according to standard diagnostic criteria for flow cytometry (CD5+, CD23−, CD200+, monoclonal B cell population), and subsequently confirmed by presence of the chromosomal translocation t(11;14) and overexpression of cyclin D1. In all cases, monoclonal B cells constituted to >90% of the total B cell population. The remaining cells were mainly monocytes and some T cells.

1. Dilute (1:1) 5 mL of MCL blood sample with 5 mL of Roswell Park Memorial Institute medium 1640 (RPMI) (Figure 1A).
2. Invert the density gradient media (Table of Materials) several times before use to ensure thorough mixing.
3. Add 5 mL of density gradient media into a 15 mL centrifuge tube using a pipette aspirator.
4. Gently layer the diluted blood sample over the density gradient media in the 15 mL centrifuge tube using an aspirator (the blood to density gradient media ratio should be 2:1, e.g., 10 mL of blood to 5 mL of density gradient media). Care should be taken to not mix the two layers (Figure 1B, C).
5. Centrifuge the sample at 400 x g for 40 min at room temperature with the centrifuge brakes turned off.
6. Using a sterile Pasteur pipette, gently aspirate the middle layer (whitish, thin ring marked with an arrow (Figure 1D, E)) containing the mononuclear cells and transfer them into a clean tube.
7. Washing of mononuclear cells
   1. Estimate the volume of the transferred cell suspension and add to it 3 volumes of sterile 1x phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA).
   2. Mix the suspension by pipetting up and down several times.
   3. Centrifuge the suspension at 400 - 500 x g for 10 - 15 min at room temperature.
   4. Discard the supernatant to obtain the cell pellet and repeat the wash step again.
   5. Remove the supernatant and resuspend the cells in the desired volume, e.g., 1 or 2 mL of PBS.
   6. Count the number of cells by an automated cell counter or manually using a hemocytometer (Neubauer chamber).

2. Enrichment of B Cells by B Cell Negative Selection

1. Dilute the cells in PBS to a final concentration of 50 x 10⁶ cells/mL.
2. Transfer the cells to a 5 mL round bottom polystyrene tube. Add 100 µL of antibody cocktail from the B cell enrichment kit to 2 mL of cell suspension, and mix gently by pipetting up and down (Figure 2-1).
3. Incubate the cells at room temperature for 10 min.
4. Briefly vortex the magnetic particles provided in the B cell enrichment kit for 30 s (Figure 2-2). Add 150 µL of the magnetic particles to 2 mL of the sample .
5. Incubate the sample for another 5 min at room temperature.
6. Bring the sample volume to 2.5 mL with 1x PBS containing 2% fetal calf serum (FCS) and 1 mM ethylenediaminetetraacetic acid (EDTA). Place the tube in the magnet and incubate the tube for another 3 - 5 min (Figure 2-3).
7. Pick up the magnet with the tube in place, and in one continuous motion, decant the tube so that the enriched cell suspension is collected in a fresh tube (Figure 2-4). Count the cells by an automated cell counter or manually using a hemocytometer to obtain the total yield of B cells.
   NOTE: Typically, the yield is ~10⁵ B cells/mL for normal B cells and 5 - 50 x 10⁶ cells/mL for MCL.
8. Confirming the purity of the B cell enrichment
   1. Take ~ 50,000 cells and incubate the cells with anti-CD19/CD20 antibodies (1:20 dilution) and anti-CD45 antibody coupled to different fluorochromes (1:10 dilution) at room temperature for 15 - 30 min.
   2. Wash with 1 mL of 1x PBS + 1% BSA, centrifuge the cells at 400 - 500 x g for 5 min, and resuspend the cells in ~200 µL of PBS.
   3. Acquire the cell samples by flow cytometry and analyze for the purity of B cells. Purity is mostly >90% by this method (Figure 3).

3. Development of the Xenograft Mouse Model

1. Suspend ~40 - 60 x 10⁶ cells in 150 - 200 µL of 1x PBS (volume required to inject per mouse).
2. Inject 150 µL of the cell suspension intravenously (i.v.) into each tail vein of 6 - 7 week old immunodeficient NOD scid gamma (NSG) mice irrespective of the gender (Figure 4A).
3. Allow the mice to develop human lymphoma for ~10 weeks.
   NOTE: Primary human lymphoma cells take much longer to develop tumor compared to human lymphoma cell lines (~3 weeks).
4. When the animals start to show symptoms of fatal illness like weight loss, ruffled hair, decreased activity, hind limb paralysis, etc., sacrifice the mice by CO₂ asphyxiation followed by cardiac heart puncture to obtain blood.
5. Collect different organs like bone marrow, spleen, and liver by dissecting the mice and collect peripheral blood by drawing blood from the heart at the time of sacrifice for tumor engraftment analysis (Figure 4B - F).

4. Chimerism Analysis of Tumor Cell Engrafted in Different Organs

1. After collecting the different organs, process them by mechanical disruption to generate single cell suspensions following red blood cell (RBC) lysis using 200 µL of 1x ammonium chloride potassium buffer for blood and bone marrow, 500 µL for spleen, and 2 mL for liver.
2. Count and stain the cells using human B cell specific antibodies, i.e., anti-CD19, anti-CD20, and anti-CD45.
   NOTE: NSG mice lack B cells and hence for analysis, cells are stained with human specific B cell markers.
3. Analyze the cells derived from each organ by flow cytometry by gating on B cells (CD19+, CD20+, CD45+).
4. Quantify the engrafted tumor cells in different organs derived from MCL injected mice based on the gated cells as indicated in step 4.3 (Figure 4G) .
   NOTE: See Table of Materials for details.

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 )