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
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&#8722;, 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 &#62;90% of the total B cell population. The remaining cells were mainly monocytes and some T cells.
<ol>
	<li>Dilute (1:1) 5 mL of MCL blood sample with 5 mL of Roswell Park Memorial Institute medium 1640 (RPMI) (Figure 1A).</li>
	<li>Invert the density gradient media (Table of Materials) several times before use to ensure thorough mixing.</li>
	<li>Add 5 mL of density gradient media into a 15 mL centrifuge tube using a pipette aspirator.</li>
	<li>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).</li>
	<li>Centrifuge the sample at 400 x g for 40 min at room temperature with the centrifuge brakes turned off.</li>
	<li>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.</li>
	<li>Washing of mononuclear cells
	<ol>
		<li>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).</li>
		<li>Mix the suspension by pipetting up and down several times.</li>
		<li>Centrifuge the suspension at 400 - 500 x g for 10 - 15 min at room temperature.</li>
		<li>Discard the supernatant to obtain the cell pellet and repeat the wash step again.</li>
		<li>Remove the supernatant and resuspend the cells in the desired volume, e.g., 1 or 2 mL of PBS.</li>
		<li>Count the number of cells by an automated cell counter or manually using a hemocytometer (Neubauer chamber).</li>
	</ol>
	</li>
</ol>
2. Enrichment of B Cells by B Cell Negative Selection
<ol>
	<li>Dilute the cells in PBS to a final concentration of 50 x&#160;106 cells/mL.</li>
	<li>Transfer the cells to a 5 mL round bottom polystyrene tube. Add 100 &#181;L 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).</li>
	<li>Incubate the cells at room temperature for 10 min.</li>
	<li>Briefly vortex the magnetic particles provided in the B cell enrichment kit for 30 s (Figure 2-2). Add 150 &#181;L of the magnetic particles to 2 mL of the sample .</li>
	<li>Incubate the sample for another 5 min at room temperature.</li>
	<li>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).</li>
	<li>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&#160;106 cells/mL for MCL.</li>
	<li>Confirming the purity of the B cell enrichment
	<ol>
		<li>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.</li>
		<li>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 &#181;L of PBS.</li>
		<li>Acquire the cell samples by flow cytometry and analyze for the purity of B cells. Purity is mostly &#62;90% by this method (Figure 3).</li>
	</ol>
	</li>
</ol>
3. Development of the Xenograft Mouse Model
<ol>
	<li>Suspend ~40 - 60 x 106 cells in 150 - 200 &#181;L of 1x PBS (volume required to inject per mouse).</li>
	<li>Inject 150 &#181;L 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).</li>
	<li>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).</li>
	<li>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.</li>
	<li>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).</li>
</ol>
4. Chimerism Analysis of Tumor Cell Engrafted in Different Organs
<ol>
	<li>After collecting the different organs, process them by mechanical disruption to generate single cell suspensions following red blood cell (RBC) lysis using 200 &#181;L of 1x ammonium chloride potassium buffer for blood and bone marrow, 500 &#181;L for spleen, and 2 mL for liver.</li>
	<li>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.</li>
	<li>Analyze the cells derived from each organ by flow cytometry by gating on B cells (CD19+, CD20+, CD45+).</li>
	<li>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.</li>
</ol>

<ol>
	<li>Baggiolini, M., Dewald, B., Moser, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chemokines:+an+update.">Human chemokines: an update.</a> Annu Rev Immunol. 15, 675-705 (1997).</li><li>Baggiolini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+and+leukocyte+traffic.">Chemokines and leukocyte traffic.</a> Nature. 392, 565-568 (1998).</li><li>Janeway, C. A., Travers, P., Walport, M., Shlomchik, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunobiology: The Immune System in Health and Disease. , (2001).</li><li>De Silva, N. S., Klein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+B+cells+in+germinal+centres.">Dynamics of B cells in germinal centres.</a> Nat Rev Immunol. 15 (3), 137-148 (2015).</li><li>Cohen, P. L., Kurtin, P. J., Donovan, K. A., Hanson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+and+peripheral+blood+involvement+in+mantle+cell+lymphoma.">Bone marrow and peripheral blood involvement in mantle cell lymphoma.</a> Br. J. Haematol. 101, 302-310 (1998).</li><li>Meusers, P., Hense, J., Brittinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mantle+cell+lymphoma:+diagnostic+criteria,+clinical+aspects+and+therapeutic+problems.">Mantle cell lymphoma: diagnostic criteria, clinical aspects and therapeutic problems.</a> Leukemia (Baltimore). 11 (Suppl 2), S60-S64 (1997).</li><li>Herrmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+overall+survival+in+advanced+stage+mantle+cell+lymphoma.">Improvement of overall survival in advanced stage mantle cell lymphoma.</a> J Clin Oncol. 27 (4), 511-518 (2008).</li><li>Martin, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intensive+treatment+strategies+may+not+provide+superior+outcomes+in+mantle+cell+lymphoma:+overall+survival+exceeding+7+years+with+standard+therapies.">Intensive treatment strategies may not provide superior outcomes in mantle cell lymphoma: overall survival exceeding 7 years with standard therapies.</a> Ann Oncol. 19 (7), 1327-1330 (2008).</li><li>Iyengar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+human+primary+mantle+cell+lymphoma+engraftment+in+NSG+mice.">Characteristics of human primary mantle cell lymphoma engraftment in NSG mice.</a> Br J Haematol. 173 (1), 165-169 (2016).</li><li>Klco, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+of+genetically+defined+subclones+in+acute+myeloid+leukemia.">Functional heterogeneity of genetically defined subclones in acute myeloid leukemia.</a> Cancer cell. 25 (3), 379-392 (2014).</li><li>Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+tumor+xenografts+as+predictive+preclinical+models+for+anticancer+drug+activity+in+humans:+better+than+commonly+perceived-but+they+can+be+improved.">Human tumor xenografts as predictive preclinical models for anticancer drug activity in humans: better than commonly perceived-but they can be improved.</a> Cancer Biol Ther. 2 (4 Suppl 1), S134-S139 (2003).</li><li>Johnson, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+drug+activity+in+NCI+preclinical+in+vitro+and+in+vivo+models+and+early+clinical+trials.">Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials.</a> Br. J. Cancer. 84, 1424-1431 (2001).</li><li>Scholz, C. C., Berger, D. P., Winterhalter, B. R., Henss, H., Fiebig, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+drug+response+in+patients+and+in+the+clonogenic+assay+with+solid+human+tumour+xenografts.">Correlation of drug response in patients and in the clonogenic assay with solid human tumour xenografts.</a> Eur. J. Cancer. 26, 901-905 (1990).</li><li>Do&#241;ate, C., Vijaya Kumar, A., Imhof, B. A., Matthes, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frontline+Science:+Anti-JAM-C+therapy+eliminates+tumor+engraftment+in+a+xenograft+model+of+mantle+cell+lymphoma.">Frontline Science: Anti-JAM-C therapy eliminates tumor engraftment in a xenograft model of mantle cell lymphoma.</a> J Leukoc Biol. 100 (5), 843-853 (2016).</li></ol>

The authors declare that they have no competing financial interests.

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, one of the most immune deficient mouse models to date in order to achieve greater success of lymphoma engraftment. NSG mice lack mature T cells, B cells, and NK cells. They also have impaired cytokine signaling and have defects in innate immune response.
Lymphocyte preparation from the primary MCL sample by density gradient is an important step to remove other cell types like red blood cells and platelets. Traditionally, a density gradient centrifugation allows successful isolation of lymphocytes. Further processing of these lymphocytes to enrich the B cell population is attained by the use of a B cell enrichment kit following the protocol as described within this manuscript. Care should be taken to attain a high purity of B lymphocyte populations to reach optimal engraftment of the lymphoma cells. The MCL cells are injected intravenously (at least 40 - 60 x 106 cells/mouse) through the lateral tail vein, and mice were allowed to develop lymphoma for up to 10 weeks. The mice were closely examined every day for symptoms of illness including, weight loss, ruffled hair, decreased activity, hind limb paralysis, etc. Analysis of different organs like spleen, liver, bone marrow, and blood by flow cytometry revealed MCL engraftment. Recently, it has been shown that primary MCL cells engraft in bone marrow and spleen of irradiated NSG mice at 20 weeks of injection9. Independent of this finding, we have successfully developed our own xenograft model of primary MCL with more rapid tumor formation.
This xenograft mouse model may become an extremely useful tool for the study of disease progression of different lymphoma types. Xenograft models also provide powerful tools to perform preclinical testing of candidate drugs for hematologic malignancies like MCL, however, with certain limitations for example, the dominant clone present at relapse in a patient is not necessarily the clone emerging on xenotransplantation10. This could be due to the different selective pressure that the xenograft undergoes in different host systems. The hematopoietic composition in this strain does not fully recapitulate human hematopoiesis, thus, limiting the long-term maintenance of human cells.The advantages in using human tumor xenografts overrides the limitations as results are obtained in a few weeks from a human tumor biopsy regarding response to therapy. Drug responses do not often correlate with clinical activities in patients11 when human cell lines instead of primary tumor cells are used as xenografts, but they help form the groundwork for possible therapeutic responses. The use of primary tumors as an orthotropic xenograft has a stronger predictive response value, particularly when a clinically relevant drug dosage is used11,12,13. Homing of lymphoma cells to their niches is an important pre-requisite in order to survive and to establish tumor engraftment. This process is majorly regulated via the lymphatic system, and the entry of circulating lymphocytes through the high endothelial venules maintains the lymphocyte homeostasis in lymph nodes.
Circulation of B lymphocytes through lymph nodes requires crossing endothelial barriers and chemoattractant-triggered cell migration by coordinated interaction with different adhesion molecules. Therefore, targeting the molecules that control the homing of lymphocytes to their survival niches could constitute a new treatment strategy for B cell lymphomas as they behave similar to normal lymphocytes. Our aim was to target the molecule JAM-C, a junctional adhesion molecule, which is present on MCL. This xenograft model will be used to study the therapeutic effects of JAM-C antibody on homing and engraftment of primary lymphoma cells to their survival niches. We have recently shown an effect of anti-JAM-C antibody in mice that received a MCL cell line, Jeko-114. This antibody had a blocking effect on homing of Jeko-1 cells and also when administered in regular intervals, it efficiently prevented the engraftment of Jeko-1 cells. In addition to its effect on homing, the antibody treatment eradicated the lymphoma engrafted in most of the organs tested in this mice14.
In the light of the scientific and clinical question being addressed, these xenografted tumors represent at present, an interesting model to study the therapeutic outcome of MCL.

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 negative B cell enrichment kit to obtain a pure cell population for xenograft injection into mice. Care should be taken to obtain maximum purity in order to have successful MCL engraftment. The purity obtained using this method is usually &#62;90%. The remaining cells were mainly monocytes and some T cells (data not shown).
Figure 2 represents the different steps of the B cell purification protocol as described in the methods section. The enriched cells are further analyzed for their purity by flow cytometer using different markers (CD45, CD19, CD20, CD23, CD200, CD5, kappa, and lambda). Sequential gating as shown in Figure 3 allows characterizing of MCL cells: CD45+, CD19+, CD20+, CD5+, CD23&#8722;, and CD200&#8722; are selected. Compensation for multicolor staining was carried out by using single stained controls for each of the fluorochromes used, according to standard cytometry set-up.
Figure 3E, G represents the typical dot plot of B cells before and after enrichment. In this case, &#62;95% of the enriched cells using this kit are B cells. The cells are suspended in PBS at a concentration of 40 - 60 &#215;106 cells in 150 - 200 &#181;L PBS as mentioned in the protocol. They were immediately injected i.v. to NSG mice. After 10 weeks, &#62;90% of the injected animals developed lymphoma shows signs of terminal illness (weight loss, ruffled hair, decreased activity, etc.). After sacrifice, spleen and liver are removed and processed to obtain single cell suspensions by mechanical disruption (Figure 4D, F); blood is drawn carefully by cardiac heart puncture and the bone marrow is processed by cutting both ends of the femur and flushing it using a 2 mL syringe filled with RPMI medium or PBS (Figure 4C). The cells are further processed by flow cytometry using human B cell specific markers like CD19/CD20 and CD45. We used human B cell specific markers here because the recipient mouse strain (NSG) lacks B Cells. Figure 4B-F represents the collection of organs after sacrifice and the steps of further processing. The degree of engraftment of MCL cells derived from two patients is represented in Figure 4G. The engraftment pattern in different organs and between patients can be compared following this technique.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56023/56023fig1.jpg" /> 
Figure 1: Isolation of PBMCs from whole blood. Blood drawn from a MCL patient is diluted 1:1 in RPMI medium (A), and placed carefully on top of the density gradient media layer (B) without mixing the blood into this density gradient media (C). Centrifugation at 400 x g for 45 min separates the mononuclear cells, which appear as whitish ring (arrow indicates the mononuclear cell layer) (D). This layer is pipetted gently without mixing with other layers into a clean tube for further processing (E). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56023/56023fig1large.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/56023/56023fig2.jpg" /> 
Figure 2: Enrichment of B cells from PBMCs. PBMCs isolated by density gradient centrifugation are washed twice with PBS. Using the negative B cell enrichment kit and by following the manufacturer&#39;s protocol, a pure population of B cells is obtained (1-4). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56023/56023fig2large.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/56023/56023fig3.jpg" /> 
Figure 3: FACS analysis of MCL cells and purity of B cells before and after enrichment. The mononuclear MCL cells are characterized by FACS analysis using different markers like CD45, CD19, CD20, CD23, CD200, CD5, kappa, and lambda. Cells that are positive for CD45, CD19, CD20, CD5, and negative for CD23 and CD200 are selected (A-F). G represents the cell population after enrichment using the negative selection kit compared to E, which is before enrichment. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56023/56023fig3large.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/56023/56023fig4.jpg" /> 
Figure 4: NSG mice as a xenograft model for engraftment of MCL cells. B cells derived from MCL patients were injected i.v. via the lateral tail vein of NSG mice (A). After allowing several days for the tumor to engraft, the mice were sacrificed, and dissected (B) to collect different organs like bone marrow (C), spleen (D), peripheral blood (E) (drawn by cardiac heart puncture), and liver (F). These organs were processed further and analyzed by flow cytometry for the presence of B cells. G represents the engraftment pattern of MCL cells in different organs derived from MCL injected NSG mice. Results from two patients are shown. Data are shown as mean &#177; SEM, n = 3. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56023/56023fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</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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Nanyang Technological University

Research

JoVE Journal

Cancer Research

11.1K Views.  University of Geneva. 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.

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

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

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 ...

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.

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.

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

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. ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...