Cells and culture
Granta-519 human mantle cell lymphoma (MCL) cell line was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ) repository (Braunschweig, Germany). Cells were cultured at 0.5-2.0 × 106 cell/ml in RPMI-1640 supplemented with 10% (v/v) fetal calf serum (FCS), 2 mM L-glutamine, 100 unit/ml penicillin and 0.1 mg/ml streptomycin. Experiments with human specimens were approved by the ethics committee of the Hospital de la Princesa. Human samples were obtained from healthy donors and from patients with different B-cell neoplasms after informed consent. Human peripheral blood (PB) and bone marrow (BM) aspirates were obtained by venipuncture and sternun puncture, respectively, and peripheral blood mononuclear cells (PBMC) were separated by ficoll density gradient centrifugation.
Murine splenocytes were obtained from NOD/SCID and NSG mice by splenectomy and separated by ficoll density gradient centrifugation.
Mouse anti-human CCR7 mAb (150503 clone, IgG2a isotype) was obtained from R&D Systems (Minneapolis, MN, USA) and was resuspended in sterile water.
Alemtuzumab was obtained from the department of pharmacy at our hospital.
For flow cytometric analysis, mouse anti-human CD19 mAb (fluorescein isothiocyanate; FITC), mouse anti-human CD20 mAb (Pacific Blue; PB), mouse anti-human CCR7 mAb (Phycoerythrin; PE) and the DNA dye 7-Actinomycin-D (7-AAD) were purchased from Becton Dickinson (BD) Biosciences (San José, CA, USA).
CCR7 expression in Granta-519 cells was assessed by flow cytometry. Briefly, 1 × 106 Granta-519 cells were washed twice with cold PBS, resuspended in 100 μl cold PBS, incubated with the PE-conjugated anti-human CCR7 mAb for 15 minutes and washed with PBS. An appropriate isotype control was included in the analysis.
For staining of primary samples, 100 μl whole PB or BM samples were incubated for 15 minutes at room temperature with PE-conjugated anti-human CCR7 mAb. This incubation was followed by the lysis of red blood cells by using ammonium chloride lysing solution (BD Biosciences) following the manufacturer’s instructions. Finally, leukocytes were resuspended on 500 μl cold PBS. Data acquisition and analysis were performed on a FACSCanto II flow cytometer using the DIVA software (BD Biosciences). In all experiments, a minimum of 5000 neoplastic B cells was acquired. Results are expressed as the percentage of CCR7 positive cells and mean fluorescence intensity (MFI) of CCR7 expression.
Chemotaxis of Granta-519 cells in response to CCL19 and to CCL21 was assayed in Transwell cell culture chambers (6.5 mm diameter, 10 μm thickness, 5 μm diameter pore size, Costar, Cambridge, MA). When required, cells were preincubated for 30 min with 2 μg/ml anti-CCR7 mAb. Briefly, 5 × 105 Granta-519 cells, resuspended in 100 μl RPMI-1640 medium with 0.5% bovine serum albumin (BSA), were added to the upper compartment of the chamber, and chemokines were added to the lower well in 600 μl of the same medium at the optimal concentration (1 μg/ml for both CCL19 and CCL21). Migration was allowed to proceed for 4 h at 37°C in 5% CO2 atmosphere. Migrated cells were recovered from the lower chamber and counted by flow cytometry for 60 s after calibrating the flow rate with Trucount tubes (BD Biosciences). Events were compared with the number of cells counted in the initial suspension of cells to calculate the percentage of input (100 × number of cells migrated/number of cells counted in the initial suspension). Each experiment was performed in duplicate.
Complement-dependent cytotoxicity (CDC)
1 × 105 Granta-519 cells (50 μl) were seeded in a 96-well round-bottom plate together with 2 μg/ml of either purified anti-human CCR7 mAb or the corresponding isotype control (IC). After 30 min incubation at 37°C, the cells were centrifuged and washed. Then, baby rabbit complement (Serotec, Oxford, UK), diluted at the concentration indicated by the manufacturer (25%) in RPMI-1640 medium was added. After 1–2 h at 37°C, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD19 mAb and PB conjugated anti-CD20 mAb and with 7-AAD as a viability exclusion dye. The percentage of non-viable cells was measured and the percentage of lysis with heat-inactivated complement was used to calculate the specific lysis with the formula: Specific lysis (%) = 100 × (% dead cells with complement –% dead cells with inactivated complement) / (100–% dead cells with inactivated complement).
Antibody-dependent cell-mediated cytotoxicity (ADCC)
ADCC assays were performed using Granta-519 cell line as target cells and either human PBMC or murine splenocytes as effectors cells. The target to effector ratio was 1:10 in both cases.
Granta-519 cell were washed and resuspended at 1 × 106 cells/ml in PBS containing 5 μg/ml calcein-UV Cell Tracker (Invitrogen, OR, USA) and incubated at 37°C for 30 minutes. Cells were then washed twice and resuspended in RPMI-1640 supplemented with 10% FCS, in presence or absence of 100 μg/ml of either isotype control, anti-CCR7 mAb, or alemtuzumab for 30 minutes. Granta-519 cells were washed again and 1 × 105 cells were plated with human PBMC or murine splenocytes. After 24 hours, the cells were stained with 7-AAD and analyzed by flow cytometry (FACSCanto II, BD Biosciences). The percentage of Granta-519 cells killed by antibody-mediated cytotoxicity was calculated substracting the percentage of dead cells in the presence of control isotype mAb.
NOD/SCID and NSG female mice were housed in the animal facility of the Instituto de Investigaciones Biomédicas “Alberto Sols” and in the facilities of Vivotecnia, under standard sterile conditions in air-filtered containers, according to protocols approved by European directives and Spanish laws (European Directives 86/609/EEC/2003/65/EC Spanish Law RD 1201/2005). NSG mice were from Charles River (Barcelona, Spain). The in vivo experimental procedures were approved by the pertinent ethic committees and carried out in accordance with the guidelines of the European directives and Spanish laws.
Only those animals that met the inclusion criteria (20% of the mean body weight) were included in the study and distributed into the different experimental groups according to the body weight stratification method.
In vivo anti-tumor activity of anti-human CCR7 mAb in NOD/SCID mice
To evaluate the anti-tumor efficacy of the anti-human CCR7 mAb, NOD/SCID mice were xenografted with the Granta-519 human MCL cell line. All mice used in the experiment were females and were 8 ± 1 weeks old.
We have used two inoculation vias: (1) cells were subcutaneously injected resulting in a localized tumor and (2) cells were intravenously injected resulting over time in a disseminated lymphoma.
The subcutaneous model was developed by inoculating a group of 5 mice with 5 × 106 viable cells subcutaneously (in the right hind flank). The number of inoculated cells to establish the subcutaneous model was chosen on the basis of previous experiments to determine the number of Granta-519 cells required to develop palpable tumors in the mouse in around one week.
This subcutaneous model was used as an early-treatment model of the lymphoma and therefore the mice were intraperitoneally injected with 200 μg anti-human CCR7 mAb two days after inoculation of Granta-519 cells. This treatment was repeated on day 6 and 10. As a control group we inoculated a group of 5 mice with sterile PBS on days 2, 6 and 10.
The disseminated model involved inoculating mice intravenously (in the tail vein) with 0.5 × 106 cells. The number of Granta-519 cells inoculated in the intravenous model was chosen on the basis of previous experiments done to establish the number of Granta-519 cells that resulted in the development of visible signs of disease in a period of around 40–60 days. This model was split into two branches, a “peri-implantation” model, defined as the period in which tumor cells are circulating and not yet located in the target organs, in which mice were treated 2 days after the xenograft, and a “post-implantation” model, in which surviving tumor cells are expected to have reached their target organs. In this model mice were treated 7 days after the xenograft. The peri-implantation model included a group of 5 mice treated with 200 μg anti-human CCR7 mAb intraperitoneally on days 2, 6 and 10. A control group of 5 mice were inoculated with PBS on the same days of 2, 6 and 10. The post-implantation model involved 3 groups of mice. A group of 5 mice were inoculated with 200 μg anti-human CCR7 mAb intraperitoneally on days 7, 11 and 15. A second group of 5 mice were a control group inoculated with 200 μg of an isotype control intraperitoneally on days 7, 11 and 15. And a third group also a control group that received sterile PBS inoculated on days 7, 11 and 15. Mice were weighted every three days and checked for any signs of pathologies, discomfort or mortality, according to the OCDE Humane Endpoints Guidance Document.
The length and width of the tumors in the subcutaneous model were measured with a caliper three times per week or when deemed necessary once the tumor lenght was ≥ 4 mm. The largest (D) and shortest (d) diameters of the tumor were measured every third day and the tumor volume was calculated according to the formula: V = D * d2/2. The growth inhibitory rate (IR) was calculated by the formula: IR(%) = 100 ‒ [(V1/V2)] * 100, wherein V1 is the mean tumor volume in the mAb treated group, and V2 is the mean tumor volume in the control group. Animals bearing tumors with D ≥ 15 mm or with signs of ulceration were sacrificed for humane reasons.
The in vivo experiments in the subcutaneous model continued until two mice in a group developed tumor size with D ≥ 15 mm. This was observed on day 27 in the control group. Since the ethical protocol requires that these two mice should be sacrificed we decided to sacrifice all the mice to perform a comparative study.
Mice were euthanized and the tumors and organs (spleen, bone marrow, brain, lungs, liver, small intestine, ovaries and spine) were harvested and weighed. Organs were fixed in 4% neutral buffered formaldehyde for histochemistry analysis whereas a piece of fresh spleen and the bone marrow were conserved in PBS at 4°C for flow cytometry assays. Subcutaneous tumors were divided in two: one half was fixed in 4% neutral buffered formaldehyde for histochemistry analysis and the other half was conserved in PBS at 4°C for flow cytometry assays. In the intravenous model, mice were euthanized when they developed incipient signs of limb paralysis, approximately 6–9 weeks after the inoculation of the lymphoma cells.
In order to evaluate potential toxic effects of the anti-human CCR7 mAb, a third group of 3 mice were not inoculated with tumor cells but treated with the anti-human CCR7 mAb following the same administration regimen than that of the treated xenografted mice.
Flow cytometric cell analysis
Spleens and tumors were mechanically disaggregated. Cells were harvested and washed twice in cold PBS. Red cells were lysed using ammonium chloride solution (BD Biosciences), and then the remaining cells were washed twice with cold PBS, resuspended in binding buffer (BD Biosciences), and counted. One million cells from the spleen, bone marrow or tumors were incubated with PB anti-human CD20 mAb (clone 2H7, specific for human B cells, non cross reacting with murine lymphoid cells) in 50 μl of blocking solution (PBS, 2% BSA) for 15 minutes. The appropriate isotype control was included in the analysis. Analysis was performed on a FACSCanto II flow cytometer using the DIVA software (BD Biosciences).
Apoptosis assay (viability assays)
The Annexin-V-FITC assay (BD Biosciences) was used according to the manufacturer’s instructions to quantitatively determine the percentage of non-viable cells following exposure to anti-human CCR7 mAb. Briefly, tumor cells were harvested and resuspended in binding buffer (BD Biosciences). 100 μl of the cell suspension was stained with Annexin-V-FITC. After 10 minutes, 0.4 ml of binding buffer and 10 μl of 7-AAD were added, and the cells were analyzed immediately by flow cytometry. Early apoptotic cells were defined as Annexin-V+/7-AAD-, late apoptotic cells as Annexin-V+/7-AAD+, dead cells as Annexin-V-/7-AAD+ and viable cells as Annexin-V-/7-AAD-.
Organs and tissues extracted from mice were fixed in 4% formalin, dehydrated through grades of ethanol in a Microm STP 420D sample processor (Thermo Scientific, Kaklamazoo MI, USA), embedded in paraffin in EC 350–1 paraffin station (Myr, El Vendrell, Tarragona, Spain) and sliced in 4 μm sections. For human CD20 inmmunohistochemistry, the antigen retrieval was performed with citrate pH 6 in microwave. Slides were stained with anti-human CD20 antibody (clone L26, Roche) by using the VENTANA BenchMark ULTRA System (Roche).
Statistical significance between untreated and treated samples was assessed with Student’s t test for unpaired data (two tail). Differences were considered significant if P-values were < 0.05. Survival data were analyzed by the Kaplan-Meier method and the Tarone-Ware test was used to test for significance among all the groups.