The chick chorioallantoic membrane as an in vivo xenograft model for Burkitt lymphoma

The Burkitt lymphoma cell lines BL2B95 were cultured in BL-medium (RPMI 1640 medium with 10% FCS, 1% penicillin/streptomycin, 10 mM HEPES, 1 mM sodium-pyruvate, 50 μM α-thioglycerol and 20 nM BCS). Cells were cultured in cell culture flasks and incubated at 37°C and 5% CO₂. BL2-GFP (BL-2 ns-c* GFP) cells were cultured in RPMI 1640 with 10% FCS and 1% penicillin/streptomycin.

A self-inactivating lentivirus was prepared by transient transfection of 293 T cells using calcium phosphate precipitation method. Briefly, pGIPZ ns-control (Thermo Scientific, Schwerte, Germany) encoding GFP and a non-silencing control shRNA (ns-c) was co-transfected with packaging vector pCMVΔr8.91 and envelope vector pVSV-G in a ratio of 3:2:1 into 293 T cells. After harvesting and determination of titer, lentivirus supernatant was added to BL2 cells at a MOI < 1 in the presence of 10 μg/ml protamine sulfate, and samples were centrifuged for 1.5 h at 850 g and 37°C. After 2 days 1 μg/ml puromycin was added to select stably transduced cells expressing GFP. The GFP expression in puromycin-resistant cells was analyzed with a flow cytometer and, when the cells were positive, they were expanded (Additional file 1: Figure S1).

Fertilized White Leghorn chick eggs were incubated at 80% relative humidity and 37.8°C. The eggs were windowed at day 3 and the window was sealed with cellotape. At day 10 of chick development, one million BL2B95 cells/egg were applied on the CAM. Cells were resuspended in 50% BL-medium and 50% Matrigel and incubated for 30 min at 37°C, 5% CO₂ before applying them on the CAM. The tumors were dissected on day 17 of chick development. Tumors were fixed in 4% paraformaldehyde for 15 min, washed thrice in PBS and transferred into 10% sucrose for 3 h at 4°C and 30% sucrose overnight at 4°C. Tumors were then embedded in tissue freezing medium and cut with a cryotome into 4-12 μm thick sections. The experiments were performed according to the guidelines of the European Parliament (2010/63/EU) and the council for the protection of animals in science (§14 TierSchVersV).

Specific pathogen free fertilized White Leghorn chick eggs were incubated for 72 h at >80% relative humidity and 37.8°C. On developmental day 3 the eggs were cracked open and the embryo was carefully transferred into a plastic square weighing boat (89×89×25mm) and cultured until day 17 of embryonic development (Additional file 2: Figure S2 A-G). The weighing boat was placed in a tissue culture flask with a re-closable lid (Additional file 2: Figure S2 H). 13 ml of purified water (0.1% copper sulfate) were added to ensure sufficient humidity. On day 10, 10⁶ BL2-GFP cells in varying percentages of Matrigel (10-50%) or without Matrigel were inoculated on the CAM (Additional file 3: Figure S3). The embryos were incubated until day 17 in the above mentioned conditions (>80% rh, 37.8°C). Pictures were taken every 24 h with Leica MZ16FA microscope. Procedures were adopted from [13]. The experiments were performed according to the guidelines of the European Parliament (2010/63/EU) and the council for the protection of animals in science (§14 TierSchVersV).

HE, panoptic Pappenheim, Trichrome and Gomori silver staining were performed according to standard procedures [19].

Specimens with an approximate volume of 600 mm³ were fixed with Karnovsky fixative for at least two hours, washed in 0.15 M phosphate buffer for 10 min, transferred into osmium tetroxide solution and incubated for 2 h at 4°C. Then the samples were rinsed with 0.15_M phosphate buffer for 10 min and subsequently dehydrated in an ascending ethanol series of 30%, 50%, 70%, 90% and two times absolute ethanol for 10 min each. Next the samples were incubated twice in 100% propylene oxide for 10 min at 4°C. They were then incubated for 1 h at 4°C in 50% propylene oxide and 50% glycid ether, transferred into 25% propylene oxide and 75% glycid ether and incubated over night at 4°C. Then the samples were embedded in epon embedding solution and incubated for 24 h at 60°C. The embedded tissue was cut with an Ultracut E microtome (Reichert-Jung) to 90 nm sections and transferred onto formvar-coated grids. After air-drying samples were incubated 10 min in 1% uranyl acetate solution, 10 min in lead citrate (Reynolds) and rinsed with purified water. Specimens were analyzed with a Leo 906E (Zeiss) transmission electron microscope.

Immunofluorescence staining of specimens was performed by incubation for 1 h with blocking reagent (PBS, 1% BSA, 5% goat serum, 0.2% Triton X-100), 1 h incubation of primary antibody diluted in antibody solution (TBS [0.05 M, pH 7.2-7.4], 1% BSA, 0.5% Triton X-100) and 1 h incubation of secondary antibody diluted in antibody solution mixed with DAPI (1:10,000). After every step specimens were rinsed thrice with PBS. Samples were mounted with Fluoromount-G (Sigma-Aldrich) and dried over night at room temperature. Stained specimens were studied with Zeiss Axio Imager.Z1 (Carl Zeiss Goettingen) and filter sets 38HE, 43, 49 and 50. Primary antibodies were rabbit-anti-human Prox1 (Relia Tech) at a 1:500 dilution, mouse anti-human HLA A,B,C (BioLegend) at 1:200 dilution and mouse anti-Mep21 (chick CD34 homolog; M. Williams, AbLab) at dilution of 1:100. Secondary antibodies (Invitrogen) were Alexa Fluor® 594 goat anti-mouse IgG (H + L), Alexa Fluor® 488 goat anti-rabbit IgG (H + L), Alexa Fluor® 660 goat anti-rabbit IgG (H + L), highly cross-adsorbed; Alexa Fluor® 594 goat anti-mouse IgG2a (γ2a); Alexa Fluor® 488 goat anti-mouse IgG1 (γ1); at a dilution of 1:200 in antibody solution.

Cryosections were fixed in 100% methanol for 3 min, incubated for 3 min in TBS/0.1% Tween, and transferred into 3% H₂O₂. Specimens were then washed thrice in TBS/0.1% Tween, blocked with PBS/1% BSA. Subsequently anti-Ki67 antibody was added (rabbit mAb, clone D3B5, Cell Signaling Technology, Danvers, MA, USA) at a concentration of 1:200 (diluted in PBS/1% BSA) and incubated over night at 4°C on a rocking table. Specimen were then washed thrice with TBS/0.1% Tween, secondary HRP-conjugated goat-anti-rabbit antibody (St. Cruz Biotechnology, Heidelberg, Germany) was added at a concentration of 1:200 (diluted in PBS/1%BSA) and incubated for 30 min at room temperature. After that the sections were washed thrice with TBS/0.1% Tween, incubated for 5 min in TrisHCl-buffer/0.125% ammonium sulfate/0.05% DAB/0.015% H₂O₂ and washed with tab water. Section were then counterstained with 0.1% nuclear fast red-aluminum sulfate solution (Merck Millipore, Darmstadt, Germany), washed with tab water, incubated twice in 100% ethanol for 3 min, and incubated twice in xylene for 3 min. Samples were mounted with DePeX (Serva, Heidelberg, Germany). Staining with antibodies against CD20, CD19, CD10, CD5, TdT (IR604, IR656, IR648, IR082, IR001; Dako, Hamburg, Germany), and HLA A,B,C (BioLegend) was performed as described above except for the counterstaining, which was performed with hematoxylin, but omitted for CD10 and HLA.

Immunoblot analyses were performed as described previously [20]. Bcl-6 antibody was obtained from Cell Signaling, c-Myc antibody was from Abcam, alpha-Tubulin antibody was from Millipore.

Proliferation of cell lines was assessed using ³H thymidine incorporation assay as recently described [10].

BL2B95 were inoculated on the CAM of day-10 chick embryo development. The embryos were sacrificed after 7 days and BL2B95-derived tumors were dissected. We observed that BL2B95 cells formed solid tumors on the CAM in 100% of the experiments (n = 63). Tumors formed by BL2B95 assumed a lentiform shape and varied in color between reddish (highly vascularized) and whitish (sparsely vascularized) (Figure 1A). BL2-GFP cells formed tumors on the CAM, too, but showed uniformly a more whitish color (n = 11), suggesting a higher angiogenic potential of the EBV-transduced cells.

Tumor cryosections were analyzed with classical histological staining protocols including HE, panoptic Pappenheim, Trichrome and Gomori silver staining. A modified protocol of the panoptic Pappenheim stain illustrated the immigration of chick leukocytes into the tumor periphery (Figure 1A, B). Thereby, chick macrophages possessed a light-blue cytoplasm and reddish granules, whereas the BL2B95 cells stained dark-blue (basophilic) and were considerably larger than the chick leukocytes. We observed that the borders of the tumors were seamed by immigrating chick macrophages. They were discernible as lightly stained cell clusters in the dark-blue BL2B95 tumor mass. The hematoxylin and eosin staining, too, revealed a ‘starry sky’ appearance of the tumor, consisting of dark and lightly stained areas (Figure 1C). The chick macrophages were visible as light cells loaded with cellular debris and apoptotic tumor cells. This ‘starry sky’-like appearance does reflect the characteristics of human BL histology and shows that, like the primary tumors, the experimental tumors largely consist of BL2B95 cells and interacting leukocytes. Trichrome staining of the tumor specimens showed that they contained small portions of connective tissue, which, upon further analyses by silver staining, was characterized by agyrophilic fibers (Figure 1D, F). These fibers bind Ag⁺-ions during the staining procedure. Agyrophilia is a feature of type-III collagen, which is characteristic of lymphatic tissues and areas of active inflammatory reactions. Ki67 staining of the experimental tumors showed a mitotic index greater than 90% (Figure 1E). The immunohistochemical staining with established BL markers showed that the tumor cells show the characteristic pattern of classical BL. The experimental BL2B95 tumors were positive for CD20, CD19, and CD10 (Figure 2A-C). CD5 and TdT staining turned out to be negative (Figure 2D, E). HLA, which was used to differentiate between human and chick cells, clearly stained all BL cells (Figure 2F).

In order to characterize in greater detail the chick leukocytes, which immigrated into the BL2B95 tumors, we performed transmission electron microscopy (TEM)-based analyses (Figure 3A-D). The studies confirm that the tumors consisted mainly of BL2B95 cells and chick leukocytes (Figure 3A). BL2B95 cell nuclei often presented condensed or precondensed chromosomes, revealing high mitotic activity (Figure 3C, D). The nuclei of the tumor cells were mainly euchromatic with one or several prominent nucleoli. The cytoplasm possessed a lower electron density compared to the chick leukocytes and appeared therefore lighter in the TEM pictures. Lipid vesicles in the BL2B95 cell cytoplasm and plasmalemmal microvesicles were also observed, showing that the BL cells maintained their initial morphologic features in the CAM model (Figure 3A, C, D). The identification of chick leukocyte subgroups was performed according to morphological criteria and revealed the presence of dendritic cells, macrophages and heterophilic granulocytes (chick granulocytes corresponding to human neutrophils) within or in close proximity to the BL2B95 tumors (Figure 3A-D). In accordance with previous studies, lymphogenic dissemination of BL2B95 cells was also observed with this method [9, 10]. Thereby the tumor cells often filled the lumen of lymphatic vessels completely (Figure 3C, D).

The lymphatic dissemination of BL2B95 cells was analyzed by immunofluorescence staining of tumor cryosections and whole-mount specimens (Figure 4A-D). Tumor cells were visualized with anti-HLA-antibodies and lymphatic vessels with lymphatic endothelial cell (LEC)-specific anti-Prox1 antibodies (staining of LEC nuclei). The borders of the tumors appeared fuzzy and several lymphoma cells migrated into the stroma of the CAM, confirming our previous observations [9]. Our analyses of tumor sections showed that BL2B95 cells infiltrated lymphatic vessels both within the tumors (Figure 4A) and in the vicinity of the tumors. Additionally, whole-mount and cryosection staining showed that BL2B95 cells migrated long distances and were found in lymphatic vessels several millimeters apart from the primary tumors (Figure 3C, D; Figure 4D; Figure 5). The dissemination along lymphatics was even more clearly shown for the BL2-GFP cell line (Figure 6; Additional file 3: Figure S3). BL2-GFP cells were visualized with intravital GFP imaging. Pictures were taken every 24 h, beginning 48 h after the inoculation (day 12 of embryonic development). The time lapse image arrangement demonstrates the migratory routes of BL2-GFP cells along the outside of the CAM blood vessels (Figure 6; Additional file 3: Figure S3). The cells were often arranged in a characteristic pattern along the blood vessels, which overlays perfectly with the localization of the lymphatic vessels within the CAM. Immunofluorescence staining of the CAM revealed that blood vessels are often flanked by lymphatic vessels on each side (Figure 4D; Additional file 4: Figure S4). Of note, this technique allows to study the distribution of single GFP⁺ lymphoma cells within the CAM tissue in real-time. The CAM is lucent enough to trace even cells, which have migrated into deeper layers of the CAM, provided that the cells show a strong GFP expression. Thereby, the density of the Matrigel, which was used for the inoculation, was found to influence the timing of the immigration into the CAM stroma and lymphatics. BL2-GFP cells inoculated in 50% Matrigel showed much lesser infiltration of lymphatics compared to cells, which were inoculated with 10% and 0% Matrigel (Table 1). This is most probably caused by the matrix-metalloprotease (MMP)-independent (amoeboid) migratory mode of lymphoma cell lines, which impedes migration through a collagen-rich matrix.

The determination of the lymphatic and blood vessels was performed by immuno-fluorescence (IF) on experimental BL2B95 tumors. Blood vessels were detected with MEP21 (CD34 homolog) antibody staining, which is a marker for chick blood endothelial cells (BECs). The lymphatic vessels were immunostained in cryosections with anti-Prox1 antibodies, which mark the nuclei of LECs. The IF analyses revealed prominent MEP21⁺ and Prox1⁺ vessels in all BL2B95 tumors (Figure 4B, C). This clearly shows an interaction of both types of vessels with the BL2B95-derived tumors and indicates the secretion of hem- and lymphangiogenic growth factors by the tumors. The presence of blood vessels within the BL2B95 tumors was also observed in the histological stainings, shown in Figure 2. In contrast, BL2-GFP derived tumors showed a lesser degree of vascularization and appeared therefore more whitish.