Clin Exp Med (2013) 13:143–147 DOI 10.1007/s10238-012-0183-8
SHORT COMMUNICATION
B16-F10 melanoma cells contribute to the new formation of blood vessels in the chick embryo chorioallantoic membrane through vasculogenic mimicry Domenico Ribatti • Beatrice Nico • Anca Maria Cimpean Marius Raica • Enrico Crivellato • Simona Ruggieri • Angelo Vacca
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Received: 13 December 2011 / Accepted: 3 April 2012 / Published online: 19 April 2012 Ó Springer-Verlag 2012
Abstract Grafting of mammalian cells and tissues to the chick embryo chorioallantoic membrane (CAM) is a wellestablished experimental system to evaluate many different parameters of tumor growth, and B16-F10 murine melanoma cell line has been successfully used to study metastatic process in the CAM assay. The aim of this study was to demonstrate the capability of B16-F10 melanoma cells to contribute to the new formation of host blood vessels through a vasculogenic mimicry mode. Results have shown that B16-F10 melanoma cells are able to form in 4 days macroscopic tumor masses and induce a strong angiogenic response comparable to that of a well-known angiogenic cytokine, namely fibroblast growth factor-2. Moreover, tumor cells are able to cross the chorionic epithelium, and to move beneath in the mesenchyme to form tumor masses
D. Ribatti (&) B. Nico Section of Human Anatomy and Histology, Department of Basic Medical Sciences, University of Bari Medical School, Piazza G. Cesare, 11, Policlinico, 70124 Bari, Italy e-mail:
[email protected] A. M. Cimpean M. Raica Department of Histology, ‘‘Victor Babes¸ ’’ University of Medicine and Pharmacy, Timisoara, Romania E. Crivellato Section of Anatomy, Department of Medical and Morphological Research, University of Udine Medical School, Udine, Italy S. Ruggieri A. Vacca Section of Internal Medicine and Clinical Oncology, Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, Bari, Italy
immunoreactive to specific antibodies anti-S100 and antiMART-1/Melan-A. Finally, we have shown that CAMs new-formed blood vessels are lined by both pigmented melanoma cells and cells immunoreactive to MART-1/ Melan-A and PAS, suggesting the occurrence of a vasculogenic mimicry process. Keywords Angiogenesis Chorioallantoic membrane Melanoma Vasculogenic mimicry
Introduction Grafting of mammalian cells and tissues to the chick embryo chorioallantoic membrane (CAM) is a well-established experimental system to evaluate many different parameters of tumor growth [1]. All studies of mammalian neoplasms in the CAM have utilized solid tumors and cell suspensions derived from solid tumors. Tumor explants and tumor cell suspensions invade the chorionic epithelium and form visible masses within 2–5 days after implantation, and tumor cells can be identified in the CAM, as well as in the internal organs of the embryo [1]. B16-F10 murine melanoma cell line has been successfully used to study metastatic process in the CAM assay. Koop et al. [2, 3] reported a high ratio of cancer cell survival and efficiency of extravasation demonstrating by intravital microscopy that more than 80 % of the B16-F10 cells arrested in the CAM microcirculation, survived and extravasated within 24 h after injection. It is to note that this experimental assay offers the opportunity to study the biologic activities of the B16-F10 cells as in an immunocompromised animal, because the chick embryo is naturally immunodeficient and can accept various cancer cells regardless of their origin without
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specific or nonspecific immune responses. In fact, the immunocompetence of birds develops only after hatching [4] and therefore xenografts are not rejected. Human malignant melanoma is a highly vascularized and metastatic tumor in which vasculogenic mimicry occurs. In this process, described for the first time by Maniotis et al. [5], a de novo generation of blood vessels occur, in which human melanoma cells directly participate in the new formation of blood vessels independently of angiogenesis. Vasculogenic mimicry may be independent [5, 6] or dependent [7–9] of the classical angiogenic factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). The aim of this study was, by using this alternative method of tumor cell implantation in the CAM assay, to demonstrate the capability of B16-F10 melanoma cells to contribute to the new formation of host blood vessels through a vasculogenic mimicry mode by using the CAM assay.
Materials and methods Cell culture Murine melanoma B16F10 cells were maintained in culture under appropriate conditions (adherent/RPMI 1640 supplemented with 10 % FCS ? penicillin ? kanamycin) and were subcultured routinely by trypsinization (0.01 % trypsin, Sigma-Aldrich, St. Louis, MO, USA). Cells were detached from the culture flask by trypsinization, followed by trypsin inactivation with RPMI medium (Sigma, USA) containing 10 % fetal bovine serum (Sigma, USA). The cell suspension was transferred to microcentrifuge tubes and spun at 1,500 rpm for 5 min. The supernatant was replaced with serum-free RPMI medium, and the cell pellet was resuspended at a final concentration of 1–2 9 106 cells/10 ll. In vivo CAM assay Accordingly to Mangieri et al. [10], fertilized White Leghorn chicken eggs (30 per group) were incubated at 37 °C at constant humidity. On day 3 of incubation, a square window was opened in the egg shell after removal of 2–3 ml of albumen so as to detach the developing CAM from the shell. The window was sealed with a glass, and the eggs were returned to the incubator. Silicon rings (Trevigen, Gaithersburg, MD, USA) were placed on top of the growing CAM at day 8 incubation under sterile conditions. The rings were then adsorbed with 2 ll of cell suspension of 20,000 B16F10 melanoma cells or with RPMI-1640 medium alone or supplemented with FGF-2
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(200 lg/ml) (R & D Systems, Abington, UK), used as negative and positive control, respectively. The angiogenic response was evaluated on day 12 of incubation after the implants by means of a stereomicroscope connected to an image analyzer system (Olympus Italia, Rozzano, Italy). Blood vessels entering the silicon rings within the focal plane of the CAM were counted by two observers (DR and BN) in a double blind fashion at a magnification of 509. On day 12 of incubation, silicon rings were removed from the CAM surface. To determine the size of resulting CAM tumors, tumor diameter was determined with the aid of a dissecting microscope and tumors were then carefully cut away from normal CAM tissue. Thereafter, the CAM was processed for light microscopy. Briefly, the CAM was fixed in vivo in Bouin’s fluid and was removed and processed for embedding in paraffin. Seven-micrometer serial sections were cut perpendicularly to the surface of the CAM. Immunohistochemistry Immunohistochemistry was performed using deparaffined sections. The sections were incubated for 30 min in a blocking buffer [BB; phosphate-buffered saline (PBS), pH 7.4, 1 % bovine serum albumin, 2 % fetal calf serum] and for 1 h at 37 °C with primary antibodies (anti-S100 polyclonal rabbit anti-human antibody and anti-MART-1/ Melan-A clone A103 monoclonal antibody, both from Dako, Carointeria, USA). The sections were incubated overnight at 4 °C with both antibodies [1:100 in Tris buffered saline (TBS)], after prior antigen retrieval by enzymatic digestion with Ficin (Sigma, USA) in a pressure cooker for 90 s in ethylene diamine tetra acetic acid (EDTA) buffer, pH 8. Working system was for both labeled streptavidin biotin (LSAB) method, followed by 9-ethyl-3-aminocarbazole as chromogen. Preimmune mouse serum (Dako, USA) replacing the primary antibodies served as negative control. For double staining with MART-1/Melan-A-periodic acid Schiff (PAS) to demonstrate vasculogenic mimicry, after standard immunohistochemical staining for MART-1/Melan-A, the slides were rinsed with distilled water for 5 min, incubated with PAS for 15 min and counterstained with Mayer’s hematoxylin for 1 min. Statistical analysis Means ± 1 and standard deviation (SD) were evaluated for all the parameters, and the statistical significance of the differences between the counts was determined by Student’s t test for unpaired data. The Graph Pad Prism 3.0 statistical package (Graph Pad Software Inc., San Diego, CA, USA) was used for the analysis, and p \ 0.05 was considered as significant.
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Results Diffuse patches of melanoma cells could be observed in the CAM by 3 days, but tumors were not large enough to be weighed accurately until 4 days. 75 % of the embryos survived and were evaluated on day 12 of incubation, and on these, viable tumors were identified in 80 %. Tumors explants were analyzed and their average diameter was 1.5 ± 0.2 mm2. On day 12 of incubation, the murine melanoma lineage B16F10 gave rise to distinct macroscopically appreciable pigmented tumor masses on the CAM surface located inside the silicon ring (Fig. 1a). Macroscopic observation showed that the rings were surrounded by numerous allantoic
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vessels that developed radially toward the implant in a ‘‘spooked-wheel’’ pattern (mean number of blood vessels = 27 ± 4). The angiogenic response was comparable to that induced by FGF-2, a well-known angiogenic cytokine (mean number of blood vessels = 28 ± 3) (not shown). On the contrary, few blood vessels were recognizable around the silicon ring containing medium alone (mean number of blood vessels = 9 ± 2, p \ 0.001 vs. B16F10-treated CAM) (Fig. 1b). At the microscopic level, on day 12 of incubation, pigmented B16F10 melanoma cells were densely packed inside the CAM mesenchyme (Fig. 1c). This was indirect evidence that melanoma cells were able to cross the chorionic epithelium of the CAM and to move inside the mesenchyme beneath the epithelium. To confirm that the growing cells were composed of melanoma cells, sections were stained with an antibody that recognized specific markers of these cells, namely S100, and tumor cells were strongly reactive to this antibody (Fig. 1d). The analysis of the morphological characteristics of the new-formed CAM blood vessels revealed that these vessels were lined by an endothelium formed by chick embryo endothelial cells (Fig. 1e), or alternatively by brown melanoma cells, immunoreactive to the MART-1/Melan-A antibody (Fig. 1f) and to MART-1/Melan-A/PAS (Fig. 1f, inset). Tumor channels positive to both MART-1/Melan-A and PAS, which may be considered expression of vasculogenic mimicry, were found in 20 % of total number of examined vessels.
Discussion
Fig. 1 Macroscopic pictures of CAMs at day 12 of incubation, showing silicon rings containing B16F10 melanoma cells suspensions. The cells give rise to distinct appreciable pigmented tumor masses inside (a) the silicon rings and are surrounded by numerous allantoic vessels. Few blood vessels are recognizable around a silicon ring treated with vehicle alone (b). In c and d microscopic pictures of CAMs at day 12 of incubation showing that B16F10 melanoma cells are densely packed inside the CAM mesenchyme are easily distinguishable as brown pigmented tumor masses (c) or reactive to antiS100 specific antibody (d). In e and f microscopic pictures of CAMs at day 12 of incubation showing the morphological features of CAMs’ blood vessels. They are lined by chick’s endothelial cells (e), or melanoma cells showing a characteristic cytoplasmic immunoreactivity to the specific antibody anti-MART-1/Melan-A (f) or are positive to both MART-1/Melan-A/PAS (f, inset). Original magnification: a, b 950; c, d 9160; e 9400; f 9600
In this study, we have demonstrated that B16-F10 melanoma cells implanted on the CAM surface are able to form in a relative short time, 4 days, macroscopic tumor masses and induce a strong angiogenic response comparable to that of a well-known angiogenic cytokine, namely FGF-2. These data confirm the angiogenic potential of melanoma cells, which are able to secrete numerous angiogenic cytokines, such as VEGF, FGF-2, interleukin-8 (IL-8), platelet derived growth factor (PDGF) [11–14]. The presence of tumor cells immunoreactive to specific antibodies anti-S100 and anti-MART-1/Melan-A inside the intermediate CAM mesenchyme is suggestive for their migratory capacity across the chorionic epithelium, confirming the migratory potential of melanoma cells in the CAM assay, as an expression of their metastatic capacity [15]. Moreover, we have shown that CAM’s new-formed blood vessels are lined by both pigmented melanoma cells and chick endothelial cells and are positive to both MART1/Melan-A and PAS, confirming the first literature evidence
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of vasculogenic mimicry [5], which demonstrated that highly aggressive uveal melanoma can form vascular channel-like PAS-positive formed by tumor cells instead of endothelial cells. After this report, vasculogenic mimicry has been observed in some other aggressive tumors, including breast cancer, hepatocellular carcinoma, nonsmall-cell lung cancer, ovarian cancer, prostatic carcinoma, pheochromocytoma and glioblastoma [16–22]. Vasculogenic mimicry represents an alternative mechanism of tumor vascularization in which tumor cells directly participate to the formation of tumor blood vessels and may be defined by the unique ability of aggressive tumor cells to express an endothelial cell phenotype [23]. At ultrastructural level, these vascular channels were not lined by endothelial cells, as in the case of tumor angiogenesis, but were delimited by a thin basal lamina and were externally lined by tumor cells [5]. Nevertheless, other mechanisms, such as ‘‘vascular cooption’’ may be involved in tumor vascularization. Holash et al. [24] reported that tumor cells migrate to host organ blood vessels in sites of metastases, or in vascularized organs such as the brain, and initiate blood-vesseldependent tumor growth as opposed to classic angiogenesis. Tumor cells often co-opt and often grow as cuffs around adjacent vessels. Finally, bone marrow-derived stem cells may be a source of endothelial precursor cells (EPC) recruited for tumor-induced neovascularization [25]. Vasculogenic mimicry is correlated with increasing malignancy and higher aggressiveness, as it has been demonstrated in human glioma [26], and specific therapeutic approaches could be more useful. Dome et al. [23] suggested that when vasculogenic mimicry is involved in tumor vascularization, the use of vascular disrupting agents against cancer cells with endothelial phenotype, targeting the cellular pathways responsible for the development of the mechanism seems to be a more appropriate strategy. In this context, an efficient antivascular cancer therapy in tumors could be designed based on different tumor vascularization mechanisms, and it can be successful if all the possible mechanisms are targeted. Overall, these data confirm the strong angiogenic capability of B16-F10 murine melanoma cells and their migratory potential in the CAM assay and show for the first time by using this assay the presence of host’ blood vessels formed by both endothelial cells and tumor cells, suggesting the occurrence of a vasculogenic mimicry process. It is to note that compared with mammal models, where tumor growth often takes between 3 and 6 weeks, CAM assay is faster. Between 2 and 5 days after tumor cells inoculation, the tumor xenografts become visible and are penetrated by new blood vessels, following a phase of rapid growth.
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Clin Exp Med (2013) 13:143–147 Acknowledgments This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC), Investigator Grant and Special Program Molecular Clinical Oncology 5 per mille n. 9965, Milan, Italy. Conflict of interest of interest.
The authors confirm that there are no conflicts
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