Leukemia (2004) 18, 1350–1351 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $30.00 www.nature.com/leu

COMMENTARY

The first evidence of the tumor-induced angiogenesis in vivo by using the chorioallantoic membrane assay dated 1913

Domenico Ribatti1

1Department of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy

Leukemia (2004) 18, 1350–1351. doi:10.1038/sj.leu.2403411 tional characterization of the immune system in the chick Published online 17 June 2004 embryo. Early lymphoid cells deriving from the yolk sac and spleen are usually recognizable in the thymus on day 8 and in Virchow, the founder of pathological anatomy, drew attention to the bursa of Fabricius on day 11.6 Thymus cells are present by the huge number of blood vessels in a tumor mass as long ago as day 11 and cell-mediated immunity has been demonstrated by 1865. Tumor vascularization was first studied systematically by day 13–14.7 The chick embryo and the nude mouse are 1 Goldman, who described the vasoproliferative response of the immunological incompetent hosts and do not reject tissues organ in which a tumor develops as follows: ‘The normal blood from a foreign source. Indeed, the chick embryo cannot mount vessels of the organs in which the tumor is developing are an ‘immune’ response to foreign tumor cells until well after day disturbed by chaotic growth, there is a dilatation and spiralling 12, but it can respond to tumor cells by infiltration of monocytes of the affected vessels, marked capillary budding and new vessel and inflammatory-like cells such as avian heterophils. formation, particularly at the advancing border’. Another interesting finding, which is independent of the onset James B Murphy, from the Laboratories of the Rockfeller of the embryo’s immune system, concerns the relationship Institute for Medical Reseach of New York published in 1913 a between the mitotic rate of the CAM vascular endothelium and 2 paper, which described, for the first time, the angiogenic the intensity of the angiogenic response induced by tumor response induced by the Jensen rat sarcoma in the chick implants. Knighton et al8published an important paper in 1977, chorioallantoic membrane (CAM). He wrote that ‘For the which using implants of fresh Walker 256 carcinoma, described general purposes of the experiment it has been found the onset of tumor vascularization in the CAM from day 5 to 16 advantageous to use the outer membrane (fused allantois and of incubation. Chick capillaries proliferated in the vicinity of the chorion) for the reason that it lies just under the shell membrane tumor graft about 24 h after implantation, but capillary sprouts and, therefore, can be inoculated with a minimum amount of did not penetrate the graft until approximately 72 h later. During trauma. Furthermore, this membrane is the respiratory organ of the avascular interval, tumor diameter did not exceed 1 mm, but the chick at this period and is rich in lymphatics and blood grew rapidly during the first 24 h following capillaries penetra- vessels. The inoculations were made between the fifth and tion. These authors demonstrated that tumors implanted on the seventh day of incubation and were allowed to grow until the CAM of older embryos grew at a slower rate in parallel with the 18th day. Tumors resulting from such an inoculation of the reduced rate of endothelial growth; in fact, the 3H thymidine Jensen sarcoma are found in the membrane as large globular labeling index of the CAM vascular endothelium decreases with masses lying or suspended from the inner surface of the thin age, with an abrupt reduction at day 11.9 More recently, we membrane by a broad pedicle.(y) The vessels are much more have published a paper,10 which, by using the same experi- numerous in the tumors of the embryo, here occurring in two mental approach, investigated the time-course of the angiogenic forms, either ingrowths as clusters from the chick membrane or response induced by gelatin sponges soaked with plasma cells as individual scattered through out the tumor.’ obtained from patients with active multiple myeloma (MM), in Starting from the observations of Murphy, the CAM has been comparison with cells obtained from patients with nonactive established as an experimental system for research in tumor MM and, respectively, monoclonal gammopathy of undeter- 3 biology, as a test system for tumor chemosensitivity, for the mined significance (MGUS). The gelatin sponges were im- 4 study of tumor invasion and metastasis and of neovasculariza- planted on the CAM surface from day 8 to 12 of incubation and 5 tion of heterologous normal and neoplastic implants. the number of vessels, of vessel bifurcation and the intervascular Moreover, it is also important to outline that in his paper, distance were evaluated at 24, 48, 72 and 96 h after the Murphy predicted the absence of an immune response in the implants. Results show that plasma cells from patients with chick CAM at the time in which he performed the experiments. active MM induce a vasoproliferative response, significantly He wrote that: ‘Apart for the thin continuation of the chick higher in comparison with that of plasma cells from patients membrane which covers the tumor and the ingrowth of vessels with nonactive MM and respectively, with MGUS. This response with their scant accompanying stroma, there is no histological is also a function of the day of incubation: implants made from evidence of reactions on the part of the embryo to the invasion day 8 to 10 induce a strong angiogenic response, while those of foreign tissue’. This observation was confirmed after the made from day 11 to 12 do not. 1950s by a number of researches concerning the morphofunc- Finally, it is important to note that grafting of tumors onto the CAM allows us to study the morphological aspects of the interactions of the tumors with the blood vessels of the host and Correspondence: Professor D Ribatti, Department of Human Anatomy to examine the identity of the vessels that supply the grafts. In and Histology, Piazza Giulio Cesare, 11, Policlinico, I-70124 Bari, Italy; Fax: þ 39 080 5478310; E-mail: [email protected] fact, previous studies have demonstrated that the formation of Received 6 April 2004; accepted 7 May 2004; Published online 17 peripheral anastomoses between host and pre-existing donor June 2004 vessels is the main and the most common mechanism involved Commentary 1351 in the revascularization of the graft of an embryonic organ onto 6 Leene W, Duyzings MJM, Von Steeg G. Lymphoid stem cell the CAM, whereas sprouting of CAM-derived vessels into the identification in the developing thymus and bursa of Fabricius of transplants only occurs in the grafts of tumor tissue.11,12 the chick. Z Zellforsch 1973; 136: 521–533. 7 Solomon JB. Lymphocytopoiesis and ontogeny of defined immunity in birds. In Fetal and Neonatal Immunology, Frontiers References of Biology, 1971, Monograph 20, Plenum Press: New York. 8 Knighton D, Ausprunk D, Tapper D, Folkman J. Avascular and 1 Goldman E. The growth of malignant disease in man and the lower vascular phases of tumor growth in the chick embryo. Br J Cancer animals with special reference to the vascular system. Lancet 1977; 35: 347–356. 1907; ii: 1236–1240. 9 Ausprunk DH, Knighton DR, Folkman J. Differentiation of the 2 Murphy JB. Transplantability of tissues to the embryo of foreign vascular endothelium in the chick chorioallantois: a structural and species. Its bearing on questions of tissue specificity and tumor autoradiographic study. Dev Biol 1974; 38: 237–247. immunity. J Exp Med 1913; 17: 482–493. 10 Ribatti D, De Falco G, Nico B, Ria R, Crivellato E, Vacca A. In vivo 3 Kunzi-Rapp K, Westphal-Frosch C, Schneckenburger H. Test time-course of the angiogenic response induced by multiple system for human tumor cell sensitivity to drugs on chicken myeloma plasma cells in the chick embryo chorioallantoic chorioallantoic membranes. In Vitro Cell Dev Biol 1992; 28A: membrane. J Anat 2003; 203: 323–328. 565–566. 11 Ausprunk DH, Knighton DR, Folkman J. Vascularization of normal 4 Quigley JP, Armstrong PB. Tumor cell intravasation elucidated: the and neoplastic tissues grafted to the chick chorioallantois. Am J chick embryo opens the window. Cell 1998; 91: 281–284. Pathol 1976; 79: 597–618. 5 Auerbach R, Kubai L, Sidky Y. Angiogenesis induction by 12 Ausprunk DH, Folkman J. Vascular injury in transplanted tissues. tumors, embryonic tissues, and lymphocytes. Cancer Res 1976; Fine structural changes in tumour, adult, and embryonic blood 36: 3435–3440. vessels. Virchows Arch B Cell Pathol 1976; 1: 31–44.

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