venules, lined by a continuous endothelium; most had. SOLID TUMORS MUST induce a new blood supply iftheyare to growbeyond adiameter ofa few millime-.
American Journal of Pathology, Vol. 133, No. 1, October 1988 Copyright ©) American Association of Pathologists
Identfication and Characterization ofthe Blood Vessels of Solid Tumors That Are Leaky to Circulating Macromolecules From the Departments ofPathology, Beth Israel Hospital and Harvard Medical School, and the Charles A. Dana Research Institute, Beth Israel Hospital, Boston, Massachusetts
HAROLD F. DVORAK,JANICE A. NAGY, JOHN T. DVORAK, and ANN M. DVORAK
The tumor microvasculature is hyperpermeable to plasma proteins, but the specific vessels that leak have not been identified. To investigate this question, the extravasation of circulating tracers of varying size was studied by fluorescence, light, and electron microscopy in animals bearing solid transplantable carcinomas. In all five tumors studied, 70 and 150 kD fluoresceinated (FITC)-dextrans and colloidal carbon leaked extensively from the prominent vascular plexus that was induced around individual tumor nodules and at the tumor-host interface. Leaky vessels were mature veins or venules, lined by a continuous endothelium; most had
closed interendothelial celljunctions. Immature interface vessels and tumor-penetrating vessels did not leak these macromolecular tracers significantly. Three kD of FITC-dextran leaked from peripherally situated tumor veins or venules but also extravasated from tumor-penetrating vessels and capillaries supplying normal tissues. These data correlate the functional and anatomic heterogeneity of tumor vessels and provide a rationale for the distribution of circulating molecules such as monoclonal antibodies and tumoricidal drugs in solid tumors. (AmJ Pathol 1988, 133:95-109)
SOLID TUMORS MUST induce a new blood supply ifthey are to grow beyond a diameter of a few millimeters, and a great deal of attention has been focused on the mechanisms by which tumors induce angiogenesis.' Significant attention has also been devoted to the anatomy and physiology of the new blood vessels that come to supply tumors.o'1 It is generally agreed that tumor vessels are anatomically heterogeneous structures. Often they consist of relatively undifferentiated channels, lined by a simple endothelium and with fewer pericytes and smooth muscle cells than would be expected of comparably sized vessels in normal tissues. The functional properties of tumor vessels have been more controversial. For example, tumor vessels have been reported to be either more or less responsive to vasoactive mediators than normal ves-
nical reasons, it has now been demonstrated clearly that the blood vessels of a variety of syngeneic tumors in several species are several times more permeable to plasma proteins than are the blood vessels that supply adjacent normal tissues.'9-24 These findings demand explanation because of their obvious relevance to an understanding of the localization of monoclonal antibodies and tumoricidal drugs in solid tumors. Whereas small molecules pass freely through normal capillaries and other vessels with intact interendothelial cell junctions, the permeability of the normal vasculature to macromolecules is tightly regulated. Normally, macromolecules are largely retained within the circulation and the small amounts that escape are thought to do so by means of vesicular transport or by
sels.' 1-13
Supported by NIH research grants CA 28471, CA 43967, and CA 28834, and under terms of a contract from the National Foundation for Cancer Research. Accepted for publication May 20, 1988. Address reprint requests to Harold F. Dvorak, MD, Department of Pathology, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215.
One functional property of tumor vessels on which investigators do agree is that, relative to normal vessels, tumor vessels are hyperpermeable to circulating macromolecules.4"424 Although some early claims of tumor vessel hyperpermeability were faulted for tech95
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the formation of transient transcytoplasmic channels across endothelial cells.25 In inflammation, however, the escape of macromolecules is greatly increased; agonists such as histamine provoke the contraction of postcapillary endothelial cells, resulting in the formation of interendothelial cell gaps through which macromolecules and even particulates may escape.26 The present study was undertaken to identify and characterize the specific blood vessels in tumors that are responsible for leaking macromolecules. Fluoresceinated dextrans of varying size and colloidal carbon were injected intravenously as tracers into the circulation of tumor-bearing guinea pigs and mice. Sites of extravasation were identified at defined time intervals and the affected vessels were studied by fluorescence, light, and electron microscopy.
Materials and Methods Tumors and Experimental Animals Ascites variants of diethylnitrosamine-induced line 1 and line 10 bile duct carcinomas were passaged in the peritoneal cavities of syngeneic strain 2 SewallWright inbred guinea pigs of either sex at 7-10-day intervals. 8 Lewis lung (LL) carcinomas were obtained as frozen solid tumor fragments from the NCI-Frederick Cancer Research Facility, Frederick, MD, and were passaged in the subcutaneous space of syngeneic C57BL/6 mice. TA3/St and mouse ovarian tumor (MOT) ascites tumors were passaged weekly in syngeneic AJax and C3HeB/FeJ mice, respectively.23 Solid tumors were established by injection of the ascites tumor cells (guinea pigs, 3 X 106 cells per site, mice 3 X 105 cells per site) into the subcutaneous space or the thigh muscle of syngeneic animals. Diced, 1-2-mm fragments of LL carcinoma were similarly transplanted by trocar. Tumors became palpable within 2-4 days and experiments were performed 47 days after transplant, before the onset of complications such as necrosis, hemorrhage, or a cellular immune response. Vascular Mapping with Microfil The tumor microvasculature was perfused with a silicone rubber compound (Microfil white, MV- 112, Canton Bio-Medical Products, Inc., Boulder, CO). Tumor-bearing guinea pigs or mice were injected intravenously with an anticoagulant mixture'9 and killed with ether. The chest was opened and the heart and aorta were exposed. For arterial perfusions, the descending aorta was opened well above the dia-
AJP * October 1988
phragm and polyethylene tubing (PE 60 for guinea pigs, PE 50 for mice) was inserted and ligatured in place. The right atrium was transected and animals were perfused through the aorta under hand pressure with 37 C heparinized (10 units/ml) Hanks' balanced salt solution (HBSS), 12-20 ml for guinea pigs, 5-10 ml for mice, and then with similar amounts of freshly constituted Microfil, prepared according to the manufacturer's recommendations. After perfusion, the tubing was clamped and the carcass was left undisturbed for 1-2 hours at room temperature and overnight at 4 C. The next day, when the silicone had hardened, tumors and other appropriate tissue samples were dissected free, fixed and dehydrated in a sequence of ethyl alcohols, and transferred to methyl salicylate for clearing. Cleared tissues were examined intact or after hand sectioning in a Wild macroscope, equipped with dark and bright field optics. Seventeen tumors, at least three examples of each type, were studied in this manner. Tracer Studies Fluoresceinated (fluorescein isothiocyanate, FITC) dextrans of average molecular weights of 3, 70, and 150 kd were obtained from Sigma Chemical Co., St. Louis, MO. The corresponding mean diameters of these FITC-dextrans were approximately 2.4, 11.6, and 17.4 nm, respectively.27 The degree of substitution was 0.003-0.01 moles FITC per mole of glucose. Colloidal carbon (C 1 I/ 1431 A, mean particle diameter, -50 nm) was obtained from Guenther Wagner, Germany. Tumor-bearing or normal guinea pigs were injected intravenously with approximately 4 ,umol of 3, 70, or 150 kD FITC-dextran or with 0.5 ml ofundiluted colloidal carbon. Mice were injected intravenously with approximately 0.2 ,umol of the 70 or 150 kD FITCdextran, with up to 6 ,umol of the 3 kD FITC-dextran, or with 0.1 ml of a 1:5 dilution of colloidal carbon. For visualization of FITC-dextrans, alone or in combination with colloidal carbon, tissues were fixed overnight in 70% ethanol-30% formalin and dehydrated in absolute ethanol.27'28 The 3 kD FITC-dextran was partly soluble in 70% alcohol and therefore, in studies with this tracer, tissues were fixed directly in absolute ethanol. Hand-cut (
