Vasculogenic mimicry and tumor angiogenesis

Abstract

Tumors require a blood supply for growth and hematogenous dissemination. Much attention has been focused on the role of angiogenesis-the recruitment of new vessels into a tumor from pre-existing vessels. However, angiogenesis may not be the only mechanism by which tumors acquire a microcirculation. Highly aggressive and metastatic melanoma cells are capable of forming highly patterned vascular channels in vitro that are composed of a basement membrane that stains positive with the periodic acid-Schiff (PAS) reagent in the absence of endothelial cells and fibroblasts. These channels formed in vitro are identical morphologically to PAS-positive channels in histological preparations from highly aggressive primary uveal melanomas, in the vertical growth phase of cutaneous melanomas, and in metastatic uveal and cutaneous melanoma. The generation of microvascular channels by genetically deregulated, aggressive tumor cells was termed "vasculogenic mimicry" to emphasize their de novo generation without participation by endothelial cells and independent of angiogenesis. Techniques designed to identify the tumor microcirculation by the staining of endothelial cells may not be applicable to tumors that express vasculogenic mimicry. Although it is not known if therapeutic strategies targeting endothelial cells will be effective in tumors whose blood supply is formed by tumor cells in the absence of angiogenesis, the biomechanical and molecular events that regulate vasculogenic mimicry provide opportunities for the development of novel forms of tumor-targeted treatments. The unique patterning characteristic of vasculogenic mimicry provides an opportunity to design noninvasive imaging techniques to detect highly aggressive neoplasms and their metastases.

Figure 1.

Normal choroidal vessels incorporated into uveal nevi and melanomas. A: Choroidal nevus. The nevus cells encircle four pre-existing choroidal vessels. B: Choroidal melanoma. At scanning magnification, few vessels are detected within the tumor, although some vessels are identified near the tumor’s edge (within the box). C: Choroidal melanoma. Higher magnification of boxed zone from B. The vessels at the tumor’s edge are lined completely by endothelial cells and these vessels have a prominent fibrous sheath, uncharacteristic of newly formed angiogenic vessels, but characteristic of normal choroidal vessels. Despite the size of this tumor, there is no evidence of necrosis. D: Normal choroidal vessel lined by endothelial cells (arrows) and invested with a distinctive fibrous connective tissue sheath is surrounded by epithelioid and spindle melanoma cells. The vessel is not damaged, and has been incorporated into the tumor. Original magnifications: A, scale bar, 250 μm; B, scale bar, 2 mm; C, scale bar, 100 μm; D, scale bar, 25 μm. A–D, hematoxylin-eosin.

Figure 2.

Patterns in primary uveal melanoma stained by the modified PAS stain (without hematoxylin counterstaining), compared with sections stained conventionally by hematoxylin-eosin. A: Straight channel. The channel splays open and contains circulating red blood cells (arrow). B: Parallel straight channels cross-link (arrow). C: Arcs (incomplete loops) are identified at the center of the micrograph, and a cluster of back-to-back complete loops is identified at the far right. D: Networks, defined as at least three back-to-back loops. Three large back-to-back loops are evident in the center of the micrograph above the normal choroidal vessel (arrow), but smaller complete loops are present throughout the upper half of the photomicrograph. E: Three pale-staining clusters of epithelioid melanoma cells correspond to the areas of tumor delimited by the large-diameter PAS-positive loops in the section adjacent to that shown in D (the arrow indicates the normal choroidal vessel for reference). The boxed area of the loop is illustrated at higher magnification in F. F: Red blood cells are identified within a space at the edge of the loop boxed in D. Arrows point to the contour of the loop highlighted here by hematoxylin-eosin. Original magnifications: A and F, scale bar, 50 μm.; B–E, scale bar, 100 μm. A–D, modified PAS without hematoxylin counterstain; E and F, hematoxylin-eosin.

Figure 3.

PAS-positive looping patterns connect to pre-existing normal vessels without intervening angiogenesis. A: Networks are traced to the vortex vein (arrowheads). There is no evidence of angiogenesis intervening between the networks and the vortex vein. B: Higher magnification of the normal vessel identified in Figure 2, D and E ▶ . The loops, which contain red blood cells (Figure 2F) ▶ connect directly to this pre-existing normal vessel without intervening angiogenesis. Original magnifications: A and B, scale bar, 50 μm.; A and B, modified PAS without hematoxylin counterstaining (Figure 3A ▶ modified from Folberg, et al 36 ).

Figure 4.

Perfusion in vasculogenic mimicry patterns. A: Column of red blood cells in an arc without branching. Endothelial cell nuclei are not identified lining this channel. A thin layer of extracellular matrix (arrow) extends from this channel. B: Thin parallel channels that do not appear to be perfused with blood (arrows) splay open focally to reveal red blood cells in the lumen. None of these channels is lined by endothelium. Original magnifications: A, scale bar, 10 μm; B, scale bar, 50 μm; A and B, hematoxylin-eosin.

Figure 5.

Angiogenesis in retinoblastoma. A: Numerous discrete vessels are identified. Note the zones of necrosis (left and upper right). B: Higher magnification. Viable tumor surrounds vessels in a cuff. Necrosis is identified farther away from the angiogenic vessel. C: With additional magnification, endothelial cell sprouting is identified histologically within the retinoblastoma tumor. Endothelial cells (arrows) are identified by light microscopy in every vessel. Original magnifications: A, scale bar, 500 μm; B, scale bar, 100 μm; C, scale bar, 50 μm. A–C, hematoxylin-eosin.

Figure 6.

Immunohistochemical staining of primary uveal melanoma with putative endothelial cell markers. A: Factor VIII-related antigen stains normal choroidal vessels (left of the tumor mass), but does not stain the interior of the tumor. B: Same tumor in A stained by the modified PAS stain without hematoxylin counterstain. Vasculogenic mimicry patterns are identified within the tumor. C: Primary uveal melanoma stained with Ulex europaeus agglutinin I. Note the intermittent staining of this vascular channel that contains red blood cells. The material between the red blood cells (plasma) stains with the Ulex lectin. The section is counterstained with hematoxylin, and despite the long segment of the channel illustrated, no endothelial cell nuclei are identified. There are no difficulties in identifying endothelial cell nuclei lining normal vessels incorporated into primary uveal melanomas (Figure 1) ▶ . D: CD31 staining tumor cells in the vicinity of vasculogenic mimicry patterns (not illustrated). Original magnifications: A and B, scale bar, 200 μm; C and D, scale bar, 10 μm. A: Factor VIII related antigen-hematoxylin; B: PAS without hematoxylin counterstain; C: Ulex europaeus agglutinin I with hematoxylin counterstain; D: CD31 counterstained with hematoxylin.

Figure 7.

Cytokeratin expression in primary uveal melanoma and vasculogenic mimicry. A: Histological section of primary uveal melanoma. A channel containing red blood cells is lined externally by spindle melanoma cells that stain positive for pan-cytokeratin. Note the lack of endothelial cells along the inner channel wall. B–E:, Tissue cultures of metastatic uveal melanoma cell line MUM2B. B: Phase contrast showing loop encircling a small cluster of epithelioid melanoma cells. C: The same field illustrated in B photographed with fluorescence. The culture has been stained with antibody to keratins 8,18. Note the alignment of keratin-positive tumor cells alongside the looping pattern formed in vitro. D: Phase contrast of another aggressive melanoma culture showing two parallel straight channels. E: The same field illustrated in D photographed with fluorescence. The culture has been stained with antibody to keratins 8,18. Note the alignment of keratin-positive tumor cells alongside the straight channels, similar to that seen in tissue section (A). Original magnifications: A, scale bar, 10 μm; B–E, scale bar, 5 μm; A: pan-cytokeratin counterstained with hematoxylin; B and D: phase contrast; C and E: fluorescence (cultures labeled with antibody to keratins 8,18).

Figure 8.

Primary cutaneous melanoma, vertical growth phase. Networks are abundant. Original magnification: scale bar, 200 μm. PAS without hematoxylin counterstaining (tissue section courtesy of Prof. T. K. Das Gupta).

Figure 9.

Perfusion of vasculogenic mimicry patterns may simulate the appearance of angiogenesis histologically. A: Primary uveal melanoma: perfusion in parallel vasculogenic mimicry channels. This is the same field illustrated in Figure 4B ▶ . Here, the areas in which the channels splay open and contain red blood cells are highlighted with arrowheads. Because the blood column itself stains with multiple putative endothelial cells markers such as Factor VIII-related antigen (Figure 6A) ▶ and Ulex (Figure 6) ▶ , as well as CD31, CD34, and KDR, it is possible to count each focus of blood as a separate vessel if the tissue section is not counterstained with hematoxylin and one is not attuned to the existence of continuous vasculogenic mimicry patterns in the section. B and C: Co-localization of CD34 to PAS-positive loops and networks. A histological section of primary uveal melanoma containing multiple loops and networks was stained with CD34 (Texas Red chromagen) and counterstained subsequently by PAS without hematoxylin counterstaining. The tissue section was photographed Bio-Rad MRC-600 laser scanning confocal microscope (Bio-Rad, Cambridge, MA) by capturing both the direct illumination channel (for the PAS-positive patterns) and the rhodamine channel (for Texas Red). B: Back-to-back loops form networks. C: The same field illustrated in B, showing CD34 (in red) co-localizing to the loops by staining the lumen contents rather than endothelial cells. A pathologist looking only at the CD34 stain (C) might conclude erroneously that this is an angiogenic hot-spot. Original magnifications: A–C, scale bar, 50 μm; A, hematoxylin-eosin; B, CD34 and periodic acid-Schiff without hematoxylin counterstaining (direct illumination); C, CD34 and periodic acid-Schiff, rhodamine channel.

Figure 10.

Diagrammatic scheme of vasculogenic mimicry. Uveal melanomas develop in an environment devoid of lymphatics. Aggressive tumors (but not non-aggressive tumors) form looping non-endothelial cell-lined channels that are delimited by PAS-positive material. These vasculogenic mimicry channels link directly to normal vessels in the choriocapillaris, the vortex vein, or normal vessels incorporated into the tumor without evidence of angiogenesis. Channels generated by tumor cells (vasculogenic mimicry) are lined externally by tumor cells, in contrast to blood vessels which are lined internally by endothelial cells. Diagram courtesy of Dr. Dawn Kirschmann.

Figure 11.

Animal model of uveal melanoma cell line implanted subcutaneously into immunosuppressed mouse. A: Scanning magnification. A tumor measuring 9 mm at its base developed from the subcutaneous injection of aggressive uveal melanoma cells. Note the presence of necrosis centrally which is not a feature typical of primary uveal melanoma (compare with the primary uveal melanoma shown in Figure 1B ▶ , which also measured 9 mm in diameter). B: Higher magnification of the interface between viable tumor and necrosis. Dilated endothelial cell-lined vessels are present adjacent to an area of necrosis (lower portion of the micrograph). C: Higher magnification of vessels shown in B. These vessels are lined by endothelial cells. Original magnifications: A, scale bar, 1 mm; B, scale bar, 50 μm; C, scale bar, 50 μm. A–C, hematoxylin-eosin.