Comparing Vasculogenic Mimicry With Endothelial Cell-lined Vessels: Techniques for 3D Reconstruction and Quantitative Analysis of Tissue Components from Archival Paraffin Blocks

Abstract

We previously described techniques to generate 3-dimensional reconstructions of the tumor microcirculation using immunofluorescence histochemistry and laser scanning confocal microscopy on serial sections from archival formalin-fixed, paraffin-embedded tissues. By aligning sequential z-stacks in an immersive visualization environment (ImmersaDesk), the need to insert fiduciary markers into tissue was eliminated. In this study, we developed methods to stitch overlapping confocal z-series together to extend the surface area of interest well beyond that captured by the confocal microscope objective and developed methods to quantify the distribution of markers of interest in 3 dimensions. These techniques were applied to the problem of comparing the surface area of nonendothelial cell-lined, laminin-rich looping vasculogenic mimicry (VM) patterns that are known to transmit fluid, with the surface area of endothelial cell-lined vessels in metastatic uveal melanoma to the liver in 3 dimensions. After labeling sections with antibodies to CD34 and laminin, the surface area of VM patterns to vessels was calculated by segmenting out structures that labeled with laminin but not with CD34 from those structures labeling with CD34, or CD34 and laminin. In metastatic uveal melanoma tissues featuring colocalization of high microvascular density [66.4 microvessels adjusted for 0.313 mm2 area (range 56.7 to 72.7)] and VM patterning, the surface area of VM patterns was 11.6-fold greater (range 10.8 to 14.1) than the surface provided by CD34-positive vessels. These methods may be extended to visualize and quantify molecular markers in 3 dimensions in a variety of pathologic entities from archival paraffin-embedded tissues.

Extracellular matrix proteins, neural differentiation and melanoma progression

“…The LN5/integrin α5β1 positivity and the presence of mitosis suggest that these are cells which have switched to an aggressive invasive phase in the bordering cells. These findings resemble the VM looping patterns described in the 3D studies [55][56][57] where they are identifiable by the laminin positivity of the bordering cells [58][59][60][61] . Thus there is a switch over to the rapid fire vascularisation to facilitate rapid growth, mimicking embryonic conditions as the melanoma becomes poorly differentiated.…”

“…The molecular profile of aggressive cutaneous and uveal melanoma cells reveals multiple phenotypes simulating a pluripotent, embryonic-like stem cell [55][56][57] .…”

Demonstrating circulation in vasculogenic mimicry patterns of uveal melanoma by confocal indocyanine green angiography

“…Three-dimensional reconstructions of vasculogenic mimicry patterns reveal them to be sheets of extracellular matrix proteins -especially rich in laminin -that wrap around branching cylinders of tumor cells. 25,26 The patterns are generated by melanoma cells and are not lined by endothelial cells. 3,4 Because it has Eye been shown repeatedly in animal models that intravenous tracers circulate not only through blood vessels but also through the non-endothelial cell-lined vasculogenic mimicry patterns, 5,9,11 it has been proposed that fluid leaks from tumor vessels into the patterns.…”

“…The presence of an active circulation through vasculogenic mimicry patterns is significant because it has been shown recently that these patterns provide for at least an 11-fold increase in surface area over tumor blood vessels. 26 Therefore, vasculogenic mimicry patterns may provide for a more effective delivery route of therapeutic agents than endothelial cell-lined blood vessels.…”

Three-dimensional visualization and quantification for the growth and invasion of oral squamous cell carcinoma

“…In the literature, several techniques for automated 3D reconstruction using paraffin-embedded histological sections have been documented [12,[17][18][19], but each of the 3D reconstruction procedures, particularly the distortion-free image registration, yet remained as major challenges. An alternative approach to circumvent the use of serially cut sections is the reconstruction of certain tissue components (e.g., blood vessels in a stromal space) by means of laser scanning confocal microscopy (LSCM) from a single, thick, paraffin-embedded tissue sample [20].…”