Mosaic blood vessels in tumors: frequency of cancer cells in contact with flowing blood

Abstract

The presence of "mosaic" vessels in which both endothelial cells and tumor cells form the luminal surface has profound implications for metastasis, drug delivery, and antivascular therapy. Yet little is known of the frequency, and thus importance, of mosaic vessels in tumors. Using CD31 and CD105 to identify endothelial cells and endogenous green fluorescent protein labeling of tumor cells, we show that approximately 15% of perfused vessels of a colon carcinoma xenografted at two different sites in mice were mosaic vessels having focal regions where no CD31/CD105 immunoreactivity was detected and tumor cells appeared to contact the vessel lumen. These regions occupied approximately 25% of the perimeter of the mosaic vessels, or approximately 4% of the total vascular surface area in these colon carcinomas. In addition, we found similar numbers of mosaic vessels in human colon carcinoma biopsies. Our results are consistent with the observation that approximately 10(6) cells are shed daily per g of tumor. More importantly, our data offer a possible explanation for the antivascular effects of cytotoxic agents and suggest potential strategies for targeting the tumor vasculature.

Figure 1

Identification of cellular constituents of the vascular wall in LS174T tumor xenografts. (A) Summary of the method used to identify mosaic vessels. Endogenous GFP expression by the implanted tumor cells (green) along with lectin fluorescence to mark perfused vessels (blue, rhodamine pseudocolor) and CD31/CD105 immunostaining (red, Cy5) for endothelium defines the cellular composition of the tumor vessels. (B) Composite three-color representation of the vessel in A. (C and D) Individual confocal slices (1 μm) from the orthotopic (C) and the ectopic tumors (D) showing apparent tumor cell involvement in the wall. The lectin staining has been omitted for clarity. Arrows mark regions that lack detectable CD31/CD105 immunoreactivity. (Scale bar: A and C, 20 μm; D, 50 μm.)

Figure 2

Confocal slices from three different depths within one of the orthotopic tumors. C shows a region where the red CD31/CD105 immunoreactivity is no longer visible. (Scale bar, 20 μm; depth between each section, 2.5 μm.)

Figure 3

Mosaic vessels in human colon cancer. (A) Fluorescence confocal image of double-staining with anti-CD31 (visualized with Texas red), anticarcinoembryonic antigen (visualized with FITC). Arrowhead marks a region lacking detectable CD31 immunoreactivity. (B) Bright-field double-staining with anti-CD31 (visualized with diaminobenzidine), anticarcinoembryonic antigen (visualized with fast-red), and hematoxylin. (Scale bars: 50 μm.)

Figure 4

Potential mechanisms of mosaic vessel formation (endothelial cells: red; cancer cells: blue). In A, rapid vessel growth occurs via endothelial migration without sufficient endothelial proliferation, leaving cancer cells (dark blue) exposed to the lumen. In B, an endothelial cell (red) is shed from the lining, exposing underlying tumor cell(s). In C, a tumor cell (dark blue) invades the vessel, displacing an endothelial cell that may later be lost from the lining. Our morphological data are most consistent with B.