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Aims: Quantitative image analysis of histopathology slides is becoming an important technology in diagnostic pathology. To this end, it is essential to combine a robust image analysis software with the most commonly used immunohistochemical staining methods. In this investigation, we describe a practical application of NIH ImageJ software for quantitative vascular image analysis for diaminobenzene chromogen-based CD34 immunostain in breast cancer. CD34 immunostain is in a unique position to identify lymphangiogenesis and angiogenesis simultaneously in a given tumor tissue. This investigation aims at establishing a practical quantitative vascular image analysis solution for diagnostic pathologists by using ImageJ, and CD34 immunostain., Methods and Results: Tissue microarray slides containing breast cancer tissue were immunostained for CD34 for simultaneous identification of lymphatic endothelial cells (LEC) and blood vessel endothelial cells (BEC). Digital images were analyzed using NIH ImageJ software. A CD34 score was quantified for each tissue core as a percentage (CD34-positive area/area of tissue core). The mean CD34 scores were 0.24%, 0.40%, 1.30%, 2.33%, 2.64%, and 3.44% for normal breast tissue, in stage IIA, IIB, IIIA, IIIB, and IIIC breast cancer tissue cores, respectively (p<0.0001). The mean CD34 scores were 0.70% and 2.21% for lymph node-negative and lymph node-positive breast cancer patients, respectively (p<0.0001)., Conclusions: ImageJ software seems to be an attractive quantitative image analysis tool for diagnostic pathology for immunohistochemistry-based applications because of its capabilities, availability, and ease of use with most image formats. Our results show the feasibility, versatility, and ease of use of ImageJ and CD34 immunohistochemistry for vascular image analysis in breast pathology. Given the prospects of novel lymphatic and vascular endothelium-targeting therapeutics in breast oncology, the practical analysis of combined LEC and BEC density described in this report could enable diagnostic pathologists to apply quantitative vascular image analysis easily in their pathology practice and translational research., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

In this investigation, we explored whether knockout of nerve/glial antigen 2 (NG2), a pericyte component, inhibited neovascularization and growth of uveal melanoma xenografts. For this, we used multichannel laser scanning confocal microscopy and quantitative image analysis. Orthotopic human uveal melanoma (OCM-1A) xenografts were induced in NG2 knockout and wild-type mice, which were immunosuppressed with cyclosporin A. Inhibition of pericytes through NG2 proteoglycan decreased neovascularization and tumor end volume, rendering pericytes and NG2 proteoglycan potential cellular and molecular therapeutic targets in uveal melanoma., (Copyright (c) 2006 S. Karger AG, Basel.)

Bone marrow (BM)-derived cells are thought to participate in the growth of blood vessels during postnatal vascular regeneration and tumor growth, a process previously attributed to stem and precursor cells differentiating to endothelial cells. We used multichannel laser scanning confocal microscopy of whole-mounted tissues to study angiogenesis in chimeric mice created by reconstituting C57BL mice with genetically marked syngeneic BM. We show that BM-derived endothelial cells do not significantly contribute to tumor- or cytokine-induced neoangiogenesis. Instead, BM-derived periendothelial vascular mural cells were persistently detected at sites of tumor- or vascular endothelial growth factor-induced angiogenesis. Subpopulations of these cells expressed the pericyte-specific NG2 proteoglycan, or the hematopoietic markers CD11b and CD45, but did not detectably express the smooth muscle markers smooth muscle alpha-actin or desmin. Thus, the major contribution of the BM to angiogenic processes is not endothelial, but may come from progenitors for periendothelial vascular mural and hematopoietic effector cells.

Apart from tumor-driven neovascularization, a less-appreciated consequence of neurofibromatosis type 1 (NF1) is the hyperproliferation of vascular mural cells (pericytes). This study aims at establishing a role for pericytes in NF1, and determining whether interference with the function of a key pericyte component (NG2 proteoglycan) inhibits NF1 tumor neovascularization. Neovascularization in NF1 was studied in Nf+/+(control), Nf1+/-, and Nf1-/-embryos at E-10, ischemia-induced retinal angiogenesis model in 24 eyes of Nf1+/-, Nf1+/+mice, and in malignant peripheral nerve sheath tumors (MPNSTs) derived from NF1 patients (ST88-14, NMS-2PC) orthotopically grown in nude mice (Crl: nu/nu). The anti-angiogenic effect of intracorneal polymer pellets containing anti-NG2 neutralizing antibody was quantified in the nude-mouse corneal angiogenesis model in which angiogenesis was induced by xenografting NMS-2PC tumor into the corneal stroma of 22 eyes. By using confocal microscopy, immunohistochemistry, and BrdU proliferation assay, the pericyte/endothelium ratios and proliferation rates were measured. Activated pericytes were present at the leading tip of the angiogenic sprouts. Pericytes showed continuous investment of endothelium in both NMS-2PC and ST88-14 MPNST tumor xenografts. Mean corneal angiogenesis induced by NMS-2PC tumor grafts in NG2-antibody treated eyes was 1.491 and 3.186 mm2 in isotype-matched non-immunoglobulin treated eyes (control) (P=0.0002). A total of 193.8 vascular nuclei (a measure of ischemia-induced retinal angiogenesis) was present in angiogenic retinal tufts in Nf1+/- mice compared to 89.23 in Nf1+/+ mice (control) (P<0.0001). Mean pericyte/endothelium investment ratios were 1.015, 1.380, and 2.084 in control, Nf1+/-, and Nf1-/-embryos, respectively. Pericytes were 23% (control), 49% (Nf1+/-), and 69% (Nf1-/-) BrdU-positive. Endothelial cells from the same embryos were 29% (control), 47% (Nf1+/-), and 62% (Nf1-/-) BrdU-positive. Angiogenesis is accelerated in NF1 due to hyperproliferation of pericytes and endothelial cells. Mitotically activated NG2-positive pericytes, and endothelial cells may serve as potential therapeutic targets in NF1.

Immunostaining with endothelial and pericyte markers was used to evaluate the cellular composition of angiogenic sprouts in several types of tumors and in the developing retina. Confocal microscopy revealed that, in addition to conventional endothelial tubes heavily invested by pericytes, all tissues contained small populations of endothelium-free pericyte tubes in which nerve/glial antigen 2 (NG2) positive, platelet-derived growth factor beta (PDGF beta ) receptor-positive perivascular cells formed the lumen of the microvessel. Perfusion of tumor-bearing mice with FITC-dextran, followed by immunohistochemical staining of tumor vasculature, demonstrated direct apposition of pericytes to FITC-dextran in the lumen, confirming functional connection of the pericyte tube to the circulation. Transplantation of prostate and mammary tumor fragments into NG2-null mice led to the formation of tumor microvasculature that was invariably NG2-negative, demonstrating that pericytes associated with tumor microvessels are derived from the host rather than from the conversion of tumor cells to a pericyte phenotype. The existence of pericyte tubes reflects the early participation of pericytes in the process of angiogenic sprouting. The ability to study these precocious contributions of pericytes to neovascularization depends heavily on the use of NG2 and PDGF beta -receptor as reliable early markers for activated pericytes.