In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.

Scanning small-angle X-ray scattering (scanning SAXS) was applied for the first time on bone to compare results from SAXS directly with those from other position-sensitive methods, such as light and polarized light microscopy, back-scattered electron imaging, and radiographic imaging. Since scanning SAXS is a nondestructive method of investigation, images from all these techniques could be obtained from the same bone sections. Thus, it could be shown that both the collagen and the mineral crystals were predominantly aligned parallel to the trabeculae and, therefore, to principle stress directions. Moreover, the mean crystal thickness as determined by scanning SAXS was found to be different at various positions inside the trabecular and cortical structure. Finally, it could be shown that scanning SAXS is suitable for detecting local changes in bone material, e.g., due to fluoride treatment.

Pycnodysostosis is an uncommon human genetic disorder characterized by osteosclerosis of the skeleton, short stature, and bone fragility. The disease results from mutations in the cathepsin K gene, a lysosomal cysteine protease highly expressed in osteoclasts and crucial for the degradation of organic matrix from mineralized bone. Recently, interest has focused on a pharmaceutical inhibition of cathepsin K to prevent bone loss. However, little is known about the cellular activity or material quality of bone in pycnodysostosis. In the present study, transiliac bone biopsies from two affected individuals, aged 5 and 21 yr, were investigated using light microscopy, quantitative backscattered electron imaging, and small angle x-ray scattering. Results were compared with published age-matched reference data. The mutations in the cathepsin K gene of both patients were identified, including one novel defect. Both individuals had severe osteosclerosis, and their biopsies displayed multinucleated osteoclasts apposed to areas of demineralized matrix as well as bone-lining cells adjacent to this undigested collagen left over by osteoclasts. The homogeneity of the mineralized matrix was markedly disturbed due to large inclusions of mineralized cartilage residues. Histomorphometric evaluation showed a quantitative decrease in static parameters of bone formation. In contrast and despite deficient cathepsin K activity, osteoclastic parameters were close to normal range. At the nanostructural level, there was a marked increase in the mean thickness of the mineral particles, reflecting decreased bone remodeling. Examination of the trabecular structure revealed that the lamellae were highly disordered, which was also apparent from a poor alignment of mineral crystals oriented along the longitudinal axis of collagen fibrils. Taken together, these results strongly suggest that functional cathepsin K is important for balanced bone turnover, and enzyme deficiency results in a profound deterioration of bone quality with respect to trabecular architecture and lamellar arrangement, which is presumably the reason for bone fragility in pycnodysostosis.