Purpose: In vivo studies have focused on the latter stages of the bone metastatic process (osteolysis), whereas little is known about earlier events, e.g., arrival, localization, and initial colonization. Defining these initial steps may potentially identify the critical points susceptible to therapeutic intervention. Experimental Design: MDA-MB-435 human breast cancer cells engineered with green fluorescent protein were injected into the cardiac left ventricle of athymic mice. Femurs were analyzed by fluorescence microscopy, immunohistochemistry, real-time PCR, flow cytometry, and histomorphometry at times ranging from 1hour to 6 weeks. Results: Single cells were found in distal metaphyses at 1 hour postinjection and remained as single cells up to 72 hours. Diaphyseal arrest occurred rarely and few cells remained there after 24 hours. At 1week, numerous foci (2-10 cells) were observed, mostly adjacent to osteoblast-like cells. By 2 weeks, fewer but larger foci (z50 cells) were seen. Most bones had a single large mass at 4 weeks (originating from a colony or coalescing foci) which extended into the diaphysis by 4 to 6 weeks. Little change (<20%) in osteoblast or osteoclast numbers was observed at 2 weeks, but at 4 to 6 weeks, osteoblasts were dramatically reduced (8% of control), whereas osteoclasts were reduced modestly (to f60% of control). Conclusions: Early arrest in metaphysis and minimal retention in diaphysis highlight the importance of the local milieu in determining metastatic potential.These results extend the Seed and Soil hypothesis by demonstrating both intertissue and intratissue differences governing metastatic location. Ours is the first in vivo evidence that tumor cells influence not only osteoclasts, as widely believed, but also eliminate functional osteoblasts, thereby restructuring the bone microenvironment to favor osteolysis.The data may also explain why patients receiving bisphosphonates fail to heal bone despite inhibiting resorption, implying that concurrent strategies that restore osteoblast function are needed to effectively treat osteolytic bone metastases.Breast cancer has a remarkable predilection to colonize bone, with an incidence between 70% and 85% in patients (1-3). At the time of death, metastatic bone disease accounts for the bulk of tumor burden (4). For women with bone metastases, the complications-severe, often intractable pain, pathologic fractures, and hypercalcemia-are catastrophic. Despite its obvious clinical importance, very little is understood about the fundamental mechanisms responsible for breast cancer metastasis to bone. Research progress has been hampered by the dearth of, and technical difficulties inherent in, the current models.Most models of metastasis poorly recapitulate the pathogenesis of breast cancer. The ideal model would involve dissemination from an orthotopic site (i.e., mammary fat pad), colonization, and osteolysis. None of the currently available human breast xenograft models spread to bone following orthotopic implantation and only on...

Tibial dyschondroplasia (TD) is a skeletal deformity associated with rapid growth in a number of avian species. The disease is the result of a disruption in the cascade of events that occur in the epiphyseal growth plate. Whereas the incidence of TD is susceptible to genetic selection, no specific genetic defect has been identified. Although there are extensive data describing the morphological and biochemical characteristics of the lesion, the mechanism of lesion formation is unknown. However, naturally occurring or induced genetic mutations in other species can provide important clues to possible mechanisms responsible for lesion development. Disruption of normal chondrocyte differentiation by constitutive activation of the parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor, inactivation of the fibroblast growth factor receptor-3 (FGFR-3) receptor, and blocking vascular endothelial growth factor (VEGF) signaling all result in lesions that resemble TD. Impairment of vascular penetration due to the ablation of matrix metalloproteinase-9 (MMP-9) or tartrate-resistant acid phosphatase (TRAP) activity also results in similar cartilage abnormalities. We have integrated these observations with our current knowledge of TD to describe a hypothesis for the sequence of events responsible for the development of tibial dyschondroplastic lesions.

The synthesis of Calcium Green C 18 , a lipophilic fluorescent calcium-sensitive dye, and its use as a monitor of Ca 2؉ efflux from cells is described. This indicator consists of a Calcium Green-1 molecule conjugated to a lipophilic 18-carbon alkyl chain which will intercalate into cell membranes. The

Carbonic anhydrase was localized in osteoclasts by light and electron microscopy using a preembedding peroxidase-antiperoxidase staining method. Osteoclasts on the endosteal surface of the metatarsi of calcitonin-treated and untreated (control) chicks were studied. A positive staining reaction was seen in the cytosol, in the Golgi apparatus, in and on vesicles, on the plasma membrane of the ruffled border, and on the bone surface beneath control osteoclasts. After calcitonin treatment, positive staining reactions were seen at the same sites except that staining was absent on the plasma membrane and endosteal bone surface. The morphology of osteoclasts from calcitonin-treated chicks was indicative of cell inactivity. The carbonic anhydrase which was bound to the plasma membrane of the ruffled border is appropriately arrayed for hydrogen ion secretion and subsequent mineral dissolution. The presence of the enzyme within lysosomelike vesicles and on the endosteal surface beneath active cells suggests that it may be released into the resorbing zone along with the lysosomal hydrolases. The function of the extracellular enzyme is also unknown.

To investigate the functional stages of osteoclasts, the ultrastructural histochemical distribution of the lysosomal enzymes [acid phosphatase (tartrate-sensitive) and neutral phosphatase], the plasma membrane enzymes [alkaline phosphatase, Ca(++)-ATPase, and alkaline ouabain-insensitive p-nitrophenylphosphatase (alkaline p-NPPase)], and the mitochondrial enzyme (cytochrome C oxidase) was evaluated in the chicken tibial metaphysis. Both active-appearing and detached (resting) osteoclasts were studied. Serial sectioning was used to identify detached osteoclasts which were present in the perivascular space. The ultrastructure of detached osteoclasts was similar to that of active osteoclasts, except for the lack of a ruffled border and clear zone, and an altered distribution pattern of small vesicles. Small vesicles were uniformly distributed in the cytoplasm of resting osteoclasts, whereas they were concentrated beneath the ruffled border of active osteoclasts. Alkaline p-NPPase, a marker enzyme for the basal ruffled border, was also apparent on the membrane of small vesicles. However, the vesicles did not possess Ca(++)-ATPase, a marker enzyme for the apical plasma membrane. These findings support the concept that small vesicles serve as a membrane reservoir for the ruffled border membrane. Pre-osteoclasts contained abundant mitochondria and lysosomes, prominent Golgi complexes, moderately developed endoplasmic reticulum, and lacked small vesicles. Pre-osteoclasts appear to fuse with osteoclasts which are attached to the bone surface, but not with detached osteoclasts. The small vesicles, from which the ruffled border arises, are absent from pre-osteoclasts, suggesting that they develop after fusion with pre-existing osteoclasts or after attachment to the bone surface. Alkaline p-NPPase appears to be a marker for differentiation of pre-osteoclasts to mature osteoclasts.