Transfection of MDA-MB-231 human breast carcinoma cells with bone sialoprotein (BSP) stimulates migration and invasion in vitro and growth of primary and secondary tumors in nude mice

We have investigated the role of bone sialoprotein (BSP), a secreted glycoprotein normally found in bone, in breast cancer progression. To explore functions for BSP in human breast cancer invasion and metastasis, the full-length BSP cDNA was transfected into the MDA-MB-231-BAG human breast cancer cell line under the control of the CMV promoter. Clones expressing BSP and vector control clones were isolated. BSP producing clones showed increased monolayer wound healing, a faster rate of stellate outgrowth in Matrigel and increased rate of invasion into a collagen matrix when compared to control clones. Clones were also examined in models of breast cancer growth and metastasis in vivo. BSP transfected clones showed an increased rate of primary tumor growth following mammary fat pad injection of nude mice. BSP transfected clones and vector control clones metastasized to soft organs and bone at a similar rate after intra-cardiac injection as determined by real-time PCR and X-ray analysis. Although these organs were targets for both BSP transfected and non-transfected cells, the size of the metastatic lesion was shown to be significantly larger for BSP expressing clones. This was determined by real-time PCR analysis for soft organs and by X-ray analysis of bone lesions. For bone this was confirmed by intra-tibial injections of cells in nude mice. We conclude that BSP acts to drive primary and secondary tumor growth of breast cancers in vivo.

Human breast cancer cell metastasis to long bone and soft organs of nude mice : a quantitative assay

Bone is a common metastatic site in human breast cancer (HBC). Since bone metastasis occurs very rarely from current spontaneous or experimental metastasis models of HBC cells in nude mice, an arterial seeding model involving the direct injection of the cells into the left ventricle has been developed to better understand the mechanisms involved in this process. We present here a sensitive polymerase chain reaction (PCR) method to detect and quantitate bone and soft organ metastasis in nude mice which have been intracardially inoculated with Lac Z transduced HBC cells. Amplification of genomically incorporated Lac Z sequences in MDA-MB-231-BAG HBC cells enables us to specifically detect these cells in mouse organs and bones. We have also created a competitive template to use as an internal standard in the PCR reactions, allowing us to better quantitate levels of HBC metastasis. The results of this PCR detection method correlate well with cell culture detection from alternate long bones from the same mice, and are more sensitive than gross Lac Z staining with X-gal or routine histology. Comparable qualitative results were obtained with PCR and culture in a titration experiment in which mice were inoculated with increasing numbers of cells, but PCR is more quantifiable, less time consuming, and less expensive. This assay can be employed to study the molecular and cellular aspects of bone metastasis, and could easily be used in conjunction with RT-PCR-based analyses of gene products which may be involved with HBC metastasis.

LCC15-MB : a vimentin-positive human breast cancer cell line from a femoral bone metastasis

The LCC15-MB cell line was established from a femoral bone metastasis that arose in a 29-year-old woman initially diagnosed with an infiltrating ductal mammary adenocarcinoma. The tumor had a relatively high (8%) S-phase fraction and 1/23 positive lymph nodes (LN). Both the primary tumor and LN metastasis were positive for estrogen receptor (ER) and progesterone receptor (PgR), but lacked erbB2 expression. Approximately one year later, the patient presented with a 0.8 cm comedo-type intraductal mammary adenocarcinoma in the left breast that was negative for ER and PgR, but positive for erbB2. Thirty-five months after the initial diagnosis she was treated for acute skeletal metastasis, and stabilized with a hip replacement. At this time, tumor cells were removed from surplus involved bone, inoculated into cell culture, and developed into the LCC15-MB cell line. The bone metastasis was a poorly differentiated adenocarcinoma lacking ER, PgR, and erbB2, characteristics shared by the LCC15-MB cells, although ER can be re-expressed by treatment of the LCC15-MB cells for 5 days with 75 μM 5-aza-2'-deoxycytidine. The LCC15-MB cell line is tumorigenic when implanted subcutaneously in NCr nu/nu mice and produces long-bone metastases after intracardiac injection. Although the bone metastasis from which the LCC15-MB cell line was derived lacked vimentin (VIM) expression, the original primary tumor and lymph node metastasis were strongly VIM positive, as are LCC15-MB cells in vitro and in nude mice. The karyotype and isozyme profiles of LCC15-MB cells are consistent with its origin from a human female, with most chromosome counts in the hypertriploid range. Thirty-two marker chromosomes are present. These cells provide an in vitro/in vivo model in which to study the inter-relationships between ER, VIM, and bone metastasis in human breast cancer.

Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro

Due to increasing clinical demand for adipose tissue, a suitable scaffold for engineering adipose tissue constructs is needed. In this study, we have developed a three-dimensional (3-D) culture system using bone marrow-derived mesenchymal stem cells (BM-MSC) and a Pluronic F-127 hydrogel scaffold as a step towards the in vitro tissue engineering of fat. BM-MSC were dispersed into a Pluronic F-127 hydrogel with or without type I collagen added. The adipogenic differentiation of the BM-MSC was assessed by cellular morphology and further confirmed by Oil Red O staining. The BM-MSC differentiated into adipocytes in Pluronic F-127 in the presence of adipogenic stimuli over a period of 2 weeks, with some differentiation present even in absence of such stimuli. The addition of type I collagen to the Pluronic F-127 caused the BM-MSC to aggregate into clumps, thereby generating an uneven adipogenic response, which was not desirable.

Bone sialoprotein supports breast cancer cell adhesion proliferation and migration through differential usage of the αvβ3 and αvβ5 integrins

Bone sialoprotein (BSP), a secreted glycoprotein found in bone matrix, has been implicated in the formation of mammary microcalcifications and osteotropic metastasis of human breast cancer (HBC). BSP possesses an integrin-binding RGD (Arg-Gly-Asp) domain, which may promote interactions between HBC cells and bone extracellular matrix. Purified BSP, recombinant human BSP fragments and BSP-derived RGD peptides are shown to elicit migratory, adhesive, and proliferative responses in the MDA-MB-231 HBC cell line. Recombinant BSP fragment analysis localized a significant component of these activities to the RGD domain of the protein, and synthetic RGD peptides with BSP flanking sequences (BSPRGD) also conferred these responses. The fibronectin-derived RGD counterpart, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), could not support these cellular responses, emphasizing specificity of the BSP configuration. Although most of the proliferative and adhesive responses could be attributed to RGD interactions, these interactions were only partly responsible for the migrational responses. Experiments with integrin-blocking antibodies demonstrated that BSP-RGD-induced migration utilizes the αvβ3 vitronectin receptor, whereas adhesion and proliferation responses were αvβ5-mediated. Using fluorescence activated cell sorting, we selected two separate subpopulations of MDA-MB-231 cells enriched for αvβ3 or αvβ5 respectively. Although some expression of the alternate αv integrin was still retained, the αvβ5-enriched MDA-MB-231 cells showed enhanced proliferative and adhesive responses, whereas the αvβ3-enriched subpopulation was suppressed for proliferation and adhesion, but showed enhanced migratory responses to BSP-RGD. In addition, similar analysis of two other HBC cell lines showed less marked, but similar RGD-dependent trends in adhesion and proliferation to the BSP fragments. Collectively, these data demonstrate BSP effects on proliferative, migratory, and adhesive functions in HBC cells and that the RGD-mediated component differentially employs αvβ3 and αvβ5 integrin receptors.

Formation of blood clot on biomaterial implants influences bone healing

The first step in bone healing is forming a blood clot at injured bones. During bone implantation, biomaterials unavoidably come into direct contact with blood, leading to a blood clot formation on its surface prior to bone regeneration. Despite both situations being similar in forming a blood clot at the defect site, most research in bone tissue engineering virtually ignores the important role of a blood clot in supporting healing. Dental implantology has long demonstrated that the fibrin structure and cellular content of a peri-implant clot can greatly affect osteoconduction and de novo bone formation on implant surfaces. This paper reviews the formation of a blood clot during bone healing in related to the use of platelet-rich plasma (PRP) gels. It is implicated that PRP gels are dramatically altered from a normal clot in healing, resulting conflicting effect on bone regeneration. These results indicate that the effect of clots on bone regeneration depends on how the clots are formed. Factors that influence blood clot structure and properties in related to bone healing are also highlighted. Such knowledge is essential for developing strategies to optimally control blood clot formation, which ultimately alter the healing microenvironment of bone. Of particular interest are modification of surface chemistry of biomaterials, which displays functional groups at varied composition for the purpose of tailoring blood coagulation activation, resultant clot fibrin architecture, rigidity, susceptibility to lysis, and growth factor release. This opens new scope of in situ blood clot modification as a promising approach in accelerating and controlling bone regeneration.

BM18 : a novel androgen-dependent human prostate cancer xenograft model derived from a bone metastasis

BACKGROUND Androgen-dependent prostate cancer (PrCa) xenograft models are required to study PrCa biology in the clinically relevant in vivo environment. METHODS Human PrCa tissue from a femoral bone metastasis biopsy (BM18) was grown and passaged subcutaneously through male severe combined immune-deficient (SCID) mice. Human mitochondria (hMt), prostate specific antigen (PSA), androgen receptor (AR), cytokeratin-18 (CK-18), pan-cytokeratin, and high molecular weight-cytokeratin (HMW-CK) were assessed using immunohistochemistry (IHC). Surgical castration was performed to examine androgen dependence. Serum was collected pre- and post-castration for monitoring of PSA levels. RESULTS: BM18 stained positively for hMt, PSA, AR, CK-18, pan keratin, and negatively for HMW-CK, consistent with the staining observed in the original patient material. Androgen-deprivation induced tumor regression in 10/10 castrated male SCID mice. Serum PSA levels positively correlated with BM18 tumor size. CONCLUSIONS BM18 expresses PSA and AR, and rapidly regresses in response to androgen withdrawal. This provides a new clinically significant PrCa model for the study of androgen-dependent growth.

Tumor cells are the source of osteopontin and bone sialoprotein expression in human breast cancer

Bone sialoprotein (BSP) and osteopontin (OPN) are secreted glycoproteins with a conserved Arg-Gly-Asp (RGD) integrin-binding motif and are expressed predominantly in bone. The RGD tripeptide is commonly present in extracellular attachment proteins and has been shown to mediate the attachment of osteosarcoma cells and osteoclasts. To determine the origin and incidence of BSP and OPN mRNA expression in primary tumor, a cohort of archival, primary invasive breast carcinoma specimens was analyzed. BSP transcripts were detected in 65% and OPN transcripts in 77% of breast cancers examined. In general, BSP and OPN transcripts were detected in both invasive and in situ carcinoma components. The transcripts were not detected in surrounding stromal cells or in peritumoral macrophages. Despite its abundance in carcinomas, BSP expression was not detected in a panel of 11 human breast cancer cell lines (MCF-7, T47D, SK-Br-3, MDA-MB-453, MDA-MB- 231, MDA-MB-436, BT549, MCF-7(AOR), Hs578T, MDA-MB-435, and LCC15-MB) and OPN expression was detected only in two of these (MDA-MB-435 and LCC15-MB). To examine the possibility that expression of these genes was down-regulated in cell culture, several cell lines were grown as nude mouse xenografts in vivo; however, these tumors also failed to express BSP. OPN expression was identified in all cell lines grown as nude mouse xenografts. Our data suggest that in human primary breast tumors, the origin of BSP and OPN mRNA is predominantly the breast cancer cells and that expression of these transcripts is influenced by the tumor environment.

Partitioning the variation in stream fish assemblages within a spatio-temporal hierarchy

This paper describes the relative influence of: (i) landscape scale environmental and hydrological factors; (ii) local scale environmental conditions including recent flow history, and; (iii) spatial effects (proximity of sites to one another) on the spatial and temporal variation in local freshwater fish assemblages in the Mary River, south-eastern Queensland, Australia. Using canonical correspondence analysis, each of the three sets of variables explained similar amounts of variation in fish assemblages (ranging from 44 to 52%). Variation in fish assemblages was partitioned into eight unique components: pure environmental, pure spatial, pure temporal, spatially structured environmental variation, temporally structured environmental variation, spatially structured temporal variation, the combined spatial/temporal component of environmental variation and unexplained variation. The total variation explained by these components was 65%. The combined spatial/temporal/environmental component explained the largest component (30%) of the total variation in fish assemblages, whereas pure environmental (6%), temporal (9%) and spatial (2%) effects were relatively unimportant. The high degree of intercorrelation between the three different groups of explanatory variables indicates that our understanding of the importance to fish assemblages of hydrological variation (often highlighted as the major structuring force in river systems) is dependent on the environmental context in which this role is examined.

Water Quality Indices from Unbalanced Spatio-Temporal Monitoring Designs

This chapter investigates a variety of water quality assessment tools for reservoirs with balanced/unbalanced monitoring designs and focuses on providing informative water quality assessments to ensure decision-makers are able to make risk-informed management decisions about reservoir health. In particular, two water quality assessment methods are described: non-compliance (probability of the number of times the indicator exceeds the recommended guideline) and amplitude (degree of departure from the guideline). Strengths and weaknesses of current and alternative water quality methods will be discussed. The proposed methodology is particularly applicable to unbalanced designs with/without missing values and reflects the general conditions and is not swayed too heavily by the occasional extreme value (very high or very low quality). To investigate the issues in greater detail, we use as a case study, a reservoir within South-East Queensland (SEQ), Australia. The purpose here is to obtain an annual score that reflected the overall water quality, temporally, spatially and across water quality indicators for each reservoir.

Medial temporal lobe roles in human path integration

Path integration is a process in which observers derive their location by integrating self-motion signals along their locomotion trajectory. Although the medial temporal lobe (MTL) is thought to take part in path integration, the scope of its role for path integration remains unclear. To address this issue, we administered a variety of tasks involving path integration and other related processes to a group of neurosurgical patients whose MTL was unilaterally resected as therapy for epilepsy. These patients were unimpaired relative to neurologically intact controls in many tasks that required integration of various kinds of sensory self-motion information. However, the same patients (especially those who had lesions in the right hemisphere) walked farther than the controls when attempting to walk without vision to a previewed target. Importantly, this task was unique in our test battery in that it allowed participants to form a mental representation of the target location and anticipate their upcoming walking trajectory before they began moving. Thus, these results put forth a new idea that the role of MTL structures for human path integration may stem from their participation in predicting the consequences of one's locomotor actions. The strengths of this new theoretical viewpoint are discussed.

Effect of hydrophilicity of carbon nanotube arrays on the release rate and activity of recombinant human bone morphogenetic protein-2

Novel nanostructures such as vertically aligned carbon nanotube (CNT) arrays have received increasing interest as drug delivery carriers. In the present study, two CNT arrays with extreme surface wettabilities are fabricated and their effects on the release of recombinant human bone morphogenetic protein-2 (rhBMP-2) are investigated. It is found that the superhydrophilic arrays retained a larger amount of rhBMP-2 than the superhydrophobic ones. Further use of a poloxamer diffusion layer delayed the initial burst and resulted in a greater total amount of rhBMP-2 released from both surfaces. In addition, rhBMP-2 bound to the superhydrophilic CNT arrays remained bioactive while they denatured on the superhydrophobic surfaces. These results are related to the combined effects of rhBMP-2 molecules interacting with poloxamer and the surface, which could be essential in the development of advanced carriers with tailored surface functionalities.

The role of the posterior superior temporal sulcus in audiovisual processing

In this study we investigate previous claims that a region in the left posterior superior temporal sulcus (pSTS) is more activated by audiovisual than unimodal processing. First, we compare audiovisual to visual-visual and auditory-auditory conceptual matching using auditory or visual object names that are paired with pictures of objects or their environmental sounds. Second, we compare congruent and incongruent audiovisual trials when presentation is simultaneous or sequential. Third, we compare audiovisual stimuli that are either verbal (auditory and visual words) or nonverbal (pictures of objects and their associated sounds). The results demonstrate that, when task, attention, and stimuli are controlled, pSTS activation for audiovisual conceptual matching is 1) identical to that observed for intramodal conceptual matching, 2) greater for incongruent than congruent trials when auditory and visual stimuli are simultaneously presented, and 3) identical for verbal and nonverbal stimuli. These results are not consistent with previous claims that pSTS activation reflects the active formation of an integrated audiovisual representation. After a discussion of the stimulus and task factors that modulate activation, we conclude that, when stimulus input, task, and attention are controlled, pSTS is part of a distributed set of regions involved in conceptual matching, irrespective of whether the stimuli are audiovisual, auditory-auditory or visual-visual.

Disentangling the cognitive components supporting Austin Maze performance in left versus right temporal lobe epilepsy

Neuropsychological tests requiring patients to find a path through a maze can be used to assess visuospatial memory performance in temporal lobe pathology, particularly in the hippocampus. Alternatively, they have been used as a task sensitive to executive function in patients with frontal lobe damage. We measured performance on the Austin Maze in patients with unilateral left and right temporal lobe epilepsy (TLE), with and without hippocampal sclerosis, compared to healthy controls. Performance was correlated with a number of other neuropsychological tests to identify the cognitive components that may be associated with poor Austin Maze performance. Patients with right TLE were significantly impaired on the Austin Maze task relative to patients with left TLE and controls, and error scores correlated with their performance on the Block Design task. The performance of patients with left TLE was also impaired relative to controls; however, errors correlated with performance on tests of executive function and delayed recall. The presence of hippocampal sclerosis did not have an impact on maze performance. A discriminant function analysis indicated that the Austin Maze alone correctly classified 73.5% of patients as having right TLE. In summary, impaired performance on the Austin Maze task is more suggestive of right than left TLE; however, impaired performance on this visuospatial task does not necessarily involve the hippocampus. The relationship of the Austin Maze task with other neuropsychological tests suggests that differential cognitive components may underlie performance decrements in right versus left TLE.

Anterior temporal cortex and semantic memory : reconciling findings from neuropsychology and functional imaging

Studies of semantic impairment arising from brain disease suggest that the anterior temporal lobes are critical for semantic abilities in humans; yet activation of these regions is rarely reported in functional imaging studies of healthy controls performing semantic tasks. Here, we combined neuropsychological and PET functional imaging data to show that when healthy subjects identify concepts at a specific level, the regions activated correspond to the site of maximal atrophy in patients with relatively pure semantic impairment. The stimuli were color photographs of common animals or vehicles, and the task was category verification at specific (e.g., robin), intermediate (e.g., bird), or general (e.g., animal) levels. Specific, relative to general, categorization activated the antero-lateral temporal cortices bilaterally, despite matching of these experimental conditions for difficulty. Critically, in patients with atrophy in precisely these areas, the most pronounced deficit was in the retrieval of specific semantic information.