Preferential uptake of antibody targeted calcium phosphosilicate nanoparticles by metastatic triple negative breast cancer cells in co-cultures of human metastatic breast cancer cells plus bone osteoblasts.

Calcium phosphosilicate nanoparticles (CPSNPs) are bioresorbable nanoparticles that can be bioconjugated with targeting molecules and encapsulate active agents and deliver them to tumor cells without causing damage to adjacent healthy tissue. Data obtained in this study demonstrated that an anti-CD71 antibody on CPSNPs targets these nanoparticles and enhances their internalization by triple negative breast cancer cells in-vitro. Caspase 3,7 activation, DNA damage, and fluorescent microscopy confirmed the apoptotic breast cancer response caused by targeted anti-CD71-CPSNPs encapsulated with gemcitabine monophosphate, the active metabolite of the chemotherapeutic gemcitabine used to treat cancers including breast and ovarian. Targeted anti-CD71-CPSNPs encapsulated with the fluorophore, Rhodamine WT, were preferentially internalized by breast cancer cells in co-cultures with osteoblasts. While osteoblasts partially internalized anti-CD71-GemMP-CPSNPs, their cell growth was not affected. These results suggest that CPSNPs may be used as imaging tools and selective drug delivery systems for breast cancer that has metastasized to bone.

A Spontaneous 3D Bone-On-a-Chip for Bone Metastasis Study of Breast Cancer Cells.

Bone metastasis occurs at ≈70% frequency in metastatic breast cancer. The mechanisms used by tumors to hijack the skeleton, promote bone metastases, and confer therapeutic resistance are poorly understood. This has led to the development of various bone models to investigate the interactions between cancer cells and host bone marrow cells and related physiological changes. However, it is challenging to perform bone studies due to the difficulty in periodic sampling. Herein, a bone-on-a-chip (BC) is reported for spontaneous growth of a 3D, mineralized, collagenous bone tissue. Mature osteoblastic tissue of up to 85 µm thickness containing heavily mineralized collagen fibers naturally formed in 720 h without the aid of differentiation agents. Moreover, co-culture of metastatic breast cancer cells is examined with osteoblastic tissues. The new bone-on-a-chip design not only increases experimental throughput by miniaturization, but also maximizes the chances of cancer cell interaction with bone matrix of a concentrated surface area and facilitates easy, frequent observation. As a result, unique hallmarks of breast cancer bone colonization, previously confirmed only in vivo, are observed. The spontaneous 3D BC keeps the promise as a physiologically relevant model for the in vitro study of breast cancer bone metastasis.

Three-Dimensional in Vitro Model to Study Osteobiology and Osteopathology.

The bone is an amazing organ that grows and remodels itself over a lifetime. It is generally accepted that bone sculpting in response to stress and force is carried out by groups of cells contained within bone multicellular units that are coordinated to degrade existing bone and form new bone. Because of the nature of bone and the extensiveness of the skeleton, it is difficult to study bone remodeling in vivo. On the other hand, because the bone contains a complex environment of many cell types, is it possible to study bone remodeling in vitro? We propose that one can at minimum study the interaction between osteoblasts (bone formation) and osteoclasts (bone degradation) in a three dimensional (3D) "bioreactor". Furthermore, one can add bone degrading metastatic cancer cells, and study how they contribute to and take part in the bone degradation process. We have primarily cultured and differentiated MC3T3-E1 osteoblasts for long periods (2-10 months) before addition of bone marrow osteoclasts and/or metastatic (MDA-MB-231), metastasis suppressed (MDA-MB-231BRMS1) or non-metastatic (MCF-7) breast cancer cells. In the co-culture of osteoblasts and osteoclasts there was clear evidence of matrix degradation. Loss of matrix was also evident after co-culture with metastatic breast cancer cells. Tri-culture permitted an evaluation of the interaction of the three cell types. The 3D system holds promise for further studies of cancer dormancy, hormone, and cytokine effects and matrix manipulation.

In vitro mimics of bone remodeling and the vicious cycle of cancer in bone.

Bone remodeling is a natural process that enables growth and maintenance of the skeleton. It involves the deposition of mineralized matrix by osteoblasts and resorption by osteoclasts. Several cancers that metastasize to bone negatively perturb the remodeling process through a series of interactions with osteoclasts, and osteoblasts. These interactions have been described as the "vicious cycle" of cancer metastasis in bone. Due to the inaccessibility of the skeletal tissue, it is difficult to study this system in vivo. In contrast, standard tissue culture lacks sufficient complexity. We have developed a specialized three-dimensional culture system that permits growth of a non-vascularized, multiple-cell-layer of mineralized osteoblastic tissue from pre-osteoblasts. In this study, the essential properties of bone remodeling were created in vitro by co-culturing the mineralized collagenous osteoblastic tissue with actively resorbing osteoclasts followed by reinfusion with proliferating pre-osteoblasts. Cell-cell and cell-matrix interactions were determined by confocal microscopy as well as by assays for cell specific cytokines and growth factors. Osteoclasts, differentiated in the presence of osteoblasts, led to degradation of the collagen-rich extracellular matrix. Further addition of metastatic breast cancer cells to the co-culture mimicked the vicious cycle; there was a further reduction in osteoblastic tissue thickness, an increase in osteoclastogenesis, chemotaxis of cancer cells to osteoclasts and formation of cancer cells into large colonies. The resulting model system permits detailed study of fundamental osteobiological and osteopathological processes in a manner that will enhance development of therapeutic interventions to skeletal diseases.

VCAM-1-targeted nanoparticles to diagnose, monitor and treat atherosclerosis.

Vascular cell adhesion molecule-1 (VCAM-1) was identified over 2 decades ago as an endothelial adhesion receptor involved in leukocyte recruitment and cell-based immune responses. In atherosclerosis, a chronic inflammatory disease of the blood vessels that is the leading cause of death in the USA, endothelial VCAM-1 is robustly expressed beginning in the early stages of the disease. The interactions of circulating immune cells with VCAM-1 on the activated endothelial cell surface promote the uptake of monocytes and the progression of atherosclerotic lesions in susceptible vessels. Herein, we review the role of VCAM-1 in atherosclerosis and the use of VCAM-1 binding peptides, antibodies and aptamers as targeting agents for nanoplatforms for early detection and treatment of atherosclerotic disease.

Dietary selenium supplementation modifies breast tumor growth and metastasis.

The survival rate for breast cancer drops dramatically once the disease progresses to the metastatic stage. Selenium (Se) is an essential micronutrient credited with having high anticancer and chemopreventive properties. In our study, we investigated if dietary Se supplementation modified breast cancer development in vivo. Three diets supplemented with sodium selenite, methylseleninic acid (MSA) or selenomethionine (SeMet), as well as a Se-deficient and a Se-adequate diet were fed to mice before mammary gland inoculation of 4T1.2 cells. The primary tumor growth, the numbers of cancer cells present in lungs, hearts, livers, kidneys and femurs and several proinflammatory cytokines were measured. We found that inorganic selenite supplementation provided only short-term delay of tumor growth, whereas the two organic SeMet and MSA supplements provided more potent growth inhibition. These diets also affected cancer metastasis differently. Mice fed selenite developed the most extensive metastasis and had an increased incidence of kidney and bone metastasis. On the other hand, mice fed the SeMet diet showed the least amount of cancer growth at metastatic sites. The MSA diet also provided some protection against breast cancer metastasis although the effects were less significant than those of SeMet. The cytokine profiles indicated that serum levels of interlukin-2, interleukin-6, interferon γ and vascular endothelial growth factor were elevated in SeMet-supplemented mice. There was no significant difference in tumor growth and the patterns of metastasis between the Se-deficient and Se-adequate groups. Our data suggest that organic Se supplementation may reduce/delay breast cancer metastasis, while selenite may exacerbate it.

Association between plasma cyclic guanosine monophosphate levels and hemodynamic instability during liver transplantation.

The activation of cyclic guanosine monophosphate (cGMP) production in patients with end-stage liver disease (ESLD) has been associated with hemodynamic instability during orthotopic liver transplantation (OLT). The aim of this prospective, observational study was to investigate the involvement of cGMP in the mediation of profound hypotension during liver graft reperfusion. An additional objective was to determine whether preoperative cGMP levels are associated with intraoperative hemodynamic instability. Forty-four consecutive patients undergoing OLT were included in the study. Blood samples for cGMP analysis were obtained from (1) the radial artery before the surgical incision; (2) the radial artery, portal vein, and flush blood during the anhepatic phase; and (3) the radial artery 20 minutes after liver graft reperfusion. On the basis of a statistical analysis, the patients were divided into 2 groups: group 1 (preoperative cGMP level ≥ 0.05 µmol/L) and group 2 (preoperative cGMP level < 0.05 µmol/L). We demonstrated a significant correlation between the preoperative levels of cGMP and the amount of catecholamine required to maintain hemodynamic stability during reperfusion (r = 0.52, P < 0.001), the length of the hospital stay (r = 0.38, P = 0.01), and the length of the intensive care unit (ICU) stay (r = 0.44, P = 0.004). We also demonstrated a significantly higher intraoperative catecholamine requirement (P < 0.001) and a prolonged postoperative ICU stay (P = 0.02) in group 1 patients versus group 2 patients. In conclusion, this study demonstrates increased baseline cGMP production in patients with ESLD, which is significantly associated with severe hypotension during OLT. We suggest that preoperative levels of cGMP correlate with hemodynamic instability during liver graft reperfusion.

Dynamic interaction between breast cancer cells and osteoblastic tissue: comparison of two- and three-dimensional cultures.

Breast cancer cell colonization of osteoblast monolayers grown in standard tissue culture (2D) is compared to colonization of a multi-cell-layer osteoblastic tissue (3D) grown in a specialized bioreactor. Colonization of 3D tissue recapitulates events observed in clinical samples including cancer penetration of tissue, growth of microcolonies, and formation of "Single cell file" commonly observed in end-stage pathological bone tissue. By contrast, adherent cancer cell colonies did not penetrate 2D tissue and did not form cell files. Thus, it appears that 3D tissue is a more biologically (clinically) relevant model than 2D monolayers in which to study cancer cell interactions with osteoblastic tissue. This direct comparison of 2D and 3D formats is implemented using MC3T3-E1 murine osteoblasts and MDA-MB-231 human metastatic breast cancer cells, or the metastasis-suppressed line, MDA-MB-231BRMS1, for comparison. When osteoblasts were co-cultured with metastatic cells, production of osteocalcin (a mineralization marker) decreased and secretion of the pro-inflammatory cytokine IL-6 increased in both 2D and 3D formats. Cancer cell penetration of the 3D tissue coincided with a changed osteoblast morphology from cuboidal to spindle-shaped, and with osteoblasts alignment parallel to the cancer cells. Metastasis-suppressed cells did not penetrate 3D tissue, did not cause a change in osteoblast morphology or align in rows. Moreover, they proliferated much less in the 3D culture than in the 2D culture in a manner similar to their growth in bone. In both systems, the cancer cells proliferated to a greater extent with immature osteoblasts compared to more mature osteoblasts.

Release of cytokines and hemodynamic instability during the reperfusion of a liver graft.

The objectives of this prospective, observational study were (1) to determine whether a transplanted liver graft releases proinflammatory cytokines into the systemic circulation upon reperfusion and (2) to determine whether they contribute to any subsequent hemodynamic instability observed after graft reperfusion (if this release occurs). Blood samples from 17 consecutive patients undergoing liver transplantation were analyzed for cytokines, including tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), IL-2, IL-6, and IL-8. Blood samples were obtained from the radial artery, portal vein, and flush blood (a sample taken from a catheter placed above the infrahepatic inferior vena cava clamp). The amount of catecholamines necessary to maintain a mean arterial pressure between 65 and 75 mm Hg during graft reperfusion was compared with the level of cytokines. A statistical analysis was performed with the least squares method, Kendall's tau-b test, and regression analysis. We demonstrated that flush blood from the liver grafts contained a significant amount and variety of cytokines. Most of these were removed by graft irrigation. The concentration of TNF-α in samples obtained from flush blood at the end of liver irrigation was significantly higher than the concentration in samples obtained from the radial artery (P = 0.0067) or portal vein (P = 0.0003) before reperfusion. This correlated directly with the amount of catecholamines used to treat hemodynamic instability. Although there were increased levels of IL-1ß, IL-2, and IL-8 in the flush blood, there was no statistically significant correlation between the levels of these cytokines and the amount of catecholamines used.

Glucocorticoid receptor expression on human B cells in response to acute heavy resistance exercise.

OBJECTIVE: To examine glucocorticoid receptor (GCR) expression on B lymphocytes in response to an acute bout of resistance exercise. METHODS: Using a within-subject design, resistance-trained women (n = 7; age: 22.13 ± 3.09 years; height: 1.69 ± 0.084 m; body weight: 65.60 ± 10.01 kg; body mass index: 22.63 ± 2.03 kg/m²; means ± SD) and men (n = 8; age: 23.28 ± 4.26 years; height: 1.73 ± 0.086 m; body weight: 73.93 ± 12.71 kg; body mass index: 24.51 ± 2.61 kg/m²; means ± SD) performed an acute resistance exercise protocol (6 sets of 5 repetition maximum heavy squats) and a control test in a balanced, randomized order. Blood samples were obtained before, during, and immediately after exercise, and after 1, 6, and 24 h of recovery. GCR expression on circulating B lymphocytes was evaluated with flow cytometry, and circulating cortisol was assayed. RESULTS: Resting GCR expression on B lymphocytes was similar between men and women. GCR expression was elevated in anticipation of exercise (p = 0.047), decreased during exercise (p = 0.049), and increased during recovery (p = 0.05 and p = 0.03 after 1 and 6 h of recovery, respectively). Trends for gender differences were apparent before and during exercise, and after 6 h of recovery. Men had significantly higher cortisol responses during (p = 0.002) and after exercise (p = 0.094) compared to before exercise. In women, however, circulating cortisol concentrations did not significantly increase in response to the squat exercise protocol. CONCLUSIONS: GCR expression on B lymphocytes decreased during resistance exercise and increased during recovery. Circulating cortisol increased during exercise in men only. Responses were attenuated in women compared to men. Our data provide insights into the temporal interactions between the endocrine and immune systems in response to acute heavy resistance exercise in men and women.

Pre-osteoblastic MC3T3-E1 cells promote breast cancer growth in bone in a murine xenograft model.

The bones are the most common sites of breast cancer metastasis. Upon arrival within the bone microenvironment, breast cancer cells coordinate the activities of stromal cells, resulting in an increase in osteoclast activity and bone matrix degradation. In late stages of bone metastasis, breast cancer cells induce apoptosis in osteoblasts, which further exacerbates bone loss. However, in early stages, breast cancer cells induce osteoblasts to secrete inflammatory cytokines purported to drive tumor progression. To more thoroughly evaluate the role of osteoblasts in early stages of breast cancer metastasis to the bones, we used green fluorescent protein-labeled human breast cancer cell lines MDA-MB-231 and MDA-MB-435, which both induce osteolysis after intra-femoral injection in athymic mice, and the murine pre-osteoblastic cell line MC3T3-E1 to modulate osteoblast populations at the sites of breast cancer metastasis. Breast cancer cells were injected directly into the femur with or without equal numbers of MC3T3-E1 cells. Tumors grew significantly larger when co-injected with breast cancer cells and MC3T3-E1 cells than injected with breast cancer cells alone. Osteolysis was induced in both groups, indicating that MC3T3-E1 cells did not block the ability of breast cancer cells to cause bone destruction. MC3T3-E1 cells promoted tumor growth out of the bone into the extraosseous stroma. These data suggest that breast cancer cells and osteoblasts communicate during early stages of bone metastasis and promote tumor growth.

Osteoblasts are a major source of inflammatory cytokines in the tumor microenvironment of bone metastatic breast cancer.

Metastatic breast cancer cells co-opt the cells of the bone to increase their production of inflammatory cytokines. Here, we sought to identify key cytokines expressed by osteoblasts in vitro and in vivo in the presence of MDA-MB-231 metastatic breast cancer cells, including a bone-seeking variant. We hypothesized that osteoblast-derived cytokines increase in the presence of metastatic breast cancer cell conditioned medium (CM), act as chemoattractants for cancer cells, and enhance osteoclast formation. We detected increases in the concentrations of osteoblast-derived IL-6, MCP-1, VEGF, MIP-2, and KC in vitro in culture supernatants from MC3T3-E1 cells in the presence of metastatic breast cancer cell CM and from cancer-bearing femurs ex vivo. A comparison of cancer cell- and osteoblast-derived cytokines revealed that while breast cancer cells expressed the same or equivalent cytokines as the osteoblasts, the breast cancer cells only produced picogram quantities of MCP-1; osteoblasts expressed nanogram amounts. Bone-derived MCP-1 increased in the proximal metaphysis, an area where breast cancer cells preferentially trafficked following intracardiac inoculation in athymic mice. An MDA-MB-231 bone-seeking variant was not different from parental lines. Osteoblast CM was a potent chemoattractant for metastatic breast cancer cells. Furthermore, culture supernatants of osteoblasts treated with breast cancer cell CM enhanced osteoclast formation. These findings suggest that bone metastatic breast cancer cells utilize osteoblast-derived cytokines to facilitate breast cancer cell colonization and survival upon arrival in the bone microenvironment.

Breast cancer metastasis to the bone: mechanisms of bone loss.

Breast cancer frequently metastasizes to the skeleton, interrupting the normal bone remodeling process and causing bone degradation. Osteolytic lesions are the end result of osteoclast activity; however, osteoclast differentiation and activation are mediated by osteoblast production of RANKL (receptor activator for NFκB ligand) and several osteoclastogenic cytokines. Osteoblasts themselves are negatively affected by cancer cells as evidenced by an increase in apoptosis and a decrease in proteins required for new bone formation. Thus, bone loss is due to both increased activation of osteoclasts and suppression of osteoblasts. This review summarizes the current understanding of the osteolytic mechanisms of bone metastases, including a discussion of current therapies.

Ex-vivo analysis of the bone microenvironment in bone metastatic breast cancer.

In humans, breast cancer has a predilection to metastasize to the skeleton. While the mechanism for preferential metastasis is unknown, the bone microenvironment likely provides a fertile soil for metastatic breast cancer cells. In order to examine the bone microenvironment ex-vivo following the formation of breast cancer metastases, several techniques may be employed: fluorescence stereomicroscopy, magnetic resonance imaging (MRI), microCT (microCT), immunohistochemistry, and cytokine arrays, to name a few. These methods allow for a comprehensive evaluation of the bone microenvironment during bone metastatic breast cancer. By identifying alterations in the bone niche caused by metastatic breast cancer cells, it may be possible to block or disrupt these factors through the use of targeted drugs. Appropriate therapeutic treatment would allow for an improved quality of life and longer survival time for individuals with bone metastatic breast cancer.

Selenium modifies the osteoblast inflammatory stress response to bone metastatic breast cancer.

Breast cancer frequently metastasizes to the skeleton resulting in bone degradation due to osteoclast activation. Metastases also downregulate differentiation and the bone-rebuilding function of osteoblasts. Moreover, cancer cells trigger osteoblast inflammatory stress responses. Pro-inflammatory mediators such as interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), expressed by osteoblasts (MC3T3-E1) stimulated with human breast cancer cell (MDA-MB-231) conditioned medium, are pivotal to osteoclast activation and metastasis. Given that these genes are regulated by nuclear factor-kappaB (NF-kappaB), a redox-sensitive transcription factor, we hypothesized that selenium (Se) could abrogate the inflammatory response to metastatic breast cancer cells by modulating NF-kappaB. Caffeic acid phenethyl ester and parthenolide inhibited NF-kappaB activation, as seen by gel shift assays and immunoblotting for p65 in nuclear fractions, as well as decreased production of IL-6 and MCP-1. Supplementation of MC3T3-E1 with methylseleninic acid (MSA) (0.5 microM to 4 microM) reduced the activation of NF-kappaB leading to a decrease in IL-6, MCP-1, COX-2 and iNOS in response to MDA-MB-231 conditioned medium. Addition of MSA to osteoblasts for as little as 15 min suppressed activation of NF-kappaB suggesting that short-lived active metabolites might be involved. However, brief exposure to MSA also brought about an increase in selenoprotein glutathione peroxidase 1. In summary, our data indicate that the osteoblast response to metastatic breast cancer cells is regulated by NF-kappaB activation, which can be effectively suppressed by MSA either through short-lived active metabolites and/or selenoproteins. Thus, Se supplementation may prevent the osteoblast inflammatory response or dampen the vicious cycle established when breast cancer cells, osteoblasts and osteoclasts interact.

Physical Activity Plus Energy Restriction Prevents 4T1.2 Mammary Tumor Progression, MDSC Accumulation, and an Immunosuppressive Tumor Microenvironment.

Physical activity and the prevention of weight gain decrease breast cancer incidence and improve survival. Unraveling the biological mechanisms underlying these cancer prevention effects is difficult because activity and dietary restriction are often linked. The goal of this study was to determine whether physical activity (PA), preventing weight gain via energy restriction (ER), or the combination was most effective in delaying tumor growth, reducing metastatic progression, and improving survival in the 4T1.2 mammary tumor model. Furthermore, we determined whether any of these interventions prevented the expansion of protumor immunosuppressive cells and altered the tumor microenvironment (TME). Female BALB/c mice (n = 7-20/group) were randomized to sedentary (SED) or PA wheel cages and fed ad libitum (AL) or 90% of control food intake (ER). After 8 weeks on the interventions, mice were inoculated with 5 × 104 4T1.2luc cells into the 4th mammary fat pad and continued on their respective intervention. PA+ER significantly delayed primary tumor growth (final tumor volume, 0.193 ± 0.042 vs. 0.369 ± 0.049 cm3, P < 0.001), reduced metastatic burden in the lungs (0.72 ± 0.36 vs. 16.27 ± 6.98, P = 0.054) and increased survival (median survival, 68 vs 40 days, P = 0.043) compared with SED+AL mice. PA+ER also reduced the expression level of metastatic and immunosuppressive genes and resulted in favorable changes in immune cell infiltrates in the tumor. These data suggest that both PA and ER are needed to reduce tumor growth, delay metastatic progression, and improve survival, and that this protection is associated with changes in immune-mediated mechanisms.

Metastatic breast cancer cells colonize and degrade three-dimensional osteoblastic tissue in vitro.

Metastatic breast cancer cells (BCs) colonize a mineralized three-dimensional (3D) osteoblastic tissue (OT) grown from isolated pre-osteoblasts for up to 5 months in a specialized bioreactor. Sequential stages of BC interaction with OT include BC adhesion, penetration, colony formation, and OT reorganization into "Indian files" paralleling BC colonies, heretofore observed only in authentic pathological cancer tissue. BCs permeabilize OT by degrading the extra-cellular collagenous matrix (ECM) in which the osteoblasts are embedded. OT maturity (characterized by culture age and cell phenotype) profoundly affects the patterns of BC colonization. BCs rapidly form colonies on immature OT (higher cell/ECM ratio, osteoblastic phenotype) but fail to completely penetrate OT. By contrast, BCs efficiently penetrate mature OT (lower cell/ECM ratio, osteocytic phenotype) and reorganize OT. BC colonization provokes a strong osteoblast inflammatory response marked by increased expression of the pro-inflammatory cytokine IL-6. Furthermore, BCs inhibit osteoblastic bone formation by down-regulating synthesis of collagen and osteocalcin. Results strongly suggest that breast cancer disrupts the process of osteoblastic bone formation, in addition to upregulating osteoclastic bone resorption as widely reported. These observations may help explain why administration of bisphosphonates to humans with osteolytic metastases slows lesion progression by inhibiting osteoclasts but does not bring about osteoblast-mediated healing.

Influence of oral contraceptive use on growth hormone in vivo bioactivity following resistance exercise: responses of molecular mass variants.

The purpose was to examine effects of oral contraceptive (OC) use on plasma growth hormone (GH) responses to heavy resistance exercise. Sixty untrained women were placed into one of two groups: currently using OC (Ortho Tri-Cyclen) (n=25; mean+/-SD: 24.5+/-4.2y, 160.4+/-7.1cm, 64.1+/-11.3kg) or not currently using OC (NOC) (n=35; 23.6+/-4.6y, 165.9+/-6.0cm, 65.7+/-10.3kg). Participants performed an acute heavy resistance exercise test (AHRET; six sets of 10 repetition squats; 2min rest between sets) during days 2-4 of the follicular phase (NOC group) or of inactive oral contraceptive intake (OC group). Plasma was obtained before and immediately after AHRET and subsequently fractionated based on apparent molecular weight (>60kD, 30-60kD, and <30kD). GH was determined in unfractionated plasma and each plasma fraction using 4 methods: (1) Nichols Institute Diagnostics immunoradiometric assay (Nichols), (2) National Institute of Diabetes and Digestive Kidney Diseases (NIDDK) competitive radioimmunoassay, (3) DSL immunofunctional enzyme-linked immunoabsorbent assay (IFA) and (4) rat tibial line bioassay. GH increased (P<0.05) in all fractions post-AHRET for the Nichols, NIDDK, and IFA. The OC group displayed higher resting GH for the NIDDK, and higher exercise-induced GH for the IFA, Nichols, and NIDDK in unfractionated plasma and >60kD subfraction compared to NOC group. No differences were observed for the tibial line bioassay. OC use augmented immunological GH response to AHRET in unfractionated plasma and >60kD molecular weight subfraction. However, OC use only increased biological activity of GH in one of two bioassays. These data demonstrated that GH concentrations at rest and following exercise are assay-dependent.

Influence of substratum surface chemistry/energy and topography on the human fetal osteoblastic cell line hFOB 1.19: Phenotypic and genotypic responses observed in vitro.

Time-dependent phenotypic response of a model osteoblast cell line (hFOB 1.19, ATCC, and CRL-11372) to substrata with varying surface chemistry and topography is reviewed within the context of extant cell-adhesion theory. Cell-attachment and proliferation kinetics are compared using morphology as a leading indicator of cell phenotype. Expression of (alpha2, alpha3, alpha4, alpha5, alphav, beta1, and beta3) integrins, vinculin, as well as secretion of osteopontin (OP) and type I collagen (Col I) supplement this visual assessment of hFOB growth. It is concluded that significant cell-adhesion events-contact, attachment, spreading, and proliferation-are similar on all surfaces, independent of substratum surface chemistry/energy. However, this sequence of events is significantly delayed and attenuated on hydrophobic (poorly water-wettable) surfaces exhibiting characteristically low-attachment efficiency and long induction periods before cells engage in an exponential-growth phase. Results suggest that a 'time-cell-substratum-compatibility-superposition principle' is at work wherein similar bioadhesive outcomes can be ultimately achieved on all surface types with varying hydrophilicity, but the time required to arrive at this outcome increases with decreasing cell-substratum-compatibility. Genomic and proteomic tools offer unprecedented opportunity to directly measure changes in the cellular machinery that lead to observed cell responses to different materials. But for the purpose of measuring structure-property relationships that can guide biomaterial development, genomic/proteomic tools should be applied early in the adhesion/spreading process before cells have an opportunity to significantly remodel the cell-substratum interface, effectively erasing cause and effect relationships between cell-substratum-compatibility and substratum properties. IMPACT STATEMENT: This review quantifies relationships among cell phenotype, substratum surface chemistry/energy, topography, and cell-substratum contact time for the model osteoblast cell line hFOB 1.19, revealing that genomic/proteomic tools are most useful in the pursuit of understanding cell adhesion if applied early in the adhesion/spreading process.

Kinetics of metastatic breast cancer cell trafficking in bone.

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 1 hour 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 1 week, numerous foci (2-10 cells) were observed, mostly adjacent to osteoblast-like cells. By 2 weeks, fewer but larger foci (> or =50 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 approximately 60% 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.