Donna M. Sosnoski

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.

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. J. Cell. Physiol. 226: 2150–2158, 2011.

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.

Roles of osteonectin in the migration of breast cancer cells into bone.

The focus of this study was to gain insight into the role(s) of osteonectin in the preferential metastasis of breast cancer cells to bone. Osteonectin was isolated from conditioned media of several cell lines including breast cancer (MDA‐MB‐435, MDA‐MB‐468), osteoblasts (hFOB1.19), non‐neoplastic breast epithelial (hTERT‐HME1), and vascular endothelial cells isolated from a bone biopsy (HBME‐1). Chemical/physical properties of osteonectin from these five sources was analyzed to determine if unique configurations of osteonectin exist and therefore identify a chemotactic isoform. Osteonectin from all sources had a molecular weight of ∼46 kDa, N‐linked glycosylation, and undetectable phosphorylated serines, sialic acids and O‐linked oligosaccharides. The cDNA for osteonectin from the breast cancer, osteoblast, and breast epithelial cell lines was identical, while the vascular endothelial cell cDNA contained point mutations that resulted in eight amino acid substitutions. Bone‐derived osteonectin was then analyzed to assess its influence on breast cancer cell motility and migration. Although osteonectin increased undirected MDA‐MB‐231 cell motility, it did not chemoattract the same breast cancer cell line. However, the breast cancer cells did migrate toward the known chemoattractant vitronectin and to bone extracts derived from wild‐type and osteonectin‐null mice. Migration to vitronectin was enhanced when osteonectin was also present. We concluded that osteonectin was not a chemotactic factor. However, through its anti‐adhesive properties, osteonectin induced undirected breast cancer cell motility, and may have enhanced chemoattraction to vitronectin. J. Cell. Biochem.

Changes in Cytokines of the Bone Microenvironment during Breast Cancer Metastasis.

It is commonly accepted that cancer cells interact with host cells to create a microenvironment favoring malignant colonization. The complex bone microenvironment produces an ever changing array of cytokines and growth factors. In this study, we examined levels of MCP-1, IL-6, KC, MIP-2, VEGF, MIG, and eotaxin in femurs of athymic nude mice inoculated via intracardiac injection with MDA-MB-231GFP human metastatic breast cancer cells, MDA-MB-231BRMS1GFP, a metastasis suppressed variant, or PBS. Animals were euthanized (day 3, 11, 19, 27 after injection) to examine femoral cytokine levels at various stages of cancer cell colonization. The epiphysis contained significantly more cytokines than the diaphysis except for MIG which was similar throughout the bone. Variation among femurs was evident within all groups. By day 27, MCP-1, MIG, VEGF and eotaxin levels were significantly greater in femurs of cancer cell-inoculated mice. These pro-osteoclastic and angiogenic cytokines may manipulate the bone microenvironment to enhance cancer cell colonization.

3D bioprinting for drug discovery and development in pharmaceutics

Successful launch of a commercial drug requires significant investment of time and financial resources wherein late-stage failures become a reason for catastrophic failures in drug discovery. This calls for infusing constant innovations in technologies, which can give reliable prediction of efficacy, and more importantly, toxicology of the compound early in the drug discovery process before clinical trials. Though computational advances have resulted in more rationale in silico designing, in vitro experimental studies still require gaining industry confidence and improving in vitro-in vivo correlations. In this quest, due to their ability to mimic the spatial and chemical attributes of native tissues, three-dimensional (3D) tissue models have now proven to provide better results for drug screening compared to traditional two-dimensional (2D) models. However, in vitro fabrication of living tissues has remained a bottleneck in realizing the full potential of 3D models. Recent advances in bioprinting provide a valuable tool to fabricate biomimetic constructs, which can be applied in different stages of drug discovery research. This paper presents the first comprehensive review of bioprinting techniques applied for fabrication of 3D tissue models for pharmaceutical studies. A comparative evaluation of different bioprinting modalities is performed to assess the performance and ability of fabricating 3D tissue models for pharmaceutical use as the critical selection of bioprinting modalities indeed plays a crucial role in efficacy and toxicology testing of drugs and accelerates the drug development cycle. In addition, limitations with current tissue models are discussed thoroughly and future prospects of the role of bioprinting in pharmaceutics are provided to the reader. STATEMENT OF SIGNIFICANCE Present advances in tissue biofabrication have crucial role to play in aiding the pharmaceutical development process achieve its objectives. Advent of three-dimensional (3D) models, in particular, is viewed with immense interest by the community due to their ability to mimic in vivo hierarchical tissue architecture and heterogeneous composition. Successful realization of 3D models will not only provide greater in vitro-in vivo correlation compared to the two-dimensional (2D) models, but also eventually replace pre-clinical animal testing, which has their own shortcomings. Amongst all fabrication techniques, bioprinting- comprising all the different modalities (extrusion-, droplet- and laser-based bioprinting), is emerging as the most viable fabrication technique to create the biomimetic tissue constructs. Notwithstanding the interest in bioprinting by the pharmaceutical development researchers, it can be seen that there is a limited availability of comparative literature which can guide the proper selection of bioprinting processes and associated considerations, such as the bioink selection for a particular pharmaceutical study. Thus, this work emphasizes these aspects of bioprinting and presents them in perspective of differential requirements of different pharmaceutical studies like in vitro predictive toxicology, high-throughput screening, drug delivery and tissue-specific efficacies. Moreover, since bioprinting techniques are mostly applied in regenerative medicine and tissue engineering, a comparative analysis of similarities and differences are also expounded to help researchers make informed decisions based on contemporary literature.

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. J. Cell. Physiol. 229: 453–462, 2014.

NCX3 is a major functional isoform of the sodium-calcium exchanger in osteoblasts.

The calcium phosphate‐based skeleton of vertebrates serves as the major reservoir for metabolically available calcium ions. The skeleton is formed by osteoblasts which first secrete a proteinaceous matrix and then provide Ca++ for the calcification process. The two calcium efflux ports found in most cells are the plasma membrane Ca‐ATPase (PMCA) and the sodium–calcium exchanger (NCX). In osteoblasts, PMCA and NCX are located on opposing sides of the cell with NCX facing the mineralizing bone surface. Two isoforms of NCX have been identified in osteoblasts NCX1, and NCX3. The purpose of this study was to determine the extent to which each of the two NCX isoforms support delivery of Ca++ into sites of calcification and to discern if one could compensate for the other. SiRNA technology was used to knockdown each isoform separately in MC3T3‐E1 osteoblasts. Osteoblasts in which either NCX1 or NCX3 was impaired were tested for Ca++ efflux using the Ca++ specific fluorophore, fluo‐4, in a sodium‐dependent calcium uptake assay adapted for image analysis. NCX3 was found to serve as a major contributor of Ca++ translocation out of osteoblasts into calcifying bone matrix. NCX1 had little to no involvement. J. Cell. Biochem. 103: 1101–1110, 2008.

The Role of Megakaryocytes in Breast Cancer Metastasis to Bone

Little is known about how megakaryocytes may affect metastasis beyond serving as a source of platelets. In this study, we explored the functional implications of megakaryocyte accumulation in the femurs of mice after injection of metastatic or non-metastatic breast cancer cells in 4T1.2 BALB/cJ and MDA-MB-231 nude mouse models. At bone metastatic sites, but not primary growth sites, tumor growth was associated with increased megakaryopoiesis in both model systems. In the orthotopic BALB/cJ model, extramedullary hematopoiesis occurred in the spleen, resulting in a four-fold increase in megakaryocytes. In support of the hypothesis that reducing megakaryocytes may reduce metastasis, we found that thrombopoietin-deficient mice exhibited a 90% relative decrease in megakaryocytes, yet they developed more aggressive metastasis than wild-type hosts. In human clinical specimens, we observed an increase in megakaryocytes in the bone marrow of 6/8 patients with metastatic breast cancer compared with age- and gender-matched controls. Taken together, our results suggested that an increase in megakaryocytes occurring in response to metastatic cells entering the bone marrow confers some measure of protection against metastasis, challenging present views on the role of megakaryocytes in this setting. Cancer Res; 77(8); 1942-54. ©2017 AACR.

Evaluation of bone-derived and marrow-derived vascular endothelial cells by microarray analysis

This study focused on the differential expression levels of proteins that may exist between bone‐derived and marrow‐derived vascular endothelial cells (BVEC and MVEC). The vascular cells were isolated from trabecular bone regions and central marrow cavity regions of mouse long bones. Cells were cultured for 1 week to expand the population then separated from non‐vascular cells using biotinylated isolectin B4, streptavidin‐coated metallic microbeads, and a magnetic column. After an additional week of culture time, RNA was isolated from both cell types and compared using microarray analysis. RT‐PCR was used to confirm and relatively quantitate the RNA messages. The bone‐derived cells expressed more aldehyde dehydrogenase 3A1 (ALDH3A1), Secreted Modular Calcium‐2 (SMOC‐2), CCAAT enhancer binding protein (C/EBP‐β), matrix metalloproteinase 13 (MMP‐13), and annexin 8 (ANX8) than the marrow‐derived cells. Spα and matrix GLA‐protein (MGP) were produced in greater abundance by the marrow‐derived cells. This study reveals that there are profound and unique differences between the vasculature of the metaphysis as compared to that of the central marrow cavity. The unique array of proteins expressed by the bone‐derived endothelial cells may support growth of tumors from cancer cells that frequently metastasize and lodge in the trabecular bone regions. J. Cell. Biochem. 102: 463–472, 2007.