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.

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.

Metastatic breast cancer induces an osteoblast inflammatory response.

Breast cancer preferentially metastasizes to the skeleton, a hospitable environment that attracts and allows breast cancer cells to thrive. Growth factors released as bone is degraded support tumor cell growth, and establish a cycle favoring continued bone degradation. While the osteoclasts are the direct effectors of bone degradation, we found that osteoblasts also contribute to bone loss. Osteoblasts are more than intermediaries between tumor cells and osteoclasts. We have presented evidence that osteoblasts contribute through loss of function induced by metastatic breast cancer cells. Metastatic breast cancer cells suppress osteoblast differentiation, alter morphology, and increase apoptosis. In this study we show that osteoblasts undergo an inflammatory stress response in the presence of human metastatic breast cancer cells. When conditioned medium from cancer cells was added to human osteoblasts, the osteoblasts were induced to express increased levels of IL-6, IL-8, and MCP-1; cytokines known to attract, differentiate, and activate osteoclasts. Similar findings were seen with murine osteoblasts and primary murine calvarial osteoblasts. Osteoblasts are co-opted into creating a microenvironment that exacerbates bone loss and are prevented from producing matrix proteins for mineralization. This is the first study implicating osteoblast produced IL-6, IL-8 (human; MIP-2 and KC mouse), and MCP-1 as key mediators in the osteoblast response to metastatic breast cancer cells.

Conserved cysteine-rich domain of paramyxovirus simian virus 5 V protein plays an important role in blocking apoptosis.

The paramyxovirus family includes many well-known human and animal pathogens as well as emerging viruses such as Hendra virus and Nipah virus. The V protein of simian virus 5 (SV5), a prototype of the paramyxoviruses, contains a cysteine-rich C-terminal domain which is conserved among all paramyxovirus V proteins. The V protein can block both interferon (IFN) signaling by causing degradation of STAT1 and IFN production by blocking IRF-3 nuclear import. Previously, it was reported that recombinant SV5 lacking the C terminus of the V protein (rSV5VDeltaC) induces a severe cytopathic effect (CPE) in tissue culture whereas wild-type (wt) SV5 infection does not induce CPE. In this study, the nature of the CPE and the mechanism of the induction of CPE were investigated. Through the use of DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, and propidium iodide staining assays, it was shown that rSV5VDeltaC induced apoptosis. Expression of wt V protein prevented apoptosis induced by rSV5VDeltaC, suggesting that the V protein has an antiapoptotic function. Interestingly, rSV5VDeltaC induced apoptosis in U3A cells (a STAT1-deficient cell line) and in the presence of neutralizing antibody against IFN, suggesting that the induction of apoptosis by rSV5VDeltaC was independent of IFN and IFN-signaling pathways. Apoptosis induced by rSV5VDeltaC was blocked by a general caspase inhibitor, Z-VAD-FMK, but not by specific inhibitors against caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 13, suggesting that rSV5VDeltaC-induced apoptosis can occur in a caspase 12-dependent manner. Endoplasmic reticulum stress can lead to activation of caspase 12; compared to the results seen with mock and wt SV5 infection, rSV5VDeltaC infection induced ER stress, as demonstrated by increased expression levels of known ER stress indicators GRP 78, GRP 94, and GADD153. These data suggest that rSV5VDeltaC can trigger cell death by inducing ER stress.