Venkatesh Krishnan

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.

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 5xa0months 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.

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.

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.

Osteogenesis in vitro: from pre-osteoblasts to osteocytes: a contribution from the Osteobiology Research Group, The Pennsylvania State University.

Murine calvariae pre-osteoblasts (MC3T3-E1), grown in a novel bioreactor, proliferate into a mineralizing 3D osteoblastic tissue that undergoes progressive phenotypic maturation into osteocyte-like cells. Initially, the cells are closely packed (high cell/matrix ratio), but transform into a more mature phenotype (low cell/matrix ratio) after about 5xa0mo, a process that recapitulates stages of bone development observed in vivo. The cell morphology concomitantly evolves from spindle-shaped pre-osteoblasts through cobblestone-shaped osteoblasts to stellate-shaped osteocyte-like cells interconnected by many intercellular processes. Gene-expression profiles parallel cell morphological changes, up-to-and-including increased expression of osteocyte-associated genes such as E11, DMP1, and sclerostin. X-ray scattering and infrared spectroscopy of contiguous, square centimeter-scale macroscopic mineral deposits are consistent with bone hydroxyapatite, showing that bioreactor conditions can lead to ossification reminiscent of bone formation. Thus, extended-term osteoblast culture (≤10xa0mo) in a bioreactor based on the concept of simultaneous growth and dialysis captures the full continuum of bone development otherwise inaccessible with conventional cell culture, resulting in an in vitro model of osteogenesis and a source of terminally differentiated osteocytes that does not require demineralization of fully formed bone.

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. J. Cell. Biochem. 116: 2715–2723, 2015.

Abstract 524: Interaction of metastatic breast cancer cells with osteoblasts and osteoclasts in a 3D in-vitro model

Breast cancer frequently metastasizes to bone where it causes osteolytic lesions. Although, the bone osteoclasts, not the cancer cells themselves, appear to be responsible for the bone resorbtion, we have evidence from 3D in vitro cell culture studies (bioreactor) that in the presence of breast cancer cells or their medium, the osteoblasts no longer make the proteins required for bone repair. Furthermore, they produce inflammatory cytokines, which in turn can stimulate osteoclasts. We have developed a bioreactor system that permits continuous growth of bone-accreting osteoblasts (MC3T3-E1) for > 10 months without subculture or perfusion. This results in a mineralizing, multiple-cell-layer tissue that strongly resembles normal bone. We have also shown that, by challenging this osteoblast tissue with human metastatic breast cancer cells (MDA-MB-231GFP), important hallmarks of cancer metastasis including single cell filing of cancer cells, cancer-cell penetration of tissue and colonization can be observed. We hypothesize that addition of osteoclast to this 3D mineralizing osteoblast in the bioreactor would be a relevant in vitro bone surrogate for studying stages of breast-cancer metastasis. In standard tissue culture and in the bioreactor, we have been able to differentiate osteoclasts from hematopoietic progenitor cells isolated from murine bone marrow. The osteoclasts formed by exposure to RANKL and M-CSF were multi-nucleated, TRAP positive cells. The pre-osteoclast cells when introduced onto the osteoblast grown in the bioreactor, differentiated to form multinucleated resorbing osteoclast, also positive for TRAP and actin ring staining. To further improve the bone remodeling aspect, pre-osteoblasts will be re-infused into the system and allowed to ‘re-mineralize’ the matrix resorbed by osteoclasts. Once the system is established, breast cancer cells will be added to examine the three way cell interactions. This 3D in-vitro cell culture system containing both osteoblast and osteoclast will help to further understand the cellular and molecular events that lead to successful breast cancer colonization of bone. Positive outcomes of the work will help clarify the etiology of metastasis but also create a much-needed in vitro tool for study of cancer therapeutics. This work was supported by US Army Medical and Materiel Command Breast Cancer Idea Program (W81XWH-06-1-0432). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 524.