THREE-DIMENSIONAL OSTEOBLASTIC TISSUE TO STUDY BREAST CANCER COLONIZATION OF BONE
Open Access
- Author:
- Krishnan, Venkatesh
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 14, 2011
- Committee Members:
- Andrea Marie Mastro, Dissertation Advisor/Co-Advisor
Andrea Marie Mastro, Committee Chair/Co-Chair
Erwin A Vogler, Committee Member
Kumble Sandeep Prabhu, Committee Member
Robert Paulson, Committee Member - Keywords:
- metastasis
bone-remodeling-unit
3D
breast-cancer
osteoclast
osteoblast
colonization of bone - Abstract:
- The cellular constituents of a tumor dictate the basic elements formulating a microenvironment. For example, in breast cancer colonization of bone, osteoblasts (bone building cells) and osteoclasts (bone resorbing cells) are important cell types that make up the bone microenvironment. This work concentrates on the role of bone microenvironment in the progression of breast cancer. Understanding the mechanisms that govern the process of metastatic colonization in specific secondary organs would help identify potential therapeutic targets. It is not known exactly why breast cancer cells target bone; but once colonization of bone occurs, an interconnected series of interactions between breast cancer cells and those of the bone microenvironment result in uncontrolled tumor growth. These interactions have been collectively interpreted in terms of the “vicious cycle of bone metastasis” which has become the central paradigm of cancer in bone. According to this model, cancer cells secrete pro-inflammatory factors that lead to osteoclast activation, through intermediary stimulation of osteoblasts. Osteoblasts increase production of inflammatory cytokines, which further stimulates osteoclasts to differentiate into mature, multinucleated osteoclasts that actively resorb bone leading to severe osteopathologies. A complete understanding of cancer in bone thus requires an in-depth knowledge of a network of biochemical signaling occurring between osteoblasts, osteoclasts and the breast cancer cells at tissue and cellular levels. I hypothesize that bone remodeling and its disruption during breast cancer colonization of bone can be studied in a bioreactor that permits the interaction of breast cancer cells with co-cultures of osteoblasts and osteoclasts under physiologically relevant conditions. This bioreactor can be used to create a unique in vitro system wherein biological complexity is carefully controlled by step-by-step addition of cellular components. Studying the three-cell type (osteoblast-osteoclast-breast cancer) interaction requires a shift in focus from isolated in vitro cell culture systems to a three-dimensional cell culture system that simulates major aspects of the in vivo bone microenvironment. We have recently developed and validated a novel three-dimensional in vitro cell culture system (bioreactor) that circumvents many limitations imposed by conventional cell/tissue culture technology. The bioreactor, based on the principle of simultaneous-growth-and-dialysis permits growth/maintenance of mineralizing, collagenous connective tissue from an inoculum of isolated osteoblasts over extended culture periods; i.e. at least up to 10 months. We have used this bioreactor as an in vitro tool to determine how metastatic breast cancer cells affect normal bone physiology. Outcomes of this work have led a number of important “firsts” that represent significant innovation in the aforementioned fields of endeavor. 1. Osteogenesis in vitro: from pre-osteoblasts to osteocytes: We have shown, for the first time, that macroscopic mineral deposits resembling bone (centimeter-scale pieces of white bone material chemically identical to real bone) can be grown from isolated bone cells in the bioreactor without the aid of any synthetic scaffolding materials. The cells morphology concomitantly evolve 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. 2. An in vitro mimic of bone-remodeling: We created an in vitro ‘bone-remodeling’ mimic to which metastatic cancer cells can be added and monitored over time. We have been able to add and differentiate osteoclasts from hematopoietic precursor cells isolated from mouse bone marrow. The pre-osteoclast cells when introduced onto the osteogenic tissue grown in the bioreactor, differentiated to form multinucleated mineral resorbing osteoclasts. The osteoclasts were functional and capable of digesting the osteoblast matrix. Re-infusing this co-culture system with pre-osteoblasts results in the re-filling of osteoclast resorption pits. Gene and protein expression data support the above observation that the degraded osteoblast tissue can be repaired by proliferating osteoblasts. 3. Challenging osteoblast tissue and osteoblast-osteoclasts co-cultures with breast cancer cells: Metastatic Breast cancer cells were added systematically to the osteoblast and osteoblast-osteoclast co-cultures. The osteoblast-breast cancer cell co-culture showed that important clinical hallmarks of cancer colonization of bone, including cancer cell penetration of tissue, colony formation and most importantly formation of single cell filing by cancer cells and alignment of cancer cells along the morphologically altered osteoblast tissue could be observed in vitro. When metastatic breast cancer cells were introduced onto the osteoblast-osteoclast co-culture (bone-remodeling mimic), the breast cancer cells migrated toward sites of active remodeling and clustered as an aggregation of cells that further degraded the osteoblast matrix. Systematic study of cancer cell interaction with ‘biosynthetic bone’ will yield novel insight into breast cancer colonization of bone. Apart from which, this model can also be used to study a wide array of osteopathologies such as osteoporosis and arthritis. Outcomes of the work will help clarify the etiology of cancer in bone and also create a much-needed in vitro tool for discovery of cancer therapeutics.