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

Abstract

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.

Primary Human Osteoblasts Cultured in a 3D Microenvironment Create a Unique Representative Model of Their Differentiation Into Osteocytes

“…In tissue engineering, the upregulation of ALP expression relates to osteogenesis and is a precursor to mineralization of the tissue (Golub and Boesze-Battaglia, 2007). Previous studies in microfluidic devices only showed the upregulation of ALP activity at the end of the culture, proving that those platforms promoted osteogenesis (Leclerc et al, 2006;Jang et al, 2008;Hao et al, 2018). Here, we measured the temporal evolution of ALP activity during the whole culture and it suggested that osteoblasts cultured at high density had a phase of increase of mineralization in the first week of culture and later continued the differentiation process.…”

“…These systems consisted of 3D collagen hydrogels/sponges and human osteoblast-like cells (Atkins et al, 2009;Bernhardt et al, 2019;Skottke et al, 2019) or mouse clonal cell-lines (MC3T3-E1 and IDG-SW3) (Woo et al, 2011;McGarrigle et al, 2016), with the last ones usually preferred due to the excessive number of cells required. On a micro-scale, a 2D layer of MC3T3-E1 cells spontaneously formed a mineralized collagenous matrix in long-term cultures (up to 1 month) (Hao et al, 2018). Cells were mainly present in the apical and basal layers of the matrix, possibly due to excessive proliferation of the mouse cell-line.…”

Emerging and Established Models of Bone Metastasis

“…Despite the push towards a focus on in vivo models by some, others believe that the ideal way to investigate the complex molecular mechanisms involved in this process is by advanced in vitro modeling [ 94 , 95 , 96 , 97 , 98 , 99 , 100 ]. These models consist of microfluidic models or advanced mathematical modeling, among others.…”

“…This allows for metastatic migration from a 3D origin tissue to a 3D target tissue, within a controllable fluidic environment [ 95 ]. Four models for bone metastasis in a microfluidic model have been identified in the literature [ 97 , 101 , 102 ]. Bersini and Jeong [ 101 , 102 ] used a tri-culture system, consisting of osteo-differentiated human bone marrow (h-BM) mesenchymal stem cells (MSCs), endothelial cell monolayer, and human breast cancer cells (MDA-MB-231).…”

“…Through the use of this novel model, the authors also identified that the myoblast cell line C2C12 had a protective effect against metastasis. Finally, the most recent microfluidic model to be introduced is from Hau et al [ 97 ] The authors attempted to identify weak areas in the model presented by Jeong and Bersini and the main limitation to improve upon was to allow maturation and growth of the osteoblastic cell lineage, allowing mineralization and natural collagen fiber organization that may be involved in the complex underlying metastatic mechanisms. This was performed by using a miniaturized bone-on-a-chip model consisting of two compartments.…”