Cancer stem cells (CSCs) are postulated to be central to establishment of metastases and the main challenge to the cure of cancer [1
]. Currently, however, the use of CSCs in research is limited by the small number of CSCs that can be isolated, and the spontaneous differentiation in in vitro
cultures. The challenge of in vitro
CSC culture is likely due, at least in part, to the lack of supportive microenvironmental niches [1
] in conventional two-dimensional (2D) cultures. Bone metastasis, which is the most severe complication and leading cause of morbidity and ultimately mortality in prostate cancer [6
], provides clues for recreating a supporting CSC niche environment for prostate cancer cells. Recent data from our group suggests that prostate cancer utilizes the hematopoietic stem cell (HSC) homing mechanisms to metastasize to the bone marrow and thrive in the niche [8
]. Based on this hypothesis that cancers parasitize the niche, we have developed microscale 3D spheroid culture of prostate cancer cells supported by cells from the HSC niche. Here, we describe a microfluidic 3D culture system that recapitulates the in vivo
growth behavior of malignant prostate cancer cells, specifically PC-3 cells, through construction of an in vitro
bone metastatic prostate cancer microenvironment.
To develop a supportive metastatic prostate cancer model, we hypothesized that it would be crucial to culture the cells in 3D along with the surrounding cells in the microenvironment that the metastatic prostate cancer cells reside in [10
]. For example, cells are known to proliferate at a much slower rate that is more physiological when cultured in 3D than 2D [13
]. It is also known that prostate cancer cells not only proliferate differently when co-cultured with other stromal cells or fibroblasts, but can also affect the proliferation rates of the other cell types under various in vitro
and in vivo
]. We adopted co-culture spheroids as a 3D prostate cancer niche model.
Spheroids are sphere-shaped cell colonies formed by self-assembly that allow various growth and functional studies of diverse tissues [19
]. Spheroids serve as excellent physiologic tumor models as they mimic avascular tumors and micrometastases [20
] and are known to provide more reliable and meaningful therapeutic readouts [21
]. Although these advantages of tumor spheroids has been widely recognized [22
], challenges involved in the tedious procedures required for formation, maintenance, solution exchange, and microscale cell and fluid manipulation are still holding back the industry from using the well-validated spheroid tissue model more widely.
Formation of spheroids occurs spontaneously, in environments where cell-cell interaction dominates over cell-substrate interactions. Typical methods for spheroid generation include hanging drops, culture of cells on non-adherent surfaces, spinner flask cultures, and NASA rotary cell culture systems [23
]. Recently, various groups have also developed spheroids on a chip works utilizing microscale technologies such as microwell arrays and microfluidic devices [25
]. There have also been spheroid co-culture works including co-culture of endothelial cells with fibroblasts and smooth muscle cells using hanging drops [22
]. Metastatic prostate cancer cell line PC-3 cells have been co-cultured with fibroblasts using the NASA rotary cell culture system [19
]. Many of these techniques, however, suffer from problems such as efficiency of forming spheroids, long-term culture, control of spheroid size, and uniform distribution of small numbers of co-culture cell types across all spheroids. Here, we apply a microfluidic spheroid formation technology used previously to form embryoid bodies [34
] to the formation of heterogeneous co-culture spheroids of PC-3's supported by osteoblasts and endothelial cells as a model of the niche microenvironment for prostate cancer metastasis to the bone.