Bioengineering embryonic stem cell microenvironments for exploring inhibitory effects on metastatic breast cancer cells

Abstract

The recreation of an in vitro microenvironment to understand and manipulate the proliferation and migration of invasive breast cancer cells may allow one to put a halt to their metastasis capacity. Invasive cancer cells have been linked to embryonic stem (ES) cells as they possess certain similar characteristics and gene signatures. Embryonic microenvironments have the potential to reprogram cancer cells into a less invasive phenotype and help elucidate tumorigenesis and metastasis. In this study, we explored the feasibility of reconstructing embryonic microenvironments using mouse ES cells cultured in alginate hydrogel and investigated the interactions of ES cells and highly invasive breast cancer cells in 2D, 2&1/2D, and 3D cultures. Results showed that mouse ES cells inhibited the growth and tumor spheroid formation of breast cancer cells. The mouse ES cell microenvironment was further constructed and optimized in 3D alginate hydrogel microbeads, and co-cultured with breast cancer cells. Migration analysis displayed a significant reduction in the average velocity and trajectory of breast cancer cell locomotion compared to control, suggesting that bioengineered mouse ES cell microenvironments inhibited the proliferation and migration of breast cancer cells. This study may act as a platform to open up new options to understand and harness tumor cell plasticity and develop therapeutics for metastatic breast cancer.

Introduction

Invasive breast cancer represents more than 75% of new cases diagnosed in 2009 according to American Cancer Society. As cancer cells grow, they can break away from the main malignant tumor and metastasize to other parts of the body by passage through the bloodstream and lymphatic system, so called cancer metastasis [1]. Tumor invasion and metastasis is the primary cause of death for patients with breast cancer [2]. However, it is difficult to investigate cancer metastasis in vivo [1]. In invasive breast cancer, the loss of epithelial polarity and the rupture of basement membrane occurred. Therefore, tumor cells are in direct contact with a remodeled microenvironment [3]. The surrounding microenvironment of breast cancer cells plays a critical role in tumorigenesis and metastasis [4], [5]. The cancer cell microenvironment is constituted of stroma, soluble factors, and cell–cell interactions [6]. Cross-talk between tumor and stromal cells in the breast tumor microenvironment triggers the secretion of molecules essential for basement membrane penetration and mediators of metastasis in breast cancer [7], [8], [9]. Bioengineering a tumor microenvironment, which can manipulate the proliferation and migration of breast cancer cells, has great potential to understand and inhibit breast cancer metastasis and lead to the prevention and correction of invasive breast cancer.

Metastatic cancer cells share some similarity with embryonic stem (ES) cells (e.g., high proliferative capacity versus self-renewal, tumor initiating versus teratoma formation, and expression of certain pluripotency markers) [10], [11], [12], [13]. Metastatic cancer cells interact dynamically with a microenvironment that facilitates plasticity, tumorigenicity and metastasis [7], [8], while ES cells sustain a microenvironment regulating self-renewal and differentiation [10]. Recent research has linked an ES-like gene expression signature with poorly differentiated aggressive human breast cancer cells [11]. Pluripotent stem cells have the potential to help elucidate early events during tumorigenesis and metastasis, including potential miRNA and epigenetic targets, tumor initiator and suppressor networks, which is unachievable using primary tumor-based studies [12].

It has been demonstrated that the embryonic microenvironment plays a key role in inhibiting cancer metastasis [13], [14], [15]. In particular, it has been shown that the human ES cell microenvironment could reprogram metastatic melanoma cells to assume a less invasive phenotype [16]. On the contrary, human ES cell-differentiated teratomas showed to support the growth and invasion of ovarian tumor cells [17]. This indicates that the undifferentiated embryonic stem cell microenvironment could inhibit cancer metastasis. The in vitro investigation of ES cell-cancer cell interactions will provide valuable insight into fundamental understanding of tumor progression and therapeutic development. As far as breast cancer is concerned, however, little research on the interaction between ES cells and breast cancer cells has been reported. This is partially due to the lack of a suitable in vitro model system which can be used to examine ES cell-breast cancer cell interactions.

Human breast cancer cell line culture has been widely used to investigate cell migration and some important information has been elucidated [18], [19]. With the advancement of microtechnology, cell printing and patterning techniques provide a platform for spatial control of the location of cells and observation of the cell–cell interaction [20], [21], [22], [23], [24], [25], [26], [27]. When breast cancer cell lines were cultured as monolayers on 2D plastic substrata or patterned surfaces, however, these cells could not recapture the tumor architecture and missed certain important signals in a tumor microenvironment [12], [13], [28], [29]. Better culture systems are therefore needed to better recapitulate tumor microenvironment for prolonged periods of time. It is well known that the cell culture environment can have a dramatic influence on cell behavior [14]. Bioengineered 3D in vitro models for studying cell–cell interactions can bridge the gap between 2D cell cultures and whole-animal systems [15], [30], [31]. These 3D models provide a unique perspective for stem cell and cancer research [32], [33], [34], [35], [36], [37]. 3D hydrogel-based culture systems have the potential to provide in vivo-like cell-extracellular matrix (ECM) interactions and cell-matrix adhesions [38], [39], [40], [41], [42], [43]. Bissell and colleagues have pioneered 3D gel models to reconstruct normal and malignant breast tissue architecture [44]. Normal epithelial or breast cancer cells can either be embedded in or grown on basement membrane-like gels, which are used to mimic the in vivo ECM and provide environmental cues [45], [46]. 3D hydrogel-based culture systems have also been used to bioengineer embryonic stem cell microenvironments [47], [48], [49], [50], [51], [52], [53]. In particular, the alginate hydrogel system is superior for studies of cell–cell interactions due to the lack of cell recognition signals in the alginate itself [54], [55]. Alginate hydrogel has been used to culture human breast cancer cells, in which 3D multicellular tumor spheroids were formed [56], [57], [58], and to culture mouse or human ES cells for the maintenance of the pluripotency and directed differentiation of ES cells [59], [60], [61], [62], [63], respectively.

In this study, we explored the feasibility of reconstructing embryonic stem cell microenvironments using the alginate hydrogel for the study of metastatic breast cancer cell proliferation and migration. Mouse CCE ES cells were used as a model system to reconstitute the embryonic microenvironment in vitro. Highly invasive rat mammary carcinoma MTLn3-Mena^INV cells were chosen for this study due to their highly metastatic potential [64], [65]. First, mouse ES cells and metastatic breast cancer cells were co-cultured in 2D microwells, 2&1/2D alginate hydrogel films, and 3D alginate hydrogel microbeads. Then an in vitro embryonic microenvironment was constructed by culturing mouse ES cells in 3D alginate hydrogel microbeads followed by the addition of metastatic breast cancer cells. The effects of ES microenvironments on the proliferation and migration of breast cancer cells were explored.