Microencapsulation of low-passage poorly-differentiated human mucoepidermoid carcinoma cells by alginate microcapsules: in vitro profiling of angiogenesis-related molecules

Human mucoepidermoid carcinoma (MEC) is the most common type of malignant salivary gland carcinomas (SGCs) [1]. MECs are histologically heterogeneous, including variable proportions of epidermoid, intermediate and mucinous cells, which are organized into solid or cystic patterns. Based on cellular compositions and other histopathological parameters, MECs are graded into low, intermediate and high grade [1, 2]. The tumor grade is determinant to the prognosis of MEC patients, with high-grade MECs having significantly worse survival rates and higher risk of recurrence after primary surgical resection in comparison with low-grade MECs [1, 3]. However, current curative treatments for high-grade MECs are under debate and notoriously ineffective [1, 3].

Tumor angiogenesis, an integral hallmark of cancer, has been revealed as a critical step for tumor growth and metastasis [4]. In consistent with this notion, we previously found that MECs also undergo vigorous angiogenesis possibly due to in situ proliferation of vascular endothelial cells in the three-dimensional (3D) microenvironment [5, 6]. Our results implied that MEC histological grades and stages are positively correlated with cancer neovascularization [6, 7]. Furthermore, in advanced stage and/or high-grade MECs with poor prognosis, cancer cells showed higher expression levels of inhibitors of DNA binding/differentiation protein 1 (Id-1), a key pro-angiogenic transcriptional factor, and lower expression levels of thrombospondin 1 (TSP-1), a key anti-angiogenic protein ligand [6‐8]. Therefore, elucidating molecular mechanisms underlying the pro-angiogenic ability of poorly differentiated high-grade MEC cells is critical for the understanding of high-grade MEC progression.

In vitro cell culture models using patient-derived cancer cell lines allow more detailed high-throughput studies of cancer-related properties and processes, such as tumor angiogenesis [9]. This has provided valuable insights into cancer progression and cancer therapies. However, such two-dimensional (2D) culture models using established human cancer cell lines have major deficiencies, including the lack of cellular heterogeneity reflective of the original malignancy and an improper tumor microenvironment, both of which are critical for cancer development and treatment resistance [9]. The former obstacle has begun to be tackled with the emerging use of tumorigenic low-passage cancer cell lines, which can better represent the heterogeneity and complexity of the parental cancers [10]. For the later obstacle, it is well known that in contrast with conventional 2D cultures, three-dimensional (3D) cell cultures provide a better in vitro approach to recapitulate in vivo characteristics of cancer cells, such as cell–cell and cell-extracellular matrix (ECM) interactions and to mimic in vivo 3D tumor microenvironment [9, 11]. In the present study, we modeled a 3D environment with an inert and biocompatible sodium alginate hydrogel matrix for low-passage poorly-differentiated MEC cells, profiled in vitro expression of key molecules for cancer neovascularization and compared them with traditional 2D cell culture.

When cultured in 2D, the heterogeneous low-passage poorly-differentiated human MEC cells (passage 10) were observed predominantly as polygonal epidermoid cells with variable cell sizes. Those cancer cells all exhibited a giant and rounded nucleus and showed a high karyoplasmic ratio (Fig. 1A). Some of these epidermoid cells showed mitosis (Fig. 1A). There were also some small or larger and more oval cells, presumably the intermediate cancer cells (Fig. 1A). Multinucleated giant cells or megakaryocytes were rarely observed (Fig. 1A). Moreover, some but a few of those cancer cells were revealed by mucicarmine staining as mucin-producing mucinous cells in individuals and clusters (Fig. 1B). Those mucinous cells also showed their characteristic morphology, acinar pyramidal or goblet-like (Fig. 1B). While the positive staining of cytokeratin in the cytoplasm and membrane of nearly all the MEC cells demonstrated their epithelial origin (Fig. 1C), the negative staining of vimentin of all the MEC cells exclude cells of mesenchymal origin (Fig. 1D). Those morphological and molecular characteristics were consistent with poorly-differentiated cancer cells seen in high-grade human MECs [1, 2], which implied the successful establishment and maintenance of heterogeneous low-passage poorly-differentiated human MEC cell lines.

Low-passage poorly-differentiated human MEC cells of the same batch for 2D cell culture were encapsulated in APA microcapsules (Fig. 2A) and maintained for a period of 7 days. The cell-loaded APA microcapsules have smooth and luster surfaces (Fig. 2A, B) and remained intact without dissolution or damage after the time of culture (Fig. 2B). Microencapsulated poorly-differentiated human MEC cells grew in cell microspheres and remained within the APA microcapsules (Fig. 2B). Scanning electron microscopy showed that microencapsulated poorly-differentiated human MEC cells adhered onto the inner surfaces of the APA microcapsules, which were filled with high densities of micropores of similar sizes (Fig. 2C). Those micropores would facilitate cancer cell interactions and growth [13]. In consistent with this assumption, our results showed that in comparison with 2D cell culture, poorly-differentiated human MEC cells cultured as 3D in the APA microcapsules proliferate stronger and relatively unlimited (Fig. 2D). In detail, significantly higher proliferation activities were seen for 3D cultured cancer cells during late but not early culture periods (1st day: 2D, 0.3053 ± 0.0198; 3D, 0.2493 ± 0.0170, P > 0.05; 2nd day: 2D, 0.3923 ± 0.0254; 3D, 0.4240 ± 0.0127, P > 0.05; 3rd day: 2D, 0.7423 ± 0.0272; 3D, 0.8760 ± 0.0272, P < 0.05; 4th day: 2D, 1.2430 ± 0.0481; 3D, 1.6060 ± 0.0560, P < 0.01; 5th day: 2D, 1.6330 ± 0.0412; 3D, 1.8350 ± 0.0340, P < 0.05; 6th day: 2D, 1.8200 ± 0.0443; 3D, 2.1540 ± 0.0441, P < 0.01; 7th day: 2D, 1.7280 ± 0.0512; 3D, 2.2510 ± 0.0659, P < 0.01). Moreover, while cell proliferation reached the plateau after 5 days during 2D culture, there were no signs of proliferation plateau during 3D culture. These results indicated that the APA microcapsule provides a suitable model of 3D culture for low-passage poorly-differentiated human MEC cells.

Then, we assessed protein expression of key molecules for cancer neovascularization [4] in 3D cultured low-passage poorly differentiated MEC cells and compared them with traditional 2D cell culture. There was a significant increase for 3D cultured cancer cells in the protein expression of key pro-angiogenic molecules, such as VEGF-A (3D: 2.313 ± 0.110 vs. 2D: 1.124 ± 0.111, P < 0.01) (Fig. 3a, b) and bFGF (3D: 2.687 ± 0.086 vs. 2D: 1.999 ± 0.077, P < 0.01) (Fig. 3a, c). Furthermore, the protein expression of TSP-1, a key anti-angiogenic molecule, was drastically reduced in 3D cultured cancer cells (3D: 0.197 ± 0.020 vs. 2D: 0.484 ± 0.011, P < 0.001) (Fig. 3a, d). Next, we further examined with ELISA the release of these growth factors for tumor angiogenesis into the tumor microenvironments, taking VEGF-A as an example. We found that 3D cultured cancer cells secreted significantly more VEGF-A than 2D culture cancer cells (3D: 2553 ± 40.26 pg/mL vs. 2D: 1941 ± 71.07 pg/mL, P < 0.001) (Fig. 4). Therefore, low-passage poorly differentiated MEC cells cultured in a 3D microenvironment demonstrated significant intensification of key molecular processes for tumor angiogenesis.

Next, we examined the hypoxic environment in 3D versus 2D culture, since tumor hypoxia has been recognized as a critical environment factor for tumor angiogenesis. Either western blotting (Fig. 5a, b) or immunofluorescence (Fig. 5c) for HIF1α showed that compared with 2D-grown cancer cells, 3D-grown cancer cells showed significantly higher expression of HIF1α, a molecular indicator of the hypoxic environment (for western blotting: 2D, 0.4190 ± 0.0205; 3D, 0.5287 ± 0.0297; P < 0.01). This implied that cancer cells during 3D culture experienced significant hypoxic environment. Then, we assessed the effect of hypoxia on VEGF-A gene expression during 3D culture with the use of the HIF1α inhibitor 2-MeOE2. Our results showed that specific inhibition of the HIF1α activities almost completely reversed the increased expression of VEGF-A in 3D cultured cancer cells (2D, 1.1610 ± 0.0384; 3D, 2.3220 ± 0.0383; 3D + HIF1α inhibitor, 1.0010 ± 0.0386) (Fig. 5d, e). This implied that the increased expression of VEGF-A during 3D culture would be dependent on the HIF1α activities, which were induced by hypoxia in 3D culture.

In this study, we established a 3D culture system for low-passage poorly-differentiated human MEC cells with alginate microencapsulation. Compared with conventional 2D adhesion culture, poorly-differentiated human high-grade MEC cells, cultured in the 3D microenvironment of APA microcapsules, proliferated more actively and showed significant higher potentials to promote tumor angiogenesis. This is due to a better modeling of the tumor microenvironment, such as hypoxia, during 3D culture.

3D culture techniques, instead of the well-established 2D monolayer cell culture, have been widely employed for in vitro experiments in recent cancer studies [9, 11]. It has reached a consensus that 3D culturing offers a better platform to recapitulate in vivo characteristics of cancer cells, therefore facilitating the translation of preclinical findings into clinical applications [9, 11]. When cultured in 3D microenvironments, heterotypic tumor cell spheroids allow tumor cells and stromal cells to build cell–cell and cell-ECM interactions and their surrounding microenvironment, both of which are critical parameters in tumor development and modulate tumor responses to therapeutic interventions [15]. The multicellular tumoroids can been grown in scaffold-free engineered devices that maximize cell–cell interactions and solute transport, such as tissue engineering bioreactors and microfluidic devices [11]. However, those multicellular spheroids under those low-adherence conditions do not capture the mechanical stiffness of cell substrates in vivo, which also contributes to tumor development [14]. Alternatively, tumor aggregates can be cultured when embedded in bioactive scaffolds such as matrigel or collagen I [11, 15]. However, these bioactive scaffolds have certain limitations, such as batch-to-batch variations and the undesirable interferences on cancer cell behaviors [11, 15]. For the reasons described above, we employed cell microencapsulation using alginate hydrogel as inert cell scaffolds to model a 3D microenvironment for low-passage poorly-differentiated human high-grade MEC cells.

As one of the naturally derived anionic polysaccharides, alginate hydrogel has many advantages over bioactive scaffolds, including their inert properties for cell behaviors, biocompatible for cell microencapsulation/culture, mechanical flexibility/stability and ease of cell recovery [13‐15]. Although alginate per se is typically non-adhesive to cells, most cells show the ability to grow in non-functionalized alginates [15]. In addition, with controllable diameters, porosity and stiffness, alginate hydrogel bears a structural resemblance to the ECM [14]. Originally developed for tissue engineering [16, 17], cell microencapsulation with alginate hydrogels has been recently adopted as a suitable model for 3D in vitro culture in cancer researches [11]. Previous studies showed that alginate microencapsulation enhanced cancer cell proliferation and survival, modulated cancer cell migration and invasiveness and exacerbated tumor malignancies [14, 15, 18‐21]. Our results demonstrated that alginate microencapsulation increased the proangiogenic potentials of low-passage poorly-differentiated human MEC cells. In tumor microenvironment where angiogenesis inducers override its inhibitors, the angiogenic switch is almost always activated and remains on to sustain cancer neovascularization [4]. VEGF-A and TSP-1 are the well-known angiogenesis inducer and inhibitor, respectively [4]. Accordingly, our results showed that when cultured in 3D microenvironment of APA microcapsules, low-passage poorly-differentiated human MEC cells significantly increased the autocrine production of VEGF and drastically decreased the autocrine production of TSP-1.

Hypoxia, a feature of most tumors [22], has been revealed as a critical parameter for cancer development and progression, including tumor angiogenesis [4, 22]. Tumor hypoxia simultaneously increases angiogenesis inducers, such as VEGF-A, and decreases angiogenesis inhibitors, such as TSP-1, thus activating and maintaining the angiogenic switch [4, 22]. As shown, low-passage poorly-differentiated human MEC cells in APA microcapsules formed multicellular spheroids, despite possibly the lack of a complex 3D structure seen in solid tumors. However, multicellular tumor cell aggregates enabled cancer cells to experience a controlled 3D environment seen in solid tumors, where a gradient of chemical and critical biological signals, such as nutrients and oxygen, promote the assembly of “tumors tissue type” [15]. Therefore, cancer cells in 3D are under hypoxic conditions [15, 21]. In contrast, low-passage poorly-differentiated human MEC cells in 2D monolayer culture are exposed to a uniform environment with sufficient availability of nutrients and oxygen [9, 21]. This is reflected by the differential expression of HIF1α for 2D versus 3D cultured cancer cells. As shown in the present study, hypoxia in 3D and normoxia in 2D would underlie the differences of gene expression important for cancer neovascularization. Hence, in vitro 3D culture models better present tumor environments in vivo and should be appreciated in future human MEC studies.

Another advantage of our study is the use of low-passage poorly-differentiated MEC cell lines, which would be a more physiologically and clinically relevant sources of high-grade human MEC cells [9]. While some human MEC cell lines have been established [23‐25], long-term in vitro maintenance leaves those high-passage cancer cell lines vulnerable to the selection of dominant cell populations and the accumulation of additional genomic and epigenomic alterations [9]. In fact, the most commonly used established cancer cell lines have been recently shown no correlation with original clinical samples [26]. This compromises the feasibility of those high-passage established cancer cell lines for in vitro testing [9]. It has been suggested that low-passage cancer cell lines, in contrast to high-passage counterparts, well reflect the biology of the original tumor, such as morphology, growth behavior, and mutational profile [27] and are therefore a versatile resources of cancer cells for preclinical in vitro studies. In fact, recent studies showed that tumorigenic low-passage cancer cell lines are more relevant to the parental cancers [10, 28, 29].

However, there are some limitations in our present study. First, we did not directly assess angiogenesis induced by low-passage poorly-differentiated MEC cells cultured in 2D and 3D environments, neither in vitro through cell co-culture systems [5, 30] nor in vivo through xenograft into nude mice. Second, it remains to identify the ideal microencapsulation protocols using alginate hydrogel for low-passage poorly-differentiated MEC cells. It has been suggested that cell microencapsulation conditions must be optimized for any new cell line to get high cell performance [31]. The candidate parameters includes variations in M/G content of sodium alginate, alginate hydrogel modifications, gelling ions (Ca²⁺, Ba²⁺), microspheres with solid versus hollow cores, and cell conditions with single cells versus spheroids [31]. This screening will boost preclinical in vitro studies of MEC using 3D cell culture models.

When cultured in a 3D microenvironment by alginate microencapsulation, low-passage poorly differentiated human MEC cells, derived from human patient samples of high-grade MEC, change their proliferation behavior and show significant intensification of key molecular processes for tumor angiogenesis. This is due to a better modeling of the hypoxic tumor microenvironment during 3D culture. These results reinforce the utilization of 3D cell cultures, such as alginate microencapsulation, to mimic the tumor microenvironment in vitro for performing in vitro cancer studies more relevant to clinical conditions.