A double-network poly(N_ɛ-acryloyl l-lysine)/hyaluronic acid hydrogel as a mimic of the breast tumor microenvironment

Abstract

To mimic the structure of breast tumor microenvironment, novel double-network poly(N_ɛ-acryloyl l-lysine)/hyaluronic acid (pLysAAm/HA) hydrogels were fabricated by a two-step photo-polymerization process for in vitro three-dimensional (3D) cell culture. The morphology, mechanical properties, swelling and degradation behaviors of pLysAAm/HA hydrogels were investigated. The growth behavior and function of MCF-7 cells cultured on the hydrogels and standard 2D culture plates were compared. The results showed that pLysAAm/HA hydrogels had a highly porous microstructure with a double network and that their mechanical properties, swelling ratio and degradation rate depended on the degree of methacrylation of HA. The results of in vitro studies revealed that the pLysAAm/HA hydrogels could support MCF-7 cell adhesion, promote cell proliferation, and induce the diversification of cell morphologies and overexpression of VEGF, IL-8 and bFGF. The MCF-7 cells cultured on 3D hydrogels showed significantly increased migration and invasion abilities as compared to 2D-cultured cells. Preliminary in vivo results confirmed that the 3D culture of MCF-7 cells resulted in greater tumorigenesis than their 2D culture. These results indicate that the pLysAAm/HA hydrogels can provide a 3D microenvironment for MCF-7 cells that is more representative of the in vivo breast cancer.

Statement of Significance

Traditional 2D cell cultures cannot ideally represent their in vivo physiological conditions. In this work, we reported a method for preparing double-network poly(N_ɛ-acryloyl l-lysine)/hyaluronic acid hydrogel, and demonstrated its suitability for use in mimicing breast tumor microenvironment. Results showed the prepared hydrogels had controllable mechanical properties, swelling ratio and degradation rate. The MCF-7 cells cultured in hydrogels expressed much higher levels of pro-angiogenic growth factors and displayed significantly enhanced migration and invasion abilities. The tumorigenic capability of MCF-7 cells pre-cultured in 3D hydrogels was enhanced significantly. Therefore, the novel hydrogel may provide a more physiologically relevant 3D in vitro model for breast cancer research. To our knowledge, this is the first report assessing a HA-based double-network hydrogel used as a tumor model.

Introduction

Breast cancer is the second leading cause of death after lung cancer in women worldwide, and about one in eight women will develop metastatic breast cancer in their lifetime [1]. However, the current first-line treatment drugs have their inherent shortcomings [2], and the development of new anticancer drugs is being hindered by the lack of effective tumor models that closely mimic the human disease [3]. Researchers have long relied on two-dimensional (2D) in vitro cell culture systems to study cancer cells [4], but they often fail to mimic the natural tumor microenvironment such as cell–cell communication and cell-extracellular matrix (ECM) interactions which play a key role in controlling cancer cell growth and function [5], [6]. In the case of 2D cultures, cells are forced to adopt a planar morphology [7], which alters cell proliferation, migration and invasion, and anti-apoptosis [8], [9]. As a result, cells generally display a dramatically reduced malignant phenotype when compared to the tumor in vivo [10]. Therefore, the traditional 2D cell cultures cannot ideally represent in vivo physiological conditions [11].

To overcome these limitations, three-dimensional (3D) culture systems are now increasingly developed for the tumor engineering [12], [13]. In 3D cultures, cells are often embedded within a matrix material that can mimic the tumor microenvironment (TME), where they can migrate and experience cell-matrix interactions and cell–cell contacts in all directions. Some studies have shown that the tumor cells grown in 3D cultures tend to develop the morphologies and phenotypes observed in vivo [14], [15], [16], display higher aggressiveness, overexpress pro-angiogenic growth factors and acquire increased drug resistance relative to the cells in 2D cultures [17], [18]. However, how to design and build a reasonable 3D culture model for tumor engineering is still a big challenge. An ideal 3D model (matrix) should possess the following characteristics: excellent biocompatibility, good mechanical properties matching with the tumor tissue, easy separation of the cells from the matrix, similar microstructure and chemical components to the tumor tissue, controllable degradability, favorable nutrient exchange, controlled release of bioactive substances, and allowing the vascularization of matrix in vivo. The well-designed 3D tumor co-culture models have been used for understanding tumorigenesis and the high throughput screening of novel drugs [19], [20]. In recent years, both animal-source polymers (e.g. Matrigel [21], collagen [10] and soft salmon fibrin [22]) and synthetic polymers (e.g. poly(lactic-co-glycolic acid) [16], poly(ethylene glycol) diacrylate [23]) have been used to create matrix-derived 3D tumor models. These 3D models have the potential to increase cancer cell malignancy and retain the in vivo phenotype. However, these animal-source materials can potentially transmit pathogens [24], and the synthetic materials are physiologically irrelevant [25] and can release degradation products that are toxic to cells. Therefore, these materials have limited applications in vitro tumor engineering.

Hyaluronic acid (HA) has been used for the construction of 3D tumor models due to its biodegradable, non-immunogenic and non-inflammatory characteristics. HA is not only a structural component in the tumor ECM but also a biologically active molecule that can promote tumor progression through cell signaling [26]. Additionally, HA protects tumor tissues against immune surveillance and chemotherapeutic agents [27]. These unique properties make HA an ideal matrix to prepare 3D tumor models. However, classic HA hydrogels with a single cross-linked network lack structural integrity that may lead to poor mechanical properties [28]. Some researchers have developed a few methods to strengthen HA hydrogels. For example, rigid inorganic nanoparticles such as silica [29] and hydroxyapatite [30] were employed to fill HA hydrogels. However, these nanoparticles are non-degradable or cytotoxic [31], [32]. Recently, the fabrication of double-network hydrogels is recognized as a promising method for strengthening hydrogels [33], [34]. On the other hand, it is known that poly-l-lysine (PLL), as a widely used cationic polymer, has an outstanding ability to promote cell attachment through electrostatic interactions [35]. Given that poly(N_ɛ-acryloyl l-lysine) (pLysAAm) and PLL have a similar chemical structure, they would share similar properties. Based on this hypothesis, a photo-crosslinked pLysAAm network was synthesized and used to modify HA hydrogel to create a more biomimetic microenvironment for breast tumor engineering. To our best knowledge, HA-based double-network hydrogels have not been used to fabricate 3D tumor models.

Here, we describe for the first time the preparation and in vitro biocompatibility of novel breast cancer TME-mimicking double-network pLysAAm/HA hydrogels. The double-network hydrogels were synthesized via a two-step photo-polymerization process. The effects of the degree of methacrylation of HA on the morphology, viscoelasticity, swelling and degradation properties of the pLysAAm/HA hydrogel were studied. In the present study, human breast cancer MCF-7 cells were chosen as model cells, since they have been proved to be responsive to 3D environment and extensively used in 3D in vitro breast cancer models [10], [36], [37]. The attachment, growth and function of MCF-7 cells in pLysAAm/HA hydrogels, including proliferation, morphology, migration, invasion and the secretion of pro-angiogenic growth factors VEGF, IL-8 and bFGF, were examined in vitro. In vivo tumorigenesis was also assessed by using xenograft formation in athymic nude mice. The results demonstrated that the MCF-7 cells cultured in 3D pLysAAm/HA hydrogel displayed a more malignant phenotype than those in traditional 2D cultures, indicating that the hydrogel is a promising matrix for the development of physiologically relevant in vitro 3D breast cancer models.