Photo-immobilized EGF chemical gradients differentially impact breast cancer cell invasion and drug response in defined 3D hydrogels

Abstract

Breast cancer cell invasion is influenced by growth factor concentration gradients in the tumor microenvironment. However, studying the influence of growth factor gradients on breast cancer cell invasion is challenging due to both the complexities of in vivo models and the difficulties in recapitulating the tumor microenvironment with defined gradients using in vitro models. A defined hyaluronic acid (HA)-based hydrogel crosslinked with matrix metalloproteinase (MMP) cleavable peptides and modified with multiphoton labile nitrodibenzofuran (NDBF) was synthesized to photochemically immobilize epidermal growth factor (EGF) gradients. We demonstrate that EGF gradients can differentially influence breast cancer cell invasion and drug response in cell lines with different EGF receptor (EGFR) expression levels. Photopatterned EGF gradients increase the invasion of moderate EGFR expressing MDA-MB-231 cells, reduce invasion of high EGFR expressing MDA-MB-468 cells, and have no effect on invasion of low EGFR-expressing MCF-7 cells. We evaluate MDA-MB-231 and MDA-MB-468 cell response to the clinically tested EGFR inhibitor, cetuximab. Interestingly, the cellular response to cetuximab is completely different on the EGF gradient hydrogels: cetuximab decreases MDA-MB-231 cell invasion but increases MDA-MB-468 cell invasion and cell number, thus demonstrating the importance of including cell-microenvironment interactions when evaluating drug targets.

Introduction

Breast cancer cell invasion through the stroma is influenced by signals the breast cancer cells receive from the microenvironment, including the extracellular matrix (ECM), mechanical properties, stromal cells, and cytokines, among others [[1], [2], [3], [4], [5]]. Of particular interest are gradients of chemoattractants (often cytokines) that arise in the microenvironment and guide cell invasion. In vivo, breast cancer cells have been shown to respond to and migrate towards increasing epidermal growth factor (EGF) concentrations secreted by macrophages within the microenvironment [6,7].

Epidermal growth factor receptor (EGFR) overexpression is associated with aggressive phenotypes, decreased patient survival, and occurs in approximately 6–19% of all breast cancers, and 30–52% of triple negative breast cancers – that is cells that are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) [[8], [9], [10], [11], [12]]. EGF is a known mitogen and causes increased cell motility, invasion, and proliferation through the mitogen-activated protein kinase (MAPK) and phospatidylinositol-3 kinase (PI-3K) downstream signalling pathways [[13], [14], [15], [16]]. However, EGF has also been shown to have an antitumorigenic effect on several EGFR overexpressing cancer cell lines including the triple negative breast cancer cell line MDA-MB-468 and the vulvar epidermoid cancer cell line A431, where exposure to high levels of EGF induces apoptosis [17,18]. It has been hypothesized that EGFR expression switches from a tumor promoting role in situ to a tumor inhibiting role in metastatic breast cancer [19]. It is therefore important to develop an in vitro model with EGF gradients to improve our understanding of the role of EGF and EGFR expression in breast cancer progression.

To study the role of EGF gradients on cancer cell invasion, a three-dimensional (3D) in vitro hydrogel model is needed: two-dimensional (2D) culture is inherently limited by a lack of matrix through which cells can invade and in vivo models are overly complex to study the role of defined gradients. Boyden chambers are widely used in invasion assays as the “gold standard”, but they produce poorly defined, transient gradients. Microfluidic devices have been used to form well-defined gradients; however, this strategy often lacks a matrix for cell invasion, forcing cells to migrate along hard plastic surfaces and poorly recapitulating invasion through native tissues [14]. Several studies have investigated the influence of EGF gradients on breast cancer cell invasion; however, migration was either along 2D surfaces or model compounds, such as dextran, were used as evidence of a relevant EGF gradient [14,20,21]. Truong et al. recently developed a microfluidic invasion platform to study breast cancer invasion through 3D matrices: SUM-159 breast cancer cells encapsulated in a hydrogel responded to a predicted EGF gradient with enhanced cell invasion and altered cellular morphology [20]. With this platform, diffusional gradients of EGF were predicted based on simulations with 10 kDa molecules – there was no direct characterization of the EGF gradients [20]. Wang et al. investigated EGF gradients with concentration slopes of 0.071, 0.143, and 0.286 ng mL⁻¹ μm⁻¹ to study MDA-MB-231 breast cancer cell migration, but did so along 2D surfaces that do not recapitulate invasion through ECM [14]. The current study is, to the best of our knowledge, the first to investigate the influence of well-characterized, immobilized EGF gradients on cell invasion in a 3D model.

3D hydrogels patterned with biomolecules in known concentrations and gradients have been previously designed [[22], [23], [24], [25], [26], [27], [28]]. For example, EGF gradients were patterned in hyaluronic acid (HA) hydrogels crosslinked with bismaleimide-poly (ethylene glycol) (PEG); however, these hydrogels did not allow cell invasion and matrix remodelling. To improve upon past hydrogel models, PEG crosslinkers were replaced by a matrix metalloproteinase (MMP) cleavable (GPQG↓IWGQ) peptide crosslinker (MMPx), which breast cancer cells can actively degrade through the expression of MMPs 1, 2, 3, 7, 8, 9 [[29], [30], [31]]. Furthermore, bromohydroxy coumarin (Bhc) was used as a caging molecule to facilitate EGF photopatterning: two photon irradiation cleaves Bhc, exposing a thiol that can immobilize thiol-reactive EGF [24]. However, Bhc isomerizes to form a product that quenches reactive thiols when irradiated [32,33]. When Bhc is modified with a methyl group at the endocyclic 3 position (mBhc), photoisomerization is blocked, thereby improving photopatterning efficiency over Bhc [32]. Nitrodibenzofuran (NDBF), another photocaging molecule, does not undergo photoisomerization and has been shown to efficiently uncage thiol-containing peptides with one- and two-photon irradiation [33]; however, NDBF, Bhc, and mBhc have not been directly compared in terms of photo-uncaging within 3D hydrogels. Herein, we compare NDBF, Bhc, and mBhc, and demonstrate the superior photopatterning efficiency of NDBF.

In the current study, a 3D in vitro breast cancer invasion platform was developed in NDBF-conjugated HA hydrogels crosslinked with MMPx (HA_NDBF/MMPx) (Fig. 1). HA hydrogels are ideal in studies of breast cancer cell invasion because HA is often overexpressed in the breast cancer microenvironment and provides an inherently bioactive and degradable material [[35], [36], [37], [38], [39], [40]]. Two-photon irradiation of HA_NDBF/MMPx cleaves NDBF, revealing a free thiol that subsequently reacts to immobilize maleimide-modified biomolecules into the hydrogel. To form gradients of EGF, maleimide-streptavidin (mal-streptavidin) is first patterned into HA_NDBF/MMPx. Adding biotinylated EGF modified with Alexa Fluor 555 (EGF555) for visualization of the patterns, results in EGF555 selectively binding to the immobilized streptavidin, forming a pattern of EGF555. Using this two-photon patterning approach, EGF gradients were formed in HA_NDBF/MMPx hydrogels, demonstrating the utility of NDBF for 3D photopatterning. These hydrogel platforms contain spatially defined EGF gradients that allow the role of EGF on breast cancer cell invasion to be studied.

The HA_NDBF/MMPx hydrogels containing EGF555 gradients are used to evaluate the response of breast cancer cell lines with different EGFR expression levels: MDA-MB-231, MDA-MB-468, and MCF-7 cell lines. MDA-MB-231 breast cancer cells are a highly invasive, triple negative breast cancer cell line that expresses EGFR. MDA-MB-468 breast cancer cells are an invasive, triple negative breast cancer cell line, with an intermediate invasive capacity that is lower than MDA-MB-231 cells but with very high EGFR expression. MCF-7 cells are a luminal A breast cancer cell line with low invasive potential that express low levels of EGFR [41,42]. We subsequently evaluate the response of the EGFR expressing breast cancer cells (MDA-MB-231 and MDA-MB-468) to the EGFR inhibitor, cetuximab. Cetuximab binds to the extracellular domain of EGFR at a higher affinity than EGF, preventing ligand binding and phosphorylation of the receptor [[43], [44], [45]]. Triple negative breast cancers tend to have the highest proportion of EGFR overexpression out of the breast cancer subtypes [[9], [10], [11], [12],46]. Since there are currently no hormone or receptor targeted treatment options for triple negative breast cancer, EGFR inhibitors have been evaluated clinically [47]. However, these EGFR inhibitor clinical trials have been unsuccessful; EGFR expression has not been predictive of patient response and EGFR activity has only been blocked in a minority of patients with EGFR expressing breast cancer [[48], [49], [50]]. Using breast cancer cell lines with different EGFR expression levels, we demonstrate how the same model microenvironment containing EGF gradients has dramatically different outcomes on breast cancer cell invasion and cetuximab response.