Targeted-TERS detection of integrin receptors on human cancer cells

Abstract

Membrane receptors play important roles in regulating cellular activities. Targeting membrane receptors in cancer cells and understanding their interactions with specific ligands are key for cancer prognosis and therapeutics. However, there is a need to develop new technologies to provide molecular insight into ligand-receptor binding chemistry in cell membrane. Integrin receptors are important membrane receptors that regulate cellular migration, invasion and proliferation in tumors. Integrins have a well-known affinity towards small peptide ligands containing arginine-glycine-aspartate (RGD) sequence and are therefore an attractive model system to study ligand-receptor interactions. We have recently reported a method to detect integrin receptors and study their binding chemistry with cyclic-RGDfC ligand using tip-enhanced Raman scattering (TERS). We have demonstrated that two integrins with similar structures can be differentiated in intact cell membrane, due to the differences in their RGD ligand binding sites, showing the potential of this TERS methodology to study other membrane receptors and their interactions in live cells.

While cellular membrane receptors are key molecules that initiate signaling pathways to regulate cellular activities and have been strongly associated with cancer progression, the tools available to identify relevant chemical reactions are limited. Recently we demonstrated that tip enhanced Raman scattering (TERS) may provide new insights into understanding the chemical interactions between specific extracellular molecules (so-called “ligands”).

Many overexpressed membrane receptors have been identified as hallmarks of various types of tumors, leading to diagnosis and treatment strategies that target these receptors ^{[1, 2]}. One widely studied example is integrin receptors. These receptors are important cell adhesion receptors that act as bridge molecules for cell-cell and cell-extracellular matrix (ECM) interactions. By binding with ECM ligands, integrins activate intracellular signal transduction pathways in tumors and mediate tumor cell migration and invasion ^[3]. Because of these biological roles in tumors, integrins have been recognized as molecular markers for targeted cancer imaging ^{[4, 5]}. For example, arginine-glycine-aspartate (RGD) peptide conjugated magnetic nanoparticles were found to provide significantly enhanced MRI image contrast at tumor areas, mediated through RGD-integrin interaction, in a xenograft model ^[6]. In addition, integrins have been targeted for drug developments in therapeutics of human cancers. Preclinical studies have shown that integrin antagonists, including monoclonal antibodies and RGD peptides, can inhibit tumor growth ^{[7, 8]}. For example, Cilengitide, a cyclic-RGD based drug molecule, which is a highly potent antagonist of integrins αvβ3, αvβ5 and α5β1, reached clinical phase III trials for treatments of glioblastomas ^[9], although some limitations of the drug have been reported recently ^[10].

The pharmacological potential of targeting integrin receptors has drawn great attention. To develop novel drugs with high efficacy and reduced side effects, understanding the interactions between integrins and ligand antagonists (e.g. RGD molecules) is critical ^[11]. Generally, understanding ligand-receptor binding and targeting pathological cell membrane receptors is key for regulating cellular signaling and activity ^{[12, 13]}, and for imaging and treating diverse human pathologies such as cancers ^{[14, 15]}. In recent years, significant progress has been made on the use of radiolabeled RGD peptides, as radiotracers, to recognize and monitor integrins in cancer patients ^[16]. Furthermore, interactions between RGD peptides and integrin receptors have been investigated at cellular and molecular levels using advanced microscopic techniques such as fluorescence microscopy ^[17] and atomic force microscopy ^{[18, 19]}. Although these techniques are able to visualize ligand-receptor binding events with high spatial resolution, and bring insights into binding properties like binding affinity and kinetics, none of them provide molecular structural information at ligand-receptor binding sites, which is a key factor in mediating binding properties. Chemical elucidation of ligand-receptor binding sites is typically obtained by characterizing crystal structures of purified receptors using x-ray diffraction (XRD) and nuclear magnetic resonance (NMR), and building computational models for analysis ^{[20, 21]}. New technologies are needed to provide molecular insight into ligand-receptor binding chemistry in cell membranes, where the cellular environment can impact receptor-ligand interactions.

Raman spectroscopy measures the energy of the vibrational modes associated with the structure of molecules, which provides an intrinsic contrast mechanism for identifying molecules in biological systems (e.g. cells and tissues). Raman spectra encode chemical-specific information regarding the identity (so-called “chemical fingerprints”) of the molecules. Raman bands arising from different functional groups identify biomolecules, but the unique spectra arising from the combination of biomolecules in a cell provide characteristic patterns that enable classification of different cell types. For example, Raman spectroscopy can classify benign and cancerous human lung cells in response to external stimuli ^{[22, 23]}, and monitor differentiation from stem-like cells in different culture conditions ^{[24, 25]}. However, since Raman scattering is an inefficient process by nature, spontaneous Raman spectroscopy is challenged to detect specific membrane receptors in cells.

Plasmonic nanostructures produce significant enhancements in the Raman scattering from molecules in close proximity—an effect known as surface-enhanced Raman scattering (SERS), which provides improved sensitivity for biomedical applications ^{[26, 27]}. By conjugating specific ligands onto plasmonic nanoparticles (e.g. Au, Ag), SERS can detect and locate specific receptors in cell membrane. One typical approach that has been widely applied is to construct SERS tags, where the intense and distinct spectrum of a Raman reporter molecule enhanced by the nanoparticle provides the detected signal for localization and mapping of specific membrane receptors, such as cancer markers, in vitro and in vivo ^[28–31]. For example, Fabris et al. constructed an RGDfC peptides-conjugated SERS nanotag to achieve specific targeting of integrin αvβ3 in human glioblastoma cells, by detecting the signal of a Raman-active dithiolated linking reporter molecule ^[31]. With these SERS tags, the signal from a reporter molecule specifically attached to the nanoparticle is observed, overwhelming the intrinsic vibrational signal of biomolecules themselves, which is usually much weaker than the signals from reporter molecules. In other words, Raman reporter-based SERS tags are unable to detect vibrational modes of membrane receptors and cannot provide structural information of the ligand-receptor binding pockets. In recent years, SERS studies without the application of reporter molecules have been reported for detection and mapping of ligand-receptor interactions on SERS-active substrates ^{[32, 33]}, and even on cell membrane surface ^[34]. Although this intrinsic SERS approach can provide structural information about the protein receptor, its imaging resolution is restricted by the diffraction limit, and therefore is unable to achieve single molecule imaging.

Tip-enhanced Raman scattering (TERS), utilizing a plasmonic nanostructure at the apex of a scanning probe microscopy (SPM) tip, integrates the chemical sensitivity of SERS and nanoscale spatial resolution of SPM to enable intrinsic chemical imaging of surfaces, such as biomembranes ^{[35, 36]}. Impressively, TERS was demonstrated to image components of individual molecules based on their vibrational modes ^[37], which makes it a promising technique to chemically characterize biomolecules, such as lipids and proteins, in cell membrane ^[36]. Our lab has conducted a series of studies to investigate chemistry of ligand-receptor interactions in recent years ^[38–43]. We have reported that Raman signals from immobilized protein receptors can be detected through a plasmonic coupling between a gold nanoparticle-coated TERS tip and a ligand-functionalized gold nanoparticle (GNPs) ^{[38, 40]}. Through protein mutation, we have been able to demonstrate that amino acids near the ligand binding sites are responsible for the observed TERS signal ^[42]. With controlled plasmonic coupling, we were able to obtain chemically specific information relevant to antibody-antigen interaction in cells, while achieve mapping of the binding events with spatial resolution below 50 nm ^[39]. An initial study on fixed SW620 cells (human colon cancer cell line) showed this detection mechanism can detect the amino acids involved in the specific interactions between αvβ3 integrin receptors and RGD peptides, in intact cell membrane ^[41]. These initial results suggest that TERS can provide chemical insights into cell membrane receptors, making it a promising new technology for investigating the chemical structure of membrane receptors and the chemical interactions that govern molecular recognition.

The TERS signal consists of the intrinsic Raman scattering from the amino acids closest to the nanoparticle. When binding to a ligand on the nanoparticle surface these amino acids are associated with the ligand-receptor binding site. We hypothesize that TERS is able to differentiate between different membrane receptors (e.g. αvβ3, αvβ5 and α5β1 integrins) that bind to same RGD sequences ^{[11, 44]}, due to the slight differences in their respective ligand binding sites. In a recent report, we used two integrin receptors, α5β1 and αvβ3, with reported affinity for cyclic-RGDfC (cRGD) peptide, to examine whether RGD-integrin binding can be differentiated in intact human colon cancer cells (SW480) using TERS ^[43]. With this method, cRGD peptide-conjugated gold nanoparticles (cRGD-GNPs) were used to target integrins α5β1 and αvβ3 on SW480 cells. While the gold-coated SPM tip scans on the cell surface, the plasmonic coupling between TERS tip and gold nanoparticle produce enhanced Raman signals, which can be used to identify the receptors bound to the nanoparticles. As shown in this and previous work ^{[38–41, 43, 45]}, the receptor remains in the cell membrane and does not necessarily reside in the tip-particle gap (Figure 1a).

Figure 1.

(a) Schematic illustration of TERS detection of integrin receptors in cell membrane. (b) Average SERS spectra of cRGD-GNPs (n=23), α5β1-bound cRGD-GNPs (n=12), and αvβ3-bound cRGD-GNPs (n=11). Shaded area represents standard deviation. (c) TERS map of SW480 cells incubated with cRGD-GNPs, constructed with single-peak intensity at 1003 cm⁻¹. Pixel step: 62.5 nm. (d, e) MCR score maps reflecting the localization of (d) α5β1 and (e) αvβ3 on the same cell membrane area as (c). Adapted with permission from Reference [43] (Xiao L, Wang H, Schultz ZD. Selective Detection of RGD-Integrin Binding in Cancer Cells Using Tip Enhanced Raman Scattering Microscopy. Anal Chem 2016; 88: 6547–6553). Copyright (2016) American Chemical Society.

SERS experiments were performed on the cRGD-GNPs incubated with purified integrin α5β1 or αvβ3 receptors, to generate a set of reference spectra for each receptor. Multiple spectra from just the cRGD-GNPs, α5β1-bound cRGD-GNPs, and αvβ3-bound cRGD-GNPs were acquired in 0.1X PBS solution where nanoparticle clusters formed plasmon-enhanced “hot spots”. Figure 1b shows the average SERS spectra of integrins α5β1 and αvβ3 bound with cRGD-GNPs. The observed vibrational bands can be assigned to amino acids found in the RGD binding sites of α5β1 and αvβ3 integrins ^{[46, 47]}. SERS spectra exhibit distinct differences in Raman bands (Figure 1b), arising from differences in ligand binding sites between α5β1 and αvβ3 receptors ^{[48, 49]}. The acquired SERS spectra were analyzed with multivariate curve resolution (MCR), a chemometric method that analyzes the variance in the spectra and generates pure components associated with the spectral composition. The MCR analysis produced 3 pure spectral components that can be attributed to cRGD-GNPs, α5β1-bound cRGD-GNPs, and αvβ3-bound cRGD-GNPs, respectively. This multivariate model accounted for approximately 90% of the total variance in the SERS data and was able to distinguish between spectra acquired from the distinct integrin receptors.

TERS experiments were performed on fixed SW480 colon cancer cells incubated with cRGD-GNPs. A TERS map was acquired by taking spectra every 62.5 nm over a 2 2 µm² surface area of a SW480 cell membrane. The TERS map was constructed using the peak intensity at 1003 cm⁻¹ (attributed to phenylalanine from cRGD-GNPs), in which high intensity pixels reflect the localization where the cRGD-GNPs bind, possibly to integrin receptors, on the cell membrane (Figure 1c). Given that both α5β1 and αvβ3 can be expressed in SW480 cells, the obtained TERS data were analyzed using the MCR model generated from the SERS data, in order to identify the receptors based on spectral similarity between TERS spectra and MCR pure components. MCR scores of α5β1 and αvβ3 were generated to estimate the spectral contribution from α5β1 and αvβ3 in each TERS spectrum ^[43]. Maps created from MCR scores reflect the localization of α5β1 (Figure 1d) and αvβ3 (Figure 1e) on the same cell membrane area as in Figure 1c. Upon the application of MCR regression model, the two integrin receptors were readily detected and differentiated from general TERS signals. We noticed that two adjacent pixels with high levels of α5β1 and αvβ3 (red dashed circles in Figures 1c–e) were observed in the TERS maps, suggesting the co-localization of α5β1 and αvβ3 within ~60 nm range, as they have been reported to co-localize on cell membranes to regulate different mechanochemical steps of cell-matrix adhesion ^[50]. In addition, the fact that exclusive spectral signatures are detected from co-localized integrin receptors α5β1 and αvβ3 indicates the TERS signal may arise from individual receptors.

The above findings indicate that TERS is capable of nanoscale imaging of individual integrin receptors within cell membranes, while providing chemical insight into the RGD-integrin binding structures. The chemical insight TERS provides is a new tool for studying drug targeting in vitro, which may aid in drug discovery. We successfully differentiated between two related integrin structures, suggesting the signature of other receptors could also be identified. Future direction will be applying the developed TERS methodology to provide an early stage screen to identify drug interactions with different receptors in living cells.

Abbreviations

TERS

tip-enhanced Raman scattering

RGD

arginine-glycine-aspartate

ECM

cell-extracellular matrix

MRI

magnetic resonance imaging

XRD

X-ray diffraction

NMR

nuclear magnetic resonance

SERS

surface-enhanced Raman scattering

SPM

scanning probe microscopy

GNPs

gold nanoparticles

cRGD (cRGDfC)

cyclic-arginine-glycine-aspartate-phenylalanine-cysteine

MCR

multivariate curve resolution