Process characterization of epithelial–mesenchymal transition in alveolar epithelial type II cells using surface-enhanced Raman scattering spectroscopy

Abstract

Epithelial–mesenchymal transition (EMT), a process by which epithelial cells undergo a phenotypic conversion that gives rise to fibroblasts and myofibroblasts, plays an important role in tissue repair and fibrosis, chronic inflammation, cancer invasion and metastasis. Therefore, increasing attention has focused on the study of EMT. However, it is still a challenge to distinguish the undifferentiated and differentiated epithelial cells, which are closely related and morphologically similar. In this study, we employed the transactivator of transcription (TAT)-functionalized AuNSs as intracellular surface-enhanced Raman scattering (SERS) probes for characterizing the EMT process in alveolar epithelial type II (ATII) cells, induced by bleomycin (BLM). CCK8 assay and reliable aggregation tests indicated a low cytotoxicity and high stability of the intracellular SERS probes. The internalization of the TAT-functionalized AuNSs acting on the ATII cells was verified by transmission electron microscopy (TEM), fluorescence images, and energy-dispersed spectroscopy (EDS). SERS spectra were analyzed by principal component analysis (PCA), which was able to distinguish between ATII cells at different stages of EMT and monitor the cellular biochemical composition changes in this process. In addition, the expression of epithelial and fibroblastic markers on ATII cells suggested that incubation with BLM could induce ATII cells to differentiate into fibroblasts. The SERS detection of the internalized TAT-functionalized AuNSs provides a continuous, non-invasive, and label-free characterization for the EMT process.

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1. Introduction

Epithelial–mesenchymal transition (EMT) is a process where epithelial cells gradually transform into mesenchymal-like cells losing their epithelial functionality and characteristics. EMT is thought to be involved in the pathogenesis of numerous lung diseases ranging from developmental disorders, and fibrotic tissue remodeling to lung cancer.¹ To date, EMT has been suggested to be a source of fibroblasts,² and it also plays a key role in the migration and invasion of cancer cells.³ Therefore, understanding the EMT process and its participation in pulmonary fibrosis is of critical importance. However, the discrimination between closely related cell phenotypes in a noninvasive and label-free manner is still a challenge using conventional optical microscopy. In spite of extensive previous studies on EMT, methods capable of distinguishing the differentiated epithelial cells remain limited.

Recently, surface-enhanced Raman scattering (SERS) spectroscopy has received a great deal of attention for its utility as an extremely high sensitivity technique for chemical and biomedical analysis.^4,5 SERS can provide rich structural information to identify chemicals and biological samples with the vibrational fingerprints.^6–8 SERS is capable of enhancing the Raman signals of analytes located near the surfaces of Ag or Au metallic nanostructures by as much as 6 to 14 orders of magnitude, which even allows detection of the signal of single molecules.⁹ Moreover, SERS is highly resistant to interferences of exoteric factors, indicating the SERS is suitable for studying living cells in systems containing water. Au nanoparticles (AuNPs) have been shown to be of great use and advantage for biological applications, particularly cellular detection and biomedical diagnostic. AuNPs are generally nontoxic, small in size (<100 nm) and good stability, can be delivered selectively to the cell specific sites.^10,11 AuNPs with different sizes, shapes, and compositions have been prepared for SERS detection. Of particular interest are Au nanostars (AuNSs), which had tunable plasmon bands in the NIR tissue optical window and the structure contains multiple sharp branches acting as “hot-spots” for the SERS effect.¹² Up to this date, several published reviews covered the application of SERS in cell detection. For example, Rodriguez-Lorenzo et al.¹³ reported TB-tagged AuNSs coated with a silica shell as a potential nanoconstruct for cellular SERS imaging. Wang et al.¹⁴ showed a EGF-functionalized SERS probes for direct detection of circulating tumor cells in peripheral blood. Besides, SERS has been used to identify the apoptotic and necrotic cell death.¹⁵ In our previous reported, we employed AuNSs and SERS for distinguishing the differentiation of lung resident mesenchymal stem cells.¹⁶

In this study, we characterized the EMT process in alveolar epithelial type II (ATII) cells by SERS spectroscopy. The alveolar epithelial cells were incubated with bleomycin (BLM) for differentiating into fibroblasts. Furthermore, using TAT-functionalized AuNSs as cellular SERS probes allowed us to investigate changes of cellular components during the EMT process. The difference between ATII cells at different stages of EMT have been well compared and distinguished through spectra analysis and principal component analysis (PCA) score plots analysis. The results demonstrated that SERS spectroscopy was a sensitive analysis technology for identification and differentiation of the EMT process in ATII cells.

2. Results and discussion

2.1 Characterization of SERS probes

The morphology and structure of the as-prepared AuNSs were characterized by SEM and TEM. As shown in Fig. 1A, large quantities of AuNSs with uniform diameter of about 90 nm were obtained. The yield of branched particles was extremely high, as demonstrated by TEM observation, where no other shapes were found in any of the analyzed samples (Fig. 1D). The high-magnification image further revealed that each Au nanostar was consisted of a solid gold core of about 80 nm with many short, irregular branches of 8–10 nm average size (Fig. 1B and C). AuNSs were prepared by a simple, fast and large-scale method. AuNSs have roughened surface determined by many tips and the cavities, which acted as potential ‘hot’ spots for enhancing the electromagnetic field around the particle.¹⁷

Fig. 1 SEM images of AuNSs with different scale bar: (A) 1 μm; (B) 50 nm. TEM images of AuNSs with different scale bar: (C) 200 nm; (D) 50 nm.

Herein, we developed a TAT-functionalized AuNSs as cellular SERS probes for characterization of the EMT in ATII cells. There are two main steps in the fabrication of the proposed SERS probes (Scheme 1). Firstly, SH-PEG-COOH were anchored onto the surface of AuNSs by the chemical bonds sulfhydryl group (–SH) for preventing the aggregation. Then, TAT molecules were connected to PEG-stabilized AuNSs, which was used as a cell-penetrating peptide. As shown in Fig. S1 in the ESI,† TAT molecules were successfully conjugated to the surface of AuNSs. Moreover, the stability of SERS probes was checked via a reliable aggregation test (Fig. S2†). The experiment demonstrated that SERS probes were reliable for applications in physiological media.

Scheme 1 Schematic illustration of TAT-functionalized AuNSs as intracellular SERS probes for characterization of the BLM-induced EMT in ATII cells. (A) Preparation of the SERS probes. (B) SERS measurements were performed for identification and differentiation of the EMT process in ATII cells.

2.2 In vitro cytotoxicity study

To show the potential utility of the TAT-functionalized AuNSs as a cellular diagnostic probe, an in vitro cytotoxicity of probes on ATII cells was determined by using the CCK-8 assay. According to the law of mass conservation, the gold content of this kind of probes was 50 μg mL⁻¹. Based on this, the concentration gradient was set to 0.5 μg mL⁻¹, 2.5 μg mL⁻¹, 5.0 μg mL⁻¹, 12.5 μg mL⁻¹, 25 μg mL⁻¹. The viabilities of ATII cells incubated with different concentrations of probes for 24 h were tested, as shown in Fig. 2A. The SERS probes showed no significant toxicity on ATII cells at low concentration (lower than 5 μg mL⁻¹). However, with the increased concentration of probes, the materials showed increased cytotoxicity to the cells. Moreover, when ATII cells were incubated with 5 μg mL⁻¹ probes for 72 h, the cell growth was not significantly affected compared with control (Fig. 2B). Therefore, to maximize the cellular uptake of TAT-functionalized AuNSs and minimize the chemical toxicity, the optimal concentration of probes was determined to be 5 μg mL⁻¹. No significant toxicity of the as-prepared SERS probes on ATII cells at low concentration could be due to the improved biocompatibility in the presence of PEG, which was reported as a good biocompatible capping agent.¹⁸

Fig. 2 (A) Detection of cell viabilities by CCK-8 assay of ATII cells incubated with the different concentration gradient of the SERS probes (0, 0.5, 2.5, 5, 12.5, 25 μg mL⁻¹) for 24 h (**p < 0.05). (B) The cell growth curve of ATII cells incubated with 0 (control) and 5 μg mL⁻¹ SERS probes in 72 h.

2.3 Cellular uptake and internalization

Before the detection of cells by SERS, we need to ensure sufficient intracellular TAT-functionalized SERS probes delivery. The probes were incubated with ATII cells for 24 h, and then were observed by a fluorescence microscopy. TAT peptide contained a FITC tag at the C-terminus, and the N-terminus was conjugated to the outer layer of the AuNSs polymer coating. The nucleus of ATII cells were stained with DAPI, a cell-permeant laser dye (Fig. 3A). The fluorescence image showed that the probes engulfed by ATII cells could clearly be observed as the green dots (Fig. 3B). TEM analysis was performed to investigate the morphology of nanostructures and its distribution inside the cell. The thin-section TEM images clearly showed that nanoparticles were mainly located in the cytoplasm and few nanoparticles entered into nucleus, although they were dispersed throughout the cell (Fig. 3C and D). Fig. 3E showed the high-magnification TEM images of the TAT-functionalized AuNSs inside cells. The AuNSs were clearly visible and distinct from the cellular structures due to their high electron density.¹⁹ There was no change in shape and size of AuNSs (black arrow) after being endocytosed in cells. A few probe clusters could be observed on membranous structures possibly because of internalization of the probe. It was believed that the probes could be delivered to cells primarily through actin-driven and lipid raft-mediated micropinocytosis.²⁰

Fig. 3 ATII cells incubated with SERS probes for 24 h. Fluorescence images of (A) ATII cells unloaded SERS probes and (B) ATII cells loaded SERS probes. (C and D) The thin-section and (E) high-magnification TEM images of ATII cells incubated with SERS probes. (F) SEM images and (G) corresponding EDS of ATII cells unloaded SERS probes. (H) SEM images and (I) corresponding EDS of ATII cells loaded SERS probes.

Energy dispersive spectrometer (EDS) is usually used for composition analysis and element analysis in micro area of materials. Fig. 3F and G showed the SEM images of ATII cells unloaded SERS probes and corresponding EDS. Fig. 3H and I showed the SEM images of ATII cells loaded SERS probes and corresponding EDS. The EDS spectrum of ATII cells loaded SERS probes showed strong signals from the gold atoms. The peak of Au could be attributed to products formed from HAuCl₄. As a result, the observations confirmed the elemental gold existed in the TAT-functionalized AuNSs incubated ATII cells.

2.4 SERS measurement

The SERS spectra were collected using a confocal Raman spectrometer. The measured SERS spectra of 20 cells were used to obtain cellular average SERS spectrum. Fig. 4A showed the Raman spectra of ATII cells unloaded SERS probes and ATII cells loaded SERS probes after 24 h of incubation. The ATII cells unloaded probes showed a silence of SERS signal. However, the strong Raman signals of ATII cells loaded probes could be observed. The characteristic peaks of the SERS spectrum provided direct information on the structural and chemical composition of the cells. It suggested that AuNSs substrates could be used for high sensitive detection of cells in vitro. In this study, SERS was utilized as a method to characterize the biochemical composition of ATII cells during their differentiation into fibroblasts. Fig. 4B depicted stacked SERS spectra normalized at 1296 cm⁻¹ (CH₂ deformation (protein assignment)). These spectra exhibited minor differences, indicating that ATII cells at different stages of EMT possessed very similar cellular components. The direct comparison did not reveal significant spectral variations during the EMT process. Then, differences in SERS spectra between different stages were compared, and the results were as follows.

Fig. 4 (A) ATII cells after taking up probes and incubated 24 h. Raman spectra of ATII cells unloaded SERS probes (black curve), and ATII cells loaded SERS probes (red curve) under 785 nm excitation. (B) The normalized mean SERS spectra of ATII cells during their differentiation into fibroblasts.

2.5 Difference spectra of EMT in ATII cells

The difference spectra were computed by subtracting the spectrum of ATII cells at 7 days of differentiation from the spectrum of ATII cells at 0 days of differentiation (7 days − ATII), the spectrum of ATII cells at 14 days of differentiation from the spectrum of ATII cells at 7 days of differentiation (14 days − 7 days), the spectrum of fibroblasts from the spectrum of ATII cells at 14 days of differentiation (3T3 − 14 days), the spectrum of fibroblasts from the spectrum of ATII cells at 0 days of differentiation (3T3 − ATII), respectively. By reference to the Raman band assignments in Table 1 in ESI,† we could assign the characteristic bands in four difference spectra.^21–36

From the Fig. 5A (7 days − ATII), we could learn that the positive bands at 756, 874, 1306, 1336, 1420, 1491, 1544, 1586, and 1659 cm⁻¹ were from the spectrum of ATII cells at 7 days of differentiation, indicating the relative intensity of these bands which were mainly the vibration peaks of proteins and lipids was higher in the spectrum of ATII cells at 7 days of differentiation when compared with that of 0 days. Whereas the negative bands for Fig. 5A at 674, 720, 786, 856, 941, 1006, 1128, 1171, 1222, and 1523 cm⁻¹ were from the spectrum of ATII cells at 0 days of differentiation. These peaks were mainly generated by the vibration peaks of DNA, proteins and carbohydrates. In Fig. 5B (14 days − 7 days), the positive bands at 818, 874, 1066, 1168, 1279, 1449, 1553, and 1608 cm⁻¹ were from the spectrum of ATII cells at 14 days of differentiation. The majority of these peaks could be assigned as proteins and fatty acids. The negative bands, 674, 720, 941, 1006, 1128, 1171, 1222, and 1523 cm⁻¹ from the spectrum of ATII cells at 7 days of differentiation and could be similarly assigned as amino acids and DNA. Fig. 5C (3T3 − 14 days) showed the positive bands at 852, 923, 1017, 1249, 1279, 1363, 1463, 1659 cm⁻¹ were from 3T3 spectrum, and the negative bands at 624, 652, 756, 951, 1066, 1128, 1171, 1306, 1449, 1523, 1591 cm⁻¹ were from the spectrum of ATII cells at 14 days of differentiation. These peaks have been previously assigned for Table 1.† Furthermore, the difference spectra between 3T3 cells and ATII cells at 0 days of differentiation (Fig. 5D) yielded positive bands at 852, 1033, 1249, 1279, 1363, 1429, 1462, and 1608 cm⁻¹ that were due to the 3T3 spectrum. The negative bands; 624, 674, 756, 1006, 1017, 1222, and 1306 cm⁻¹ were from the spectrum of ATII cells at 0 days of differentiation.

Fig. 5 ATII cells were incubated with BLM for 7 days and 14 days. Difference spectra were computed from ATII cells at different stages of EMT: (A) 7 days − ATII, (B) 14 days − ATII, (C) 3T3 − 14 days, and (D) 3T3 − ATII.

To further compare and quantize the differences between ATII cells at different stages of EMT, based on the normalized mean SERS spectra, we compared the relative intensity at six different Raman bands. As shown in Fig. 6, the relative intensity of Raman bands at 874 cm⁻¹, 1279 cm⁻¹, 1363 cm⁻¹, and 1659 cm⁻¹ increased significantly during the EMT process. However, the relative intensity of Raman bands at 720 cm⁻¹ and 1128 cm⁻¹ decreased with the incubation with BLM. The results were consistent with the preceding analysis about four difference spectra. During the EMT process, some biochemical compositions may have been changed, which could be deducted from the spectral differences between different stages as these spectral differences represent the differences in the composition of proteins, lipids and nucleic acids. In addition, such differences may result in the differentiation-dependent changes in morphology, structure and function.

Fig. 6 The comparison of relative intensity at six different Raman bands between ATII cells at different stages of EMT.

2.6 PCA analysis

The principal component analysis (PCA) of the pre-processed SERS spectra aid to classify the differentiation state of ATII cells. Fig. 7 showed the scatter plots of the SERS spectra for each cell projected into a two-dimensional subspace using principal component scores PC1 and PC2. It was seen that the PC1 possessed 62.9% variation, PC2 18.4% variation. Apparently, PCA plots indicated that the SERS spectra of ATII cells at different stages of EMT were distinguishable into four distinct groups. The groups of ATII cells at 0 days of differentiation and 3T3 cells groups were well separated from each other, which were mainly attributed to differences in biomolecular composition. After 7 days treatment with BLM, the groups of ATII cells at 7 days of differentiation were closer to the ATII cells at 0 days of differentiation. However, if ATII cells were induced to differentiate for 14 days, their plots their plots were far away from the groups ATII cells at 0 days of differentiation and overlapped substantially with those of either groups of 7 days or 3T3 cells. Therefore, it is likely that, during alveolar epithelial cells differentiation into fibroblasts, the cellular components (proteins, nucleic acids, carbohydrates and lipids) tended to be consistent with those in 3T3 cells, resulting in morphological, structural and functional changes of ATII cells. The results suggested that PCA was able to distinguish between ATII cells at different stages of EMT based on their specific spectral features.

Fig. 7 PCA scores plot of SERS spectra for the cells at different stages of EMT. Each cell was coded by color (ATII cells – red, 7 days – blue, 14 days – black, and 3T3 cells – green).

2.7 EMT process confirmed by biological assay

EMT is a process whereby fully differentiated epithelial cells undergo transition to a mesenchymal phenotype, including changes in the expression of epithelial markers, such as E-cadherin, some cytokeratins, and mesenchymal markers, such as vimentin, N-cadherin and α-smooth muscle actin (α-SMA), as well as matrix metallopeptidase 9 (MMP-9).^37,38 Therefore, EMT can be regarded as a complex manifestation of epithelial plasticity. As reported, one of the principle characteristics of EMT is the loss of E-cadherin expression, which participates in cell–cell adhesion and interacts with other molecules to form epithelial junctions.³⁹ And the upregulation of mesenchymal markers, such as vimentin, could support the induction of EMT as well.⁴⁰ To confirm the functional changes during the EMT process, ATII cells were incubated with BLM for 7 days and 14 days. Fig. 8A showed that epithelial marker E-cadherin was downregulated after BLM treatment for 7 and 14 days. In contrast, the fibrotic marker α-SMA was upregulated by BLM treatment (Fig. 8B). The results showed that BLM really could induce ATII cells differentiate into fibroblasts. Moreover, we investigated the expression of epithelial markers E-cadherin, CK18, and fibrotic marker collagen, α-SMA and vimentin by Western Blot to confirm the EMT process (Fig. 8C), which was consistent with the results of immunofluorescence. In conclusion, the biological assay gives a valid support for the use of SERS spectra to analyze the EMT process.

Fig. 8 The expression of epithelial and fibroblastic markers on ATII cells. ATII cells were incubated with 25 μM BLM for differentiation. Expression of (A) E-cadherin and (B) α-SMA in ATII cells following differentiation for 0 (control), 7 and 14 days were analyzed by immunofluorescent staining. E-cadherin was revealed with secondary Alex594-labeled antibody, and α-SMA was revealed with secondary Alex488-labeled antibody. (C) Expression of collagen, E-cadherin, α-SMA, vimentin, CK-18 and β-actin in ATII cells following differentiation for 0 (control), 7 and 14 days were detected by western blotting. β-Actin was used as an internal control.

3. Conclusions

In summary, employing TAT-functionalized AuNSs as intracellular SERS probes, we have been able to identify the BLM-induced EMT in ATII cells. The probes were tested on ATII cells and found to present a low level of cytotoxicity, high stability. The fluorescence images, TEM and EDS showed that the probes were readily internalized by the ATII cells. The strong SERS signals of ATII cells could be acquired as early as 24 h, indicating that the SERS probes were very suitable for cellular detection. The differences in SERS spectral between different stages revealed that the cellular biochemical components have been modulated during the EMT process. PCA achieved a successful segregation of undifferentiated from differentiated cells in alveolar epithelial type II cell line based on their SERS spectra. Finally, the biological assays confirmed that incubation with BLM could induce ATII cells to differentiate into fibroblasts. This work therefore demonstrated the potential of the AuNSs and SERS, which could facilitate non-invasive monitoring of the EMT process.

Acknowledgements

We gratefully acknowledge supports from the Chinese 973 Project (Grant: 2012CB933302), the National Natural Science Foundation of China (Grant: 21175022), National Science Foundation of Jiangsu Province of China (BK2011570), the Ministry of Science & Technology of China (Grant: 2012AA022703), Graduate Students' Scientific Research Innovation Project of Jiangsu Province Ordinary University (No. KYLX_0185).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra17022b

‡ Contributed equally.

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