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. 2026 Jun 15;6(4):916–922. doi: 10.1021/acsmeasuresciau.6c00014

Label-Free SERS Analysis of Glioma Cells to Detect Drug Resistance Using Metabolic Phenylalanine Level

Guohui Yang †, Xin Wang ‡, Jingbin Jin †, Xiaozhang Qu §, Kaizhi Zhang †,*, Shuping Xu ‡
PMCID: PMC13495487  PMID: 42631071

Abstract

Glioma is one of the most common malignant brain tumors, and its mainstream clinical treatment regimens mainly include postoperative combined with Temozolomide chemotherapy. Unfortunately, glioma cells tend to mutate when subjected to prolonged Temozolomide treatment, leading to drug resistance and significantly weakening the therapeutic effect. Delaying the resistance time of Temozolomide has become a pressing issue for clinicians to address, and the primary regimen for delaying drug resistance is the combined application of Temozolomide with other therapies. To explore new molecular diagnostic markers of drug resistance in glioma and to assess treatment stage, surface-enhanced Raman spectroscopy (SERS) was used to investigate changes in glioma cells under combined therapies including physical (electrical stimulation, ES) and chemical (cancer drug: Temozolomide) treatments. By analyzing intensity changes in the SERS band at 997 cm–1, we observed that glioma cells showed a higher phenylalanine (Phe) expression. Interestingly, dynamic variations in Phe content secreted from glioma cells were drug resistance-dependent. ES, a novel therapeutic technique that can inhibit cell proliferation by promoting glioma cell apoptosis, was combined with Temozolomide. In both simple ES and ES plus Temozolomide conditions, metabolic Phe levels in glioma cells are significantly elevated, suggesting that Phe overexpression in glioma cells can serve as a potential indicator for accelerated cancer cell apoptosis. Our study proves that ES can effectively reduce Temozolomide doses, offering an easy-to-implement approach to delaying the onset of drug resistance. This work not only reveals a possible antidrug-resistance treatment strategy for glioma but also provides important guidance on a potential spectral indicator for early diagnosis of drug resistance, which is of significance for clinical applications.

Keywords: SERS, Label-free, Phe, Electrical stimulation, Temozolomide

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1. Introduction

Glioma is currently one of the malignant tumors with the highest incidence and mortality rates in the nervous system, and its annual incidence rate shows an increasing trend year by year. Although surgical resection remains the preferred treatment option, comprehensive treatment such as postoperative chemotherapy and radiotherapy has become necessary in clinical practice. However, the existence of the blood-brain barrier severely limits the effectiveness of chemotherapy drugs for glioma, which makes the development of chemotherapy drugs for glioma one of the key difficulties in postoperative treatment. Temozolomide can easily cross the blood-brain barrier due to its lipophilic characteristics, and it has become a core drug for postoperative adjuvant chemotherapy of glioma in recent years. By methylating adenine and guanine in DNA, Temozolomide can lead to mispairing during DNA replication, resulting in the breakage of both single-stranded and double-stranded DNA. This breakage blocks the cell cycle at the G2/M phase, thereby inducing programmed cell death in tumor cells. However, due to the enhanced DNA repair capacity of glioma cells and dynamic changes in the tumor microenvironment, glioma cells gradually become resistant to Temozolomide, ultimately leading to drug resistance. If the indications of drug resistance can be identified at an early stage and timely additional treatment strategies can be implemented, it will be of great significance in improving the survival period of patients.

Electrical stimulation (ES), a simple and controllable physical therapy method, has been widely used in various fields, including pain relief, muscle rehabilitation, recovery from movement and consciousness disorders, wound healing, and drug delivery. − ES can alter membrane potential and activate ion channels, thereby triggering downstream signaling cascades such as the PI3K/AKT, MAPK, and p53 pathways, ultimately regulating cell proliferation, apoptosis, and autophagy. , In addition, electrical stimulation has been shown to induce reactive oxygen species (ROS) generation and mitochondrial dysfunction, leading to oxidative stress–mediated tumor cell death. Beyond its direct cytotoxic effects, electrical stimulation can also remodel the tumor microenvironment by promoting tumor vascular normalization and improving oxygenation, thereby enhancing drug delivery and therapeutic efficacy. Recent studies have shown that ES also has significant potential for glioma therapy. ES interferes with glioblastoma cell division and organelle assembly by delivering low-intensity alternating electric fields to the tumor. Stupp et al. compared adding tumor-treating fields to maintenance Temozolomide chemotherapy with maintenance Temozolomide alone, and the data showed a statistically significant improvement in progression-free and overall survival. Jin’s team studied the process of cell apoptosis induced by ES and found that the DNA bases (A) and the tumor cell backbone can be effectively damaged under 1.2 V for approximately 5 min. The programmed death of tumor cells induced by ES is much easier than that of normal cells, and caspase-3 plays a central role in mediating tumor cell apoptosis and inducing DNA fragmentation. These investigations indicate that ES can be an effective physical therapeutic tool for tumor cells, as well as glioma.

To address the aforementioned problems in Temozolomide resistance, this study adopted surface-enhanced Raman scattering (SERS) spectroscopy to explore the effects of different ES parameters on molecular changes from a metabolic regulatory perspective in the U87 glioma cell system, investigate cell function conditions under ES and chemotherapy, and further screen the potential apoptosis-related potential spectral indicator of glioma cells underlying a synergistic effect. As a molecular vibrational spectroscopy, SERS has been widely applied in biomedical fields due to its high sensitivity and fingerprint feature. , In recent years, SERS, owing to its high sensitivity, has attracted increasing attention as a powerful analytical tool for the in vitro detection, diagnosis, and therapeutic evaluation of glioma. , In the field of tumor metabolism, SERS has been successfully applied to monitor various metabolic processes, including glucose metabolism, amino acid metabolism, lipid metabolism, and nucleotide metabolism, thereby enabling the differentiation between tumor cells and normal cells. In addition, SERS can be employed to probe tumor microenvironment-related factors, such as pH, reactive oxygen species (ROS), and glutathione (GSH), providing valuable insights into tumor progression and treatment response. , Using this powerful analytical tool, we systematically analyze the molecular changes in glioma cell metabolites with silver nanoparticles (AgNPs) as a Raman signal enhancement substrate before and after ES treatment plus Temozolomide (Scheme ). Our results show that Temozolomide resistance can be significantly reduced under the ES of appropriate parameters. Through systematic experiments, we found that ES can significantly enhance the antitumor effect of Temozolomide by increasing phenylalanine (Phe) levels in cells and promoting glioma cell apoptosis. The key innovation of our study can be summarized as two aspects: (1) Phe levels can serve as a potential marker for assessing drug resistance in glioma cells. The expression level of Phe increases with increasing ES stimulation time and voltage intensity, as well as the Temozolomide concentration. (2) The drug dosage can be reduced by more than 50%, thereby effectively reducing the toxicity and side effects of treatment and delaying the occurrence of drug resistance. Therefore, the primary aim of this study is to develop a label-free SERS-based strategy to monitor molecular changes in glioma cells under electrical stimulation combined with Temozolomide treatment, and to evaluate the effect of electrical stimulation on apoptosis-related cellular responses. In this context, phenylalanine-associated spectral features were further investigated as potential indicators of treatment-related cellular responses. This work not only proposes a potential combination therapy strategy for glioma but also offers important technical support for the early diagnosis of drug-resistant glioma cells.

1. ES and Temozolomide Treatments Were Performed on U87 Glioma Cells, and SERS Measurements for Cell Metabolites Were Obtained .

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a (1) The cells were treated with ES. (2) The cells treated with Temozolomide for 12 h and then dealt with ES. (3) The cells were only incubated with Temozolomide.

2. Experimental Section

2.1. ES and Temozolomide Treatment of U87 Glioma Cells

U87 glioma cells were cultured in modified medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2 for passaging.

ES was performed at room temperature using an electrochemical workstation (CHI 660E) with a standard three-electrode system. Ag/AgCl (saturated with potassium chloride) and a Pt sheet were selected as the reference electrodes. A sodium chloride solution (0.9%) capable of exerting isotonic pressure on the cells was mixed with complete medium at a 4:1 volume ratio to serve as the electrolyte. Conductive Indium Tin Oxide glass (ITO, 2 cm × 2 cm, sheet resistance = 10 Ω) was used as the working electrode. Untreated U87 glioma cells were subjected to continuous electric field stimulation at the same voltage (0.5 V) for different durations (3, 5, and 7 min) and at the same duration (5 min) for different voltages (0.3, 0.5, and 0.7 V). Meanwhile, Temozolomide was diluted in DMSO to 50, 100, and 200 μmol, and cells were coincubated with these three concentrations of Temozolomide, which were electrically stimulated under the same voltage and duration conditions (0.5 V, 5 min). After treatment, the cells were washed three times with cold phosphate-buffered saline (PBS) (Scheme ). U87 glioma cells were seeded on ITO glass (1 cm2) and cultured to reach approximately 70–80% confluency before subsequent experimental treatments.

2.2. Fluorescence Staining and Imaging of Cells Treated with ES and Temozolomide

U87 cells treated with ES (under the voltages of 0, 0.3, 0.5, and 0.7 V, or different periods of 0, 3, 5, and 7 min) or Temozolomide with different doses were stained with calcein-AM (2 μM) and propidium iodide (PI) (4 μM). After 20 min, the cells were washed three times with cold PBS. Finally, the cells were observed using a Leica DMI6000B microscope equipped with a fluorescence detector and a 40× objective lens.

2.3. Measurements of SERS Spectra of Cells

U87 glioma cells were planted in a glass-bottom Petri dish, and they were cultured in modified medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2 for passaging. AgNPs prepared by citrate reduction (Figure S1) were added to coincubate with cells for 30 min. During this step, AgNPs could accumulate, especially on the cell surface. Label-free SERS spectra were recorded based on the plasmonic properties of AgNPs. To compare spectral features between glioma cells and normal cells, the SERS spectrum of AgNPs in the SVG P12 cell line, a human astrocyte cell line, was also obtained.

2.4. Monitoring Cellular Phe Expression under Different Treatment Conditions Using Label-Free SERS

As shown in Scheme , the label-free SERS method employing AgNPs as an enhancement substrate was used to perform SERS analysis on five groups of cells grown on ITO glass: (1) untreated cells as controls, (2) cells stimulated under different voltages (0.3, 0.5, and 0.7 V) but with the same duration (5 min), (3) cells treated under the same voltage (0.5 V) but with different durations (3, 5, and 7 min), (4) cells incubated under different Temozolomide concentrations (50, 100, and 200 μmol), and (5) cells treated with combinations of different Temozolomide concentrations (50, 100, and 200 μmol) and ES (0.5 V, 5 min). After washing the above cell groups with PBS, the cells were fixed with formaldehyde and stored in saline to prevent oxidation. The AgNPs were added, and SERS spectra of the cells under different treatment conditions were collected and analyzed. Statistical analysis was performed using GraphPad Prism (version 10.0). All data are presented as mean ± standard deviation (SD). Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p-value <0.05 was considered statistically significant.

2.5. SERS Measurements

The SERS spectra were collected using a confocal Raman system (LabRAM, HORIBA Jobin-Yvon, USA) with a 7.1 mW/633 nm laser, and SERS detection via a thermoelectrically cooled charge-coupled device (CCD) camera. Laser excitation and collection of Raman scattered light were performed using a 50× microscope objective lens (LMLFLN, Olympus, Japan). The confocal Raman spectra were acquired between 400–1700 cm–1, with an integration time of 10 s and a single accumulation. Each spectrum was averaged from nine separate cells. The peak intensity at 997 cm–1 was extracted for comparison to detect Phe content.

2.6. Caspase-3 Activity Assay

To further evaluate the mechanism of electrical stimulation-induced effects on glioma cells, caspase-3 activity was measured using a colorimetric assay kit according to the manufacturer’s instructions. U87 glioma cells were seeded on ITO glass and divided into two groups: a control group without any treatment and an electrical stimulation (ES) group treated at 0.7 V for 5 min. Each group contained six samples. After treatment, the cells were incubated for 4 h, then collected and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cell pellet was resuspended in lysis buffer. After lysis, the samples were centrifuged again, and the supernatant was collected for further analysis. The supernatant was incubated with the caspase-3-specific substrate Ac-DEVD-pNA at 37 °C for the required reaction time. The release of p-nitroaniline was measured by detecting absorbance at 405 nm using a microplate reader. Caspase-3 activity was expressed as absorbance values. Data are presented as mean ± standard deviation (mean ± SD), and statistical analysis was performed using an unpaired t test (p < 0.05 was considered statistically significant).

3. Results and Discussion

3.1. SERS Spectra Obtained from AgNPs Incubated with Cells

SERS was used to detect spectral changes in U87 glioma cells during the ES process. AgNPs (1.0 mM, 1.0 mL) as the enhancement substrate were added to the cell culture medium. Some of them tend to approach the cell membranes (Figure A). The SERS spectra were collected on the AgNPs. It is worth noting that citrate adsorbed onto AgNPs can yield detectable SERS signals (Figure S2). Table S1 lists the band assignments. The characteristic peak at 997 cm–1 was assigned to Phe. , The intensity of this peak indicates changes in Phe overexpression in U87 cells. To confirm this, we also recorded the SERS spectrum of the AgNPs incubated with the SVG P12 cell line (bottom curve), a human astrocyte cell line. As shown in Figure B, the SERS band at 997 cm–1 of SVG P12 cells (bottom curve) is much weaker than that of U87 cells (top curve), confirming the clear Phe feature in malignant tumor systems. Phe is generally highly expressed in tumor cells, and its accumulation results from metabolic reprogramming (enhanced uptake, enzyme dysregulation) and microenvironmental stress. Löding et al. showed that high levels of Phe and metabolites are associated with glioma progression by comparing prediagnostic plasma samples from glioma patients with matched healthy controls. Similarly, in the U87 glioma cell system, we observed higher Phe levels.

1.

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(A) Image of U87 glioma cells cocultured with AgNPs. (B) SERS spectra of the glioma cell (top curve), U87 cell line, and the human astrocyte cell (bottom curve), SVG P12 cell line.

3.2. Apoptosis during ES

ES of U87 glioma cells at different voltages (0.3, 0.5, and 0.7 V) and durations (3, 5, and 7 min) (as described in (1) in Scheme ) were investigated. First, we stained U87 cells from the above groups with Calcein AM/PI to identify live/dead cells. As shown in Figure , at a low voltage of less than 0.3 V, most cells were alive, as indicated by the almost no stained cells observed in the PI channel. When the voltage increased to 0.5 V, the cells’ morphology became rounded, suggesting a sick state. At 0.7 V, many dead cells were stained with PI, indicating they had entered an apoptosis stage. Similar to optimizing the ES voltage, we also optimized the ES period and found that 5 min of ES was sufficient to induce cell apoptosis. These imaging data show that the number of apoptotic cells gradually increased with increasing voltage and time.

2.

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Calcein AM/PI-stained confocal fluorescence images of U87 cells stimulated with (A) different voltages (0, 0.3, 0.5, and 0.7 V) and (B) for various periods (0, 3, 5, and 7 min) at 0.5 V. The scale bar is 25 μm.

We next detected SERS spectra in U87 glioma cells under different voltage and time conditions using label-free SERS, with AgNPs serving as signal amplifiers. The results are shown in Figure . As stimulation voltage and stimulation time increase, the intensity of the characteristic peak at 997 cm–1 increases, indicating higher Phe expression. This suggests that during the ES process, the rise in Phe and the duration of ES are positively correlated, indicating that Phe metabolic levels can serve as a molecular indicator of cell apoptosis.

3.

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(A) SERS spectra of U87 glioma cells under ES at the same time, but under different voltage conditions. (B) Plot of SERS intensity at 997 cm–1 under different voltage conditions. (C) SERS spectra of U87 glioma cells under ES at the same voltage but different time conditions. (D) Plot of SERS intensity at 997 cm–1 under the same voltage but different time conditions. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.)

Phe is an essential aromatic amino acid that is primarily converted into tyrosine (Tyr) by Phe hydroxylase (PAH), with the assistance of tetrahydrobiopterin (BH4). As an intermediate product, Tyr participates in numerous critical physiological processes, including the synthesis of catecholamine neurotransmitters such as dopamine, norepinephrine, and epinephrine, as well as the production of melanin. Tyr can also enter the tricarboxylic acid cycle through a series of metabolic reactions, thereby contributing to energy metabolism. Thus, the Phe metabolic pathway is not only vital for maintaining normal amino acid balance but also closely linked to neurological function, pigment synthesis, and energy regulation. Additionally, emerging research indicates that Phe plays a significant role in cell apoptosis. Huang et al. have shown that Phe induces apoptosis by activating the Fas/FasL signaling pathway. Moustafa R. K. et al., also revealed that elevated levels of Phe in the plasmonic photothermal therapy group trigger mitochondrial-dependent apoptosis via both the Rho/ROCK pathway and the Fas/FasL ligand-mediated apoptotic pathway. These findings align with the results of this study, which demonstrate that ES enhances tumor cell apoptosis by promoting an increase in intracellular Phe levels.

3.3. Therapeutic Effect Evaluation of Temozolomide

The therapeutic effect of Temozolomide in glioma has been confirmed in clinical practice, with significant implications for prolonging patients’ survival and improving their quality of life. To study the mechanism of Temozolomide-induced apoptosis of glioma cells (as described in (3) in Scheme ), we used SERS technology to detect the changes in glioma cells treated under different concentrations of Temozolomide (Figure A). Studies show that as the concentration of Temozolomide increases, the spectral intensity of the SERS characteristic peak of Phe in glioma cells gradually increases (Figure B). This demonstrates that as the Temozolomide concentration increases, glioma cell apoptosis is accelerated, which can be clearly identified by an increase in Phe content.

4.

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(A) SERS spectra of U87 glioma cells under different Temozolomide concentrations. (B) Plot of SERS intensity at 997 cm–1 under different Temozolomide concentrations. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.)

3.4. Therapeutic Effect Evaluation for the Case of Temozolomide Combined with ES

The above results demonstrate that both Temozolomide and ES can accelerate glioma cell apoptosis, and the inhibitory effect of Temozolomide on glioma proliferation has been clinically confirmed. However, Temozolomide has inevitable limitations in clinical application. , With prolonged medication and increased dosage, glioma cells may mutate, leading to reduced Temozolomide inhibition of glioma proliferation and, thus, drug resistance. To reduce the possibility of drug resistance, the combination use of ES and Temozolomide was suggested. The application of this combined treatment scheme can reduce the Temozolomide dosage, thereby delaying the onset of glioma drug resistance, prolonging patients’ survival, and improving quality of life. We assessed the combination therapeutic effect by label-free SERS to monitor the Phe level during this process. Under conditions in which different concentrations of Temozolomide were added to U87 cells incubated for 12 h, followed by ES treatment at a consistent voltage (0.5 V) and duration (5 min). We then evaluated Phe levels using SERS.

Figure A and B show the SERS profiles and the characteristic peak intensities at 997 cm–1 for ES plus Temozolomide at various Temozolomide concentrations, in comparison with Temozolomide alone (Figure B). It can be observed that with the application of ES to glioma cells treated with Temozolomide, the intensity of Phe is significantly higher than under Temozolomide alone. When Temozolomide was administered after ES acting on glioma cells, the expression level of Phe increased significantly. ES in combined Temozolomide can more effectively promote cell apoptosis even at lower Temozolomide doses.() To validate this viewpoint, we performed Calcein AM/PI staining on U87 glioma cells treated with ES in combination with Temozolomide, or Temozolomide alone (Figure C). The results show that the number of apoptotic U87 glioma cells significantly increased when Temozolomide was combined with ES treatment. These data support our expectation that ES plus Temozolomide reduces Temozolomide dosage, potentially delaying the development of drug resistance in glioma cells. By tracing Phe levels, we can infer that ES helps reduce Temozolomide dosages. For instance, the trial with 50 μM of Temozolomide plus ES (0.5 V) shows a killing effect comparable to that of 100 μM of Temozolomide, indicating that the combination treatment is a practical way to overcome Temozolomide resistance.

5.

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(A) SERS spectra of U87 glioma cells under different concentrations of Temozolomide combined with ES (0.5 V). (B) Plot of the 997 cm–1 band intensity under different concentrations of Temozolomide combined ES, in comparison to individual Temozolomide therapy. (C) Calcein AM/PI-stained U87 glioma cells treated with ES combined with Temozolomide or with Temozolomide alone. The scale bar is 25 μm. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.)

To further evaluate apoptosis-related responses of U87 glioma cells under ES, caspase-3 activity was measured using a colorimetric assay. As shown in Figure S2, compared with the untreated control group, the ES-treated group (0.7 V, 5 min) exhibited a significant increase in caspase-3 activity (p < 0.05). These results indicate that electrical stimulation promotes apoptosis-related responses in glioma cells under the present experimental conditions. In addition, the trend of caspase-3 activity is consistent with the variation observed in the SERS signal at 997 cm–1, suggesting a potential association between the spectral response and apoptosis-related processes.

4. Conclusions

This study used label-free SERS to evaluate the therapeutic effect of the first-line chemotherapy drug Temozolomide, when combined with ES physical therapy, in delaying the onset of Temozolomide resistance in glioma. The band at 997 cm–1, assigned to Phe, was strongly correlated with cell apoptosis, suggesting it could serve as a potential spectral indicator of the therapeutic effect revealed by label-free SERS. Thus, changes in Phe expression levels were quantitatively analyzed. This SERS-based assay is easy to implement and can provide an in situ, nondestructive phenylalanine assay, compared with other amino acid assays, such as mass spectrometry. The expression level of Phe increases with increasing ES stimulation time and voltage intensity, as well as with increasing Temozolomide concentration. The expression level of Phe in the ES-Temozolomide combined treatment group was significantly higher than that in the single Temozolomide treatment group. These findings confirm that the ES-Temozolomide combination therapy can enhance tumor cell apoptosis by promoting Phe overexpression while reducing Temozolomide dosage, thereby effectively inhibiting glioma cell activity and delaying the onset of drug resistance. It is worth noting that, compared with drug intervention, ES treatment has the advantage of fewer side effects. This study not only explores a combined treatment effect but also demonstrates that the mechanism of tumor cell apoptosis is associated with the Phe metabolic pathway. This work provides an important basis for an in-depth understanding of the antitumor mechanism of ES and also lays a theoretical foundation for clinical transformation and application, with significant prospects for early clinical warning of drug resistance based on metabolic Phe levels.

Supplementary Material

tg6c00014_si_001.pdf (284.1KB, pdf)

Acknowledgments

This work was supported by National Natural Science Foundation of China NSFC (No. 22373041), the Fundamental Research Funds for the Central Universities, Department of Finance of Jilin Province (2025SCZ45), Development and Reform Commission of Jilin Province (2015Y030-7), National Key Research and Development Program of China (2023YFC3706002), and the Innovative Research Project of State Key Laboratory of Supramolecular Structure and Materials (2024, 2025)

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmeasuresciau.6c00014.

  • Materials, apparatus, preparation, and characterization of AgNPs, figures, and tables (PDF)

#.

Guohui Yang and Shuping Xu contributed equally to this work. Guohui Yang performed the cell experiments and Raman measurements, acquired the data, and wrote the manuscript. Xin Wang assisted with spectral data processing and statistical analysis. Jingbin Jin performed cell culture and experiments. Xiaozhang Qu contributed to the design of the experimental setup and interpretation of SERS results. Kaizhi Zhang and Shuping Xu conceived and supervised the project, guided data interpretation, and revised the manuscript. All authors discussed the results and approved the final version of the manuscript.

The authors declare no competing financial interest.

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