The Anxiolytic Drug Tandospirone Enhances Cisplatin Efficacy by Reducing Tumor Cell Cholesterol Levels: Dual Roles in Emotional Distress Management and Chemosensitization for NSCLC Patients

The Anxiolytic Drug Tandospirone Enhances Cisplatin Efficacy by Reducing Tumor Cell Cholesterol Levels: Dual Roles in Emotional Distress Management and Chemosensitization for NSCLC Patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The Anxiolytic Drug Tandospirone Enhances Cisplatin Efficacy by Reducing Tumor Cell Cholesterol Levels: Dual Roles in Emotional Distress Management and Chemosensitization for NSCLC Patients Xichun Qin, Bo Cheng, Shangshang Ma, Kun Li, Yongfei Fan, Mingjun Li, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7149599/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Nov, 2025 Read the published version in npj Digital Medicine → Version 1 posted 9 You are reading this latest preprint version Abstract Patients undergoing lung cancer surgery face a significantly increased risk of emotional distress (ED), including anxiety and depression. This distress not only severely impairs their quality of life but may also negatively affect treatment outcomes. Therefore, effective ED management is crucial in postoperative care. This study systematically evaluated the mental health status of 1,185 non-small cell lung cancer (NSCLC) patients and identified chemotherapy as an independent risk factor for ED development. Additionally, we investigated the safety and therapeutic effects of commonly used anti-ED drugs in cellular and animal models. These drugs did not promote tumor cell growth, and notably, tandospirone unexpectedly enhanced the efficacy of cisplatin. Mechanistically, tandospirone reduces cholesterol levels in tumor cells, thereby promoting apoptosis and increasing their susceptibility to chemotherapy. Our findings highlight the importance of ED management in lung cancer patients and propose tandospirone as a promising dual-purpose agent for both psychological support and chemotherapy sensitization, potentially enabling more comprehensive and effective treatment strategies. Biological sciences/Cancer Biological sciences/Drug discovery Health sciences/Oncology Lung cancer Emotional distress Tandospirone Chemotherapy sensitization Cholesterol metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Lung cancer remains one of the most prevalent and deadly malignancies worldwide, posing a severe threat to public health [ 1 – 2 ]. Despite substantial advancements in diagnosis and treatment, surgical resection remains the primary therapeutic approach for early-stage non-small cell lung cancer (NSCLC) [ 3 ]. However, postoperative patients often experience a range of physical and psychological challenges [ 4 ]. Emotional distress (ED), commonly manifesting as depression and/or anxiety, is a frequent psychological complication following lung cancer surgery. The incidence of ED in lung cancer patients is significantly higher than that in the general population, adversely affecting their quality of life and potentially influencing treatment outcomes and prognosis [ 5 – 6 ]. Therefore, identifying effective interventions for ED in postoperative lung cancer patients is of great clinical significance. The underlying mechanisms of ED are complex and involve physiological, psychological, and social factors. Postoperative pain, physical limitations, fear of disease recurrence, and uncertainty about the future all contribute to ED [ 7 – 8 ]. Furthermore, many early-stage lung cancer patients undergo adjuvant therapies such as chemotherapy, immunotherapy, and targeted therapy post-surgery, which may exacerbate ED symptoms [ 9 – 11 ]. However, research on the interaction between anti-ED medications and lung cancer treatment remains limited. Clinically, anti-ED drugs are widely prescribed for various anxiety and depression disorders. However, for postoperative lung cancer patients, especially those receiving adjuvant therapy, their safety profile and potential impact on tumor growth remain critical concerns. While some epidemiological studies have suggested that anti-ED drugs may influence tumor progression through direct effects on cancer cells or by modulating neurotransmitter levels and the immune microenvironment [ 12 – 14 ], their specific mechanisms and clinical implications remain unclear. Therefore, effective ED management in postoperative lung cancer patients requires not only psychological interventions but also a thorough evaluation of pharmacological treatments' feasibility and safety. Given this context, we conducted a large-scale clinical survey involving 1,185 patients undergoing lung cancer surgery. Our goal was to systematically evaluate postoperative mental health and examine the correlation between ED and adjuvant therapies. Our findings revealed a significantly higher incidence of anxiety and depression in patients receiving chemotherapy than in other treatment groups, indicating that chemotherapy may be a major risk factor for ED. To explore potential pharmacological interventions, we systematically evaluated the effects of commonly used anti-anxiety and anti-depressant drugs in combination with cisplatin chemotherapy in both cell and animal models. Among these, tandospirone lowers cholesterol levels in tumor cells, thereby enhancing their susceptibility to apoptosis. These findings suggest that tandospirone may serve a dual function: alleviating ED symptoms and enhancing chemotherapy efficacy, providing a novel avenue for integrative lung cancer treatment strategies. 2. Methods Study design and ethics statement This single-center, cross-sectional study was approved by the Ethics Committee of the Shanghai Pulmonary Hospital affiliated with Tongji University (K22-184Y). The study protocol was in accordance with the relevant guidelines and regulations. This study followed the tenets of the Declaration of Helsinki. This study was anonymous, and the questionnaire used in the study did not collect any identifying information (e.g., name, hospital number), and the data were used only for statistical analyses. The Ethics Committee of Shanghai Pulmonary Hospital (Shanghai, China) waived off the requirement for obtaining informed consent. Study participants This study involved 220 healthy volunteers and 1,500 NSCLC patients who underwent surgery at the Shanghai Pulmonary Hospital between June 2022 and January 2024. The patient exclusion criteria included a history of neoadjuvant therapy, other malignancies within the past 3 years, psychiatric disorders, and any current antidepressant or anti-anxiety treatment. ED was assessed 6-month post-surgery. Ultimately, 192 volunteers and 1,185 NSCLC patients were included in the final analyses. Clinical characteristics Patient data were collected and analyzed, including demographics (age, sex), medical history (hypertension, diabetes), lifestyle factors (smoking, drinking), education level, BMI, and postoperative medications (chemotherapy, targeted therapy, immunotherapy, etc.). Evaluation of ED ED generally refers to depression and/or anxiety symptoms. PHQ-9 and GAD-7 were used to screen these symptoms in cancer patients, with assessments conducted 6 months post-surgery [ 15 – 16 ]. ED was defined as a PHQ-9 or GAD-7 score of ≥ 5 based on clinical thresholds [ 5 ]. Cell culture Human NSCLC cell lines (A549 and H1299) were obtained from Procell (Wuhan, China) and cultured in DMEM (BasalMedia, L110 KJ, China) supplemented with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (BasalMedia, S110JV) and incubated at 37°C under a 5% CO₂ atmosphere. Cell viability analysis A549 and H1299 cells were seeded into 96-well plates and incubated under standard culture conditions. Anti-ED drugs, cholesterol (14606, Sigma-Aldrich) or methyl-β-cyclodextrin (MβCD, 332615, Sigma-Aldrich) or/and cisplatin, were added at varying concentrations based on the experimental protocol and cultured for 48 h. Next, fresh medium mixed with CCK-8 reagent (Beyotime, C0041) at a 10:1 ratio was added, and the cells were incubated at 37°C for another 2 h. Absorbance was measured at 450 nm using a microplate reader (Infinite 200 PRO, Tecan). Cell viability was determined based on the average optical density (OD) values from at least three replicate wells. Apoptosis analysis with Flow Cytometer The experimental cells were plated into 12-well plates at a density of 3 × 10⁵ cells/well and treated with anti-ED drugs, MβCD, and/or cisplatin. After 48 h of incubation, apoptosis was evaluated using an Annexin V-FITC/PI apoptosis detection kit (KeyGEN BioTECH, KGA1102) according to the manufacturer’s protocol. Briefly, the cells were collected, washed thrice with PBS, and resuspended in 500 µL of binding buffer. Then, 5 µL of Annexin V-FITC and 5 µL of PI were added, and the mixture was incubated in the dark at room temperature for 15 min. Flow cytometry data were obtained using the CytoFLEX S cytometer (Beckman, USA) and analyzed with FlowJo software (version 10). Colony formation assay Cells (500–1000/well) were seeded into six-well plates and incubated at 37°C in a humidified atmosphere with 5% CO₂. Colonies were collected and washed with phosphate-buffered saline, fixed in 4% paraformaldehyde for 20 min, and stained with 0.5% crystal violet for 30 min. Images were captured using the Pannoramic MIDI system. Cell invasion, migration, and wound-healing assays For the migration assay, A549 or H1299 (2 × 10⁴) cells were seeded in a serum-free medium in the upper chamber (Corning Inc., Corning, NY, USA). For the invasion assay, the upper chamber was pre-coated with Matrigel (diluted 1:10; BD Biosciences, San Jose, CA, USA), and 5 × 10⁵ cells were seeded in serum-free medium. The cells that migrated or invaded through the membrane were stained with 0.04% crystal violet and enumerated. Animal models BALB/c nude mice (4–6 weeks old) were obtained from Gempharmatech Co., Ltd. (Nanjing, China) and housed in a specific pathogen-free (SPF) facility at Tongji University (Shanghai, China). No sex-based selection was performed for the mice used in these experiments. To evaluate the in vivo tumor growth and metastatic potential, both subcutaneous xenograft and lung metastasis models were established. For the subcutaneous xenograft model, A549 cells (1 × 10⁵ cells/injection) were injected into the left flank of the mice. Drug treatments were initiated 2 weeks after tumor establishment, and the tumor size was measured weekly. After 4 weeks, the tumors were harvested for volumetric analyses. In the lung metastasis model, A549 cells (1 × 10⁵ cells/dose) were injected via the tail vein. After 4 weeks, the mice were euthanized, and lung metastases were examined through hematoxylin and eosin (H&E) staining. Immunohistochemistry Immunohistochemical (IHC) staining for Ki67 was conducted on paraffin-embedded tumor tissue sections in accordance with the experimental protocol. Images were captured using an inverted microscope (Olympus). RNA sequencing RNA high-throughput sequencing was performed using the Illumina HiSeq platform (Shanghai, China). Total RNA was first depleted of rRNA with the NEBNext rRNA Depletion Kit (New England Biolabs, Massachusetts, USA) following the manufacturer’s instructions. RNA libraries were then constructed using the NEBNext® UltraTM II Directional RNA Library Prep Kit (New England Biolabs) according to the provided protocol. Quality control and library quantification were assessed using the BioAnalyzer 2100 (Agilent Technologies, USA). Sequencing was conducted on the Illumina HiSeq system with 150 bp paired-end reads. Isolation of lipid rafts The experimental cells were seeded, treated with specified drugs, and harvested. They were then lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.5% Triton X-100 for 30 min. The lysates were then centrifuged at 16,000 g at 4°C for 30 min, and the supernatants were collected as non-lipid raft fractions. The remaining insoluble pellets were resuspended in 200 µL of buffer (0.5% SDS, 2 mM DTT) and incubated for 10 min. After another round of centrifugation under the same conditions, the supernatants were transferred into separate tubes as lipid raft fractions. The cholesterol levels were measured using the Amplex® Red Cholesterol Assay Kit (A12216, Invitrogen, Carlsbad, CA, USA) [ 17 ]. Total RNAoifd extraction and real‑time quantitative polymerase chain reaction (RT‑qPCR) Total RNA was extracted using TRIzol reagent, and 2 µg of RNA was used for reverse transcription. RT-qPCR was conducted following the protocol provided in the UltraSYBR one-step RT-qPCR kit (CWBIO, Beijing, China). The primers used for the analysis are detailed in Supplementary Table 1. Western blotting Proteins from A549 and H1299 cells were extracted by using a total protein extraction kit. Protein concentrations were measured with a BCA protein assay kit. Proteins were separated via SDS-PAGE and transferred onto 0.45-µm polyvinylidene fluoride (PVDF) membranes (MilliporeSigma). The membranes were blocked with 5% non-fat dry milk for 1 h, then incubated overnight at 4°C with primary antibodies targeting HMGCR (ab242315, Abcam), SREBP2 (ab30682, Abcam), and GAPDH (60004-1-Ig, Proteintech). The next day, membranes were incubated with the appropriate secondary antibody for 60 min at room temperature. Statistical analysis Continuous variables were expressed as the mean ± standard deviation. The χ 2 test was performed for the categorical comparison of demographic variables between different groups, whereas the Student’s t -test and/or non-parametric test were applied for a continuous comparison. Skewness continuous variables were analyzed using median and interquartile range (IQR) and compared using the Mann–Whitney U-test. A logistic regression model was applied to evaluate factors affecting the occurrence of ED. The α-value < 0.05 on both sides was considered statistically significant. All statistical analyses were performed using SPSS 25.0 (SPSS, Inc., Chicago, IL, USA). 3. Results 3.1 NSCLC patients have a significantly increased risk of ED after surgery A total of 1500 NSCLC patients who underwent surgical resection and 220 healthy volunteers were recruited from our center for a comprehensive questionnaire-based assessment. Patients with incomplete data, a history of neoadjuvant therapy, other malignancies within the past 3 years, pre-existing psychiatric disorders, or those currently receiving antidepressant or anti-anxiety treatment were excluded from the study. The final analysis included 1,185 NSCLC patients and 192 healthy controls (Fig. 1 ). Demographic characteristics were similar between the healthy population and postoperative patients (Table 1 ). However, the prevalence of ED among healthy individuals was only 8.9%, whereas it was significantly higher in postoperative NSCLC patients, reaching 32.2% (P < 0.001). These findings underscore a markedly increased risk of ED following lung cancer surgery. Table 1 Demographics and characteristics of patient. *Statistically significant values. Variables Total (n = 1377) Healthy (n = 192) Surgery (n = 1185) P value Age, years (%) < 65 ≥65 1196 (86.9) 181 (13.1) 168 (87.5) 24 (12.5) 1028 (86.8) 157 (13.2) 0.908 Sex (male/female) 551/826 78/114 473/712 0.874 Smoking history (%) 369 (26.8) 49 (25.5) 320 (27.0) 0.726 Drinking history (%) 233 (16.9) 33 (17.2) 200 (16.9) 0.917 Hypertension (%) 336 (24.4) 42 (21.9) 294 (24.8) 0.416 Diabetes (%) BMI < 24 ≥24 Educational level (%) <High school ≥High school Emotional distress (%) 106 (7.7) 731 (53.1) 646 (46.9) 1041 (75.6) 336 (24.4) 399 (29.0) 13 (6.8) 106 (55.2) 86 (44.8) 137 (71.4) 55 (28.6) 17 (8.9) 93 (7.8) 625 (52.7) 560 (47.3) 904 (76.3) 281 (23.7) 382 (32.2) 0.770 0.534 0.148 < 0.001* 3.2 Chemotherapy is an independent risk factor for increased ED in NSCLC patients after surgery To identify key risk factors associated with ED, we stratified postoperative NSCLC patients into ED and non-ED groups. Compared with the non-ED group, patients in the ED group were older, had a higher proportion of females, a greater prevalence of diabetes, a lower education level and a higher lymph node metastasis positivity. However, no statistically significant differences were observed in hypertension status, smoking history, and tumor T stage (Table 2 ). According to the type of postoperative adjuvant therapy, 602 patients received no treatment, 80 patients received chemotherapy, 246 patients received chemotherapy combined with other treatments (such as targeted therapy or immunotherapy), and 253 patients received other treatments without chemotherapy (Fig. 2 A). Further analysis of treatment modalities revealed a significant association between chemotherapy and increased ED incidence (Fig. 2 B and Supplement Table 2 ). Multivariate regression analysis confirmed chemotherapy as an independent risk factor for ED (Supplement Table 3 and Supplement Table 4). These findings suggest that patients undergoing chemotherapy after NSCLC surgery face an elevated risk of psychological distress. Table 2 Demographics and characteristics of ED in NSCLC patients. *Statistically significant values. Variables Total (n = 1185) ED (n = 382) No ED (n = 803) P value Age, years (%) < 65 ≥65 1028 (86.8) 157 (13.2) 316 (82.7) 66 (17.3) 712 (88.7) 91 (11.3) 0.009* Sex (male/female) 473/712 131/251 342/461 0.006* Smoking history (%) 320 (27.0) 93 (24.3) 227 (28.3) 0.162 Drinking history (%) 200 (16.9) 58 (15.2) 142 (17.7) 0.319 Hypertension (%) 294 (24.8) 102 (26.7) 192 (24.0) 0.314 Diabetes (%) BMI < 24 ≥24 Educational level (%) <High school ≥High school 93 (7.8) 625 (52.7) 560 (47.3) 904 (76.3) 281 (23.7) 41 (10.7) 193 (50.5) 189 (49.5) 313 (81.9) 69 (18.1) 52 (6.5) 432 (53.8) 371 (46.2) 591 (73.6) 212 (26.4) 0.001* 0.319 0.002* Tumor stage (%) I II III IV Lymph node metastasis (%) Negative Positive Distant metastasis (%) M0 M1 946 (79.8) 221 (18.7) 18 (1.5) 0 902 (76.1) 283 (23.9) 1185 (100.0) 0 294 (77.0) 81 (21.2) 7 (1.8) 0 275 (72.0) 107 (28.0) 382 (100.0) 0 652 (81.2) 140 (17.4) 11 (1.4) 0 627 (78.1) 176 (21.9) 803 (100.0) 0 0.232 0.024* N/A 3.3 Common anti-ED drugs do not promote tumor cell progression and may enhance chemotherapy effects in patients with ED We systematically evaluated the impact of six commonly prescribed anti-ED drugs on lung cancer cell proliferation and apoptosis. The selected medications included paroxetine and sertraline in selective serotonin reuptake inhibitors (SSRIs); bupropion in serotonin and norepinephrine reuptake inhibitors (SNRIs); amitriptyline in tricyclic antidepressants (TcAs); and other drugs such as tandospirone and agomelatine (Fig. 3 A). First, we conducted CCK-8 assays to assess the effects of these drugs on the proliferation of two NSCLC cell lines (A549 and H1299) across a concentration range of 0–200 µg/mL. None of the six medications significantly promoted tumor cell proliferation. Instead, all displayed a mild inhibitory effect on0 cell growth at higher concentrations (Fig. 3 B, C). This result suggests that these anti-ED drugs slightly inhibit lung cancer cell progression within the commonly used clinical dose range. To further explore their influence on tumor cell survival, we standardized the drug concentration to 100 µg/mL and performed flow cytometry analysis to assess apoptosis induction (Fig. 3 D, E). All six drugs induced some degree of apoptosis in A549 and H1299 cells, with tandospirone and sertraline exhibiting the most pronounced effects (apoptosis rates of 7.2% and 9.9%, respectively). We then examined the interactions between these anti-ED drugs and cisplatin chemotherapy. None of the medications reduced the sensitivity of A549 or H1299 cells to cisplatin; rather, they increased chemotherapy efficacy to varying degrees (Fig. 3 F, G). This suggests that these anti-ED drugs do not reduce the chemotherapeutic effectiveness of cisplatin. Notably, tandospirone exhibited a significant synergistic effect, markedly increasing cisplatin-induced cytotoxicity in both cell lines. These results indicate that tandospirone may have an unexpected function of enhancing chemotherapy. 3.4 Tandospirone enhances the anti-tumor effects of cisplatin Tandospirone is a non-benzodiazepine anxiolytic that alleviates anxiety and depressive symptoms by modulating the serotonin (5-HT) system [ 18 ]. It is widely used for the long-term treatment of generalized anxiety disorder and depression [ 19 ]. However, its role in tumor biology remains unexplored. To investigate the therapeutic potential of tandospirone in augmenting cisplatin’s antitumor effects, we conducted a comprehensive evaluation using both in vitro and in vivo models. The cloning sphere experiment revealed that NSCLC cells treated with the tandospirone-cisplatin combination exhibited significantly reduced clonogenic capacity compared with the cisplatin monotherapy group. The number and size of tumor cell colonies were significantly diminished, suggesting that the combination therapy effectively impairs the self-renewal ability of cancer stem-like cells (Fig. 4 A). Furthermore, flow cytometry analysis demonstrated that the cell apoptosis rate was significantly higher in cells treated with the tandospirone-cisplatin combination than in those treated with cisplatin alone (Fig. 4 B). These results indicate that tandospirone enhances cisplatin-induced apoptosis, thereby increasing tumor cell susceptibility to chemotherapy. To further assess its impact on tumor progression, we conducted Transwell invasion and scratch wound-healing assays. The combination treatment significantly suppressed tumor cell migration and invasion, as evidenced by a substantial reduction in both the number of transmembrane invasive cells and wound closure rates (Fig. 4 C, D) These findings suggest that tandospirone exerts a synergistic effect in limiting tumor cell dissemination. To validate these findings in an animal model, we established a subcutaneous xenograft model and a lung metastasis model using A549 cells injected via the tail vein of nude mice (Fig. 4 E). In the subcutaneous tumor model, mice receiving the tandospirone-cisplatin combination exhibited significantly slower tumor growth compared with those treated with cisplatin alone. Final tumor weight was also significantly lower in the combination therapy group than in the cisplatin monotherapy group, indicating that tandospirone enhances the inhibitory effect of cisplatin (Fig. 4 F, H). The lung metastasis model further corroborated these results, showing a marked reduction in metastatic nodules in the combination treatment group compared with the cisplatin monotherapy group, suggesting that tandospirone not only inhibits primary tumor growth but also plays a crucial role in preventing distant metastases (Fig. 4 I, J). To assess the impact of combined therapy on tumor cell proliferation, we performed Ki67 IHC staining and quantitative analysis on mouse tumor tissues. Quantitative analysis revealed a significant decrease in Ki67-positive cells in the combination treatment group compared with the cisplatin monotherapy group. This reduction in proliferative activity provides further evidence of the enhanced anti-tumor efficacy of tandospirone in combination with cisplatin (Fig. 4 K, I). 3.5 Tandospirone reduces cholesterol levels in tumor cells To elucidate the molecular mechanism by which tandospirone enhances cisplatin sensitivity, we conducted RNA-seq transcriptomic analysis on A549 cells treated with tandospirone. The analysis identified 272 differentially expressed genes (screening condition: |fold change| ≥ 1.0, Padjust < 0.05) (Fig. 5 A, B). Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed a significant enrichment of cholesterol synthesis-related pathways (Fig. 5 C, D and Supplementary Fig. 2A). Additionally, Gene Set Enrichment Analysis further confirmed that cholesterol biosynthesis pathways and steroid biosynthesis were markedly altered in response to tandospirone treatment (Fig. 5 E and Supplementary Fig. 2B). These findings suggest that tandospirone may exert its chemotherapy-sensitizing effects by modulating cholesterol metabolism. To validate this hypothesis, we measured intracellular cholesterol levels in A549 and H1299 cells following tandospirone treatment. Compared with untreated controls, tandospirone significantly reduced both total intracellular cholesterol and membrane-associated cholesterol (Fig. 5 F, G). This indicates that tandospirone effectively disrupts cholesterol homeostasis in tumor cells. To further explore the regulatory mechanisms underlying this cholesterol depletion, we examined key genes involved in cholesterol biosynthesis. qPCR and western blot analyses (Fig. 5 H–K and Supplementary Fig. 3A, B) demonstrated that tandospirone treatment led to a significant upregulation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol synthesis [ 20 ], and sterol regulatory element binding protein 2 (SREBP2), a key upstream transcription factor regulating cholesterol metabolism [ 21 ]. This suggests that tumor cells may have initiated a feedback regulation mechanism because of the reduced cholesterol levels. Tandospirone may consume cholesterol in tumor cells and induce abnormal activation of the cholesterol synthesis pathway, thereby modifying the cell metabolic state, affecting the cell's survival and proliferation, and improving cisplatin’s antitumor effect. 3.6 Reducing cholesterol levels in NSCLC cells can increase their sensitivity to apoptosis Building on our experimental results, we speculate that reducing cholesterol levels may render tumor cells more susceptible to chemotherapy. To validate this hypothesis, we conducted additional experiments to assess the role of cholesterol depletion in tandospirone-induced cisplatin sensitization. We first supplemented A549 and H1299 cells with exogenous cholesterol to determine whether restoring cholesterol levels would counteract the sensitizing effect of tandospirone (Fig. 6 B). Although exogenous cholesterol marginally promoted tumor cell proliferation (Fig. 6 A), it significantly attenuated the enhanced cytotoxicity of cisplatin observed in the presence of tandospirone (Fig. 6 B). To further investigate whether cholesterol depletion alone could enhance cisplatin’s antitumor effects, we treated A549 and H1299 cells with methyl-β-cyclodextrin (MβCD), a cholesterol scavenger [ 22 ]. Our results revealed that cholesterol depletion significantly increased cell death, indicating that reduced cholesterol levels independently contribute to enhanced chemotherapy sensitivity (Fig. 6 C, D). This finding was reinforced by clonogenic assays, where MβCD-treated cells exhibited a significantly reduced capacity for colony formation, an effect that was further amplified when combined with cisplatin (Fig. 6 E). Flow cytometry analysis confirmed these observations, showing a substantial increase in apoptosis in cholesterol-depleted cells, with an even more pronounced effect when co-administered with cisplatin (Fig. 6 F). Furthermore, Transwell invasion and scratch assays demonstrated that cholesterol depletion also inhibited tumor cell migration and invasion. Specifically, MβCD treatment significantly reduced the number of cells crossing the membrane and slowed wound healing, with these effects being further intensified when combined with cisplatin (Supplementary Fig. 4A, B). These findings suggest that cholesterol depletion not only enhances cisplatin-induced apoptosis but also impairs tumor cell motility and metastatic potential. To explore whether cholesterol depletion broadly sensitizes tumor cells to other forms of cell death, we tested its effects in combination with various chemotherapeutic drugs and targeted inhibitors, including cisplatin, pemetrexed, docetaxel, bortezomib (a proteasome inhibitor) [ 23 ], and ML162 (a GPX4 inhibitor) [ 24 ]. Remarkably, MβCD treatment significantly enhanced the cytotoxicity of all tested agents, as evidenced by a pronounced decrease in cell viability (Fig. 6 G). Likewise, tandospirone also exhibited a strong synergistic effect when combined with these apoptosis-inducing agents, reinforcing the notion that cholesterol depletion serves as a metabolic vulnerability that can be exploited to enhance cancer therapy. 4. Discussion In this study, we conducted a comprehensive analysis of ED in postoperative lung cancer patients, investigating its prevalence, risk factors, and potential therapeutic interventions. Additionally, we explored the impact of anti-ED drugs on tumor biology and chemotherapy sensitivity, yielding significant findings at multiple levels (Fig. 6 H). Our results not only provide a scientific foundation for clinical psychological interventions but also introduce a new approach to tumor metabolic regulation that warrants further investigation. Lung cancer remains one of the most aggressive malignancies worldwide, with high morbidity and mortality rates [ 2 , 25 ]. Despite advances in treatment, surgical resection remains the primary curative option for early- and mid-stage lung cancer patients. However, postoperative recovery extends beyond physical healing; patients frequently experience the adverse side effects of chemotherapy, fear of disease recurrence, and a decline in overall quality of life, all of which contribute to a high incidence of ED [ 5 , 26 ]. Our findings indicate that ED significantly increases following lung cancer surgery and is strongly associated with adjuvant treatment, identifying chemotherapy as an independent risk factor. While anti-ED medications are commonly prescribed for patients experiencing anxiety and depression concerns persist regarding their potential interference with chemotherapy efficacy. To address this issue, we systematically screened 6 widely used anti-ED drugs, assessing their effects on cell proliferation, invasion, and migration in A549 and H1299 lung cancer cell lines. Contrary to common concerns, our results demonstrated that these drugs did not promote tumor progression. Instead, certain anti-ED medications, particularly tandospirone, exhibited unexpected tumor-suppressive effects. Notably, tandospirone not only failed to diminish the cytotoxicity of cisplatin but significantly reduced its IC50, indicating a pronounced chemosensitizing effect. Tandospirone, a well-established anxiolytic that functions as a partial agonist of 5-HT1A receptors [ 18 ], has not been previously studied for its impact on tumor metabolism. Through RNA-seq analysis, we identified that tandospirone induces significant alterations in cholesterol metabolism, particularly by activating cholesterol synthesis pathways and significantly reducing intracellular cholesterol levels. qPCR and western blot analyses confirmed the upregulation of key cholesterol synthesis regulatory factors such as SREBP2 and HMGCR, in response to tandospirone treatment. This metabolic perturbation, characterized by "cholesterol depletion-feedback activation", likely disrupts tumor cell lipid homeostasis, rendering cancer cells more vulnerable to cisplatin-induced apoptosis. Emerging evidence highlights the crucial role of cholesterol in tumor progression and therapy resistance [ 27 , 28 ]. Cholesterol is not only a fundamental component of cell membranes but also regulates key signaling pathways, membrane microdomains, and apoptotic resistance mechanisms [ 29 ]. Studies have suggested that lung cancer cells frequently upregulate cholesterol synthesis to evade chemotherapy-induced cell death [ 30 , 31 ]. Our findings suggest that tandospirone, by depleting cholesterol and inducing metabolic stress, sensitizes tumor cells to chemotherapy. This effect was further corroborated by experiments showing that cholesterol depletion, whether induced by tandospirone or the cholesterol scavenger MβCD, enhanced the efficacy of multiple chemotherapeutic agents such as cisplatin, docetaxel, pemetrexed, bortezomib, and ML162. These results suggest that metabolic interventions targeting cholesterol depletion may serve as a broadly applicable chemosensitization strategy. From a clinical perspective, this study holds dual significance. First, it provides critical safety evidence supporting the use of anti-ED medications in postoperative lung cancer patients, demonstrating that these drugs do not compromise chemotherapy efficacy. Second, our findings introduce a novel concept of ​​"metabolic depletion-sensitization therapy," where an anxiolytic agent like tandospirone not only alleviates ED but also enhances chemotherapy effectiveness. This positions tandospirone as a promising candidate for integrated cancer care, particularly for patients experiencing both psychological distress and chemotherapy resistance. This work has several limitations. This study found that chemotherapy is an independent risk factor for ED, but potential confounding factors may be associated with the results. Of note, other treatments can also increase patients' ED levels. Many studies have pointed out that ED affects the immune system, and it is unclear how anti-ED drugs affect immunotherapy in NSCLC patients. In the future, a larger sample size and corresponding clinical studies are warranted to further explore the safety and effectiveness of anti-ED drugs in combination with other treatments. 5. Conclusion Our study revealed that the ED status of postoperative lung cancer patients is closely related to adjuvant chemotherapy, and chemotherapy is an independent risk factor for ED. In addition to improving ED symptoms, common anti-ED drugs may improve lung cancer progression. Additional studies have found that tandospirone is safe and can significantly improve the antitumor effect of cisplatin and promote cell apoptosis. Mechanistic exploration studies revealed that tandospirone reduces cholesterol levels in tumor cells, activates cholesterol synthesis-related genes such as SREBP2 and HMGCR, induces a "cholesterol depletion-death stress" state, and thus augments the killing effect of cisplatin. These findings offer a new scientific basis for the emotional management of postoperative lung cancer patients and lay a theoretical foundation for the rational use of anti-ED drugs in tumor treatment. Declarations Acknowledgements We thank all the researchers who contributed to this study; We thank BioRender for providing the tools used to create the illustrations in this study. Author contributions X. Q.: Investigation, Methodology, Data curation, Formal analysis, Writing - original draft. B. C.: Investigation, Methodology, Data curation, Formal analysis, Writing - original draft. S. M.: Methodology, Data curation, Formal analysis. K. L.: Investigation, Methodology, Data curation, Writing - review & editing. Y. F.: Investigation, Data curation, Formal analysis. M. L.: Data curation, Formal analysis. R. W.: Methodology, Investigation. X. A.: Methodology, Investigation. L. W.: Investigation, Methodology, Data curation, Writing - review & editing. X. L.: Funding acquisition, Investigation, Writing - review & editing. X. W.: Funding acquisition, Conceptualization, Investigation, Supervision. D. X.: Funding acquisition, Investigation, Supervision, Writing - review & editing. All authors reviewed the manuscript. Funding This research was supported by the National Key Research and Development Program of China (2024YFA1212000), the National Natural Science Foundation of China (82372098; 82200999), the Shanghai Committee of Science and Technology China (21Y11913400), the Key Project of Jiangsu Provincial Health Commission (K2024044), the Open Projects of Jiangsu Provincial Key Laboratories (XZSYSKF2022012), the Key Project of Xuzhou Medical Science and Technology Innovation Project (XWKYHT20230034). Data availability No datasets were generated or analyzed during the current study. Ethics approval and consent to participate All animal studies have been approved by the Review Board and Ethics Committee of Shanghai Pulmonary Hospital (ID: K22-184Y). Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Leiter Amanda, Veluswamy Rajwanth R, Wisnivesky Juan P, The global burden of lung cancer: current status and future trends.[J].Nat Rev Clin Oncol, 2023, 20: 624–639. Zhang Yanting, Vaccarella Salvatore, Morgan Eileen et al. Global variations in lung cancer incidence by histological subtype in 2020: a population-based study.[J].Lancet Oncol, 2023, 24: 1206–1218. Hendriks Lizza E L, Remon Jordi, Faivre-Finn Corinne et al. Non-small-cell lung cancer.[J].Nat Rev Dis Primers, 2024, 10: 71. Li Juan, Li Chan, Puts Martine et al. 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Andersen Barbara L, Rowland Julia H, Somerfield Mark R, Screening, assessment, and care of anxiety and depressive symptoms in adults with cancer: an american society of clinical oncology guideline adaptation.[J].J Oncol Pract, 2015, 11: 133–4. Pan Zhenzhen, Wang Kai, Wang Xiniao et al. Cholesterol promotes EGFR-TKIs resistance in NSCLC by inducing EGFR/Src/Erk/SP1 signaling-mediated ERRα re-expression.[J].Mol Cancer, 2022, 21: 77. Huang Xuefei, Yang Jing, Yang Sijin et al. Role of tandospirone, a 5-HT1A receptor partial agonist, in the treatment of central nervous system disorders and the underlying mechanisms.[J].Oncotarget, 2017, 8: 102705–102720. Yamada Kazuo, Yagi Gohei, Kanba Shigenobu, Clinical efficacy of tandospirone augmentation in patients with major depressive disorder: a randomized controlled trial.[J].Psychiatry Clin Neurosci, 2003, 57: 183–7. Lu Xiao-Yi, Shi Xiong-Jie, Hu Ao et al. Feeding induces cholesterol biosynthesis via the mTORC1-USP20-HMGCR axis.[J].Nature, 2020, 588: 479–484. Trindade Bruno C, Ceglia Simona, Berthelette Alyssa et al. The cholesterol metabolite 25-hydroxycholesterol restrains the transcriptional regulator SREBP2 and limits intestinal IgA plasma cell differentiation.[J].Immunity, 2021, 54: 2273–2287.e6. Guilbaud Emma, Barouillet Thibault, Ilie Marius et al. Cholesterol efflux pathways hinder KRAS-driven lung tumor progenitor cell expansion.[J].Cell Stem Cell, 2023, 30: 800–817.e9. Tan Carlyn Rose C, Abdul-Majeed Saif, Cael Brittany et al. Clinical Pharmacokinetics and Pharmacodynamics of Bortezomib.[J].Clin Pharmacokinet, 2019, 58: 157–168. Ma Furong, Li Yulong, Cai Maohua et al. ML162 derivatives incorporating a naphthoquinone unit as ferroptosis/apoptosis inducers: Design, synthesis, anti-cancer activity, and drug-resistance reversal evaluation.[J].Eur J Med Chem, 2024, 270: 116387. Li Yuting, Yan Bingshuo, He Shiming, Advances and challenges in the treatment of lung cancer.[J].Biomed Pharmacother, 2023, 169: 115891. Ghoneim Mohamed M, O'Hara Michael W, Depression and postoperative complications: an overview.[J].BMC Surg, 2016, 16: 5. Huang Binlu, Song Bao-Liang, Xu Chenqi, Cholesterol metabolism in cancer: mechanisms and therapeutic opportunities.[J].Nat Metab, 2020, 2: 132–141. Kuzu Omer F, Noory Mohammad A, Robertson Gavin P,The Role of Cholesterol in Cancer.[J].Cancer Res, 2016, 76: 2063–70. Luo Jie, Yang Hongyuan, Song Bao-Liang, Mechanisms and regulation of cholesterol homeostasis.[J].Nat Rev Mol Cell Biol, 2020, 21: 225–245. Wu Yufeng, Si Ruirui, Tang Hong et al. Cholesterol reduces the sensitivity to platinum-based chemotherapy via upregulating ABCG2 in lung adenocarcinoma.[J].Biochem Biophys Res Commun, 2015, 457: 614–20. Daya Tasvi, Breytenbach Andrea, Gu Liang et al. Cholesterol metabolism in pancreatic cancer and associated therapeutic strategies.[J].Biochim Biophys Acta Mol Cell Biol Lipids, 2025, 1870: 159578. Additional Declarations No competing interests reported. Supplementary Files Supplementmaterials.pdf Cite Share Download PDF Status: Published Journal Publication published 17 Nov, 2025 Read the published version in npj Digital Medicine → Version 1 posted Editorial decision: Revision requested 01 Aug, 2025 Reviews received at journal 31 Jul, 2025 Reviews received at journal 30 Jul, 2025 Reviewers agreed at journal 24 Jul, 2025 Reviewers agreed at journal 22 Jul, 2025 Reviewers invited by journal 22 Jul, 2025 Editor assigned by journal 22 Jul, 2025 Submission checks completed at journal 21 Jul, 2025 First submitted to journal 17 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7149599","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":489138511,"identity":"f733825b-c988-4ae0-a940-b7b4cb460fd6","order_by":0,"name":"Xichun Qin","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Xichun","middleName":"","lastName":"Qin","suffix":""},{"id":489138512,"identity":"1f82d180-7a02-4b09-a648-1d62267d3144","order_by":1,"name":"Bo Cheng","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Cheng","suffix":""},{"id":489138513,"identity":"3f6ee482-b954-4edd-ab9d-59d91d69b6fb","order_by":2,"name":"Shangshang Ma","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Shangshang","middleName":"","lastName":"Ma","suffix":""},{"id":489138514,"identity":"90280d15-9a76-43b0-a932-ebf034ef409b","order_by":3,"name":"Kun Li","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Li","suffix":""},{"id":489138515,"identity":"b690041f-9cd7-44cd-af15-3ce2392e529f","order_by":4,"name":"Yongfei Fan","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Yongfei","middleName":"","lastName":"Fan","suffix":""},{"id":489138516,"identity":"4af46329-6f32-4d8d-9a03-f1395c5d3aac","order_by":5,"name":"Mingjun Li","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Mingjun","middleName":"","lastName":"Li","suffix":""},{"id":489138517,"identity":"d29693b6-99f0-4b6e-b229-55e551f5697a","order_by":6,"name":"Rangrang Wang","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Rangrang","middleName":"","lastName":"Wang","suffix":""},{"id":489138518,"identity":"1aab1a44-6c59-406b-a30c-419cab61c3dd","order_by":7,"name":"Xuefeng Ai","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Xuefeng","middleName":"","lastName":"Ai","suffix":""},{"id":489138520,"identity":"c6893bd6-f230-43cd-9c50-7161293cc3f6","order_by":8,"name":"Leilei Wu","email":"","orcid":"","institution":"Hangzhou Institute of Medicine (HIM), Zhejiang Cancer Hospital, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Leilei","middleName":"","lastName":"Wu","suffix":""},{"id":489138522,"identity":"94abb9e2-668e-4804-b194-90bec7d7d4f8","order_by":9,"name":"Xiucheng Liu","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Xiucheng","middleName":"","lastName":"Liu","suffix":""},{"id":489138523,"identity":"e6c4f325-be53-4a7e-84c3-20a2e8e559bc","order_by":10,"name":"Xiaojin Wu","email":"","orcid":"","institution":"Xuzhou Central Hospital, Xuzhou Clinical School of Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaojin","middleName":"","lastName":"Wu","suffix":""},{"id":489138524,"identity":"6aaf44e2-0805-4c73-a76b-de8f56be3aa1","order_by":11,"name":"Dong Xie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYDACZgYGCSAlB+ZIGACJA0RqMeYhXgsDREtiD5xLSIvBceaDNz7uqE3fz957+IVFAYMc340Exs8FeLRINrMlW848czy3h+dcmgXQYcaSNxKYpWfg0cLPzGMmzdt2LLdHIsfMAKglccONBDZmHjxa2Jj5v4G0pPNAtdQT1AK0hQ2opSYBqMX4AVBLggEhLUC/GFvObDtg2HPmjBkwkCUMZ5552CyNT4vB+cMPb3xsq5Nnb+8x/izxx0ae73jywc/4tEDBYbC/pCXAccTYQFgDA0MdiGD++IEYtaNgFIyCUTDiAAD6xEPkNQt3pwAAAABJRU5ErkJggg==","orcid":"","institution":"Tongji University","correspondingAuthor":true,"prefix":"","firstName":"Dong","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2025-07-17 13:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7149599/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7149599/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41746-025-02029-8","type":"published","date":"2025-11-17T15:57:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87703363,"identity":"fc076936-0c54-4c86-aeda-943be042e0d3","added_by":"auto","created_at":"2025-07-28 07:40:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4514234,"visible":true,"origin":"","legend":"\u003cp\u003eFlow chart of the selected population in this study.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7149599/v1/9d28e0983750ec0a856a0b4c.png"},{"id":87701616,"identity":"59e2ca61-e3ef-4ecc-b8a3-27268ae436e2","added_by":"auto","created_at":"2025-07-28 07:24:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1220682,"visible":true,"origin":"","legend":"\u003cp\u003ePatients receiving chemotherapy have a significantly increased risk of ED. (A) Waffle chart of statistics on postoperative adjuvant therapy for NSCLC patients. (B) Statistics of ED patients and non-ED patients according to postoperative adjuvant treatment methods.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7149599/v1/845f099f1f3729e29bf636e2.png"},{"id":87701620,"identity":"d63296df-4e73-4ffd-8c46-752547a515cc","added_by":"auto","created_at":"2025-07-28 07:24:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5646895,"visible":true,"origin":"","legend":"\u003cp\u003eDrugs commonly used by ED patients do not promote tumor growth or affect chemotherapy. (A) Anti-ED drugs include SSRIs (paroxetine, sertraline), SNRIs (bupropion), TcAs (amitriptyline), and others such as tandospirone and agomelatine. Heat map display of cell viabilities\u003cbr\u003e\n\u003cbr\u003e\nof A549 (B) and H1299 (C) cells after incubation with different concentrations of anti-ED drugs for 48 h. Effects of various anti-ED drugs (100 μg/mL) on\u003cbr\u003e\n\u003cbr\u003e\napoptosis of A549 (D) and H1299 (E) cells after 48 h of incubation. Effects of various anti-ED drugs (100 μg/mL) combined with different concentrations of cisplatin (DDP) on the viability of A549 (F) and H1299 (G) cells.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7149599/v1/32cbe1f90e58e7af5a390433.png"},{"id":87701634,"identity":"fb0b6d5d-789c-4e90-a030-04991a68095c","added_by":"auto","created_at":"2025-07-28 07:24:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":29382423,"visible":true,"origin":"","legend":"\u003cp\u003eTandospirone enhances the anti-tumor effect of cisplatin.(A) A colony formation assay was performed to determine the effect of tandospirone (100 μg/mL) on the proliferation of cisplatin (DDP)-treated A549 and H1299 cells. Quantitative analysis is presented on the right (n = 3). (B) Flow cytometry was performed to determine the effect of tandospirone on the apoptosis of DDP-treated A549 and H1299 cells. Quantitative analysis is presented on the right (n = 3). (C) Migration and invasion of A549 and H1299 cells treated with tandospirone combined with DDP. Quantitative analysis is presented on the right (n = 3), Scale bar = 100 μm. (D) The wound healing assay revealed that tandospirone enhances DDP-induced inhibition of A549 and H1299 cell migration. Quantitative analysis is presented on the right (n = 3). (E) Schematic diagram of \u003cem\u003ein vitro\u003c/em\u003e experimental model. (F–H) The effects of tandospirone combined with cisplatin on tumor size, weight, and volume are assessed using the mouse xenograft model (n = 5).(I–J) HE staining and quantitative analysis of lung metastatic nodules in xenograft tumors. (K–L) IHC staining and Ki67 quantification in xenograft tumors, Scale bar = 200 μm. Data are expressed as the mean ±SD, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7149599/v1/3e7971ef3c92ec02c57f762d.png"},{"id":87702284,"identity":"dc62c6b1-0eac-4594-aecd-6f1db782dbf3","added_by":"auto","created_at":"2025-07-28 07:32:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3099330,"visible":true,"origin":"","legend":"\u003cp\u003eTandospirone decreases the cholesterol levels in tumor cells. (A) Heat map display of the top 50 genes of A549 cells after tandospirone (100 μg/mL) treatment. (B) Volcano plot depicting differentially expressed genes (|fold change| ≥ 1.0, padjust \u0026lt;0.05) in the TDSgroup \u003cem\u003evs.\u003c/em\u003e Ctrl group. (C–D) GO and KEGG pathway enrichment analysis was conducted on differentially expressed genes. (E) GSEA of TDS and Ctrl groups by using a cholesterol biosynthetic process signature. (F–G)The cholesterol level in the cytoplasm or lipid rafts of A549 and H1299 cells. (H–I) The levels of HMGCR and SREBP2 were determined by qRT-PCR analysis of A549 and H1299 cells. (J–K) Western blotting revealed that the levels of HMGCR and SREBP2 are increased in A549 cells treated with TDS. Data are expressed as the mean ±SD, n = 3, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7149599/v1/7523c7aa4bba59b88fe87aeb.png"},{"id":87702285,"identity":"a3648e07-e716-4acc-8521-593fdc5a8e2b","added_by":"auto","created_at":"2025-07-28 07:32:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6181586,"visible":true,"origin":"","legend":"\u003cp\u003eLowering cholesterol levels in NSCLC cells can enhance their apoptosis sensitivity.(A) Cell viability values of A549 and H1299 cells after incubation with different concentrations of cholesterol for 48 h. (B) Cell viability values ​​showed the effect of cholesterol (5 μM) on the TDS effect of DDP. (C) Cell viability values of A549 and H1299 cells after incubation with different concentrations of MβCD for 48 h. (D) Cell viability values ​​show the effect of MβCD (2.5 mM) on the efficacy of DDP. (E) A colony formation assay was performed to determine the effect of MβCD (2.5 mM) on the proliferation of DDP-treated A549 and H1299 cells. Quantitative analysis is presented on the right (n = 3). (B) Flow cytometry was performed to determine the effect of MβCD (2.5 mM) on apoptosis of DDP-treated A549 and H1299 cells. Quantitative analysis is presented on the right (n = 3).(G) A heat map illustrating cell viability values (A549) showing the impact of cholesterol-depletion combined with various chemotherapeutic drugs and cell death-inducing agents, such as cisplatin, pemetrexed, docetaxel, bortezomib, and ML162. (H) The schematic diagram illustrates that chemotherapy increases the probability of ED in NSCLC patients and that tandospirone can enhance the efficacy of chemotherapy by regulating the cholesterol level.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7149599/v1/b36e45b767d7db816b921c4c.png"},{"id":96650176,"identity":"2ca1b1f6-6143-4e3c-af18-32b1df579bfa","added_by":"auto","created_at":"2025-11-24 16:09:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":44436870,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7149599/v1/fc7f1860-e9fa-4b51-a0ea-926ad4761a5d.pdf"},{"id":87701625,"identity":"8a805156-ef7d-4b21-8da7-c5da7640ec24","added_by":"auto","created_at":"2025-07-28 07:24:22","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":704527,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementmaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7149599/v1/a50c832fd4ffcccdae69f69f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Anxiolytic Drug Tandospirone Enhances Cisplatin Efficacy by Reducing Tumor Cell Cholesterol Levels: Dual Roles in Emotional Distress Management and Chemosensitization for NSCLC Patients","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLung cancer remains one of the most prevalent and deadly malignancies worldwide, posing a severe threat to public health [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite substantial advancements in diagnosis and treatment, surgical resection remains the primary therapeutic approach for early-stage non-small cell lung cancer (NSCLC) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, postoperative patients often experience a range of physical and psychological challenges [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Emotional distress (ED), commonly manifesting as depression and/or anxiety, is a frequent psychological complication following lung cancer surgery. The incidence of ED in lung cancer patients is significantly higher than that in the general population, adversely affecting their quality of life and potentially influencing treatment outcomes and prognosis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, identifying effective interventions for ED in postoperative lung cancer patients is of great clinical significance.\u003c/p\u003e\u003cp\u003eThe underlying mechanisms of ED are complex and involve physiological, psychological, and social factors. Postoperative pain, physical limitations, fear of disease recurrence, and uncertainty about the future all contribute to ED [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, many early-stage lung cancer patients undergo adjuvant therapies such as chemotherapy, immunotherapy, and targeted therapy post-surgery, which may exacerbate ED symptoms [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, research on the interaction between anti-ED medications and lung cancer treatment remains limited. Clinically, anti-ED drugs are widely prescribed for various anxiety and depression disorders. However, for postoperative lung cancer patients, especially those receiving adjuvant therapy, their safety profile and potential impact on tumor growth remain critical concerns. While some epidemiological studies have suggested that anti-ED drugs may influence tumor progression through direct effects on cancer cells or by modulating neurotransmitter levels and the immune microenvironment [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], their specific mechanisms and clinical implications remain unclear. Therefore, effective ED management in postoperative lung cancer patients requires not only psychological interventions but also a thorough evaluation of pharmacological treatments' feasibility and safety.\u003c/p\u003e\u003cp\u003eGiven this context, we conducted a large-scale clinical survey involving 1,185 patients undergoing lung cancer surgery. Our goal was to systematically evaluate postoperative mental health and examine the correlation between ED and adjuvant therapies. Our findings revealed a significantly higher incidence of anxiety and depression in patients receiving chemotherapy than in other treatment groups, indicating that chemotherapy may be a major risk factor for ED. To explore potential pharmacological interventions, we systematically evaluated the effects of commonly used anti-anxiety and anti-depressant drugs in combination with cisplatin chemotherapy in both cell and animal models. Among these, tandospirone lowers cholesterol levels in tumor cells, thereby enhancing their susceptibility to apoptosis. These findings suggest that tandospirone may serve a dual function: alleviating ED symptoms and enhancing chemotherapy efficacy, providing a novel avenue for integrative lung cancer treatment strategies.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e\u003cb\u003eStudy design and ethics statement\u003c/b\u003e\u003c/p\u003e\u003cp\u003e This single-center, cross-sectional study was approved by the Ethics Committee of the Shanghai Pulmonary Hospital affiliated with Tongji University (K22-184Y). The study protocol was in accordance with the relevant guidelines and regulations. This study followed the tenets of the Declaration of Helsinki. This study was anonymous, and the questionnaire used in the study did not collect any identifying information (e.g., name, hospital number), and the data were used only for statistical analyses. The Ethics Committee of Shanghai Pulmonary Hospital (Shanghai, China) waived off the requirement for obtaining informed consent.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStudy participants\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study involved 220 healthy volunteers and 1,500 NSCLC patients who underwent surgery at the Shanghai Pulmonary Hospital between June 2022 and January 2024. The patient exclusion criteria included a history of neoadjuvant therapy, other malignancies within the past 3 years, psychiatric disorders, and any current antidepressant or anti-anxiety treatment. ED was assessed 6-month post-surgery. Ultimately, 192 volunteers and 1,185 NSCLC patients were included in the final analyses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eClinical characteristics\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePatient data were collected and analyzed, including demographics (age, sex), medical history (hypertension, diabetes), lifestyle factors (smoking, drinking), education level, BMI, and postoperative medications (chemotherapy, targeted therapy, immunotherapy, etc.).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluation of ED\u003c/b\u003e\u003c/p\u003e\u003cp\u003eED generally refers to depression and/or anxiety symptoms. PHQ-9 and GAD-7 were used to screen these symptoms in cancer patients, with assessments conducted 6 months post-surgery [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. ED was defined as a PHQ-9 or GAD-7 score of \u0026ge;\u0026thinsp;5 based on clinical thresholds [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHuman NSCLC cell lines (A549 and H1299) were obtained from Procell (Wuhan, China) and cultured in DMEM (BasalMedia, L110 KJ, China) supplemented with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (BasalMedia, S110JV) and incubated at 37\u0026deg;C under a 5% CO₂ atmosphere.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell viability analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA549 and H1299 cells were seeded into 96-well plates and incubated under standard culture conditions. Anti-ED drugs, cholesterol (14606, Sigma-Aldrich) or methyl-β-cyclodextrin (MβCD, 332615, Sigma-Aldrich) or/and cisplatin, were added at varying concentrations based on the experimental protocol and cultured for 48 h. Next, fresh medium mixed with CCK-8 reagent (Beyotime, C0041) at a 10:1 ratio was added, and the cells were incubated at 37\u0026deg;C for another 2 h. Absorbance was measured at 450 nm using a microplate reader (Infinite 200 PRO, Tecan). Cell viability was determined based on the average optical density (OD) values from at least three replicate wells.\u003c/p\u003e\u003cp\u003e\u003cb\u003eApoptosis analysis with Flow Cytometer\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe experimental cells were plated into 12-well plates at a density of 3 \u0026times; 10⁵ cells/well and treated with anti-ED drugs, MβCD, and/or cisplatin. After 48 h of incubation, apoptosis was evaluated using an Annexin V-FITC/PI apoptosis detection kit (KeyGEN BioTECH, KGA1102) according to the manufacturer\u0026rsquo;s protocol. Briefly, the cells were collected, washed thrice with PBS, and resuspended in 500 \u0026micro;L of binding buffer. Then, 5 \u0026micro;L of Annexin V-FITC and 5 \u0026micro;L of PI were added, and the mixture was incubated in the dark at room temperature for 15 min. Flow cytometry data were obtained using the CytoFLEX S cytometer (Beckman, USA) and analyzed with FlowJo software (version 10).\u003c/p\u003e\u003cp\u003e\u003cb\u003eColony formation assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCells (500\u0026ndash;1000/well) were seeded into six-well plates and incubated at 37\u0026deg;C in a humidified atmosphere with 5% CO₂. Colonies were collected and washed with phosphate-buffered saline, fixed in 4% paraformaldehyde for 20 min, and stained with 0.5% crystal violet for 30 min. Images were captured using the Pannoramic MIDI system.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell invasion, migration, and wound-healing assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor the migration assay, A549 or H1299 (2 \u0026times; 10⁴) cells were seeded in a serum-free medium in the upper chamber (Corning Inc., Corning, NY, USA). For the invasion assay, the upper chamber was pre-coated with Matrigel (diluted 1:10; BD Biosciences, San Jose, CA, USA), and 5 \u0026times; 10⁵ cells were seeded in serum-free medium. The cells that migrated or invaded through the membrane were stained with 0.04% crystal violet and enumerated.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnimal models\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBALB/c nude mice (4\u0026ndash;6 weeks old) were obtained from Gempharmatech Co., Ltd. (Nanjing, China) and housed in a specific pathogen-free (SPF) facility at Tongji University (Shanghai, China). No sex-based selection was performed for the mice used in these experiments. To evaluate the \u003cem\u003ein vivo\u003c/em\u003e tumor growth and metastatic potential, both subcutaneous xenograft and lung metastasis models were established. For the subcutaneous xenograft model, A549 cells (1 \u0026times; 10⁵ cells/injection) were injected into the left flank of the mice. Drug treatments were initiated 2 weeks after tumor establishment, and the tumor size was measured weekly. After 4 weeks, the tumors were harvested for volumetric analyses. In the lung metastasis model, A549 cells (1 \u0026times; 10⁵ cells/dose) were injected via the tail vein. After 4 weeks, the mice were euthanized, and lung metastases were examined through hematoxylin and eosin (H\u0026amp;E) staining.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunohistochemistry\u003c/b\u003e\u003c/p\u003e\u003cp\u003eImmunohistochemical (IHC) staining for Ki67 was conducted on paraffin-embedded tumor tissue sections in accordance with the experimental protocol. Images were captured using an inverted microscope (Olympus).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRNA high-throughput sequencing was performed using the Illumina HiSeq platform (Shanghai, China). Total RNA was first depleted of rRNA with the NEBNext rRNA Depletion Kit (New England Biolabs, Massachusetts, USA) following the manufacturer\u0026rsquo;s instructions. RNA libraries were then constructed using the NEBNext\u0026reg; UltraTM II Directional RNA Library Prep Kit (New England Biolabs) according to the provided protocol. Quality control and library quantification were assessed using the BioAnalyzer 2100 (Agilent Technologies, USA). Sequencing was conducted on the Illumina HiSeq system with 150 bp paired-end reads.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIsolation of lipid rafts\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe experimental cells were seeded, treated with specified drugs, and harvested. They were then lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.5% Triton X-100 for 30 min. The lysates were then centrifuged at 16,000 \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 30 min, and the supernatants were collected as non-lipid raft fractions. The remaining insoluble pellets were resuspended in 200 \u0026micro;L of buffer (0.5% SDS, 2 mM DTT) and incubated for 10 min. After another round of centrifugation under the same conditions, the supernatants were transferred into separate tubes as lipid raft fractions. The cholesterol levels were measured using the Amplex\u0026reg; Red Cholesterol Assay Kit (A12216, Invitrogen, Carlsbad, CA, USA) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eTotal RNAoifd extraction and real‑time quantitative polymerase chain reaction (RT‑qPCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted using TRIzol reagent, and 2 \u0026micro;g of RNA was used for reverse transcription. RT-qPCR was conducted following the protocol provided in the UltraSYBR one-step RT-qPCR kit (CWBIO, Beijing, China). The primers used for the analysis are detailed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWestern blotting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eProteins from A549 and H1299 cells were extracted by using a total protein extraction kit. Protein concentrations were measured with a BCA protein assay kit. Proteins were separated via SDS-PAGE and transferred onto 0.45-\u0026micro;m polyvinylidene fluoride (PVDF) membranes (MilliporeSigma). The membranes were blocked with 5% non-fat dry milk for 1 h, then incubated overnight at 4\u0026deg;C with primary antibodies targeting HMGCR (ab242315, Abcam), SREBP2 (ab30682, Abcam), and GAPDH (60004-1-Ig, Proteintech). The next day, membranes were incubated with the appropriate secondary antibody for 60 min at room temperature.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eContinuous variables were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The χ\u003csup\u003e2\u003c/sup\u003e test was performed for the categorical comparison of demographic variables between different groups, whereas the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test and/or non-parametric test were applied for a continuous comparison. Skewness continuous variables were analyzed using median and interquartile range (IQR) and compared using the Mann\u0026ndash;Whitney U-test. A logistic regression model was applied to evaluate factors affecting the occurrence of ED. The α-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 on both sides was considered statistically significant. All statistical analyses were performed using SPSS 25.0 (SPSS, Inc., Chicago, IL, USA).\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e3.1 NSCLC patients have a significantly increased risk of ED after surgery\u003c/h2\u003e\u003cp\u003eA total of 1500 NSCLC patients who underwent surgical resection and 220 healthy volunteers were recruited from our center for a comprehensive questionnaire-based assessment. Patients with incomplete data, a history of neoadjuvant therapy, other malignancies within the past 3 years, pre-existing psychiatric disorders, or those currently receiving antidepressant or anti-anxiety treatment were excluded from the study. The final analysis included 1,185 NSCLC patients and 192 healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Demographic characteristics were similar between the healthy population and postoperative patients (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, the prevalence of ED among healthy individuals was only 8.9%, whereas it was significantly higher in postoperative NSCLC patients, reaching 32.2% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These findings underscore a markedly increased risk of ED following lung cancer surgery.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDemographics and characteristics of patient. *Statistically significant values.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal (n\u0026thinsp;=\u0026thinsp;1377)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHealthy (n\u0026thinsp;=\u0026thinsp;192)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSurgery (n\u0026thinsp;=\u0026thinsp;1185)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge, years (%)\u003c/p\u003e\u003cp\u003e\u0026lt;\u0026thinsp;65\u003c/p\u003e\u003cp\u003e\u0026ge;65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1196 (86.9)\u003c/p\u003e\u003cp\u003e181 (13.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e168 (87.5)\u003c/p\u003e\u003cp\u003e24 (12.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1028 (86.8)\u003c/p\u003e\u003cp\u003e157 (13.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.908\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSex (male/female)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e551/826\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e78/114\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e473/712\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.874\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSmoking history (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e369 (26.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e49 (25.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e320 (27.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.726\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDrinking history (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e233 (16.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33 (17.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e200 (16.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.917\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHypertension (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e336 (24.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42 (21.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e294 (24.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.416\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDiabetes (%)\u003c/p\u003e\u003cp\u003eBMI\u003c/p\u003e\u003cp\u003e\u0026lt;\u0026thinsp;24\u003c/p\u003e\u003cp\u003e\u0026ge;24\u003c/p\u003e\u003cp\u003eEducational level (%)\u003c/p\u003e\u003cp\u003e\u0026lt;High school\u003c/p\u003e\u003cp\u003e\u0026ge;High school\u003c/p\u003e\u003cp\u003eEmotional distress (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e106 (7.7)\u003c/p\u003e\u003cp\u003e731 (53.1)\u003c/p\u003e\u003cp\u003e646 (46.9)\u003c/p\u003e\u003cp\u003e1041 (75.6)\u003c/p\u003e\u003cp\u003e336 (24.4)\u003c/p\u003e\u003cp\u003e399 (29.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13 (6.8)\u003c/p\u003e\u003cp\u003e106 (55.2)\u003c/p\u003e\u003cp\u003e86 (44.8)\u003c/p\u003e\u003cp\u003e137 (71.4)\u003c/p\u003e\u003cp\u003e55 (28.6)\u003c/p\u003e\u003cp\u003e17 (8.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e93 (7.8)\u003c/p\u003e\u003cp\u003e625 (52.7)\u003c/p\u003e\u003cp\u003e560 (47.3)\u003c/p\u003e\u003cp\u003e904 (76.3)\u003c/p\u003e\u003cp\u003e281 (23.7)\u003c/p\u003e\u003cp\u003e382 (32.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.770\u003c/p\u003e\u003cp\u003e0.534\u003c/p\u003e\u003cp\u003e0.148\u003c/p\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Chemotherapy is an independent risk factor for increased ED in NSCLC patients after surgery\u003c/h2\u003e\u003cp\u003eTo identify key risk factors associated with ED, we stratified postoperative NSCLC patients into ED and non-ED groups. Compared with the non-ED group, patients in the ED group were older, had a higher proportion of females, a greater prevalence of diabetes, a lower education level and a higher lymph node metastasis positivity. However, no statistically significant differences were observed in hypertension status, smoking history, and tumor T stage (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). According to the type of postoperative adjuvant therapy, 602 patients received no treatment, 80 patients received chemotherapy, 246 patients received chemotherapy combined with other treatments (such as targeted therapy or immunotherapy), and 253 patients received other treatments without chemotherapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Further analysis of treatment modalities revealed a significant association between chemotherapy and increased ED incidence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Supplement Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Multivariate regression analysis confirmed chemotherapy as an independent risk factor for ED (Supplement Table\u0026nbsp;3 and Supplement Table\u0026nbsp;4). These findings suggest that patients undergoing chemotherapy after NSCLC surgery face an elevated risk of psychological distress.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDemographics and characteristics of ED in NSCLC patients. *Statistically significant values.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVariables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal (n\u0026thinsp;=\u0026thinsp;1185)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eED (n\u0026thinsp;=\u0026thinsp;382)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo ED (n\u0026thinsp;=\u0026thinsp;803)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge, years (%)\u003c/p\u003e\u003cp\u003e\u0026lt;\u0026thinsp;65\u003c/p\u003e\u003cp\u003e\u0026ge;65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1028 (86.8)\u003c/p\u003e\u003cp\u003e157 (13.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e316 (82.7)\u003c/p\u003e\u003cp\u003e66 (17.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e712 (88.7)\u003c/p\u003e\u003cp\u003e91 (11.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.009*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSex (male/female)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e473/712\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e131/251\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e342/461\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.006*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSmoking history (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e320 (27.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e93 (24.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e227 (28.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.162\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDrinking history (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e200 (16.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e58 (15.2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e142 (17.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.319\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHypertension (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e294 (24.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e102 (26.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e192 (24.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.314\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDiabetes (%)\u003c/p\u003e\u003cp\u003eBMI\u003c/p\u003e\u003cp\u003e\u0026lt;\u0026thinsp;24\u003c/p\u003e\u003cp\u003e\u0026ge;24\u003c/p\u003e\u003cp\u003eEducational level (%)\u003c/p\u003e\u003cp\u003e\u0026lt;High school\u003c/p\u003e\u003cp\u003e\u0026ge;High school\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e93 (7.8)\u003c/p\u003e\u003cp\u003e625 (52.7)\u003c/p\u003e\u003cp\u003e560 (47.3)\u003c/p\u003e\u003cp\u003e904 (76.3)\u003c/p\u003e\u003cp\u003e281 (23.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e41 (10.7)\u003c/p\u003e\u003cp\u003e193 (50.5)\u003c/p\u003e\u003cp\u003e189 (49.5)\u003c/p\u003e\u003cp\u003e313 (81.9)\u003c/p\u003e\u003cp\u003e69 (18.1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e52 (6.5)\u003c/p\u003e\u003cp\u003e432 (53.8)\u003c/p\u003e\u003cp\u003e371 (46.2)\u003c/p\u003e\u003cp\u003e591 (73.6)\u003c/p\u003e\u003cp\u003e212 (26.4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.001*\u003c/p\u003e\u003cp\u003e0.319\u003c/p\u003e\u003cp\u003e0.002*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTumor stage (%)\u003c/p\u003e\u003cp\u003eI\u003c/p\u003e\u003cp\u003eII\u003c/p\u003e\u003cp\u003eIII\u003c/p\u003e\u003cp\u003eIV\u003c/p\u003e\u003cp\u003eLymph node metastasis (%)\u003c/p\u003e\u003cp\u003eNegative\u003c/p\u003e\u003cp\u003ePositive\u003c/p\u003e\u003cp\u003eDistant metastasis (%)\u003c/p\u003e\u003cp\u003eM0\u003c/p\u003e\u003cp\u003eM1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e946 (79.8)\u003c/p\u003e\u003cp\u003e221 (18.7)\u003c/p\u003e\u003cp\u003e18 (1.5)\u003c/p\u003e\u003cp\u003e0\u003c/p\u003e\u003cp\u003e902 (76.1)\u003c/p\u003e\u003cp\u003e283 (23.9)\u003c/p\u003e\u003cp\u003e1185 (100.0)\u003c/p\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e294 (77.0)\u003c/p\u003e\u003cp\u003e81 (21.2)\u003c/p\u003e\u003cp\u003e7 (1.8)\u003c/p\u003e\u003cp\u003e0\u003c/p\u003e\u003cp\u003e275 (72.0)\u003c/p\u003e\u003cp\u003e107 (28.0)\u003c/p\u003e\u003cp\u003e382 (100.0)\u003c/p\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e652 (81.2)\u003c/p\u003e\u003cp\u003e140 (17.4)\u003c/p\u003e\u003cp\u003e11 (1.4)\u003c/p\u003e\u003cp\u003e0\u003c/p\u003e\u003cp\u003e627 (78.1)\u003c/p\u003e\u003cp\u003e176 (21.9)\u003c/p\u003e\u003cp\u003e803 (100.0)\u003c/p\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.232\u003c/p\u003e\u003cp\u003e0.024*\u003c/p\u003e\u003cp\u003eN/A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.3 Common anti-ED drugs do not promote tumor cell progression and may enhance chemotherapy effects in patients with ED\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe systematically evaluated the impact of six commonly prescribed anti-ED drugs on lung cancer cell proliferation and apoptosis. The selected medications included paroxetine and sertraline in selective serotonin reuptake inhibitors (SSRIs); bupropion in serotonin and norepinephrine reuptake inhibitors (SNRIs); amitriptyline in tricyclic antidepressants (TcAs); and other drugs such as tandospirone and agomelatine (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). First, we conducted CCK-8 assays to assess the effects of these drugs on the proliferation of two NSCLC cell lines (A549 and H1299) across a concentration range of 0\u0026ndash;200 \u0026micro;g/mL. None of the six medications significantly promoted tumor cell proliferation. Instead, all displayed a mild inhibitory effect on0 cell growth at higher concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). This result suggests that these anti-ED drugs slightly inhibit lung cancer cell progression within the commonly used clinical dose range. To further explore their influence on tumor cell survival, we standardized the drug concentration to 100 \u0026micro;g/mL and performed flow cytometry analysis to assess apoptosis induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). All six drugs induced some degree of apoptosis in A549 and H1299 cells, with tandospirone and sertraline exhibiting the most pronounced effects (apoptosis rates of 7.2% and 9.9%, respectively).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe then examined the interactions between these anti-ED drugs and cisplatin chemotherapy. None of the medications reduced the sensitivity of A549 or H1299 cells to cisplatin; rather, they increased chemotherapy efficacy to varying degrees (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G). This suggests that these anti-ED drugs do not reduce the chemotherapeutic effectiveness of cisplatin. Notably, tandospirone exhibited a significant synergistic effect, markedly increasing cisplatin-induced cytotoxicity in both cell lines. These results indicate that tandospirone may have an unexpected function of enhancing chemotherapy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Tandospirone enhances the anti-tumor effects of cisplatin\u003c/h2\u003e\u003cp\u003eTandospirone is a non-benzodiazepine anxiolytic that alleviates anxiety and depressive symptoms by modulating the serotonin (5-HT) system [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It is widely used for the long-term treatment of generalized anxiety disorder and depression [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, its role in tumor biology remains unexplored. To investigate the therapeutic potential of tandospirone in augmenting cisplatin\u0026rsquo;s antitumor effects, we conducted a comprehensive evaluation using both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models. The cloning sphere experiment revealed that NSCLC cells treated with the tandospirone-cisplatin combination exhibited significantly reduced clonogenic capacity compared with the cisplatin monotherapy group. The number and size of tumor cell colonies were significantly diminished, suggesting that the combination therapy effectively impairs the self-renewal ability of cancer stem-like cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Furthermore, flow cytometry analysis demonstrated that the cell apoptosis rate was significantly higher in cells treated with the tandospirone-cisplatin combination than in those treated with cisplatin alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results indicate that tandospirone enhances cisplatin-induced apoptosis, thereby increasing tumor cell susceptibility to chemotherapy. To further assess its impact on tumor progression, we conducted Transwell invasion and scratch wound-healing assays. The combination treatment significantly suppressed tumor cell migration and invasion, as evidenced by a substantial reduction in both the number of transmembrane invasive cells and wound closure rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D) These findings suggest that tandospirone exerts a synergistic effect in limiting tumor cell dissemination.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo validate these findings in an animal model, we established a subcutaneous xenograft model and a lung metastasis model using A549 cells injected via the tail vein of nude mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). In the subcutaneous tumor model, mice receiving the tandospirone-cisplatin combination exhibited significantly slower tumor growth compared with those treated with cisplatin alone. Final tumor weight was also significantly lower in the combination therapy group than in the cisplatin monotherapy group, indicating that tandospirone enhances the inhibitory effect of cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, H). The lung metastasis model further corroborated these results, showing a marked reduction in metastatic nodules in the combination treatment group compared with the cisplatin monotherapy group, suggesting that tandospirone not only inhibits primary tumor growth but also plays a crucial role in preventing distant metastases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, J). To assess the impact of combined therapy on tumor cell proliferation, we performed Ki67 IHC staining and quantitative analysis on mouse tumor tissues. Quantitative analysis revealed a significant decrease in Ki67-positive cells in the combination treatment group compared with the cisplatin monotherapy group. This reduction in proliferative activity provides further evidence of the enhanced anti-tumor efficacy of tandospirone in combination with cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK, I).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Tandospirone reduces cholesterol levels in tumor cells\u003c/h2\u003e\u003cp\u003eTo elucidate the molecular mechanism by which tandospirone enhances cisplatin sensitivity, we conducted RNA-seq transcriptomic analysis on A549 cells treated with tandospirone. The analysis identified 272 differentially expressed genes (screening condition: |fold change| \u0026ge; 1.0, Padjust\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed a significant enrichment of cholesterol synthesis-related pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D and Supplementary Fig.\u0026nbsp;2A). Additionally, Gene Set Enrichment Analysis further confirmed that cholesterol biosynthesis pathways and steroid biosynthesis were markedly altered in response to tandospirone treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and Supplementary Fig.\u0026nbsp;2B). These findings suggest that tandospirone may exert its chemotherapy-sensitizing effects by modulating cholesterol metabolism.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo validate this hypothesis, we measured intracellular cholesterol levels in A549 and H1299 cells following tandospirone treatment. Compared with untreated controls, tandospirone significantly reduced both total intracellular cholesterol and membrane-associated cholesterol (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, G). This indicates that tandospirone effectively disrupts cholesterol homeostasis in tumor cells. To further explore the regulatory mechanisms underlying this cholesterol depletion, we examined key genes involved in cholesterol biosynthesis. qPCR and western blot analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH\u0026ndash;K and Supplementary Fig.\u0026nbsp;3A, B) demonstrated that tandospirone treatment led to a significant upregulation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol synthesis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and sterol regulatory element binding protein 2 (SREBP2), a key upstream transcription factor regulating cholesterol metabolism [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This suggests that tumor cells may have initiated a feedback regulation mechanism because of the reduced cholesterol levels. Tandospirone may consume cholesterol in tumor cells and induce abnormal activation of the cholesterol synthesis pathway, thereby modifying the cell metabolic state, affecting the cell's survival and proliferation, and improving cisplatin\u0026rsquo;s antitumor effect.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Reducing cholesterol levels in NSCLC cells can increase their sensitivity to apoptosis\u003c/h2\u003e\u003cp\u003eBuilding on our experimental results, we speculate that reducing cholesterol levels may render tumor cells more susceptible to chemotherapy. To validate this hypothesis, we conducted additional experiments to assess the role of cholesterol depletion in tandospirone-induced cisplatin sensitization. We first supplemented A549 and H1299 cells with exogenous cholesterol to determine whether restoring cholesterol levels would counteract the sensitizing effect of tandospirone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Although exogenous cholesterol marginally promoted tumor cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), it significantly attenuated the enhanced cytotoxicity of cisplatin observed in the presence of tandospirone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate whether cholesterol depletion alone could enhance cisplatin\u0026rsquo;s antitumor effects, we treated A549 and H1299 cells with methyl-β-cyclodextrin (MβCD), a cholesterol scavenger [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Our results revealed that cholesterol depletion significantly increased cell death, indicating that reduced cholesterol levels independently contribute to enhanced chemotherapy sensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). This finding was reinforced by clonogenic assays, where MβCD-treated cells exhibited a significantly reduced capacity for colony formation, an effect that was further amplified when combined with cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Flow cytometry analysis confirmed these observations, showing a substantial increase in apoptosis in cholesterol-depleted cells, with an even more pronounced effect when co-administered with cisplatin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Furthermore, Transwell invasion and scratch assays demonstrated that cholesterol depletion also inhibited tumor cell migration and invasion. Specifically, MβCD treatment significantly reduced the number of cells crossing the membrane and slowed wound healing, with these effects being further intensified when combined with cisplatin (Supplementary Fig.\u0026nbsp;4A, B). These findings suggest that cholesterol depletion not only enhances cisplatin-induced apoptosis but also impairs tumor cell motility and metastatic potential.\u003c/p\u003e\u003cp\u003eTo explore whether cholesterol depletion broadly sensitizes tumor cells to other forms of cell death, we tested its effects in combination with various chemotherapeutic drugs and targeted inhibitors, including cisplatin, pemetrexed, docetaxel, bortezomib (a proteasome inhibitor) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and ML162 (a GPX4 inhibitor) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Remarkably, MβCD treatment significantly enhanced the cytotoxicity of all tested agents, as evidenced by a pronounced decrease in cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Likewise, tandospirone also exhibited a strong synergistic effect when combined with these apoptosis-inducing agents, reinforcing the notion that cholesterol depletion serves as a metabolic vulnerability that can be exploited to enhance cancer therapy.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we conducted a comprehensive analysis of ED in postoperative lung cancer patients, investigating its prevalence, risk factors, and potential therapeutic interventions. Additionally, we explored the impact of anti-ED drugs on tumor biology and chemotherapy sensitivity, yielding significant findings at multiple levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Our results not only provide a scientific foundation for clinical psychological interventions but also introduce a new approach to tumor metabolic regulation that warrants further investigation.\u003c/p\u003e\u003cp\u003eLung cancer remains one of the most aggressive malignancies worldwide, with high morbidity and mortality rates [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Despite advances in treatment, surgical resection remains the primary curative option for early- and mid-stage lung cancer patients. However, postoperative recovery extends beyond physical healing; patients frequently experience the adverse side effects of chemotherapy, fear of disease recurrence, and a decline in overall quality of life, all of which contribute to a high incidence of ED [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Our findings indicate that ED significantly increases following lung cancer surgery and is strongly associated with adjuvant treatment, identifying chemotherapy as an independent risk factor. While anti-ED medications are commonly prescribed for patients experiencing anxiety and depression concerns persist regarding their potential interference with chemotherapy efficacy. To address this issue, we systematically screened 6 widely used anti-ED drugs, assessing their effects on cell proliferation, invasion, and migration in A549 and H1299 lung cancer cell lines. Contrary to common concerns, our results demonstrated that these drugs did not promote tumor progression. Instead, certain anti-ED medications, particularly tandospirone, exhibited unexpected tumor-suppressive effects. Notably, tandospirone not only failed to diminish the cytotoxicity of cisplatin but significantly reduced its IC50, indicating a pronounced chemosensitizing effect. Tandospirone, a well-established anxiolytic that functions as a partial agonist of 5-HT1A receptors [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], has not been previously studied for its impact on tumor metabolism.\u003c/p\u003e\u003cp\u003eThrough RNA-seq analysis, we identified that tandospirone induces significant alterations in cholesterol metabolism, particularly by activating cholesterol synthesis pathways and significantly reducing intracellular cholesterol levels. qPCR and western blot analyses confirmed the upregulation of key cholesterol synthesis regulatory factors such as SREBP2 and HMGCR, in response to tandospirone treatment. This metabolic perturbation, characterized by \"cholesterol depletion-feedback activation\", likely disrupts tumor cell lipid homeostasis, rendering cancer cells more vulnerable to cisplatin-induced apoptosis. Emerging evidence highlights the crucial role of cholesterol in tumor progression and therapy resistance [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Cholesterol is not only a fundamental component of cell membranes but also regulates key signaling pathways, membrane microdomains, and apoptotic resistance mechanisms [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Studies have suggested that lung cancer cells frequently upregulate cholesterol synthesis to evade chemotherapy-induced cell death [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our findings suggest that tandospirone, by depleting cholesterol and inducing metabolic stress, sensitizes tumor cells to chemotherapy. This effect was further corroborated by experiments showing that cholesterol depletion, whether induced by tandospirone or the cholesterol scavenger MβCD, enhanced the efficacy of multiple chemotherapeutic agents such as cisplatin, docetaxel, pemetrexed, bortezomib, and ML162. These results suggest that metabolic interventions targeting cholesterol depletion may serve as a broadly applicable chemosensitization strategy.\u003c/p\u003e\u003cp\u003eFrom a clinical perspective, this study holds dual significance. First, it provides critical safety evidence supporting the use of anti-ED medications in postoperative lung cancer patients, demonstrating that these drugs do not compromise chemotherapy efficacy. Second, our findings introduce a novel concept of ​​\"metabolic depletion-sensitization therapy,\" where an anxiolytic agent like tandospirone not only alleviates ED but also enhances chemotherapy effectiveness. This positions tandospirone as a promising candidate for integrated cancer care, particularly for patients experiencing both psychological distress and chemotherapy resistance.\u003c/p\u003e\u003cp\u003eThis work has several limitations. This study found that chemotherapy is an independent risk factor for ED, but potential confounding factors may be associated with the results. Of note, other treatments can also increase patients' ED levels. Many studies have pointed out that ED affects the immune system, and it is unclear how anti-ED drugs affect immunotherapy in NSCLC patients. In the future, a larger sample size and corresponding clinical studies are warranted to further explore the safety and effectiveness of anti-ED drugs in combination with other treatments.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur study revealed that the ED status of postoperative lung cancer patients is closely related to adjuvant chemotherapy, and chemotherapy is an independent risk factor for ED. In addition to improving ED symptoms, common anti-ED drugs may improve lung cancer progression. Additional studies have found that tandospirone is safe and can significantly improve the antitumor effect of cisplatin and promote cell apoptosis. Mechanistic exploration studies revealed that tandospirone reduces cholesterol levels in tumor cells, activates cholesterol synthesis-related genes such as SREBP2 and HMGCR, induces a \"cholesterol depletion-death stress\" state, and thus augments the killing effect of cisplatin. These findings offer a new scientific basis for the emotional management of postoperative lung cancer patients and lay a theoretical foundation for the rational use of anti-ED drugs in tumor treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all the researchers who contributed to this study;\u0026nbsp;We thank BioRender for providing the tools used to create the illustrations in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX. Q.: Investigation, Methodology, Data curation, Formal analysis, Writing - original draft. B. C.: Investigation, Methodology, Data curation, Formal analysis, Writing - original draft. S. M.: Methodology, Data curation, Formal analysis. K. L.: Investigation, Methodology, Data curation, Writing - review \u0026amp; editing. Y. F.: Investigation, Data curation, Formal analysis. M. L.: Data curation, Formal analysis. R. W.: Methodology, Investigation. X. A.: Methodology, Investigation. L. W.: Investigation,\u0026nbsp;Methodology, Data curation, Writing - review \u0026amp; editing. X. L.: Funding acquisition, Investigation, Writing - review \u0026amp; editing. X. W.: Funding acquisition, Conceptualization, Investigation, Supervision. D. X.: Funding acquisition, Investigation, Supervision, Writing - review \u0026amp; editing. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the\u0026nbsp;National Key Research and Development Program of China (2024YFA1212000), the National Natural Science Foundation of China (82372098; 82200999), the Shanghai Committee of Science and Technology China (21Y11913400), the Key Project of Jiangsu Provincial Health Commission (K2024044), the Open Projects of Jiangsu Provincial Key Laboratories (XZSYSKF2022012), the Key Project of Xuzhou Medical Science and Technology Innovation Project (XWKYHT20230034).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal studies have been approved by the Review Board and Ethics Committee of Shanghai Pulmonary Hospital (ID: K22-184Y).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLeiter Amanda, Veluswamy Rajwanth R, Wisnivesky Juan P, The global burden of lung cancer: current status and future trends.[J].Nat Rev Clin Oncol, 2023, 20: 624\u0026ndash;639.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang Yanting, Vaccarella Salvatore, Morgan Eileen et al. 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Cholesterol metabolism in pancreatic cancer and associated therapeutic strategies.[J].Biochim Biophys Acta Mol Cell Biol Lipids, 2025, 1870: 159578.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-digital-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjdigitalmed","sideBox":"Learn more about [npj Digital Medicine](http://www.nature.com/npjdigitalmed/)","snPcode":"41746","submissionUrl":"https://submission.springernature.com/new-submission/41746/3","title":"npj Digital Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lung cancer, Emotional distress, Tandospirone, Chemotherapy sensitization, Cholesterol metabolism","lastPublishedDoi":"10.21203/rs.3.rs-7149599/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7149599/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePatients undergoing lung cancer surgery face a significantly increased risk of emotional distress (ED), including anxiety and depression. This distress not only severely impairs their quality of life but may also negatively affect treatment outcomes. Therefore, effective ED management is crucial in postoperative care. This study systematically evaluated the mental health status of 1,185 non-small cell lung cancer (NSCLC) patients and identified chemotherapy as an independent risk factor for ED development. Additionally, we investigated the safety and therapeutic effects of commonly used anti-ED drugs in cellular and animal models. These drugs did not promote tumor cell growth, and notably, tandospirone unexpectedly enhanced the efficacy of cisplatin. Mechanistically, tandospirone reduces cholesterol levels in tumor cells, thereby promoting apoptosis and increasing their susceptibility to chemotherapy. Our findings highlight the importance of ED management in lung cancer patients and propose tandospirone as a promising dual-purpose agent for both psychological support and chemotherapy sensitization, potentially enabling more comprehensive and effective treatment strategies.\u003c/p\u003e","manuscriptTitle":"The Anxiolytic Drug Tandospirone Enhances Cisplatin Efficacy by Reducing Tumor Cell Cholesterol Levels: Dual Roles in Emotional Distress Management and Chemosensitization for NSCLC Patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 07:24:17","doi":"10.21203/rs.3.rs-7149599/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-01T04:57:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-31T14:32:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-30T06:59:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"205969030458647105438832943704629655016","date":"2025-07-24T10:19:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"160056861473428739857782364800183447113","date":"2025-07-22T11:09:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-22T09:03:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-22T05:00:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-21T12:32:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Digital Medicine","date":"2025-07-17T13:31:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-digital-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjdigitalmed","sideBox":"Learn more about [npj Digital Medicine](http://www.nature.com/npjdigitalmed/)","snPcode":"41746","submissionUrl":"https://submission.springernature.com/new-submission/41746/3","title":"npj Digital Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c0cc647b-b5c8-4ac6-87a3-946bbec355fa","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51928554,"name":"Biological sciences/Cancer"},{"id":51928555,"name":"Biological sciences/Drug discovery"},{"id":51928556,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-11-24T16:02:41+00:00","versionOfRecord":{"articleIdentity":"rs-7149599","link":"https://doi.org/10.1038/s41746-025-02029-8","journal":{"identity":"npj-digital-medicine","isVorOnly":false,"title":"npj Digital Medicine"},"publishedOn":"2025-11-17 15:57:21","publishedOnDateReadable":"November 17th, 2025"},"versionCreatedAt":"2025-07-28 07:24:17","video":"","vorDoi":"10.1038/s41746-025-02029-8","vorDoiUrl":"https://doi.org/10.1038/s41746-025-02029-8","workflowStages":[]},"version":"v1","identity":"rs-7149599","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7149599","identity":"rs-7149599","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}