Chronic Stress Enhances Glycolysis and Promotes Tumorigenesis

Chronic Stress Enhances Glycolysis and Promotes Tumorigenesis | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chronic Stress Enhances Glycolysis and Promotes Tumorigenesis Qiufeng Qin, Shuying Li, yixuan Zhong, Jing Bai, Lin An, Lei Yang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3957233/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Depression is a well-known risk factor for tumors, but the mechanisms other than inflammation are unclear. Aerobic glycolysis is considered to be a critical element in the reprogramming of energy metabolism in malignant tumors, and impaired glycolysis has been reported in the brains of chronic stress mice. Therefore, this study aimed to explore the role of glycolysis in which depression promotes tumorigenesis. We examined the impacts of chronic unpredictable mild stress (CUMS) on the growth and metastasis of breast cancer (BC) and lung cancer (LC). The findings showed that both CUMS and tumors induced depressive-like behavior, neuronal damage, and impaired synaptic plasticity in mice, while CUMS also enhanced tumor development and metastasis in both BC and LC. In the brain, both CUMS and tumor alone and in combination significantly reduced glycolytic products and enzyme levels. However, CUMS significantly enhanced the levels of aerobic glycolytic products and enzymes in tumor tissue. Collectively, our results provide insights into that down-regulated glycolysis in the brain, leading to depression-like behavior, and how depression, in turn, enhanced glycolysis and promoted tumorigenesis. CUMS metastasis tumorigenesis glycolysis orthotopic Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key messages Both CUMS and tumor-induced depressive-like behavior, neuronal damage, and impaired synaptic plasticity in mice, CUMS can enhance tumor development and metastasis in both BC and LC. In the brain, both CUMS and tumor alone and in combination significantly reduced glycolytic products and enzyme levels. In tumor tissue, CUMS significantly enhanced the levels of aerobic glycolytic products and enzymes. This study validated the potential mechanism of glycolysis in depression-promoted tumorigenesis and development. Introduction Patients diagnosed with cancer often concurrently suffer from depression [ 1 , 2 ], the negative emotions induced by this psychological condition can potentially stimulate tumor growth and metastasis of tumors [ 3 – 5 ]. Past studies have unequivocally shown that chronic stress significantly affects every stage of cancer development in patients, ranging from carcinogenesis to angiogenesis stimulation and metastatic dissemination [ 6 ]. Chronic stress-induced inadequate coping, negative emotional responses, or a diminished quality of life are associated with an increased risk of cancer incidence [ 7 ]. Annually, approximately one million new cancer cases are diagnosed in young individuals aged 20–39 years, primarily attributed to chronic stress [ 8 ]. A poor prognosis and elevated mortality are observed among cancer patients concurrently suffering from comorbid depression [ 6 ]. Cancer patients exhibit a depression morbidity of approximately 12.5%, which is up to fourfold higher than that of the general population [ 9 ]. Chronic stress has been demonstrated in studies to raise plasma catecholamine levels, including epinephrine and noradrenaline, and accelerate the aggressive development of BC, ovarian carcinoma, and gastric cancer [ 10 , 11 ]. However, the research concerning the relationship between chronic stress and cancer remains limited. Otto Warburg and colleagues ascertained in the 1920s that tumor cells, under aerobic conditions, exhibit a preference for generating energy via a process termed aerobic glycolysis. This is manifested by excessive glucose absorption and lactate accumulation, a phenomenon known as the Warburg effect [ 12 , 13 ]. To fulfill uncontrolled biosynthesis and energy demands, cancer cells frequently tend to perform aerobic glycolysis [ 13 ]. In numerous cancer patients, clinical investigations utilizing the imaging technique of positron emission tomography (PET) with the glucose analog tracer 18fludeoxyglucose (FDG) 6–8 have unambiguously demonstrated a substantial elevation in glucose absorption in the majority of metastatic and primary human cancer patients [ 14 ]. Tumor cells facilitate the formation of new blood vessels through the secretion of VEGF, induced by lactate, which supplies sufficient oxygen and nutrients for proliferation [ 15 ]. Persistent chronic stress triggers a decrease in lactate levels and leads to depression. Intriguingly, stress-induced epinephrine enhances breast cancer stem-like characteristics through LDHA (lactate dehydrogenase A) - dependent metabolic reprogramming [ 11 ]. Lactate performs antidepressant functions by maintaining normal neuron function, and modulating levels and activity of histone deacetylases in the hippocampal region[ 16 ]. Meanwhile, LDHA regulats neuronal excitability to inhibit depressive-like behavior through lactate homeostasis [ 17 ]. However, the research on lactate levels in depression and cancer models has been a gap in the literature. To address this, we developed an animal model of breast cancer-related depression (BCRD) by in situ injection of BC cells into CUMS mice and a model of lung cancer-related depression (LCRD) by injecting LC cells into the tail veins of CUMS mice. Behavioral assessments and synapse plasticity detection were used for depression evaluation, while tumor volume and H&E staining were performed to examine tumorigenesis. Metabolites and key functional enzymes were detected to unveal the role of glycolysis in depression-promoted tumor glycolysis. The findings of this research highlight the potential role of chronic stress in exacerbating tumorigenesis and metastasis through the stimulation of glycolysis. Material and Methods Cell culture Luciferase gene-tagged Lewis murine lung cancer cell (LLC-Luc) and luciferase gene-tagged 4T1 murine breast cancer cell (4T1-Luc) were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). LLC-Luc cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL, Grand Island, NY, USA); the 4T1-Luc cells were cultured in RPMI-1640 medium (Gibco-BRL, Grand Island, NY, USA); all mediums were supplemented with 10% fetal bovine serum and cultured at 37°C in a humidified incubator containing 5% CO 2 . Animals Six–week–old female BALB / c mice and male C57BL / 6 mice were purchased from the Guangdong Medical Laboratory Animal Center (Guangdong, China), and housed in special-pathogen-free ventilation facilities. 12 h / 12 h light/dark cycle was carried out, with ambient temperatures of 20–26 o C and relative humidity of 40–70%, 5 mice per cage, and eating and drinking freely. The Ethics Committee of Guangzhou University of Chinese Medicine has authorized a laboratory animal protocol. Chronic unpredictable mild stress experiment Chronic unpredictable mild stress (CUMS) might mimic the onset of depression produced by various pressures in human daily life [ 5 , 18 ]. Mice were exposed to CUMS for 8 weeks, which included day-night reversal (24 h), cold-water swimming (10°C ± 1°C, 3 min), crowd-feeding (24 h), water and food deprivation (24 h), an empty water bottle (24 h), a 45 ° cage tilt (24 h), a tail clamp (1 cm from the tail end, 60 s), a self-made plastic seal tube (3 h), and a wet pad (24 h). 2 or 3 stimulation modalities were chosen randomly each day to ensure no repetition within 2 days. Establishment of tumor orthotopic transplantation 5 × 10 5 4T1-Luc cells were injected into the right second mammary fat pad of BALB/c mice for orthotopic transplantation of BC. CUMS mice were inoculated with 4T1-Luc cells to develop BCRD. The tumor size of BC was measured with an vernier caliper every two days. It was calculated as follows: tumor volume (mm 3 ) = [length × width 2 ] / 2. 1 × 10 6 LLC - Luc cells were injected into the tail vein of C57BL/6 mice for orthotopic transplantation of LC, and CUMS mice adopted LLC-Luc cells to develop LCRD. The tumor size was evaluated every week using IVIS (PerkinElmer, Boston, United States) with 150 mg/kg of D-luciferin potassium salt (PerkinElmer, Boston, United States) given intraperitoneally. Behavior assessments The sucrose preference test (SPT) is done for anhedonia assessment. Mice were fed in solitary cages preferentially. Two bottles containing 1% sucrose solution were placed at each side of the cages for 24 hours as an adaption phase. The next day, one bottle was replaced with water and left for another 24 hours, and two bottles of solution were changed the position at 12 hours to avoid the error caused by location preference. Then, mice were formally tested after 24 hours of freely eating without water, one bottle of water and one bottle of 1% sucrose solution were placed on each side of the cage and switched at 12 hours. 24 hours later, weigh each bottle and calculate sucrose preference (%) = sucrose consumption / (water consumption + sucrose consumption) × 100% [ 19 ]. An open field test (OFT) was also performed. BALB/c mice were set up in the center of the blackboard, while C57BL/6 mice were on a whiteboard and 10 minutes for each mouse. The open-field arena is 50 × 50 × 40 cm (length × width × height). The mice’s activity was filmed using a digital camera, and the total distance, center distance, and center time were calculated using software. The tail suspension test (TST) was done as follows, mice were suspended 30 cm above the ground with wide tape on the tail-suspension device for 6 minutes and recorded with a digital camera recorded the mice's activity. The first 2 minutes are considered familiarity time and only the last 4 minutes are counted. Mice were placed in cylindrical containers filled with room-temperature water at a depth of 30 cm for forced swim test (FST). Each mouse swam for 6 minutes and was videotaped, and then blow-dried the mice hair and put them back. The first 2 minutes are considered familiarity time and only the last 4 minutes are counted. The Y-Maze test was considered evaluable for working memory and exploratory behavior in mice. Mice were placed on either of three identical arms (arm length: 35 cm, arm width: 5 cm, wall height: 10 cm) and were allowed to explore freely for 8 minutes. The percentage of spontaneous alternation was calculated as alternation (%) = (number of correct alternations) / (number of total arm entries-2) x 100% [ 20 ]. 1.5.5 Elevated plus-maze test (EPM) was used to investigate the anxiety-related behavior in rodents The maze consists of four arms: a pair of open arms and a pair of closed arms (35 cm long, 5 cm wide, and 10 cm high), which were connected by a central platform. The maze rises 50 cm above the ground. Gently place the mouse in the central area facing the open arm and track the mouse's movements within the elevated cross-maze instrument. Record the number of entries and the time spent in each arm [ 21 ]. Western Blot Tissue protein was extracted with ice RIPA and was detected with a BCA kit following the guidelines. The protein samples were split using SDS-PAGE, moved to a PVDF membrane, and then treated with primary antibodies such as HKII (hexokinase II), PFKP (phosphofructokinase platelet type), PKM2 (pyruvate kinase isozyme type M2), PDH (pyruvate dehydrogenase), and LDHA overnight at 4°C. After incubation with enzyme-linked secondary antibody, the target protein level was detected with a super-ECL detection reagent. Glycolytic metabolites measurement The contents of the ATP colorimetric kit (Cat.#A095-1-1), pyruvate test kit (Cat.#A081-1-1), and lactic acid assay kit (Cat.#A019-2) were obtained from Nanjing Jiancheng, Nanjing, China and followed the manufacturer's instructions. Hematoxylin and eosin staining (H&E), Nissl staining After being fixed with 4% paraformaldehyde for 24 hours, the tissue samples were paraffin-embedded and sliced into 4 µm. The tissue sections were deparaffinized with xylene and graded alcohol. According to the standard protocol of H&E staining eosin dye for 3 minutes hematoxylin dye for 10 minutes. Standard Nissl's staining method was performed. Immunohistochemistry Tissue slices were deparaffinized, and antigen recovery was performed in citrate buffer (pH = 6). Then slices were incubated with the specific antibody HKII (AB227198) in the wet box at 4°C overnight. The DAB detection kit was used as a color developer after the secondary antibody was combined, and finally, the percentage of positive cells was estimated with Image J analysis software [ 22 ]. All images were taken using an optical microscope. Statistical Analysis All data are presented as mean ± standard error of the mean (SEM). All charts show relevant data for at least 3 independent tests. SPSS 26.0 software was used to analyze the statistical analyses. Student’s t-test was used for between-group comparisons followed by a Tukey post-hoc test when an ANOVA revealed significance. A two (± tumor) by two (± CUMS) ANOVA with time as a repeated measure was used for the comparison of four experimental groups. Significance is defined as P <0.05 for all analysis. Results CUMS induces depression-like behavior in BALB/c mice and C57BL/6 mice The experimental flow chart is shown in Fig. 1 A. In CUMS group mice, sucrose preference decreased (Fig. 1 B, 1 H), total locomotion diminished in OFT (Fig. 1 C, 1 I), prolonged immobility time (Fig. 1 E, 1 F, 1 K, 1 L), and lower body weights (Fig. 1 G, 1 M) compared to the control group. These results indicate that the depression model was successfully established in BALB/c mice and C57BL/6 mice. The combination of tumors and CUMS exacerbated depression-like behavior in mice. To investigate whether the mice in the tumor group exhibited anxiety-like and depressive-like behaviors and whether depression-like behaviors were more severe in the BCRD and LCRD[ 20 ], we performed behavioral assessments on tumor-bearing mice separately. In BALB/c mice, tumor-bearing decreased sucrose preference (Fig. 2 A), prolonged immobility time of TST (Fig. 2 D), and impaired spatial cognition (Fig. 2 E), compared to the control group. Compared to the BC group, BCRD mice displayed more decreased sucrose preference in SPT (Fig. 2 A), more diminished total locomotion in OFT (Fig. 2 B&C), more prolonged immobility time (Fig. 2 D), and severe spatial cognitive dysfunction (Fig. 2 E). No significance in the number and stay time of mice entering the open arm between tumor-bearing mice and control mice. Meanwhile, we also assessed the change of SPT and TST of each mouse before and after bearing the tumor. The data showed both control and CUMS mice occurred obvious anhedonia and prolonged immobility time after tumor-bearing, and CUMS induced more individuals and greater variation depression in tumor-bearing mice (Fig. 2 H, 2 I). In C57BL/6 mice, as the same, tumor-bearing decreased sucrose preference (Fig. 2 K), diminished total locomotion (Fig. 2 L), and reduced open arm entries and dwell time (Fig. 2 N, 2 O) compared to the control group. Compared to the LC group, LCRD mice displayed more decreased sucrose preference in SPT (Fig. 2 K), more diminished total locomotion in OFT (Fig. 2 L), decreased the number and stay time of mice entering the open arm (Fig. 2 N, 2 O). Whether comparing between groups or comparing before and after tumor-bearing, CUMS led to more severe depression-like behavior(Fig. 2 P), consistent with breast cancer. Chronic stress-induced impairment of hippocampal neurons in tumor-bearing mice. It is reported that hippocampal neurons play a crucial role in depression [ 5 , 23 ], and Chronic stress causes pathophysiological changes in the hippocampus, which can induce depression [ 11 , 24 ]. As seen in Fig. 3 , the results of Nissl and H&E staining showed that hippocampal neurons were full and clear with a tight and neat cellular arrangement in the control group mice. The Nissl bodies were clear, and no obvious neuron degeneration. The hippocampus neurons were damaged, irregularly arranged, and sparsely distributed, with a widened interstitium in CA1, CA2, and CA3 regions in both CUMS group and two tumor groups (BC and LC). There was a significant decrease in the number of Nissl bodies and a tendency for them to spread to the outer layer. In addition, CUMS aggravated irregular arrangement and sparse distribution in CA2 and CA3 hippocampal regions in tumor-bearing mice, and Nissl bodies were absent and there were obvious neuronal“escapes” and ablation. These results suggest that CUMS aggravates hippocampal neuronal damage in tumor-bearing mice. Western blotting results showed that PSD-95, GAP-43, and Syn were significantly lower in the CUMS group and the BC and LC group compared with the control group. However the interaction between the control group and the BCRD and LCRD group was not significant(Fig C and D, G and H). Chronic stress accelerated tumor tumorigenesis and metastasis Here, we used live mouse bioluminescence imaging to track tumor growth and metastasis in both models of mice. Fluorescence intensity obtained by in vivo imaging, and tumor visual morphology in vitro showed that the tumors in tumor-bearing mice suffered from CUMS were more severe than those without CUMS (Fig. 4 A, 4 B, 4 E, 4 J, 4 K, 4 N). At the same time, the volume of the mammary glands the weight of the BCRD mice, and the weight of the lungs of the LCRD mice increased significantly (Fig. 4 C, 4 D, 4 L). Tissue sections were prepared for pathologic analysis. H&E staining showed that more numerous and larger tumor foci with more dense tumor cells were observed both in BCRD and LCRD mice, and the core of the tumor showed a necrosis-like structure due to lack of nutrients. More scattered tumor-infiltrating cells were also observed in other residual normal tissue cells (Fig. 4 H, 4 M). The 4T1-Luc cells are reported to be a highly metastatic breast cancer cell line with a tendency to metastasize to the lung in vivo [ 25 ]. this phenomenon has indeed been observed by in vivo imaging (Fig. 4 G). Furthermore, a greater number and size of metastases were discovered in breast cancer mice exposed to CUMS (Fig. 4 F). The liver is a common priority metastasis site of lung cancer [ 26 , 27 ]. and the bioluminescence imaging results were confirmed (Fig. 4 O). As similar, more liver metastases were also observed in LCRD (Fig. 4 P). Histopathologic results also showed that the cancer cell morphology in BCRD and LCRD mice metastases was more serious and deteriorated (Fig. 4 I, 4 Q). The above results indicated that chronic stress was a high-risk factor for tumor metastasis. Chronic stress led to a reduction in glycolysis within the brain tissues of tumor-bearing mice. Studies have shown that brain plasticity is one of the pathogenic mechanisms of depression, and glycolytic metabolism is closely related to synaptic plasticity [ 28 – 30 ]. In BALB/c mice, BCRD group glucose metabolites ATP and pyruvate were significantly less than BC, while no difference in lactic acid (Fig. 6 A-C). Glycolytic metabolic enzymes in the brain (HKI, PFKP, PKM2, PDH, and LDHA) were detected with the Western blot test. The levels of HKI and LDHA were decreased in CUMS mice, and tumor mice compared to the control group. However, there were no significant changes observed in PFKP and PKM2 expression, and the interaction between the control group and the BCRD group was not significant (Fig. 6 D, 6 E). In C57BL/6 mice, LCRD group glucose metabolites ATP and lactic acid were significantly less than LC, there was no significant difference in pyruvate levels (Fig. 6 F-H). Western blot results showed that HKI and LDHA were significantly lower in the CUMS group and the LC group compared with the control group. The expression level of PFKP was not significant in the four groups of mice, and the interaction between the control group and the LCRD group was not significant (Fig. 6 I, 6 J). Overall, those results showed that CUMS may induce a decrease in glycolytic enzymes in the brains of mice. Chronic stress augmented aerobic glycolysis within tumor tissues Warburg effect is critical for the metabolic reprogramming of tumor cells [ 31 , 32 ]. the uptake of glucose was enhanced, leading to an increase in lactate production and extracellular acidification rates [ 33 ]. It has been reported that CUMS induces adrenergic activation of LDHA to produce lactate, which promotes the growth of breast cancer [ 10 , 11 ]. Therefore, we investigated the regulatory role of CUMS in glycolysis. As shown in Fig. 7 , the tumor tissues of tumor-bearing mice(both BCRD and LCRD) pretreated by CUMS exhibited elevated levels of glycolysis products and key catalytic enzymes(Fig. 6 A-C, 6 E, 6 F, 6 G-I, 6 K, 6 L). However, IHC detection results revealed the distribution pattern of HKII in BC and LC tissues is different. The distribution of HKII in BC tissues is relatively uniform, and HKII is mainly confined to superficial tumor tissues, while HKII in LC tissues is diffusely dispersed in tumor cells and surrounding tissues of normal lung tissues. Discussion It has been reported that approximately 280 million people worldwide suffer from depression, and 800,000 of them die from depression each year [ 34 – 36 ]. Nearly 80% of depression patients are not diagnosed in time [ 36 ]. Animal and clinical studies have shown a link between emotional dysfunction and tumorigenesis [ 37 ]. Approximately 20%-30% of patients with advanced cancer develop clinically significant depression, and 15% suffer from severe anxiety disorders [ 38 ]. In this study, it was demonstrated that chronic stress can cause depression-like behavior and facilitated tumor genesis and metastasis of BC and LC, meanwhile, all the tumor-bearing mice exhibited depression-like behavior to different extents. In addition, Chronic stress is not only associated with depression but also with burnout and cognitive impairment [ 39 ]. Among 102 cancer survivors aged 25–79 years, approximately one-third had cognitive failures in daily life [ 40 – 42 ]. Our results suggest that chronic stress induces the production of depressive-like behaviors, reduces spatial cognition in mice, and induces mice to exhibit more severe depressive-like behaviors and cognitive dysfunction. These results are consistent with those reported in the literature [ 43 ]. Dysregulation of neuroplasticity and neuronal cell damage are thought to be key mediators in the pathogenesis of depression [ 30 , 44 , 45 ]. Our results also confirmed that CUMS induced neuronal damage and synaptic plasticity reduction in mice, which is consistent with the literature [ 46 , 47 ]. Furthermore, cancer is intricately linked with neurological remodeling and dysfunction neurological-cancer interactions have the potential to modulate tumor growth, invasion, and metastasis dissemination[ 48 ]. tumor growth has been associated with substantial alterations in the hippocampus and a reduction in neuronal cell count, ultimately contributing to depressive symptoms [ 49 ]. Notably, Chronic stress is correlated with the activation of the neuroendocrine system, specifically the hypothalamic-pituitary-adrenal axis, and the sympathetic nervous system. Additionally, it leads to the release of stress hormones such as catecholamines and glucocorticoids [ 50 ]. The microenvironment is changed by the disruption of stress, neurotransmitters, and immune cells and promotes tumorigenesis and progression through a variety of mechanisms [ 37 ]. It was reported that the stimulation of β2-adrenergic receptors in PDAC cancer cells by noradrenaline induced by chronic stress leads to the production of autocrine and paracrine effects, thereby promoting tumor growth [ 51 ]. Our findings also validate the notion that tumors can induce neuronal cell damage and diminish synaptic plasticity in mice. Chronic stress exacerbates neuronal impairment and reduces the survival rate of mice with tumors. The Warburg effect is considered to be a critical element in the reprogramming of energy metabolism in malignant tumors [ 31 ]. Chronic stress also plays an important role [ 10 ], and it promotes tumor growth and metastasis through multiple mechanisms [ 37 ]. HKII is the first irreversible enzyme of glycolysis that inhibits the activity of the PDH complex by phosphorylating S1 of PDHA293 and promotes the Warburg effect [ 52 ]. In addition to serving as an energetic metabolism substrate, lactate can be transported to neurons thereby sustaining neuronal function and exerting antidepressant effects [ 53 , 54 ]. Additionally, the augmented Warburg effect in the hippocampus contributes to enhanced synaptic plasticity [ 55 ]. The glycolytic metabolites in the brain of each group of mice revealed a significant reduction in glycolytic activity within the brains of tumor-bearing mice compared to the control group, and chronic stress further exacerbated the impairment in glycolysis. Recent studies have shown that chronic stress accelerates the progression of colorectal cancer by boosting glycolysis via the CREB1/2-AR signaling pathway [ 10 ]. In a mouse model, chronic stress boosts adrenaline levels and encourages tumor growth by activating the LDHA/USP28/MYC/SLUG signaling axis [ 11 ]. our results also show that chronic stress induced higher expression of glycolytic metabolites in tumor tissue. In conclusion, our findings consistently indicate chronic stress-induced anxiety-like and depression-like behaviors in mice, while also promoting the overproduction of lactic acid through increased aerobic glycolytic enzymes in tumor tissue. extracellular acidification to maintain the invasive growth of cancer cells and further exacerbate the progression and metastasis of BC and LC. The findings of this study contribute to a deeper understanding of the impact of metabolic remodeling on the pathogenesis of cancer-related depression. However, It was challenging to establish a strong connection between CUMS and glycolysis because of the small sample size, which limited the experiment's findings to examining changes in glycolysis in tumor progression under chronic stress rather than delving into the underlying mechanisms in detail. As a result, future related studies should concentrate on assessing glycolysis's significant contribution to the phenomenon of cancer-associated depression. In the meantime, because the cancer cell lines used in this study came from different places, different strains of mice were given different injections of cancer cells. It was discovered that this caused variations in the immune responses and stress levels in the male and female mice, which in turn led to variations in the model's experiment outcomes. Consequently, we plan to inject the cancer cells into the same strains of mice in future related studies. Declarations Acknowledgments We thank the Natural Science Foundation of Guang Dong Province for the generous funding of our studies. Moreover, we thank the National Natural Science Foundation of China for the generous funding of our studies. Funding This study was supported by the Natural Science Foundation of Guang Dong Province (2023A1515011343) and the National Natural Science Foundation of China (81903943). Author Contributions Conceptualization: H.Q.L, R.Z, and S.S.B. Primary writing and original draft preparation: Q.F.Q and S.Y.L. Editing: Y.X.Z, J.B, S.S.B. Visualization and supervision: L.A, L.Y, W.G, D.D, J.L.Z. All authors agreed to the version of the manuscript. Data availability Data generated or analyzed during this study are included in this published article (and its supplementary information files). Ethics approval The animal study was reviewed and approved by Guangzhou University of Chinese Medicine. Competing interests The authors report no conflicts of interest. References Panjwani AA, Li ME (2021) Recent trends in the management of depression in persons with cancer. Current Opinion in Psychiatry 34: 448-459. 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DOI 10.1146/annurev-neuro-110920-040422 Appelbaum LG, Shenasa MA, Stolz L, Daskalakis Z (2023) Synaptic plasticity and mental health: methods, challenges and opportunities. Neuropsychopharmacol 48: 113-120. DOI 10.1038/s41386-022-01370-w Winkler F, Venkatesh HS, Amit M, Batchelor T, Demir IE, Deneen B, Gutmann DH, Hervey-Jumper S, Kuner T, Mabbott D, et al. (2023) Cancer neuroscience: State of the field, emerging directions. Cell 186: 1689-1707. DOI 10.1016/j.cell.2023.02.002 Zhu Q, Meng P, Han Y, Yang H, Yang Q, Liu Z, Wang Y, Long M (2022) Luteolin Induced Hippocampal Neuronal Pyroptosis Inhibition by Regulation of miR-124-3p/TNF-α/TRAF6 Axis in Mice Affected by Breast-Cancer-Related Depression. Evidence-based complementary and alternative medicine : eCAM 2022: 2715325. DOI 10.1155/2022/2715325 Tian W, Liu Y, Cao C, Zeng Y, Pan Y, Liu X, Peng Y, Wu F (2021) Chronic Stress: Impacts on Tumor Microenvironment and Implications for Anti-Cancer Treatments. Frontiers in cell and developmental biology 9: 777018. DOI 10.3389/fcell.2021.777018 Hanahan D, Monje M (2023) Cancer hallmarks intersect with neuroscience in the tumor microenvironment. Cancer Cell 41: 573-580. DOI 10.1016/j.ccell.2023.02.012 Luo F, Li Y, Yuan F, Zuo J (2019) Hexokinase II promotes the Warburg effect by phosphorylating alpha subunit of pyruvate dehydrogenase. Chinese journal of cancer research = Chung-kuo yen cheng yen chiu 31: 521-532. DOI 10.21147/j.issn.1000-9604.2019.03.14 Powell CL, Davidson AR, Brown AM (2020) Universal Glia to Neurone Lactate Transfer in the Nervous System: Physiological Functions and Pathological Consequences. Biosensors-Basel 10. DOI ARTN 183 10.3390/bios10110183 Carrard A, Elsayed M, Margineanu M, Boury-Jamot B, Fragnière L, Meylan EM, Petit JM, Fiumelli H, Magistretti PJ, Martin JL (2018) Peripheral administration of lactate produces antidepressant-like effects. Mol Psychiatry 23: 392-399. DOI 10.1038/mp.2016.179 Yang SQ, Tang YY, Zeng D, Tian Q, Wei HJ, Wang CY, Zhang P, Chen YJ, Zou W, Tang XQ (2022) Sodium hydrosulfide reverses β(2)-microglobulin-induced depressive-like behaviors of male Sprague-Dawley rats: Involving improvement of synaptic plasticity and enhancement of Warburg effect in hippocampus. Behavioural brain research 417: 113562. DOI 10.1016/j.bbr.2021.113562 Supplementary Files Westernbolt.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 02 Sep, 2024 Reviewers invited by journal 26 Feb, 2024 Editor assigned by journal 14 Feb, 2024 First submitted to journal 13 Feb, 2024 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 In Review Editorial Policies 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-3957233","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275265231,"identity":"186e2351-804a-40c4-9a6c-be96496ebc27","order_by":0,"name":"Qiufeng Qin","email":"","orcid":"","institution":"Guangzhou University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qiufeng","middleName":"","lastName":"Qin","suffix":""},{"id":275265232,"identity":"8dc34251-b3cc-4c15-a6bd-f178e3501fa1","order_by":1,"name":"Shuying Li","email":"","orcid":"","institution":"Guangzhou University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shuying","middleName":"","lastName":"Li","suffix":""},{"id":275265233,"identity":"fdac9ca6-1ee5-43b5-bad0-95a69396187c","order_by":2,"name":"yixuan Zhong","email":"","orcid":"","institution":"Guangzhou University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"yixuan","middleName":"","lastName":"Zhong","suffix":""},{"id":275265234,"identity":"652b2e4a-5bf9-487b-95dc-730e055880e4","order_by":3,"name":"Jing Bai","email":"","orcid":"","institution":"Guangzhou University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Bai","suffix":""},{"id":275265235,"identity":"c158d183-43da-4722-b486-0ae496774aed","order_by":4,"name":"Lin An","email":"","orcid":"","institution":"Guangzhou University of Chinese 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Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shasha","middleName":"","lastName":"Bai","suffix":""}],"badges":[],"createdAt":"2024-02-15 00:38:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3957233/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3957233/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51836548,"identity":"2da657ea-c3be-4ecc-a4ea-a757151a70e5","added_by":"auto","created_at":"2024-02-29 21:42:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5178881,"visible":true,"origin":"","legend":"\u003cp\u003eEstablishment of the CUMS model in BALB/c mice and C57BL/6 mice. (A) Flowchart of the experiment. (B, H) CUMS decreasedthe percentage of sucrose preference. (C, I) CUMS decreased the total distance in the OFT. (D, J) The representative movement trajectories of each group mouse in the OFT. (E, F, K, L) CUMS increased immobility time in TST and FST. (G, M) The body weight curve of all group mice during the whole experiment. Data are represented as the mean value ± SEM, n = 16, \u003cem\u003e* p＜0.05,* * p＜0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957233/v1/bd46500f0ede6caa83cda989.jpg"},{"id":51836550,"identity":"15ade0c3-e939-464e-8591-af93420707cb","added_by":"auto","created_at":"2024-02-29 21:42:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5418289,"visible":true,"origin":"","legend":"\u003cp\u003eChronic stress aggravated depression-like behavior in tumor-bearing mice. (A, K) The percentage of sucrose preference in the SPT. (B, L) Results of the total distance in the OFT. (C, M) The representative movement trajectories of each group in the OFT. (D) Results of immobility time in the TST. (E) Results for the number of alternations in the Y-maze. (F, G, N, O) Open arm entry number and residence time in the EPM. (H, I, P) Self-behavioral comparison. (J, Q) The body weight curve. Data are represented as the mean value±SEM (BALB/c: n= 8, C57BL/6: n= 6).\u003cem\u003e * p\u0026lt;0.05, ** p\u0026lt;0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957233/v1/2524787faedbf6042d1807ef.jpg"},{"id":51836552,"identity":"695306e1-19bc-4346-9fb7-33816f078f3b","added_by":"auto","created_at":"2024-02-29 21:42:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31206040,"visible":true,"origin":"","legend":"\u003cp\u003eCUMS aggravated hippocampal neuron damage in tumor-bearing mice. (A, D) Nissl staining of the hippocampus in BALB/c mice and C57BL/6 mice. (B, F) H\u0026amp;E staining of the hippocampus in BALB/c mice and C57BL/6 mice. (C and D, G and H) Western blotting was used to detect the expression of brain plasticity enzymes. Data are represented as the mean value±SEM (C and D, G and H, n= 4).\u003cem\u003e * p\u0026lt;0.05, ** p\u0026lt;0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957233/v1/32de6e7daf75d3260a0dc906.jpg"},{"id":51836841,"identity":"08a92bd3-a9f0-4a07-ab2b-af8730d70780","added_by":"auto","created_at":"2024-02-29 21:50:39","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20910265,"visible":true,"origin":"","legend":"\u003cp\u003eTumor growth and metastasis were aggravated by chronic stress. (A, E, F, G, J, N, O, P) The representative pictures of vivo imaging assay and tumors in each group. (B, C, D, K, L) The fluorescence intensity values, Tumor weight, and growth curves. (H, I, M, Q) The representative pictures of tumor tissues and metastasis were stained with H\u0026amp;E staining. Data are represented as the mean value±SEM (BALB/c: n= 8, C57BL/6: n= 6).\u003cem\u003e * p\u0026lt;0.05, ** p\u0026lt;0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957233/v1/3c4da4721a9fc4d3e1a39797.jpg"},{"id":51836546,"identity":"51120496-c7dc-4f11-8db3-45912fa66a9a","added_by":"auto","created_at":"2024-02-29 21:42:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4838279,"visible":true,"origin":"","legend":"\u003cp\u003eChronic stress induced a decreased glycolysis in the brain of tumor-bearing mice.\u003cstrong\u003e \u003c/strong\u003e(A-C, F-H) Glycolytic metabolites (ATP, pyruvate, lactic acid) concentration. (D, E, I, J) The expression of glycolysis enzymes was detected with western blot in mice brains. Data are represented as the mean value±SEM.\u003cem\u003e * p\u0026lt;0.05, ** p\u0026lt;0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957233/v1/838cbcfa60031d3bbd568d0f.jpg"},{"id":51836554,"identity":"1b8b82d7-ce50-46df-92cc-43e60da1e226","added_by":"auto","created_at":"2024-02-29 21:42:40","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":11787323,"visible":true,"origin":"","legend":"\u003cp\u003eAerobic glycolysis enhanced both in BCRD and LCRD. (A-C, G-I) Glycolytic metabolites in tumor tissue. (D, J) HKII distribution in tumor tissue was detected with IHC. (E and F, K and L) Western blotting was used to detect the expression of glycolysis enzymes in the tumor. Data are represented as the mean value±SEM.\u003cem\u003e * p\u0026lt;0.05, ** p\u0026lt;0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957233/v1/1594fa6a516d03ab648da14c.jpg"},{"id":51836553,"identity":"6c8d96d5-5ee7-48a8-9a5e-19769fdbb4fb","added_by":"auto","created_at":"2024-02-29 21:42:40","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":251096,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe chronic stress-induced depression-like behavior in mice exacerbated tumorigenesis and metastasis by augmenting glycolysis in tumor cells.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957233/v1/d135b5aa921454d9d537097d.jpg"},{"id":51836953,"identity":"1661dd6c-47cb-4292-a345-a20df51d67c4","added_by":"auto","created_at":"2024-02-29 21:58:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1859186,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3957233/v1/94ef4611-c14b-46f4-ab8f-76440f4f0c07.pdf"},{"id":51836551,"identity":"e2ca1b58-23a7-425b-938d-b38c9a9a8fbf","added_by":"auto","created_at":"2024-02-29 21:42:40","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":364806,"visible":true,"origin":"","legend":"","description":"","filename":"Westernbolt.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3957233/v1/f2a243215eabee890cbc2a91.pdf"}],"financialInterests":"","formattedTitle":"Chronic Stress Enhances Glycolysis and Promotes Tumorigenesis","fulltext":[{"header":"Key messages","content":"\u003cul\u003e\n \u003cli\u003eBoth CUMS and tumor-induced depressive-like behavior, neuronal damage, and impaired synaptic plasticity in mice,\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCUMS can enhance tumor development and metastasis in both BC and LC.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eIn the brain, both CUMS and tumor alone and in combination significantly reduced glycolytic products and enzyme levels.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eIn tumor tissue, CUMS significantly enhanced the levels of aerobic glycolytic products and enzymes.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eThis study validated the potential mechanism of glycolysis in depression-promoted tumorigenesis and development.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003ePatients diagnosed with cancer often concurrently suffer from depression [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], the negative emotions induced by this psychological condition can potentially stimulate tumor growth and metastasis of tumors [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Past studies have unequivocally shown that chronic stress significantly affects every stage of cancer development in patients, ranging from carcinogenesis to angiogenesis stimulation and metastatic dissemination [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Chronic stress-induced inadequate coping, negative emotional responses, or a diminished quality of life are associated with an increased risk of cancer incidence [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Annually, approximately one million new cancer cases are diagnosed in young individuals aged 20\u0026ndash;39 years, primarily attributed to chronic stress [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A poor prognosis and elevated mortality are observed among cancer patients concurrently suffering from comorbid depression [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Cancer patients exhibit a depression morbidity of approximately 12.5%, which is up to fourfold higher than that of the general population [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Chronic stress has been demonstrated in studies to raise plasma catecholamine levels, including epinephrine and noradrenaline, and accelerate the aggressive development of BC, ovarian carcinoma, and gastric cancer [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the research concerning the relationship between chronic stress and cancer remains limited.\u003c/p\u003e \u003cp\u003eOtto Warburg and colleagues ascertained in the 1920s that tumor cells, under aerobic conditions, exhibit a preference for generating energy via a process termed aerobic glycolysis. This is manifested by excessive glucose absorption and lactate accumulation, a phenomenon known as the Warburg effect [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To fulfill uncontrolled biosynthesis and energy demands, cancer cells frequently tend to perform aerobic glycolysis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In numerous cancer patients, clinical investigations utilizing the imaging technique of positron emission tomography (PET) with the glucose analog tracer 18fludeoxyglucose (FDG)\u003csup\u003e6\u0026ndash;8\u003c/sup\u003e have unambiguously demonstrated a substantial elevation in glucose absorption in the majority of metastatic and primary human cancer patients [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Tumor cells facilitate the formation of new blood vessels through the secretion of VEGF, induced by lactate, which supplies sufficient oxygen and nutrients for proliferation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Persistent chronic stress triggers a decrease in lactate levels and leads to depression. Intriguingly, stress-induced epinephrine enhances breast cancer stem-like characteristics through LDHA (lactate dehydrogenase A) - dependent metabolic reprogramming [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Lactate performs antidepressant functions by maintaining normal neuron function, and modulating levels and activity of histone deacetylases in the hippocampal region[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Meanwhile, LDHA regulats neuronal excitability to inhibit depressive-like behavior through lactate homeostasis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, the research on lactate levels in depression and cancer models has been a gap in the literature. To address this, we developed an animal model of breast cancer-related depression (BCRD) by in situ injection of BC cells into CUMS mice and a model of lung cancer-related depression (LCRD) by injecting LC cells into the tail veins of CUMS mice. Behavioral assessments and synapse plasticity detection were used for depression evaluation, while tumor volume and H\u0026amp;E staining were performed to examine tumorigenesis. Metabolites and key functional enzymes were detected to unveal the role of glycolysis in depression-promoted tumor glycolysis.\u003c/p\u003e \u003cp\u003eThe findings of this research highlight the potential role of chronic stress in exacerbating tumorigenesis and metastasis through the stimulation of glycolysis.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eLuciferase gene-tagged Lewis murine lung cancer cell (LLC-Luc) and luciferase gene-tagged 4T1 murine breast cancer cell (4T1-Luc) were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). LLC-Luc cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Gibco-BRL, Grand Island, NY, USA); the 4T1-Luc cells were cultured in RPMI-1640 medium (Gibco-BRL, Grand Island, NY, USA); all mediums were supplemented with 10% fetal bovine serum and cultured at 37\u0026deg;C in a humidified incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eSix\u0026ndash;week\u0026ndash;old female BALB / c mice and male C57BL / 6 mice were purchased from the Guangdong Medical Laboratory Animal Center (Guangdong, China), and housed in special-pathogen-free ventilation facilities. 12 h / 12 h light/dark cycle was carried out, with ambient temperatures of 20\u0026ndash;26 \u003csup\u003eo\u003c/sup\u003e C and relative humidity of 40\u0026ndash;70%, 5 mice per cage, and eating and drinking freely. The Ethics Committee of Guangzhou University of Chinese Medicine has authorized a laboratory animal protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eChronic unpredictable mild stress experiment\u003c/h2\u003e \u003cp\u003eChronic unpredictable mild stress (CUMS) might mimic the onset of depression produced by various pressures in human daily life [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Mice were exposed to CUMS for 8 weeks, which included day-night reversal (24 h), cold-water swimming (10\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 3 min), crowd-feeding (24 h), water and food deprivation (24 h), an empty water bottle (24 h), a 45 \u0026deg; cage tilt (24 h), a tail clamp (1 cm from the tail end, 60 s), a self-made plastic seal tube (3 h), and a wet pad (24 h). 2 or 3 stimulation modalities were chosen randomly each day to ensure no repetition within 2 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of tumor orthotopic transplantation\u003c/h2\u003e \u003cp\u003e5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e 4T1-Luc cells were injected into the right second mammary fat pad of BALB/c mice for orthotopic transplantation of BC. CUMS mice were inoculated with 4T1-Luc cells to develop BCRD. The tumor size of BC was measured with an vernier caliper every two days. It was calculated as follows: tumor volume (mm\u003csup\u003e3\u003c/sup\u003e) = [length \u0026times; width\u003csup\u003e2\u003c/sup\u003e] / 2.\u003c/p\u003e \u003cp\u003e1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e LLC - Luc cells were injected into the tail vein of C57BL/6 mice for orthotopic transplantation of LC, and CUMS mice adopted LLC-Luc cells to develop LCRD. The tumor size was evaluated every week using IVIS (PerkinElmer, Boston, United States) with 150 mg/kg of D-luciferin potassium salt (PerkinElmer, Boston, United States) given intraperitoneally.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBehavior assessments\u003c/h2\u003e \u003cp\u003eThe sucrose preference test (SPT) is done for anhedonia assessment. Mice were fed in solitary cages preferentially. Two bottles containing 1% sucrose solution were placed at each side of the cages for 24 hours as an adaption phase. The next day, one bottle was replaced with water and left for another 24 hours, and two bottles of solution were changed the position at 12 hours to avoid the error caused by location preference. Then, mice were formally tested after 24 hours of freely eating without water, one bottle of water and one bottle of 1% sucrose solution were placed on each side of the cage and switched at 12 hours. 24 hours later, weigh each bottle and calculate sucrose preference (%)\u0026thinsp;=\u0026thinsp;sucrose consumption / (water consumption\u0026thinsp;+\u0026thinsp;sucrose consumption) \u0026times; 100% [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAn open field test (OFT) was also performed. BALB/c mice were set up in the center of the blackboard, while C57BL/6 mice were on a whiteboard and 10 minutes for each mouse. The open-field arena is 50 \u0026times; 50 \u0026times; 40 cm (length \u0026times; width \u0026times; height). The mice\u0026rsquo;s activity was filmed using a digital camera, and the total distance, center distance, and center time were calculated using software.\u003c/p\u003e \u003cp\u003eThe tail suspension test (TST) was done as follows, mice were suspended 30 cm above the ground with wide tape on the tail-suspension device for 6 minutes and recorded with a digital camera recorded the mice's activity. The first 2 minutes are considered familiarity time and only the last 4 minutes are counted.\u003c/p\u003e \u003cp\u003eMice were placed in cylindrical containers filled with room-temperature water at a depth of 30 cm for forced swim test (FST). Each mouse swam for 6 minutes and was videotaped, and then blow-dried the mice hair and put them back. The first 2 minutes are considered familiarity time and only the last 4 minutes are counted.\u003c/p\u003e \u003cp\u003eThe Y-Maze test was considered evaluable for working memory and exploratory behavior in mice. Mice were placed on either of three identical arms (arm length: 35 cm, arm width: 5 cm, wall height: 10 cm) and were allowed to explore freely for 8 minutes. The percentage of spontaneous alternation was calculated as alternation (%) = (number of correct alternations) / (number of total arm entries-2) x 100% [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e1.5.5 Elevated plus-maze test (EPM) was used to investigate the anxiety-related behavior in rodents The maze consists of four arms: a pair of open arms and a pair of closed arms (35 cm long, 5 cm wide, and 10 cm high), which were connected by a central platform. The maze rises 50 cm above the ground. Gently place the mouse in the central area facing the open arm and track the mouse's movements within the elevated cross-maze instrument. Record the number of entries and the time spent in each arm [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot\u003c/h2\u003e \u003cp\u003eTissue protein was extracted with ice RIPA and was detected with a BCA kit following the guidelines. The protein samples were split using SDS-PAGE, moved to a PVDF membrane, and then treated with primary antibodies such as HKII (hexokinase II), PFKP (phosphofructokinase platelet type), PKM2 (pyruvate kinase isozyme type M2), PDH (pyruvate dehydrogenase), and LDHA overnight at 4\u0026deg;C. After incubation with enzyme-linked secondary antibody, the target protein level was detected with a super-ECL detection reagent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGlycolytic metabolites measurement\u003c/h2\u003e \u003cp\u003eThe contents of the ATP colorimetric kit (Cat.#A095-1-1), pyruvate test kit (Cat.#A081-1-1), and lactic acid assay kit (Cat.#A019-2) were obtained from Nanjing Jiancheng, Nanjing, China and followed the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin and eosin staining (H\u0026amp;E), Nissl staining\u003c/h2\u003e \u003cp\u003eAfter being fixed with 4% paraformaldehyde for 24 hours, the tissue samples were paraffin-embedded and sliced into 4 \u0026micro;m. The tissue sections were deparaffinized with xylene and graded alcohol. According to the standard protocol of H\u0026amp;E staining eosin dye for 3 minutes hematoxylin dye for 10 minutes. Standard Nissl's staining method was performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eTissue slices were deparaffinized, and antigen recovery was performed in citrate buffer (pH\u0026thinsp;=\u0026thinsp;6). Then slices were incubated with the specific antibody HKII (AB227198) in the wet box at 4\u0026deg;C overnight. The DAB detection kit was used as a color developer after the secondary antibody was combined, and finally, the percentage of positive cells was estimated with Image J analysis software [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. All images were taken using an optical microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). All charts show relevant data for at least 3 independent tests. SPSS 26.0 software was used to analyze the statistical analyses. Student\u0026rsquo;s t-test was used for between-group comparisons followed by a Tukey post-hoc test when an ANOVA revealed significance. A two (\u0026plusmn;\u0026thinsp;tumor) by two (\u0026plusmn;\u0026thinsp;CUMS) ANOVA with time as a repeated measure was used for the comparison of four experimental groups. Significance is defined as \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 for all analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCUMS induces depression-like behavior in BALB/c mice and C57BL/6 mice\u003c/h2\u003e \u003cp\u003eThe experimental flow chart is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. In CUMS group mice, sucrose preference decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), total locomotion diminished in OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), prolonged immobility time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL), and lower body weights (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM) compared to the control group. These results indicate that the depression model was successfully established in BALB/c mice and C57BL/6 mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe combination of tumors and CUMS exacerbated depression-like behavior in mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether the mice in the tumor group exhibited anxiety-like and depressive-like behaviors and whether depression-like behaviors were more severe in the BCRD and LCRD[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e20\u003c/span\u003e], we performed behavioral assessments on tumor-bearing mice separately. In BALB/c mice, tumor-bearing decreased sucrose preference (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), prolonged immobility time of TST (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), and impaired spatial cognition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), compared to the control group. Compared to the BC group, BCRD mice displayed more decreased sucrose preference in SPT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), more diminished total locomotion in OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026amp;C), more prolonged immobility time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), and severe spatial cognitive dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). No significance in the number and stay time of mice entering the open arm between tumor-bearing mice and control mice. Meanwhile, we also assessed the change of SPT and TST of each mouse before and after bearing the tumor. The data showed both control and CUMS mice occurred obvious anhedonia and prolonged immobility time after tumor-bearing, and CUMS induced more individuals and greater variation depression in tumor-bearing mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eIn C57BL/6 mice, as the same, tumor-bearing decreased sucrose preference (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK), diminished total locomotion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL), and reduced open arm entries and dwell time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO) compared to the control group. Compared to the LC group, LCRD mice displayed more decreased sucrose preference in SPT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK), more diminished total locomotion in OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL), decreased the number and stay time of mice entering the open arm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO). Whether comparing between groups or comparing before and after tumor-bearing, CUMS led to more severe depression-like behavior(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eP), consistent with breast cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eChronic stress-induced impairment of hippocampal neurons in tumor-bearing mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIt is reported that hippocampal neurons play a crucial role in depression [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and Chronic stress causes pathophysiological changes in the hippocampus, which can induce depression [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the results of Nissl and H\u0026amp;E staining showed that hippocampal neurons were full and clear with a tight and neat cellular arrangement in the control group mice. The Nissl bodies were clear, and no obvious neuron degeneration. The hippocampus neurons were damaged, irregularly arranged, and sparsely distributed, with a widened interstitium in CA1, CA2, and CA3 regions in both CUMS group and two tumor groups (BC and LC). There was a significant decrease in the number of Nissl bodies and a tendency for them to spread to the outer layer. In addition, CUMS aggravated irregular arrangement and sparse distribution in CA2 and CA3 hippocampal regions in tumor-bearing mice, and Nissl bodies were absent and there were obvious neuronal\u0026ldquo;escapes\u0026rdquo; and ablation. These results suggest that CUMS aggravates hippocampal neuronal damage in tumor-bearing mice. Western blotting results showed that PSD-95, GAP-43, and Syn were significantly lower in the CUMS group and the BC and LC group compared with the control group. However the interaction between the control group and the BCRD and LCRD group was not significant(Fig C and D, G and H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eChronic stress accelerated tumor tumorigenesis and metastasis\u003c/h2\u003e \u003cp\u003eHere, we used live mouse bioluminescence imaging to track tumor growth and metastasis in both models of mice. Fluorescence intensity obtained by in vivo imaging, and tumor visual morphology \u003cem\u003ein vitro\u003c/em\u003e showed that the tumors in tumor-bearing mice suffered from CUMS were more severe than those without CUMS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN). At the same time, the volume of the mammary glands the weight of the BCRD mice, and the weight of the lungs of the LCRD mice increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). Tissue sections were prepared for pathologic analysis. H\u0026amp;E staining showed that more numerous and larger tumor foci with more dense tumor cells were observed both in BCRD and LCRD mice, and the core of the tumor showed a necrosis-like structure due to lack of nutrients. More scattered tumor-infiltrating cells were also observed in other residual normal tissue cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM).\u003c/p\u003e \u003cp\u003eThe 4T1-Luc cells are reported to be a highly metastatic breast cancer cell line with a tendency to metastasize to the lung \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. this phenomenon has indeed been observed by in vivo imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Furthermore, a greater number and size of metastases were discovered in breast cancer mice exposed to CUMS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). The liver is a common priority metastasis site of lung cancer [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. and the bioluminescence imaging results were confirmed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO). As similar, more liver metastases were also observed in LCRD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eP). Histopathologic results also showed that the cancer cell morphology in BCRD and LCRD mice metastases was more serious and deteriorated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eQ). The above results indicated that chronic stress was a high-risk factor for tumor metastasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eChronic stress led to a reduction in glycolysis within the brain tissues of tumor-bearing mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eStudies have shown that brain plasticity is one of the pathogenic mechanisms of depression, and glycolytic metabolism is closely related to synaptic plasticity [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR31\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn BALB/c mice, BCRD group glucose metabolites ATP and pyruvate were significantly less than BC, while no difference in lactic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). Glycolytic metabolic enzymes in the brain (HKI, PFKP, PKM2, PDH, and LDHA) were detected with the Western blot test. The levels of HKI and LDHA were decreased in CUMS mice, and tumor mice compared to the control group. However, there were no significant changes observed in PFKP and PKM2 expression, and the interaction between the control group and the BCRD group was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eIn C57BL/6 mice, LCRD group glucose metabolites ATP and lactic acid were significantly less than LC, there was no significant difference in pyruvate levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-H). Western blot results showed that HKI and LDHA were significantly lower in the CUMS group and the LC group compared with the control group. The expression level of PFKP was not significant in the four groups of mice, and the interaction between the control group and the LCRD group was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Overall, those results showed that CUMS may induce a decrease in glycolytic enzymes in the brains of mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eChronic stress augmented aerobic glycolysis within tumor tissues\u003c/h2\u003e \u003cp\u003eWarburg effect is critical for the metabolic reprogramming of tumor cells [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. the uptake of glucose was enhanced, leading to an increase in lactate production and extracellular acidification rates [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. It has been reported that CUMS induces adrenergic activation of LDHA to produce lactate, which promotes the growth of breast cancer [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, we investigated the regulatory role of CUMS in glycolysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the tumor tissues of tumor-bearing mice(both BCRD and LCRD) pretreated by CUMS exhibited elevated levels of glycolysis products and key catalytic enzymes(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-I,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). However, IHC detection results revealed the distribution pattern of HKII in BC and LC tissues is different. The distribution of HKII in BC tissues is relatively uniform, and HKII is mainly confined to superficial tumor tissues, while HKII in LC tissues is diffusely dispersed in tumor cells and surrounding tissues of normal lung tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt has been reported that approximately 280\u0026nbsp;million people worldwide suffer from depression, and 800,000 of them die from depression each year [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR38\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Nearly 80% of depression patients are not diagnosed in time [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Animal and clinical studies have shown a link between emotional dysfunction and tumorigenesis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Approximately 20%-30% of patients with advanced cancer develop clinically significant depression, and 15% suffer from severe anxiety disorders [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this study, it was demonstrated that chronic stress can cause depression-like behavior and facilitated tumor genesis and metastasis of BC and LC, meanwhile, all the tumor-bearing mice exhibited depression-like behavior to different extents. In addition, Chronic stress is not only associated with depression but also with burnout and cognitive impairment [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Among 102 cancer survivors aged 25\u0026ndash;79 years, approximately one-third had cognitive failures in daily life [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR45\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our results suggest that chronic stress induces the production of depressive-like behaviors, reduces spatial cognition in mice, and induces mice to exhibit more severe depressive-like behaviors and cognitive dysfunction. These results are consistent with those reported in the literature [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDysregulation of neuroplasticity and neuronal cell damage are thought to be key mediators in the pathogenesis of depression [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our results also confirmed that CUMS induced neuronal damage and synaptic plasticity reduction in mice, which is consistent with the literature [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Furthermore, cancer is intricately linked with neurological remodeling and dysfunction neurological-cancer interactions have the potential to modulate tumor growth, invasion, and metastasis dissemination[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. tumor growth has been associated with substantial alterations in the hippocampus and a reduction in neuronal cell count, ultimately contributing to depressive symptoms [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Notably, Chronic stress is correlated with the activation of the neuroendocrine system, specifically the hypothalamic-pituitary-adrenal axis, and the sympathetic nervous system. Additionally, it leads to the release of stress hormones such as catecholamines and glucocorticoids [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The microenvironment is changed by the disruption of stress, neurotransmitters, and immune cells and promotes tumorigenesis and progression through a variety of mechanisms [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It was reported that the stimulation of β2-adrenergic receptors in PDAC cancer cells by noradrenaline induced by chronic stress leads to the production of autocrine and paracrine effects, thereby promoting tumor growth [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Our findings also validate the notion that tumors can induce neuronal cell damage and diminish synaptic plasticity in mice. Chronic stress exacerbates neuronal impairment and reduces the survival rate of mice with tumors.\u003c/p\u003e \u003cp\u003eThe Warburg effect is considered to be a critical element in the reprogramming of energy metabolism in malignant tumors [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Chronic stress also plays an important role [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and it promotes tumor growth and metastasis through multiple mechanisms [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. HKII is the first irreversible enzyme of glycolysis that inhibits the activity of the PDH complex by phosphorylating S1 of PDHA293 and promotes the Warburg effect [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In addition to serving as an energetic metabolism substrate, lactate can be transported to neurons thereby sustaining neuronal function and exerting antidepressant effects [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Additionally, the augmented Warburg effect in the hippocampus contributes to enhanced synaptic plasticity [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The glycolytic metabolites in the brain of each group of mice revealed a significant reduction in glycolytic activity within the brains of tumor-bearing mice compared to the control group, and chronic stress further exacerbated the impairment in glycolysis. Recent studies have shown that chronic stress accelerates the progression of colorectal cancer by boosting glycolysis via the CREB1/2-AR signaling pathway [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In a mouse model, chronic stress boosts adrenaline levels and encourages tumor growth by activating the LDHA/USP28/MYC/SLUG signaling axis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. our results also show that chronic stress induced higher expression of glycolytic metabolites in tumor tissue.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings consistently indicate chronic stress-induced anxiety-like and depression-like behaviors in mice, while also promoting the overproduction of lactic acid through increased aerobic glycolytic enzymes in tumor tissue. extracellular acidification to maintain the invasive growth of cancer cells and further exacerbate the progression and metastasis of BC and LC. The findings of this study contribute to a deeper understanding of the impact of metabolic remodeling on the pathogenesis of cancer-related depression. However, It was challenging to establish a strong connection between CUMS and glycolysis because of the small sample size, which limited the experiment's findings to examining changes in glycolysis in tumor progression under chronic stress rather than delving into the underlying mechanisms in detail. As a result, future related studies should concentrate on assessing glycolysis's significant contribution to the phenomenon of cancer-associated depression. In the meantime, because the cancer cell lines used in this study came from different places, different strains of mice were given different injections of cancer cells. It was discovered that this caused variations in the immune responses and stress levels in the male and female mice, which in turn led to variations in the model's experiment outcomes. Consequently, we plan to inject the cancer cells into the same strains of mice in future related studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWe thank\u003c/em\u003e the Natural Science Foundation of Guang Dong Province for the generous funding of our studies. Moreover, we thank the National Natural Science Foundation of China for the generous funding of our studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Natural Science Foundation of Guang Dong Province (2023A1515011343) and the National Natural Science Foundation of China (81903943).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: H.Q.L,\u0026nbsp;R.Z, and S.S.B.\u0026nbsp;Primary writing and original\u0026nbsp;draft preparation:\u0026nbsp;Q.F.Q\u0026nbsp;and\u0026nbsp;S.Y.L.\u0026nbsp;Editing:\u0026nbsp;Y.X.Z, J.B, S.S.B.\u0026nbsp;Visualization and supervision:\u0026nbsp;L.A, L.Y, W.G, D.D, J.L.Z.\u0026nbsp;All authors agreed to the version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData generated or analyzed during this study are included in this published article (and its supplementary information files).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e The animal study was reviewed and approved by Guangzhou University of Chinese Medicine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePanjwani AA, Li ME (2021) Recent trends in the management of depression in persons with cancer. 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DOI 10.1016/j.bbr.2021.113562\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"CUMS, metastasis, tumorigenesis, glycolysis, orthotopic","lastPublishedDoi":"10.21203/rs.3.rs-3957233/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3957233/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDepression is a well-known risk factor for tumors, but the mechanisms other than inflammation are unclear. Aerobic glycolysis is considered to be a critical element in the reprogramming of energy metabolism in malignant tumors, and impaired glycolysis has been reported in the brains of chronic stress mice. Therefore, this study aimed to explore the role of glycolysis in which depression promotes tumorigenesis. We examined the impacts of chronic unpredictable mild stress (CUMS) on the growth and metastasis of breast cancer (BC) and lung cancer (LC). The findings showed that both CUMS and tumors induced depressive-like behavior, neuronal damage, and impaired synaptic plasticity in mice, while CUMS also enhanced tumor development and metastasis in both BC and LC. In the brain, both CUMS and tumor alone and in combination significantly reduced glycolytic products and enzyme levels. However, CUMS significantly enhanced the levels of aerobic glycolytic products and enzymes in tumor tissue. Collectively, our results provide insights into that down-regulated glycolysis in the brain, leading to depression-like behavior, and how depression, in turn, enhanced glycolysis and promoted tumorigenesis.\u003c/p\u003e","manuscriptTitle":"Chronic Stress Enhances Glycolysis and Promotes Tumorigenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-29 21:42:34","doi":"10.21203/rs.3.rs-3957233/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-09-02T10:07:22+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-26T21:12:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-14T06:13:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Medicine","date":"2024-02-14T00:52:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmme","sideBox":"Learn more about [Journal of Molecular Medicine](https://www.springer.com/journal/109)","snPcode":"109","submissionUrl":"https://submission.nature.com/new-submission/109/3","title":"Journal of Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7251ee41-8528-4b3d-bcbf-92f934c4334b","owner":[],"postedDate":"February 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-02-29T21:42:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-29 21:42:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3957233","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3957233","identity":"rs-3957233","version":["v1"]},"buildId":"pf3fE39SIOqb-0xH_OWvX","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}