Extracorporeal shock waves effectively suppressed the proliferation and growth of colorectal cancer

Extracorporeal shock waves effectively suppressed the proliferation and growth of colorectal cancer | 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 Extracorporeal shock waves effectively suppressed the proliferation and growth of colorectal cancer Xiaoli Zhang, Chun Ran, Qingzhi Song, Guoqing Lv This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4956573/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Shock waves are widely used to treat various diseases and are garnering further attention for medical applications. Growing evidence suggests that the application of extracorporeal shock waves (ESV) could substantially inhibit tumor growth. However, the therapeutic efficacy of ESV in colorectal cancer and the underlying mechanisms remain elusive. Using colorectal cancer cell lines HT29 and SW620, we generated xenograft mouse models, and examined the therapeutic effect of a stepwise increase in ESV energy on tumor growth. In vivo , the application of 60 mJ ESV significantly delayed xenograft growth compared with 120 and 240 mJ ESV, with no impact on body weight or hepatic and renal function. Transcriptome analysis revealed that 60 mJ ESV suppressed colorectal cancer cell proliferation and induced cell apoptosis and ferroptosis; these findings were further confirmed by immunohistochemical staining and western blotting. Mechanistically, ESV suppressed cell proliferation and induced cell apoptosis and ferroptosis by activating the p53 signaling pathway, as evidenced in vitro study. In conclusion, we revealed that 60 mJ ESV could substantially inhibit colorectal cancer growth by activating p53 pathway-related proliferation inhibition and cell death. These findings suggest that ESV therapy could be a promising therapeutic strategy for colorectal cancer. Physical sciences/Physics/Biological physics Biological sciences/Cancer/Cancer therapy Biological sciences/Cell biology/Cell death/Apoptosis Biological sciences/Cell biology/Cell signalling Health sciences/Gastroenterology/Gastrointestinal diseases/Gastrointestinal cancer/Colorectal cancer extracorporeal shock waves colorectal cancer p53 signaling pathway xenograft mouse models ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Colorectal cancer (CRC) is a prevalent malignancy of the digestive tract and has the third morbidity and the second highest mortality rate globally 1 . Surgery is the most effective treatment for CRC, especially during the early tumor stages 2 . However, a substantial of patients are diagnosed at an advanced disease stage, frequently after metastasis to distant sites in the body or are otherwise unresectable 3 . Chemotherapy and ionizing radiation are occasionally used to treat high-risk CRC 4 . However, these therapeutic regimens tend to have limited efficacy owing to their severe side effects and toxicity 5 . Moreover, chemotherapeutic drugs tend to indiscriminately kill cancer and normal cells. Despite the considerable advances in diagnosis and treatment, the mortality rate among patients with CRC remains high and poses a major health burden worldwide 6 . To improve the survival rate and prognosis of patients with CRC, there is an urgent need to explore safe and relatively efficacious therapeutic options. Shock waves are supersonic pressure waves generated by shock tubes or lasers. Shock waves can deliver a sequence of transient pressure disturbances characterized by high amplitude, fast pressure rise, short pulse duration, and rapid propagation 7 . The shock wave technique is a non-invasive, targeted, and extracorporeal therapeutic strategy with numerous medical applications 8 . Shock wave therapy has been established as a tolerable and effective method for the disintegration of urinary calculi and treatment of chronic tendinitis, bone and wound healing, and ischemic heart disease, eliciting promising results 7 , 9 , 10 . Furthermore, shock waves can increase cell membrane permeability and have been successfully applied in drug delivery and gene transfer 11 . However, the application of shock-wave therapy in oncology remains poorly explored. In pre-clinical models, shock waves, especially in combination with several antineoplastic drugs, were found to exert substantial effects. Shock waves are found to enhance the antineoplastic effect of molecular iodine supplements in breast cancer 12 , and effectively suppress the proliferation and growth of tongue squamous cell carcinoma combined with 5-fluorouracil (5-FU) 13 . Approximately 30 years ago, shock waves were reported to increase the chemocytotoxicity of 5-FU in CRC cells by inducing cavitation 14 . However, research on the effects of shock waves in CRC has been limited to in vitro cytotoxicity and lacks comprehensive investigations into the underlying mechanisms. Moreover, the therapeutic effects of shock waves alone on CRC remain unknown. p53 , also known as the guardian of the genome, is a classic tumor suppressor that limits cellular proliferation by inducing cell cycle arrest, apoptosis and ferroptosis 15 – 17 . p53 is activated by various stress signals, including DNA damage or oncogene activation, and orchestrates a plethora of downstream responses, such as DNA repair, cell cycle arrest, senescence, metabolism, and cell death 17 , 18 . Several chemotherapy regimens and radiotherapy rely on p53 activation and its downstream responses to impede cancer cell growth. The activation of p53 for induction of p 53 -related outcomes, such as cell death in response to cancer therapy, has been explored to improve clinical outcomes 19 , 20 . Therefore, identifying therapies capable of targeting p53 may be a promising direction for treating CRC. Accordingly, we speculate whether shock waves can activate p53 -related cancer inhibition. In the present study, we investigated the therapeutic effect of extracorporeal shock waves (ESV) alone in CRC using animal experiments. We further explored the potential molecular mechanisms of ESV-mediated inhibiting of CRC growth. 2. Results 2.1 Impact of stepwise increased dosage of ESV energy on CRC xenograft tumor growth To determine the therapeutic effect of ESV in vivo , we established a mouse model of subcutaneous tumors. Nude mice were subcutaneously injected with HT29 cells and observed daily for tumor growth (Fig. 1 A). When the average tumor volume reached approximately 50 mm 3 , ESV therapy (60, 120, and 240mJ for 2000 shots) was administered every third day (Fig. 1 B, Fig. S1 ). Body weight and tumor volume were measured at different time points (i.e., on days 0, 3, 6, and 9 after ESV therapy). There were no significant changes in body weight (Fig. 1 C). In the control group, tumors grew by 262.8% on day 9. Administration of 60 mJ ESV significantly suppressed tumor growth, resulting in growth of only 141% ( p <0.01). Administration of 120 mJ ESV generated a weak and non-significant inhibition, eliciting tumor growth of 197%, while 240 mJ ESV exerted no suppressive effect on tumor growth (249.3%, Fig. 1 E). Upon examination of animals after the last treatment, animals in the 240 mJ ESV group exhibited bruises, suggesting significant vascular lesions (Fig. 1 D). After screening for ESV energy in the CRC xenograft mouse, we selected 60 mJ for subsequent in vivo experiments. 2.2 Administration of 60 mJ ESV delayed the growth of CRC-derived xenografts To further validate the therapeutic impact of 60 mJ ESV on CRC, we extended the experimental endpoint (i.e., by days 0, 3, 6, 9, 12, and 15 after ESV therapy), and examined the side effects using various methods. In the second set of experiments, the weight of mice was unaltered, consistent with the results of the previous experiment (Fig. 2 A). Plasma alanine aminotransferase (ALT) and creatinine (Cr) levels are biomarkers of hepatic and renal function, respectively. Herein, there were no differences in serum ALT and Cr levels between the control and 60 mJ ESV groups, suggesting that 60 mJ ESV did not induce notable toxicity (Fig. 2 B-C). Mice administered ESV had slower tumor growth than mice in the control group (Fig. 2 D). At the experimental endpoint (day 15), mice in the 60 mJ ESV group had smaller tumors than mice in the control group (Fig. 2 E). Pathological examination revealed the absence of notable abnormalities in the livers, indicating no liver metastasis (Fig. 2 F). In addition, the administration of 60 mJ ESV suppressed the growth of SW620-derived xenografts, another colorectal cancer cell line (Fig. S2 ). Collectively, these results strongly suggested that 60 mJ ESV could effectively suppress CRC growth in vivo without inducing toxicity. 2.3 Administration of 60 mJ ESV blocks CRC cell proliferation To elucidate the mechanisms through which shock waves inhibit CRC growth in transplant models, transcriptome analysis was performed using HT29-derived tumor tissues from the control (CON) and 60 mJ group. RNA-Seq captures the transcriptional information of cells in a fixed state and provides a snapshot of the DNA expression profile at a certain moment. RNA-Seq revealed that 60 mJ ESV reprogrammed the transcriptome, with 1748 upregulated genes and 2021 downregulated genes (Fig. 3 A-B). Metascape was used to analyze differentially expressed genes between the two groups. As shown in Fig. 3 C, downregulated differentially expressed genes were mainly enriched in “DNA metabolic process” and “mitotic cell cycle.” Mitosis is the basis for cell proliferation. Typically, tumor cells have abnormally active mitotic abilities and can proliferate rapidly. According to the RNA-seq data, administration of 60 mJ ESV could suppress the proliferation of CRC cells. Ki-67 staining was performed to evaluate cell proliferation using IHC staining. In the 60 mJ ESV group, the tumor tissues showed decreased Ki-67 staining (Fig. 3 D, Fig. S3). These results indicated that ESV may exert a therapeutic effect by suppressing the proliferation of CRC cells. 2.4 ESV induces apoptosis and ferroptosis in CRC cells Cell death and cell cycle arrest contribute to the inhibition of cell proliferation. To explore the mechanism through which ESV blocks CRC cell growth, we performed a Gene Set Enrichment Analysis (GSEA). Genes upregulated in the ESV group were enriched in pathways associated with apoptosis (Fig. 4 A). Based on western blot analysis, the application of ESV elevated the expression of cleaved-PARP and cleaved-caspase3, both apoptosis biomarkers (Fig. 4 B). IHC staining also verified the upregulated expression of cleaved-caspase3 in the ESV group (Fig. 4 C, Fig. S3A). Moreover, the ferroptosis biomarkers, AKR1C1 and COX2, were increased in the ESV group than in the control group (Fig. 4 D-E, Fig. S3B). These findings indicated that ESV could substantially induce apoptosis and ferroptosis in CRC tissues. 2.5 Effect of ESV on cell proliferation and death is related to the p53 pathway To determine the potential pathway involved in ESV-mediated CRC inhibition, Metascape and GSEA analyses were applied to the RNA-seq data. The results revealed the enrichment of the p53 signaling pathway gene set in the ESV group (Fig. 5 A-B). Given that the p53 signaling pathway is a classic tumor suppressor that plays an important role in tumor regulation, we speculated that ESV could inhibit CRC growth via this pathway. We detected the p53 mRNA and protein levels in HT29-derived tumors using real-time PCR and western blot, respectively. ESV-treated mice exhibited upregulated p53 expression when compared with control mice; this enhanced expression was confirmed by IHC staining for p53 (Fig. 5 C-E, Fig. S3C). Taken together, these results strongly suggested that ESV could suppress cell proliferation and induce apoptosis and ferroptosis by upregulating p53 expression in vivo . 2.6 ESV inhibits proliferation and promotes death of CRC cells in vitro In addition to the above in vivo animal experiments, we performed in vitro cell culture experiments. The CCK-8 assay demonstrated that 60 mJ ESV markedly suppressed the proliferation of HT29 cells in vitro (Fig. 6 A). Flow cytometric analysis revealed that ESV-treated CRC cells had a greater proportion of cells in the G0/G1 phase (77.3%) than control cells (60.6%). ESV-treated cells were arrested at the G0/G1 phase of the cell cycle, and the percentage of cells in the S phase was reduced (Fig. 6 B). Furthermore, flow cytometric analysis showed that treatment with ESV significantly increased the percentage of dead HT29 cells (PI + cells, Fig. 6 C). Treatment with ESV increased the levels of P53 protein in vitro , along with those of cleaved-PARP, cleaved-caspase3, AKR1C1 and COX2 (Fig. 6 D). Collectively, these findings indicated that ESV could impede CRC growth via p53 pathway-mediated cell cycle arrest and cell death, both in vivo and in vitro . 3. Discussion Herein, we investigated the therapeutic effects of ESV monotherapy on blocking CRC growth both in vivo and in vitro , which elicited notable preclinical implications. First, animal experiments revealed that the administration of 60 mJ ESV decreased tumor growth without affecting body weight or liver and kidney functions. Second, in vivo and in vitro studies revealed that the administration of ESV not only suppressed CRC cell proliferation but also induced cell apoptosis and ferroptosis. Third, ESV therapy could exert tumor-suppressive effect by reactivating the p53 signaling pathway. These results strongly indicate that ESV therapy may be an effective therapeutic modality for suppressing CRC growth. Currently, effective and safe methods to treat patients with advanced-stage or unresectable cancer are lacking 21 . ESV, which is non-invasive and targeted, could be valuable for tumor treatment. Therefore, we evaluated the therapeutic effects of ESV in CRC inhibition. The most important finding of this study was that administration of ESV alone could significantly reduce the tumor volume in nude mice with CRC transplanted subcutaneously. However, colorectal tumors are located in the abdominal cavity rather than on the body surface. Further studies exploring the effects of ESV therapy using spontaneous CRC models are needed, for example, the APC min/+ mouse or azoxymethane/dextran sulfate sodium-induced mouse colitis-associated carcinogenesis model 22 . Moreover, the characteristics of the ESV can best be applied to relatively superficial tissues and organs. ESV was found to effectively suppress the proliferation and growth of tongue squamous cell carcinoma and breast cancer, which can be considered relatively superficial types of cancer in the human body 12 , 13 . Importantly, specific instruments to administer shock-wave therapy in patients with breast cancer have been developed and are undergoing preliminary clinical trials. As early as 30 years ago, scientists explored the potential of shock waves for tumor treatment. Shock waves can shatter tumor cells and injure the microvasculature, raising concerns regarding the risk of metastasis 23 , 24 . Subsequent studies revealed that the effect of shock waves on tumors depended on the shockwave energy. Low-dose shock waves were shown to enhance free radical generation, ATP release, or membrane instability, which could induce apoptotic mechanisms, ultimately reducing tumor growth and diminishing the potential for tumor metastasis 12 , 25 . Consistently, our study investigated three different shock wave energies, demonstrating that low-energy (60 mJ) shock waves could remarkably block CRC growth with no liver metastasis. In contrast, 240 mJ not only failed to inhibit tumor growth but also caused hematoma. In addition to energy, other mechanical parameters of shock waves, including peak pressure, rise time, and shock wave impulse, can also impact therapeutic outcomes. Schmidt et al. conducted in vitro experiments to examine the effects of shock wave on U87 brain cancer cells. The authors found that when the incident peak pressure exceeded a lethal level, shock waves could induce substantial cell damage 26 . Liao et al. found that shock waves with higher impulses led to decreased cell viability, whereas shock waves with similar peak pressures exerted distinct effects on cell viability 27 . Herein, we specifically focused on the effects of shock waves of different energies on CRC growth. Additional studies are needed to investigate the effects of the other mechanical characteristics of shock waves on CRC cell viability. In conclusion, we present new evidence demonstrating a novel method for treating CRC both in vivo and in vitro . The use of 60 mJ ESV could markedly suppress CRC growth by blocking the proliferation of cancer cells and inducing apoptosis and ferroptosis. Mechanistically, ESV may activate the p53 signaling pathway to inhibit CRC growth. Our study highlights that appropriate ESV energy may serve as an accessory option (i.e., “give it a try”) in those patients with terminal-stage CRC who are refractory to conventional therapy. 4. Materials and Methods All experimental methods and protocols were performed in accordance with the relevant guidelines and regulations of the Beijing Neurosurgical Institute, and the protocols for animal procedures were approved by the ethics committee of Beijing Neurosurgical Institute Ethics Committee of Capital Medical University (BNI202304002). 4.1 Mouse Xenograft Studies Six-to-eight-week-old male BALB/c nude mice were subcutaneously injected with 1 × 10 6 HT29 in 100 µL. The mice were observed daily for tumor growth. When the average tumor volume reached approximately 50 mm 3 , the mice were divided randomly into four groups, including control (Ctrl), 60mJ, 120mJ, and 240mJ shock waves groups. The control group without any treatment, shock waves groups accepted ESV therapy with stepwise increased energy (60, 120, 240 mJ, 2000 shots, 12Hz) at days 0, 3, 6, 9 and 12 after therapy initiation. At the same time, the tumor volumes were measured once every 3 days to determine the effect of each therapeutic regimen. The lengths (L) and widths (W) of the tumors were measured, and the tumor volumes were calculated as (L × W 2 )/2. 4.2 ESV application and definition The ESV machine, called lattice shock wave physiotherapy instrument (J Gendica, China), was utilized in the present study. In detail, the ESV energy could be adjusted prior to applying for the in vitro and in vivo studies. After a determination of machine parameters, the machine was switched on. The automatic shots to the specific tissues/cells were started on. We defined the ESV energy applied to animals as 60mJ, 120mJ, and 240mJ (2000 shots, 12Hz). Additionally, we defined the ESV energy applied to HT29 cell line as 60mJ, 2000 shots, 12Hz. 4.3 RNA Sequencing RNA samples were prepared from three biological replicates of HT29-derived xenograft tissues using TRIzol reagent. RNA quality was quantified using RNA integrity number. Sequencing libraries were constructed using the NEB Next Ultra RNA Library Prep Kit for Illumina and quantified using the KAPA Library Quantification kit (KAPA Biosystems). The libraries were then sequenced on an Illumina HiSeq sequencer. 150bp paired-end reads were mapped to human hg19 genome. Cuffdiff was used to analyze differentially expressed genes between the samples. 4.4 Immunohistochemistry (IHC) analysis Paraffin-embedded xenografts were cut into 4 mm sections. The sections were dewaxed and hydrated, and then blocked and incubated with primary antibodies against Ki67 (Cell Signaling Technology, 12202, 1:200), P53 (Proteintech, 60283-2-Ig, 1:1000), cleaved-caspase3 (Cell Signaling Technology, 9664T, 1:1000) at 4°C overnight. The signals were visualized using a two-step plus®Poly-HRP Anti-Mouse/Rabbit IgG Detection System (OriGene, PV9000). Counterstaining was performed using hematoxylin. The sections were digitally scanned under a microscope with an FL-20 cooled camera, and images were analyzed using the Mosaic™ V2.1 software. 4.5 Reverse Transcription-Quantitative Polymerase Chain Reaction The total RNA content was isolated from cells or the tumor using TRIzol™ reagent (Invitrogen), treated with DNase I (Thermo Fisher Scientific, Cat# EN0521), and then reverse transcribed into cDNA using a Maximal First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Cat# K1622). Real-time polymerase chain reaction (PCR) was performed using an S1000 PCR instrument (Bio-Rad, Hercules, CA, USA) to quantify gene expression. The PCR data were normalized to glyceraldehyde-3-phosphate dehydrogenase expression at the mRNA level. The primers used are listed in Supplementary Table S1 . 4.6 Western blotting Proteins were extracted from tumor tissues or cells with RIPA buffer (0.01% EDTA, 0.1% TritonX-100 and 10% proteinase inhibitor cocktail). Protein concentrations were quantified with a protein assay kit (Bio-Rad). 100 µg lysate were separated on 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. The membranes were probed overnight at 4°C with primary antibody against human P53 (Cell Signaling Technology, 48818S, 1:1000), β-Actin (Sigma, 1:10000), cleaved-caspase3 (Cell Signaling Technology, 9664T, 1:1000), cleaved-PARP (Cell Signaling Technology, 9541T, 1:1000), AKR1C1 (Abcam, ab192785, 1:1000), COX2 (Abcam, ab179800, 1:1000), followed by incubation with peroxidase-conjugated secondary antibody (Cell Signaling Technology, 1:10000) for 1.5 hours. The signal was visualized with ECL (Millipore). 4.7 Statistical analysis All experiments were independently repeated at least three times unless stated otherwise. Statistical analyses were performed using the GraphPad Prism software (version 8.0). Data are presented as the mean ± standard deviation (SD). Two groups were compared using the Student’s t-test for unpaired data. Comparisons among more than two groups were performed using ANOVA and Tukey’s test. For all tests, P values ≤ .05, were considered statistically significant ( ∗ P < .05 ). Declarations Funding This work was supported by the National Nature Science Foundation of China (82203229); the Shenzhen Science and Technology Program (JCYJ20220531094005012), and the opening project of State Key Laboratory of Explosion Science and Technology (Beijing Institute of Technology, KFJJ22-16M). Contributions XLZ and QZS performed the experiments. XLZ and CR designed experimental protocols, interpreted data obtained from the experiments, and wrote the manuscript. GQL edited the manuscript. Data availability All raw data used to generate the results of this study are available from the corresponding author by request. Competing interests statement The authors declare no competing interests. Ethic statement Our studies did not include human participants, human data or human tissue. All the animal protocols containing all the procedures were approved by the ethics committee of Beijing Neurosurgical Institute Ethics Committee of Capital Medical University. The experimental protocols were conducted in accordance with the ARRIVE guidelines. Conflict of interest The authors declare no competing interests. References Sung, H. et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 71 , 209–249. 10.3322/caac.21660 (2021). Zhang, X. et al. Glutathione peroxidase 4 as a therapeutic target for anti-colorectal cancer drug-tolerant persister cells. Front. Oncol. 12 , 913669. 10.3389/fonc.2022.913669 (2022). Cañellas-Socias, A. et al. Metastatic recurrence in colorectal cancer arises from residual EMP1(+) cells. Nature . 611 , 603–613. 10.1038/s41586-022-05402-9 (2022). 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Supplementary Files SupplementaryInformation1.pdf SupplementaryInformation2.pdf Cite Share Download PDF Status: Published Journal Publication published 21 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 04 Feb, 2025 Reviews received at journal 26 Jan, 2025 Reviewers agreed at journal 20 Jan, 2025 Reviews received at journal 09 Jan, 2025 Reviewers agreed at journal 07 Jan, 2025 Reviewers invited by journal 08 Oct, 2024 Editor assigned by journal 08 Oct, 2024 Editor invited by journal 03 Sep, 2024 Submission checks completed at journal 02 Sep, 2024 First submitted to journal 22 Aug, 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 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-4956573","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":357921268,"identity":"c180c47f-fa26-4191-8929-06931b62bd93","order_by":0,"name":"Xiaoli Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYPACCSBmbGBgqJCQkydRyxkLY8MGkixjbKtIZDhAQJE5+9lj0rxtFvL8EsmNHz7Ok0hgbGB++OgGHi2WPXlp0jxnJAxn9hxslpy5TSKPnYHN2DgHjxaDAzlm0jwVEowbjjc2SPNukyhmbOBhk8ar5fwboBYDCfsNhxmbf/POkUhsOEBIyw2ILYlAW9qkeRuI0vLG2HLOGYlkoF/aLGcckzA2bCbkl/M5hjfettXZ9kukP77xoaZOTp69+eFjfFqAgEUClc+MXzlYyQfCakbBKBgFo2BEAwDh00fAHYEOOAAAAABJRU5ErkJggg==","orcid":"","institution":"Beijing Neurosurgical Institute","correspondingAuthor":true,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"Zhang","suffix":""},{"id":357921269,"identity":"91fd7c8f-9e43-4616-8dbe-7e9dbd859df6","order_by":1,"name":"Chun Ran","email":"","orcid":"","institution":"China Ordnance Society","correspondingAuthor":false,"prefix":"","firstName":"Chun","middleName":"","lastName":"Ran","suffix":""},{"id":357921270,"identity":"3d00d4ea-cc03-4739-a226-01cdf34749c8","order_by":2,"name":"Qingzhi Song","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qingzhi","middleName":"","lastName":"Song","suffix":""},{"id":357921271,"identity":"ccf82911-c1de-41da-a294-101ffbc816ba","order_by":3,"name":"Guoqing Lv","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Guoqing","middleName":"","lastName":"Lv","suffix":""}],"badges":[],"createdAt":"2024-08-22 08:51:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4956573/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4956573/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-94386-3","type":"published","date":"2025-03-21T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":65859137,"identity":"e2ae2b55-4dae-4e92-83a0-d42899175cc3","added_by":"auto","created_at":"2024-10-03 15:41:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":301492,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of stepwise increased dosage of ESV energy on CRC xenograft tumor growth.(A) Establishment of HT29-derived transplant tumor model in nude mice. (B) Schematic representation of increased dosages of ESV energy (60 mJ, 120 mJ and 240 mJ, 2000 shots, 12 Hz) at different time (every third day) in CRC xenograft model. (C) Body weights of control (Ctrl), 60 mJ, 120 mJ, and 240 mJ mice (n = 5). (D) The anatomy view of a mouse in 240 mJ group. (E) Relative tumor volumes at day 0, day 3, day 6, day 9 after the first ESV treatment (n = 5). Tumor volume = (width² × length)/2. Statistical significance was determined using one-way ANOVA with Tukey’s test.\u003cem\u003e **p \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage127.png","url":"https://assets-eu.researchsquare.com/files/rs-4956573/v1/6e8379febcba931139dd04c7.png"},{"id":65859131,"identity":"ff976bd4-03f4-4ff4-8000-4ba7a284a35a","added_by":"auto","created_at":"2024-10-03 15:41:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":53896,"visible":true,"origin":"","legend":"\u003cp\u003e60 mJ ESV suppresses the growth of HT29-derived xenografts. (A) Body weights of control (CON) and 60 mJ mice (n = 4). (B-C) Serum alanine aminotransferase (ALT) and creatinine (Cr) in plasma (n = 4). (D) Relative tumor volumes at day 0, day 3, day 6, day 9, day 12 and day 15 after the first ESV treatment (n = 4). (E) Images of tumors at the end of the experiment (n = 4). (F) Mouse livers of 60 mJ ESV group at the end of the experiment (n =2). Data are presented as means ±SD of three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s test. \u003cem\u003e*p \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage26.png","url":"https://assets-eu.researchsquare.com/files/rs-4956573/v1/256c6bb9be442ba0a5822991.png"},{"id":65859133,"identity":"a6f24693-f535-4f47-8f46-8c4c0401e6c4","added_by":"auto","created_at":"2024-10-03 15:41:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":213506,"visible":true,"origin":"","legend":"\u003cp\u003e60 mJ ESV blocks the proliferation of CRC cells. (A) Volcano plot representing transcriptome differences between CON and 60 mJ ESV group. (B) The number of differentially expressed genes between CON and 60 mJ ESV group. (C) Metascape analysis of downregulated genes in 60 mJ ESV group. (D) Ki67-staining (Scale: 100 μm).\u003c/p\u003e","description":"","filename":"Onlinefloatimage35.png","url":"https://assets-eu.researchsquare.com/files/rs-4956573/v1/61fc772159568d50e80230dd.png"},{"id":65859365,"identity":"450252a7-9494-4f31-932d-8f2d5f71c8e1","added_by":"auto","created_at":"2024-10-03 15:49:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":206593,"visible":true,"origin":"","legend":"\u003cp\u003e60 mJ ESV induces apoptosis and ferroptosis in CRC cells. (A) Gene Set Enrichment Analysis plots for apoptosis. NES, normalized enrichment score; Black bars represent genes contained within the specific pathway ranked from positively expressed (left) to negatively expressed (right) based on log 2-fold change (60 mJ ESV \u003cem\u003evs\u003c/em\u003e. CON). (B) Western blot analysis of cleaved-PARP and cleaved-caspase3. (C) cleaved-caspase3 staining (Scale: 100 μm). (D) The relative mRNA levels of \u003cem\u003eAKR1C1\u003c/em\u003eand \u003cem\u003eCOX2\u003c/em\u003e measured by real-time PCR. (E) Western blot analysis of AKR1C1 and COX2.\u003cem\u003e ***p \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage45.png","url":"https://assets-eu.researchsquare.com/files/rs-4956573/v1/213b4e9636035bd574a8e3ce.png"},{"id":65859136,"identity":"28f3b48b-dc59-43e7-8882-d3b5ad2a650d","added_by":"auto","created_at":"2024-10-03 15:41:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":213412,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of 60 mJ ESV on cell proliferation and death is related to \u003cem\u003ep53\u003c/em\u003e pathway. (A) Metascape analysis of upregulated genes in 60mJ ESV group. (B) Gene set enrichment analysis plots for \u003cem\u003ep53\u003c/em\u003e pathway. (C) The relative mRNA level of \u003cem\u003ep53 \u003c/em\u003emeasured by real-time PCR. (D) Western blot analysis of P53. (E) P53 staining (Scale: 100 μm). Statistical significance was determined using one-way ANOVA with Tukey’s test. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage53.png","url":"https://assets-eu.researchsquare.com/files/rs-4956573/v1/c4e17020a6cd43b6d05f7b5a.png"},{"id":65859130,"identity":"a0e968fc-fa2a-43dd-b0d2-3ec83d734371","added_by":"auto","created_at":"2024-10-03 15:41:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":64149,"visible":true,"origin":"","legend":"\u003cp\u003e60 mJ ESV inhibits proliferation and promotes death of CRC cells \u003cem\u003ein vitro\u003c/em\u003e. (A) HT29 cell viability at day 3 and day 6 after treatment with 60 mJ ESV, 2000 shots, 12 Hz. (B) Cells were analyzed by flow cytometry to determine the percentage of cells in the indicated cell cycle phases. (C) Cells were analyzed by flow cytometry to determine the percentage of death cells. (D) Western blot analysis of P53, cleaved-PARP, cleaved-caspase3, AKR1C1 and COX2. Data are presented as means ±SD of three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s test. \u003cem\u003e*p \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage65.png","url":"https://assets-eu.researchsquare.com/files/rs-4956573/v1/0e502ac7347f6fc7f3b55245.png"},{"id":79120415,"identity":"ac6a479f-2bc3-433f-acb5-0ac47e3f33cf","added_by":"auto","created_at":"2025-03-24 16:07:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1553215,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4956573/v1/9dbfa42a-e045-4c64-b053-a0aaa0cc210c.pdf"},{"id":65860202,"identity":"a760feb3-abc5-4176-9fc2-948e2578172f","added_by":"auto","created_at":"2024-10-03 15:57:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":345195,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4956573/v1/1bbd21d19edad913759b175d.pdf"},{"id":65859364,"identity":"1c89c80d-4178-475f-afd2-859349e5bdee","added_by":"auto","created_at":"2024-10-03 15:49:57","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":547242,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4956573/v1/b20125b1df9589452c20b3ec.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Extracorporeal shock waves effectively suppressed the proliferation and growth of colorectal cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is a prevalent malignancy of the digestive tract and has the third morbidity and the second highest mortality rate globally\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Surgery is the most effective treatment for CRC, especially during the early tumor stages\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, a substantial of patients are diagnosed at an advanced disease stage, frequently after metastasis to distant sites in the body or are otherwise unresectable\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Chemotherapy and ionizing radiation are occasionally used to treat high-risk CRC\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, these therapeutic regimens tend to have limited efficacy owing to their severe side effects and toxicity\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Moreover, chemotherapeutic drugs tend to indiscriminately kill cancer and normal cells. Despite the considerable advances in diagnosis and treatment, the mortality rate among patients with CRC remains high and poses a major health burden worldwide\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. To improve the survival rate and prognosis of patients with CRC, there is an urgent need to explore safe and relatively efficacious therapeutic options.\u003c/p\u003e \u003cp\u003eShock waves are supersonic pressure waves generated by shock tubes or lasers. Shock waves can deliver a sequence of transient pressure disturbances characterized by high amplitude, fast pressure rise, short pulse duration, and rapid propagation\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The shock wave technique is a non-invasive, targeted, and extracorporeal therapeutic strategy with numerous medical applications\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Shock wave therapy has been established as a tolerable and effective method for the disintegration of urinary calculi and treatment of chronic tendinitis, bone and wound healing, and ischemic heart disease, eliciting promising results \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Furthermore, shock waves can increase cell membrane permeability and have been successfully applied in drug delivery and gene transfer\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, the application of shock-wave therapy in oncology remains poorly explored. In pre-clinical models, shock waves, especially in combination with several antineoplastic drugs, were found to exert substantial effects. Shock waves are found to enhance the antineoplastic effect of molecular iodine supplements in breast cancer\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and effectively suppress the proliferation and growth of tongue squamous cell carcinoma combined with 5-fluorouracil (5-FU)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Approximately 30 years ago, shock waves were reported to increase the chemocytotoxicity of 5-FU in CRC cells by inducing cavitation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, research on the effects of shock waves in CRC has been limited to \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity and lacks comprehensive investigations into the underlying mechanisms. Moreover, the therapeutic effects of shock waves alone on CRC remain unknown.\u003c/p\u003e \u003cp\u003e \u003cem\u003ep53\u003c/em\u003e, also known as the guardian of the genome, is a classic tumor suppressor that limits cellular proliferation by inducing cell cycle arrest, apoptosis and ferroptosis\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ep53\u003c/em\u003e is activated by various stress signals, including DNA damage or oncogene activation, and orchestrates a plethora of downstream responses, such as DNA repair, cell cycle arrest, senescence, metabolism, and cell death\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Several chemotherapy regimens and radiotherapy rely on \u003cem\u003ep53\u003c/em\u003e activation and its downstream responses to impede cancer cell growth. The activation of \u003cem\u003ep53\u003c/em\u003e for induction of p\u003cem\u003e53\u003c/em\u003e-related outcomes, such as cell death in response to cancer therapy, has been explored to improve clinical outcomes\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Therefore, identifying therapies capable of targeting \u003cem\u003ep53\u003c/em\u003e may be a promising direction for treating CRC. Accordingly, we speculate whether shock waves can activate \u003cem\u003ep53\u003c/em\u003e-related cancer inhibition.\u003c/p\u003e \u003cp\u003eIn the present study, we investigated the therapeutic effect of extracorporeal shock waves (ESV) alone in CRC using animal experiments. We further explored the potential molecular mechanisms of ESV-mediated inhibiting of CRC growth.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Impact of stepwise increased dosage of ESV energy on CRC xenograft tumor growth\u003c/h2\u003e \u003cp\u003eTo determine the therapeutic effect of ESV \u003cem\u003ein vivo\u003c/em\u003e, we established a mouse model of subcutaneous tumors. Nude mice were subcutaneously injected with HT29 cells and observed daily for tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). When the average tumor volume reached approximately 50 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, ESV therapy (60, 120, and 240mJ for 2000 shots) was administered every third day (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Body weight and tumor volume were measured at different time points (i.e., on days 0, 3, 6, and 9 after ESV therapy). There were no significant changes in body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In the control group, tumors grew by 262.8% on day 9. Administration of 60 mJ ESV significantly suppressed tumor growth, resulting in growth of only 141% (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Administration of 120 mJ ESV generated a weak and non-significant inhibition, eliciting tumor growth of 197%, while 240 mJ ESV exerted no suppressive effect on tumor growth (249.3%, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Upon examination of animals after the last treatment, animals in the 240 mJ ESV group exhibited bruises, suggesting significant vascular lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). After screening for ESV energy in the CRC xenograft mouse, we selected 60 mJ for subsequent \u003cem\u003ein vivo\u003c/em\u003e experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Administration of 60 mJ ESV delayed the growth of CRC-derived xenografts\u003c/h2\u003e \u003cp\u003eTo further validate the therapeutic impact of 60 mJ ESV on CRC, we extended the experimental endpoint (i.e., by days 0, 3, 6, 9, 12, and 15 after ESV therapy), and examined the side effects using various methods. In the second set of experiments, the weight of mice was unaltered, consistent with the results of the previous experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Plasma alanine aminotransferase (ALT) and creatinine (Cr) levels are biomarkers of hepatic and renal function, respectively. Herein, there were no differences in serum ALT and Cr levels between the control and 60 mJ ESV groups, suggesting that 60 mJ ESV did not induce notable toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). Mice administered ESV had slower tumor growth than mice in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). At the experimental endpoint (day 15), mice in the 60 mJ ESV group had smaller tumors than mice in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Pathological examination revealed the absence of notable abnormalities in the livers, indicating no liver metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). In addition, the administration of 60 mJ ESV suppressed the growth of SW620-derived xenografts, another colorectal cancer cell line (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Collectively, these results strongly suggested that 60 mJ ESV could effectively suppress CRC growth \u003cem\u003ein vivo\u003c/em\u003e without inducing toxicity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Administration of 60 mJ ESV blocks CRC cell proliferation\u003c/h2\u003e \u003cp\u003eTo elucidate the mechanisms through which shock waves inhibit CRC growth in transplant models, transcriptome analysis was performed using HT29-derived tumor tissues from the control (CON) and 60 mJ group. RNA-Seq captures the transcriptional information of cells in a fixed state and provides a snapshot of the DNA expression profile at a certain moment. RNA-Seq revealed that 60 mJ ESV reprogrammed the transcriptome, with 1748 upregulated genes and 2021 downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). Metascape was used to analyze differentially expressed genes between the two groups. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, downregulated differentially expressed genes were mainly enriched in \u0026ldquo;DNA metabolic process\u0026rdquo; and \u0026ldquo;mitotic cell cycle.\u0026rdquo; Mitosis is the basis for cell proliferation. Typically, tumor cells have abnormally active mitotic abilities and can proliferate rapidly. According to the RNA-seq data, administration of 60 mJ ESV could suppress the proliferation of CRC cells. Ki-67 staining was performed to evaluate cell proliferation using IHC staining. In the 60 mJ ESV group, the tumor tissues showed decreased Ki-67 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, Fig. S3). These results indicated that ESV may exert a therapeutic effect by suppressing the proliferation of CRC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 ESV induces apoptosis and ferroptosis in CRC cells\u003c/h2\u003e \u003cp\u003eCell death and cell cycle arrest contribute to the inhibition of cell proliferation. To explore the mechanism through which ESV blocks CRC cell growth, we performed a Gene Set Enrichment Analysis (GSEA). Genes upregulated in the ESV group were enriched in pathways associated with apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Based on western blot analysis, the application of ESV elevated the expression of cleaved-PARP and cleaved-caspase3, both apoptosis biomarkers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). IHC staining also verified the upregulated expression of cleaved-caspase3 in the ESV group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Fig. S3A). Moreover, the ferroptosis biomarkers, AKR1C1 and COX2, were increased in the ESV group than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E, Fig. S3B). These findings indicated that ESV could substantially induce apoptosis and ferroptosis in CRC tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Effect of ESV on cell proliferation and death is related to the p53 pathway\u003c/h2\u003e \u003cp\u003eTo determine the potential pathway involved in ESV-mediated CRC inhibition, Metascape and GSEA analyses were applied to the RNA-seq data. The results revealed the enrichment of the \u003cem\u003ep53\u003c/em\u003e signaling pathway gene set in the ESV group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). Given that the \u003cem\u003ep53\u003c/em\u003e signaling pathway is a classic tumor suppressor that plays an important role in tumor regulation, we speculated that ESV could inhibit CRC growth via this pathway. We detected the \u003cem\u003ep53\u003c/em\u003e mRNA and protein levels in HT29-derived tumors using real-time PCR and western blot, respectively. ESV-treated mice exhibited upregulated p53 expression when compared with control mice; this enhanced expression was confirmed by IHC staining for p53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-E, Fig. S3C). Taken together, these results strongly suggested that ESV could suppress cell proliferation and induce apoptosis and ferroptosis by upregulating p53 expression \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 ESV inhibits proliferation and promotes death of CRC cells in vitro\u003c/h2\u003e \u003cp\u003eIn addition to the above \u003cem\u003ein vivo\u003c/em\u003e animal experiments, we performed \u003cem\u003ein vitro\u003c/em\u003e cell culture experiments. The CCK-8 assay demonstrated that 60 mJ ESV markedly suppressed the proliferation of HT29 cells \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Flow cytometric analysis revealed that ESV-treated CRC cells had a greater proportion of cells in the G0/G1 phase (77.3%) than control cells (60.6%). ESV-treated cells were arrested at the G0/G1 phase of the cell cycle, and the percentage of cells in the S phase was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Furthermore, flow cytometric analysis showed that treatment with ESV significantly increased the percentage of dead HT29 cells (PI\u003csup\u003e+\u003c/sup\u003e cells, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Treatment with ESV increased the levels of P53 protein \u003cem\u003ein vitro\u003c/em\u003e, along with those of cleaved-PARP, cleaved-caspase3, AKR1C1 and COX2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Collectively, these findings indicated that ESV could impede CRC growth via p53 pathway-mediated cell cycle arrest and cell death, both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eHerein, we investigated the therapeutic effects of ESV monotherapy on blocking CRC growth both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e, which elicited notable preclinical implications. First, animal experiments revealed that the administration of 60 mJ ESV decreased tumor growth without affecting body weight or liver and kidney functions. Second, \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e studies revealed that the administration of ESV not only suppressed CRC cell proliferation but also induced cell apoptosis and ferroptosis. Third, ESV therapy could exert tumor-suppressive effect by reactivating the p53 signaling pathway. These results strongly indicate that ESV therapy may be an effective therapeutic modality for suppressing CRC growth.\u003c/p\u003e \u003cp\u003eCurrently, effective and safe methods to treat patients with advanced-stage or unresectable cancer are lacking\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. ESV, which is non-invasive and targeted, could be valuable for tumor treatment. Therefore, we evaluated the therapeutic effects of ESV in CRC inhibition. The most important finding of this study was that administration of ESV alone could significantly reduce the tumor volume in nude mice with CRC transplanted subcutaneously. However, colorectal tumors are located in the abdominal cavity rather than on the body surface. Further studies exploring the effects of ESV therapy using spontaneous CRC models are needed, for example, the \u003cem\u003eAPC\u003c/em\u003e\u003csup\u003e\u003cem\u003emin/+\u003c/em\u003e\u003c/sup\u003e mouse or azoxymethane/dextran sulfate sodium-induced mouse colitis-associated carcinogenesis model\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Moreover, the characteristics of the ESV can best be applied to relatively superficial tissues and organs. ESV was found to effectively suppress the proliferation and growth of tongue squamous cell carcinoma and breast cancer, which can be considered relatively superficial types of cancer in the human body\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Importantly, specific instruments to administer shock-wave therapy in patients with breast cancer have been developed and are undergoing preliminary clinical trials.\u003c/p\u003e \u003cp\u003eAs early as 30 years ago, scientists explored the potential of shock waves for tumor treatment. Shock waves can shatter tumor cells and injure the microvasculature, raising concerns regarding the risk of metastasis\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Subsequent studies revealed that the effect of shock waves on tumors depended on the shockwave energy. Low-dose shock waves were shown to enhance free radical generation, ATP release, or membrane instability, which could induce apoptotic mechanisms, ultimately reducing tumor growth and diminishing the potential for tumor metastasis\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Consistently, our study investigated three different shock wave energies, demonstrating that low-energy (60 mJ) shock waves could remarkably block CRC growth with no liver metastasis. In contrast, 240 mJ not only failed to inhibit tumor growth but also caused hematoma.\u003c/p\u003e \u003cp\u003eIn addition to energy, other mechanical parameters of shock waves, including peak pressure, rise time, and shock wave impulse, can also impact therapeutic outcomes. Schmidt \u003cem\u003eet al.\u003c/em\u003e conducted \u003cem\u003ein vitro\u003c/em\u003e experiments to examine the effects of shock wave on U87 brain cancer cells. The authors found that when the incident peak pressure exceeded a lethal level, shock waves could induce substantial cell damage\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Liao \u003cem\u003eet al.\u003c/em\u003e found that shock waves with higher impulses led to decreased cell viability, whereas shock waves with similar peak pressures exerted distinct effects on cell viability\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Herein, we specifically focused on the effects of shock waves of different energies on CRC growth. Additional studies are needed to investigate the effects of the other mechanical characteristics of shock waves on CRC cell viability.\u003c/p\u003e \u003cp\u003eIn conclusion, we present new evidence demonstrating a novel method for treating CRC both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. The use of 60 mJ ESV could markedly suppress CRC growth by blocking the proliferation of cancer cells and inducing apoptosis and ferroptosis. Mechanistically, ESV may activate the p53 signaling pathway to inhibit CRC growth. Our study highlights that appropriate ESV energy may serve as an accessory option (i.e., \u0026ldquo;give it a try\u0026rdquo;) in those patients with terminal-stage CRC who are refractory to conventional therapy.\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cp\u003eAll experimental methods and protocols were performed in accordance with the relevant guidelines and regulations of the Beijing Neurosurgical Institute, and the protocols for animal procedures were approved by the ethics committee of Beijing Neurosurgical Institute Ethics Committee of Capital Medical University (BNI202304002).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Mouse Xenograft Studies\u003c/h2\u003e \u003cp\u003eSix-to-eight-week-old male BALB/c nude mice were subcutaneously injected with 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e HT29 in 100 \u0026micro;L. The mice were observed daily for tumor growth. When the average tumor volume reached approximately 50 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, the mice were divided randomly into four groups, including control (Ctrl), 60mJ, 120mJ, and 240mJ shock waves groups. The control group without any treatment, shock waves groups accepted ESV therapy with stepwise increased energy (60, 120, 240 mJ, 2000 shots, 12Hz) at days 0, 3, 6, 9 and 12 after therapy initiation. At the same time, the tumor volumes were measured once every 3 days to determine the effect of each therapeutic regimen. The lengths (L) and widths (W) of the tumors were measured, and the tumor volumes were calculated as (L \u0026times; W\u003csup\u003e2\u003c/sup\u003e)/2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.2 ESV application and definition\u003c/h2\u003e \u003cp\u003eThe ESV machine, called lattice shock wave physiotherapy instrument (J Gendica, China), was utilized in the present study. In detail, the ESV energy could be adjusted prior to applying for the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies. After a determination of machine parameters, the machine was switched on. The automatic shots to the specific tissues/cells were started on. We defined the ESV energy applied to animals as 60mJ, 120mJ, and 240mJ (2000 shots, 12Hz). Additionally, we defined the ESV energy applied to HT29 cell line as 60mJ, 2000 shots, 12Hz.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.3 RNA Sequencing\u003c/h2\u003e \u003cp\u003eRNA samples were prepared from three biological replicates of HT29-derived xenograft tissues using TRIzol reagent. RNA quality was quantified using RNA integrity number. Sequencing libraries were constructed using the NEB Next Ultra RNA Library Prep Kit for Illumina and quantified using the KAPA Library Quantification kit (KAPA Biosystems). The libraries were then sequenced on an Illumina HiSeq sequencer. 150bp paired-end reads were mapped to human hg19 genome. Cuffdiff was used to analyze differentially expressed genes between the samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Immunohistochemistry (IHC) analysis\u003c/h2\u003e \u003cp\u003eParaffin-embedded xenografts were cut into 4 mm sections. The sections were dewaxed and hydrated, and then blocked and incubated with primary antibodies against Ki67 (Cell Signaling Technology, 12202, 1:200), P53 (Proteintech, 60283-2-Ig, 1:1000), cleaved-caspase3 (Cell Signaling Technology, 9664T, 1:1000) at 4\u0026deg;C overnight. The signals were visualized using a two-step plus\u0026reg;Poly-HRP Anti-Mouse/Rabbit IgG Detection System (OriGene, PV9000). Counterstaining was performed using hematoxylin. The sections were digitally scanned under a microscope with an FL-20 cooled camera, and images were analyzed using the Mosaic\u0026trade; V2.1 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Reverse Transcription-Quantitative Polymerase Chain Reaction\u003c/h2\u003e \u003cp\u003eThe total RNA content was isolated from cells or the tumor using TRIzol\u0026trade; reagent (Invitrogen), treated with DNase I (Thermo Fisher Scientific, Cat# EN0521), and then reverse transcribed into cDNA using a Maximal First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Cat# K1622). Real-time polymerase chain reaction (PCR) was performed using an S1000 PCR instrument (Bio-Rad, Hercules, CA, USA) to quantify gene expression. The PCR data were normalized to glyceraldehyde-3-phosphate dehydrogenase expression at the mRNA level. The primers used are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Western blotting\u003c/h2\u003e \u003cp\u003eProteins were extracted from tumor tissues or cells with RIPA buffer (0.01% EDTA, 0.1% TritonX-100 and 10% proteinase inhibitor cocktail). Protein concentrations were quantified with a protein assay kit (Bio-Rad). 100 \u0026micro;g lysate were separated on 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. The membranes were probed overnight at 4\u0026deg;C with primary antibody against human P53 (Cell Signaling Technology, 48818S, 1:1000), β-Actin (Sigma, 1:10000), cleaved-caspase3 (Cell Signaling Technology, 9664T, 1:1000), cleaved-PARP (Cell Signaling Technology, 9541T, 1:1000), AKR1C1 (Abcam, ab192785, 1:1000), COX2 (Abcam, ab179800, 1:1000), followed by incubation with peroxidase-conjugated secondary antibody (Cell Signaling Technology, 1:10000) for 1.5 hours. The signal was visualized with ECL (Millipore).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were independently repeated at least three times unless stated otherwise. Statistical analyses were performed using the GraphPad Prism software (version 8.0). Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Two groups were compared using the Student\u0026rsquo;s t-test for unpaired data. Comparisons among more than two groups were performed using ANOVA and Tukey\u0026rsquo;s test. For all tests, \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026le;\u0026thinsp;.05, were considered statistically significant ( \u003csup\u003e\u003cem\u003e\u0026lowast;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05 ).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the National Nature Science Foundation of China (82203229); the Shenzhen Science and Technology Program (JCYJ20220531094005012), and the opening project of State Key Laboratory of Explosion Science and Technology (Beijing Institute of Technology, KFJJ22-16M).\u003c/p\u003e\n\u003ch2\u003eContributions\u003c/h2\u003e\n\u003cp\u003eXLZ and QZS performed the experiments. XLZ and CR designed experimental protocols, interpreted data obtained from the experiments, and wrote the manuscript. GQL edited the manuscript.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eAll raw data used to generate the results of this study are available from the corresponding author by request.\u003c/p\u003e\n\u003ch2\u003eCompeting interests statement\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eEthic statement\u003c/h2\u003e\n\u003cp\u003eOur studies did not include human participants, human data or human tissue. All the animal protocols containing all the procedures were approved by the ethics committee of Beijing Neurosurgical Institute Ethics Committee of Capital Medical University. The experimental protocols were conducted in accordance with the ARRIVE guidelines.\u003c/p\u003e\n\u003ch2\u003eConflict of interest\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung, H. et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCA Cancer J. Clin.\u003c/em\u003e \u003cb\u003e71\u003c/b\u003e, 209\u0026ndash;249. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3322/caac.21660\u003c/span\u003e\u003cspan address=\"10.3322/caac.21660\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, X. et al. Glutathione peroxidase 4 as a therapeutic target for anti-colorectal cancer drug-tolerant persister cells. \u003cem\u003eFront. 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Shock wave impact on the viability of MDA-MB-231 cells. \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e15\u003c/b\u003e, e0234138. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0234138\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0234138\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"extracorporeal shock waves, colorectal cancer, p53 signaling pathway, xenograft mouse models, ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-4956573/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4956573/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eShock waves are widely used to treat various diseases and are garnering further attention for medical applications. Growing evidence suggests that the application of extracorporeal shock waves (ESV) could substantially inhibit tumor growth. However, the therapeutic efficacy of ESV in colorectal cancer and the underlying mechanisms remain elusive. Using colorectal cancer cell lines HT29 and SW620, we generated xenograft mouse models, and examined the therapeutic effect of a stepwise increase in ESV energy on tumor growth. \u003cem\u003eIn vivo\u003c/em\u003e, the application of 60 mJ ESV significantly delayed xenograft growth compared with 120 and 240 mJ ESV, with no impact on body weight or hepatic and renal function. Transcriptome analysis revealed that 60 mJ ESV suppressed colorectal cancer cell proliferation and induced cell apoptosis and ferroptosis; these findings were further confirmed by immunohistochemical staining and western blotting. Mechanistically, ESV suppressed cell proliferation and induced cell apoptosis and ferroptosis by activating the p53 signaling pathway, as evidenced \u003cem\u003ein vitro\u003c/em\u003e study. In conclusion, we revealed that 60 mJ ESV could substantially inhibit colorectal cancer growth by activating p53 pathway-related proliferation inhibition and cell death. These findings suggest that ESV therapy could be a promising therapeutic strategy for colorectal cancer.\u003c/p\u003e","manuscriptTitle":"Extracorporeal shock waves effectively suppressed the proliferation and growth of colorectal cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-03 15:41:52","doi":"10.21203/rs.3.rs-4956573/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-04T06:27:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-26T07:24:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"303824093070973656391663305839301578472","date":"2025-01-20T08:04:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-09T08:01:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84721592577318370495717682196676831211","date":"2025-01-07T08:45:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-08T13:22:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-08T13:21:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-09-03T17:28:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-02T12:44:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-08-22T08:48:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e0d57f4f-6878-4fc7-baaa-90d447e95199","owner":[],"postedDate":"October 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":38075983,"name":"Physical sciences/Physics/Biological physics"},{"id":38075984,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":38075985,"name":"Biological sciences/Cell biology/Cell death/Apoptosis"},{"id":38075986,"name":"Biological sciences/Cell biology/Cell signalling"},{"id":38075987,"name":"Health sciences/Gastroenterology/Gastrointestinal diseases/Gastrointestinal cancer/Colorectal cancer"}],"tags":[],"updatedAt":"2025-03-24T16:00:15+00:00","versionOfRecord":{"articleIdentity":"rs-4956573","link":"https://doi.org/10.1038/s41598-025-94386-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-03-21 15:57:14","publishedOnDateReadable":"March 21st, 2025"},"versionCreatedAt":"2024-10-03 15:41:52","video":"","vorDoi":"10.1038/s41598-025-94386-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-94386-3","workflowStages":[]},"version":"v1","identity":"rs-4956573","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4956573","identity":"rs-4956573","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}