Effect of growth hormone on cell proliferation and biological behavior of adamantinomatous craniopharyngioma cells

Effect of growth hormone on cell proliferation and biological behavior of adamantinomatous craniopharyngioma cells | 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 Research Article Effect of growth hormone on cell proliferation and biological behavior of adamantinomatous craniopharyngioma cells Zhiwei Xiong, Kai Li, Milai Yu, Zijing Wang, Yihan Wang, Rongjun Chen, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4282785/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: This study aims to investigate the impact of growth hormone therapy on the postoperative management of craniopharyngioma and to evaluate the direct influence of GH on craniopharyngioma. Methods: The research involved the application of varying concentrations of growth hormone to stimulate and cultivate the ACP cell line. It focused on assessing the influence of growth hormone on the growth, proliferation, and apoptosis of ACP cells, and explored changes in transcription levels following growth hormone treatment of ACP cells through transcriptome sequencing. The results shed light on the effects and underlying mechanisms through which growth hormone impacts ACP cells. Results: The CCK8 cell viability assay's results indicate that growth hormone stimulation does not significantly increase ACP cell proliferation. Furthermore, sequencing data analysis suggests that growth hormone does not promote the activation of pathways associated with craniopharyngioma proliferation. Immunofluorescence analysis confirmed the presence of GHR receptors on ACP cells. Moreover, flow cytometry experiments focusing on cell cycle and apoptosis in ACP cells show that growth hormone does not affect cell cycle progression or apoptosis. Conclusion: The findings suggest that growth hormone can impact craniopharyngioma directly but it didn’t promote the growth of ACP.. craniopharyngioma growth hormone cell proliferation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Craniopharyngiomas are benign epithelial tumors primarily found in the intrasellar/suprasellar area. [1] Pituitary hormone deficiency is a common issue linked with craniopharyngioma and/or its treatment. [2, 3]Before surgery, many craniopharyngioma patients show signs of pituitary hormone deficiency, with a significant portion of children exhibiting at least one defect in the hypothalamic-pituitary axis at diagnosis. Growth hormone replacement therapy (GHRT) is frequently used as a supplemental treatment to improve growth and prognosis in pediatric patients with craniopharyngioma. [4, 5] Recent studies have shown that GHRT can aid in the recovery of endocrine functions following hypothalamic damage and restore hypothalamic functionality.[6] The concern regarding the safety of GHRT in individuals with craniopharyngioma arises from the growth-promoting and anti-apoptotic properties of growth hormone and insulin-like growth factor-1 (IGF-1). [6,7] Research has indicated that growth hormone has the potential to facilitate the advancement of breast cancer, prostate cancer, and other types of cancer cells. [8, 9] Furthermore, a comprehensive longitudinal study involving individuals who received human pituitary growth hormone during childhood and early adulthood revealed a heightened susceptibility to mortality from cancer, notably colorectal cancer and Hodgkin's disease. The safety implications of GHRT in patients with craniopharyngioma are a matter of particular significance. [10，11] Clinical studies have found that growth hormone does not increase the recurrence rate of craniopharyngioma and is safe for patients. [12, 13] Despite the safety of GHRT demonstrated in clinical studies, the conservative approach to its clinical practice following craniopharyngioma suggests initiating treatment with a low dose. Higher doses of GHRT may result in elevated side effects and a heightened risk of tumor recurrence. [14, 15] It is not clear whether the non-increase in recurrence rate of craniopharyngioma after growth hormone treatment is related to the direct effect of growth hormone on craniopharyngioma. This study employed various concentrations of growth hormone to stimulate and culture the ACP cell line, observing changes in ACP cell growth and examining the influence of growth hormone on ACP cells and its potential mechanisms. Methods Cell Culture ACP samples designated for primary cell culture were cut into 1 mm 3 fragments after rinsing in phosphate-buffered saline (PBS) solution (Gibco/Thermo Fisher Scientific, Waltham, MA, USA). These pieces were then digested for 30 minutes at 37°C using 0.25% trypsin (Gibco/Thermo Fisher Scientific) before being transferred into Dulbecco’s modified Eagle’s medium (DMEM, Gibco/Thermo Fisher Scientific) supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS, Gibco/Thermo Fisher Scientific). After digestion, the cells were centrifuged, resuspended, and incubated at 37°C in a humidified 5% CO 2 atmosphere. The filtered residue was spread into a flask and then placed in an incubator without additional reagents. After 5–6 passages, CnT-Prime Epithelial Culture Medium was used for continued cultivation. RNA Preparation for High-Throughput RNA-Sequencing Cells subjected to various concentrations of growth hormone and physiological saline were rapidly washed once with PBS buffer. Add 1ml of TRIzol reagent per 10 cm 2 culture area (equivalent to one well of a six-well plate or a 35mm diameter culture dish). Using a 1ml pipette, pipette the reagent repeatedly to ensure TRIzol contacts all cell surfaces in the culture flasks, thoroughly lysing them; then transfer to an RNase-free 1.5 ml or 2 ml centrifuge tube. RNA purity was verified using a NanoPhotometer® spectrophotometer, and RNA concentration was determined with a Qubit® RNA Assay Kit in a Qubit® 2.0 Fluorometer. RNA integrity was evaluated using the RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Library Preparation and Sequencing RNA samples from three biological replicates were purified using poly-T oligonucleotide-linked magnetic beads. The prepared DNB was loaded onto the microarray chip (Patterned Array) and sequenced using combined probe anchored polymerization technology (cPAS, Combinatorial Probe-Anchor Synthesis), anchoring sequencing primer molecules and fluorescent probes onto the DNA nanospheres. After the polymerization reaction, a high-resolution imaging system collected, read, and identified the light signal to acquire single base sequence information, proceeding to the next cycle to obtain subsequent base sequence information. Gene Expression Analysis The RNA-seq reads were aligned to the reference genome using Hisat2 v2.0.5. Feature Counts v1.5.0-\p3 was utilized to tally the reads mapped to each gene. Differential expression analysis was conducted using the DESeq2 method (version 1.10.1). The resulting p value was adjusted using the Benjamini and Hochberg method to control the false discovery rate ( p < 0.05). Genes with an adjusted p value <0.05, as identified by DESeq2, between different groups were deemed differentially expressed. Functional Enrichment Analysis of Differentially Expressed Genes The differentially expressed genes (DEGs) between groups (PEL_day 1 vs. control, PEL_day 7 vs. control) with an adjusted p value <0.05 were analyzed using the Database for Annotation, Visualization and Integrated Discovery. Gene ontology (GO) terms and KEGG pathways were identified based on cluster analysis with Benjamini multiple test correction ( p < 0.05). The gene set enrichment analysis (GSEA), GO analysis, and KEGG pathway analysis results were visualized using the R package clusterProfiler. GSVA analysis Gene set variation analysis (GSVA), a non-parametric, unsupervised approach to gene set enrichment analysis, evaluates the gene set enrichment outcomes for each transcriptome sample. The process involves two main steps: First, the expression matrix of genes across different samples is transformed into a matrix of gene set expressions across different samples using the GSVA package. Then, based on the official recommendations, the limma package is employed to determine if significant differences exist in the enrichment scores of each functional pathway between different groups. CCK-8 assay To assess ACP cell proliferation, a CCK-8 kit (Dojindo) was used. A total of 1,000 cells were cultured in 100 μL per well in five replicate wells within a 96-well plate using CnT medium. The CCK-8 reagent (10 μL) was then mixed with 90 μL DMEM to create a working solution. This working solution (100 μL) was added to each well, and the plate was incubated for 1.5 h at various time points: 0 h, 24 h, 48 h, and 72 h. The 50% inhibitory concentration (IC 50 ) value was calculated using GraphPad (San Diego, CA). Cell cycle detection Cells in the logarithmic growth phase were dissociated with 0.25% trypsin, suspended in PBS, and fixed in ice-cold 70% ethanol overnight at 4°C. After fixation, the cells were centrifuged at 1,000 rpm for 5 min, resuspended in 50 μL of RNase A, and incubated at 37°C for 30 min. Then, 400 μL of propidium iodide (PI) was added to the cell suspension for an additional 30 min, and the mixture was analyzed using ModFit LT (Verity Software House, USA). Apoptosis assay Apoptosis was evaluated using the Annexin V FITC kit (Keygentec, Nanjing, China) and analyzed with the Cellometer (Nexcelom, Lawrence, MA). Briefly, 2 × 10 5 cells/well were seeded into each well of 6-well plates, incubated overnight, and then treated with SIM at IC 50 concentrations for 24h and 48h. Afterward, the cells were collected, washed with PBS, and resuspended in 100 μL binding buffer. Then, 1 μL of Annexin V-FITC (100 μg/mL) and 0.5 μL of propidium iodide (2 mg/mL) were added to the binding buffer, and the mixture was incubated in the dark for 30 min. The samples were immediately analyzed using the Cellometer. To ensure consistency in responses, all experiments were conducted in triplicate. Western blot analysis Cellular and fresh tissue lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. These membranes were then blocked with 5% fat-free milk and incubated overnight at 4°C with primary antibodies, as specified in Supplemental Table 2. Immunoreactivity was detected using an enhanced chemiluminescence kit (Millipore), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the loading control. Relative protein expression levels were normalized to the expression level of β-Tubulin. Immunohistochemical and immunofluorescence staining Specimens were fixed in 10% formalin immediately post-surgery and embedded in paraffin 24 hours later. Sections 4 μm thick were placed on poly-L-lysine-coated slides. The slides underwent deparaffinization, rehydration, and soaking in 10 mm sodium citrate buffer, pH 6.0, before pretreatment in a microwave oven for 15 minutes. After blocking with 3% hydrogen peroxide for 10 minutes at room temperature, the slides were incubated with primary antibodies against GHR overnight at 4°C. Subsequently, a 2-step application of Poly-HRP Anti-Rabbit IgG detection system (ZSGB-Bio, Beijing, China) was employed. For immunofluorescence staining of cells, cells were plated on confocal dishes at a density of 5w/ml, allowed to adhere overnight, and fixed with 4% formaldehyde for 10 minutes. Post-fixation, the cells were washed with PBST three times for 5 minutes each, blocked with goat serum for one hour, and incubated with GHR antibody overnight at 4°C. After washing with PBST three times for 5 minutes each, the cells were incubated with goat anti-rabbit 488 secondary antibody at 37°C for one hour, followed by three more PBST washes. Stained samples were imaged using an Olympus BX63 fluorescence microscope. Results 1. Effects of different concentrations of growth hormone stimulation on the growth of craniopharyngioma cells We tested various concentrations and durations of growth hormone to study its impact on ACP cell growth. ACP cells were stimulated with various concentrations of growth hormone for 24h, 48h, and 72h[16] (Fig. 1 A-C). Compared to the control group, growth hormone did not promote ACP cell growth. High concentrations of growth hormone inhibited ACP cell growth at 48 hours, but this inhibitory effect disappeared after 72 hours. Our findings indicate that growth hormone does not impact ACP cell proliferation. 2. The differential gene enrichment pathways in ACP cells after growth hormone stimulation are not related to tumor progression. After observing no effect in the CCK8 experiment, we aimed to study the transcriptional changes in ACP cells following growth hormone stimulation.RNA sequencing was conducted on both the growth hormone-treated group and the saline-treated group. The sequencing results indicated that 129 genes were up-regulated and 391 genes were down-regulated following treatment with 10ug of growth hormone (Fig. 2A). GO and KEGG enrichment analyses revealed that the top 15 enriched pathways were primarily involved in inflammatory responses and cell adhesion (Fig. 2B-C). After treatment with 0.01ug of growth hormone, 104 genes were up-regulated and 254 genes were down-regulated (Fig. 2A). The GO and KEGG enrichment analyses indicated that the top 15 enriched pathways were mainly related to inflammatory response and cell-to-cell signaling (Fig. 2B-C). The pathways significantly enriched in these analyses are not associated with Wnt, cell cycle, apoptosis, or other pathways that promote malignant transformation or proliferation of craniopharyngioma cells. Therefore, growth hormone does not facilitate the proliferation of craniopharyngioma cells. 3. There is no change in the transcriptome levels of ACP cell proliferation and apoptosis after growth hormone stimulation. The WNT-β-catenin pathway, implicated in the malignant phenotypic transformation of craniopharyngioma, showed reduced expression of Wnt pathway activator (WNT4) and no significant change or a slight reduction in the expression of inhibitors (SFRP1, DKK3, AXIN1, and APC). The gene expression of activator (TCF7), target genes (WISP2 and CDH1), and Wnt regulator (TP53) was reduced (Fig. 3A). GSEA enrichment indicated that ACP cells are enriched in the WNT-β-catenin pathway after growth hormone treatment, but with no significant difference (Fig. 3B). This suggests that growth hormone does not activate the WNT-β-catenin pathway in ACP cells. Furthermore, GSVA analysis indicated that growth hormone stimulation does not affect the proliferation and apoptosis of ACP cells, but it reduces the inflammatory response in ACP cells (Fig. 3C-D). 4. Growth hormone does not stimulate the growth of craniopharyngioma cells Immunofluorescence staining was performed on craniopharyngioma specimens, revealing that growth hormone receptors (GHR) are highly expressed in craniopharyngioma (Fig. 4A). Similarly, when culturing the ACP cell line, it was observed that GHR is also highly expressed in ACP cells (Fig. 4B). However, upon treating the ACP cell line with growth hormone, it was found that concentrations of 10ug, 1ug, and 0.01ug/ml of growth hormone could promote an increase in STAT5 expression in craniopharyngioma cells (Fig. 4C). 5. Growth hormone has no effect on ACP cell cycle Stimulation of craniopharyngioma with various concentrations of growth hormone showed that growth hormone has no impact on the cell cycle of craniopharyngioma (Fig. 5A). Furthermore, no effect was observed on CD44 expression after stimulation with different concentrations of growth hormone (Fig. 5B). 6. Growth hormone has no effect on ACP cell apoptosis Upon stimulation of craniopharyngioma with different concentrations of growth hormone, it was found that growth hormone had no effect on the apoptosis of craniopharyngioma cells (Fig. 6A). Apoptosis-related proteins BCL-2 and P53 were also tested, and no differences in their expression levels were found, confirming that growth hormone does not influence the apoptosis of craniopharyngioma cells (Fig. 6B). Discussion The increased clinical focus on growth hormone deficiency (GHD) in both pediatric and adult populations has led to widespread discussion about the safety of GHRT. [4] Guidelines from the European Society of Endocrinology and Pediatric Endocrinology, as well as the American Society of Pediatric Endocrinology, support the long-term use of GHRT in GHD patients with a history of childhood cancer and intracranial tumors, considering it safe based on extensive clinical evidence. Currently, there is a paucity of foundational experimental research on the potential link between growth hormone and the proliferation activity of craniopharyngioma cells. In this study, various concentrations of growth hormone were used to stimulate and culture the ACP cell line. Results showed that growth hormone stimulation did not increase the proliferation of ACP cells. Moreover, sequencing results suggested that growth hormone might reduce the inflammatory response of ACP cells. These findings indicate that GHRT (GH-RT) may be administered safely to growth hormone deficient (GHD) patients with craniopharyngioma regarding tumor progression. Growth hormone influences proliferation, angiogenesis, and anti-apoptosis [17], with GH and GHR expressed in both tumor cells and the tumor microenvironment [18]. GHR expression has been observed in craniopharyngioma cells, and increased GHR expression may lead to more invasive craniopharyngioma [19]. Limited research has been conducted on the stimulation of ACP cells by growth hormone, with some researchers using growth hormone to stimulate primary craniopharyngioma ACP cells and noting growth stimulation in craniopharyngioma cells as a result [20]. Instead of the keratinocyte medium used in this study, we used CnT-Prime Epithelial Proliferation Medium to grow primary ACP cells. Our culture method can maintain the characteristics of ACP cells during the passage process, ensuring the reliability of our experiment. [21] Culturing primary craniopharyngioma cells is challenging, and different culturing methods may yield ACP cells with diverse characteristics. Our cultured primary cells closely resemble tumor tissue and effectively mimic tumors in vivo. At the same time, although craniopharyngioma is a benign tumor, there are still differences between tumor specimens of different individuals, and differences between specimens may lead to different experimental results. The mechanism of direct stimulation of growth hormone on ACP cells remains unclear. Activation of GHR leads to tyrosine phosphorylation of Janus kinase-2 and other substrates like STAT5, involved in genomic GH actions and potentially oncogenesis. [22]GH also activates MAPK/ERK, Ras-like GTPases, INS receptor substrate/PI3K/Akt, and interacts with focal adhesion kinases to propagate the signal through various intracellular pathways.[23] Our findings indicate that growth hormone can activate the STAT5 pathway in ACP cells, but further research is needed to fully understand the mechanism. sequencing of craniopharyngioma tissue has revealed that the CTNNB1 mutation in adamantinomatous craniopharyngioma (ACP) activates the Wnt signaling pathway, thereby facilitating the proliferation of craniopharyngioma cells [24-26]. Our sequencing analysis revealed enrichment of the Wnt signaling pathway in ACP cells, while growth hormone stimulation did not result in the upregulation of related pathways. Previous research has shown that elevated expression of IGF1R can exacerbate ACP inflammation, leading to compromised pituitary function and prognosis [27]. Additionally, our enrichment analysis demonstrated a downregulation of the inflammatory response pathway in craniopharyngioma cells following growth hormone stimulation, despite the common association of craniopharyngioma with severe inflammatory response. Our findings provide additional evidence supporting the safety and efficacy of GHRT for postoperative patients with craniopharyngioma. Previous clinical research has shown that the serum concentration of growth hormone exceeds 10ng/ml following a single dose [28], aligning with the dosages administered in our study. In contrast to a conservative approach involving low-dose initiation of recombinant human GHRT, personalized dosing regimens may more effectively address the individualized needs of patients. Timely and appropriate administration of growth hormone in pediatric populations has been shown to significantly enhance short-term height gain and ultimate adult stature in both growth hormone deficient (GHD) and non-GHD individuals. [29] Furthermore, numerous clinical studies have demonstrated that GHRT does not increase the risk of craniopharyngioma recurrence, consistent with the outcomes of our investigation [30-31]. While in vitro cell experiments suggest that growth hormone treatment does not stimulate the growth of craniopharyngioma cells, there is a lack of appropriate in vivo experiments to confirm the impact of growth hormone. However, the available animal models for adamantinomatous craniopharyngioma (ACP) primarily consist of the Hesx1Cre/+/Ctnnb1+/lox(ex3) embryonic ACP model [32] and the Sox2++CreERT2/+; Ctnnb1+/lox(ex3) adult ACP model [33]. These models have led to the development of tumors resembling human ACP through the targeting of CTNNB1 mutations. Nevertheless, these tumors lacked key characteristics of human ACP, including calcification and wet keratin, and were confined to the sellar region without infiltration into the hypothalamus. Thus, the establishment of in vivo models that accurately replicate human ACP is imperative for a more comprehensive investigation into growth hormone's mechanism of action. In conclusion, the in vitro cell experiments conducted in this study suggest that growth hormone treatment does not stimulate the growth of craniopharyngioma cells. These results align with previous research findings and offer additional support for the notion that GHRT does not increase the risk of tumor progression in craniopharyngioma patients. Declarations Statement of Ethics All materials were approved by the Ethics Committee of Nanfang Hospital (No. NFEC201096), and written informed consent was obtained. Conflict of Interest Statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work; there is no professional or other personal interest of any nature or kind in any product, service, and/or company that could be construed as influencing the position presented in, or the review of, this manuscript. Funding Sources This study is financially supported by the Startup fund from the Natural Science Foundation of Guangdong Province (Grant No. 2019A1515012140 and No. 2021A1515011371). Author Contributions Junxiang Peng, Danling Li provided the concept and designed the study. Zhiwei Xiong, Kai Li, and Milai Yu conducted the analyses. Zijing Wang and Yihan Wang wrote the manuscript. ,Rongjun Chen, Weizhao Li,Keying Zhang, Yucong Lin and Zhixuan Zhang participated in data analysis. All authors read and approved the final manuscript. 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Losa, M., et al., Growth Hormone Therapy Does Not Increase the Risk of Craniopharyngioma and Nonfunctioning Pituitary Adenoma Recurrence. J Clin Endocrinol Metab, 2020. 105(5). Gaston-Massuet, C., et al., Increased Wingless (Wnt) signaling in pituitary progenitor/stem cells gives rise to pituitary tumors in mice and humans. Proc Natl Acad Sci U S A, 2011. 108(28): p. 11482-7. Andoniadou, C.L., et al., Sox2(+) stem/progenitor cells in the adult mouse pituitary support organ homeostasis and have tumor-inducing potential. Cell Stem Cell, 2013. 13(4): p. 433-45. Supplemental Table Supplemental Table 2 is not available with this version. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-4282785","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":292683442,"identity":"12813b5c-39a7-442b-9ba8-ea5644bc91d0","order_by":0,"name":"Zhiwei Xiong","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhiwei","middleName":"","lastName":"Xiong","suffix":""},{"id":292683443,"identity":"77663558-8186-41d2-9a02-3a1ecdf62fe8","order_by":1,"name":"Kai Li","email":"","orcid":"","institution":"Southern Medical 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University","correspondingAuthor":false,"prefix":"","firstName":"Danling","middleName":"","lastName":"Li","suffix":""},{"id":292683464,"identity":"6415882f-4c16-4985-90e0-d88377a677db","order_by":11,"name":"Junxiang Peng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIie2QvQrCMBSFI0InSzaJKPYVUjqUgg/TvkUnySCd+gCKr+DQ0fGEgFOkq+CiuCoILg4K/rRzm1Ew35IznI97bwixWH4SdFCFLnBNzRRSK04i59p0TvX0AuXODOphR3Pc0+k4pKsrXEE82kezEolNLHPtBNH8UmCwJv5iGTcrHApws15S7LYFfE1ivm9VpJDPjL0VfUCSGSkKys14UpQ5gTRRPreokY4DvnO4FJq13xIy7R/P7x/jpTrdHunEo8O2xRjqxL5N1lz/KlTUiaKhZrFYLH/NC+nCVAiymimUAAAAAElFTkSuQmCC","orcid":"","institution":"Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Junxiang","middleName":"","lastName":"Peng","suffix":""}],"badges":[],"createdAt":"2024-04-17 15:18:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4282785/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4282785/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55190800,"identity":"aa5f639b-5792-49d2-b10e-a7f8f9820be0","added_by":"auto","created_at":"2024-04-23 19:41:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":71299,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth of ACP cell in respond to growth hormone.\u003c/p\u003e\n\u003cp\u003e(A) ACP cells were treated with different concentrations of growth hormone (0.01, 0.05, 0.1, 0.5, 1, 10 μg/ml) for 24 (A), 48 (B), and 72 hours (C). Data are presented as mean ± SEM (n = 6). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 by two-tailed Student's t-test or one-way ANOVA.\u003c/p\u003e","description":"","filename":"fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4282785/v1/c82570f0bc9d68dad6df63fc.png"},{"id":55190802,"identity":"8c26ac5f-aaa0-4ed4-9f7e-3556c4df59b4","added_by":"auto","created_at":"2024-04-23 19:41:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":389467,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential gene enrichment pathways in ACP cells following growth hormone stimulation are not associated with tumor progression.\u003c/p\u003e\n\u003cp\u003e（A）Volcano plots display the number of differentially expressed genes identified from 10 μg vs control and 10 ng vs control.\u003c/p\u003e\n\u003cp\u003e（B）Bubble map shows GO pathway enrichment data for significantly different genes.\u003c/p\u003e\n\u003cp\u003e（C）Bubble map shows KEGG pathway enrichment data for significantly different genes.\u003c/p\u003e","description":"","filename":"fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4282785/v1/381316945e76f9b80fb887f1.png"},{"id":55191124,"identity":"d53a7628-07a3-484c-9738-94893a182f6a","added_by":"auto","created_at":"2024-04-23 19:49:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":401073,"visible":true,"origin":"","legend":"\u003cp\u003eNo change in transcriptome levels of ACP cell proliferation and apoptosis after growth hormone stimulation.\u003c/p\u003e\n\u003cp\u003e（A）The heatmap illustrates the expression of genes related to the WNT-β-catenin pathway.\u003c/p\u003e\n\u003cp\u003e（B）The gene set enrichment map indicates the enrichment of the WNT-β-catenin pathway in ACP cells treated with growth hormone.\u003c/p\u003e\n\u003cp\u003e（C）GSVA of 0.01 μg rGH vs control.\u003c/p\u003e\n\u003cp\u003e（D）GSVA of 10 μg rGH vs control. Blue and red denote significantly activated and inhibited biological processes, respectively.\u003c/p\u003e","description":"","filename":"fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4282785/v1/c083b1eeb131e229329e9033.png"},{"id":55190799,"identity":"f30949f0-90e8-4efc-91de-1b3fcd898efb","added_by":"auto","created_at":"2024-04-23 19:41:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3009005,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth hormone does not stimulate the growth of craniopharyngioma cells\u003c/p\u003e\n\u003cp\u003e（A）Immunofluorescence staining of ACP tissue samples, showing representative images of GHR staining. Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e（B）Immunofluorescence staining of primary ACP cells, showing representative images of GHR staining. Scale bar, 50 μm.\u003cbr\u003e\n（C）The expression level of P-STST5 protein in ACP cells after treatment with 0.01 μg, 1 μg, and 10 μg rGH. Data are presented as mean ± SEM (n = 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 by two-tailed Student’s t-test or one-way ANOVA.\u003c/p\u003e","description":"","filename":"fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4282785/v1/acf66e96141364c56f12d787.png"},{"id":55190798,"identity":"152c173f-6312-4e5a-b641-874851a41744","added_by":"auto","created_at":"2024-04-23 19:41:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":308004,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth hormone has no effect on ACP cell cycle\u003c/p\u003e\n\u003cp\u003e(A) Representative FACS plots of cell cycle analysis in ACP cells after treatment with different concentrations of rGH.\u003c/p\u003e\n\u003cp\u003e(B) The expression level of CD44 protein in ACP cells after treatment with 0.01 μg, 1 μg, and 10 μg rGH. Data are presented as mean ± SEM (n = 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 by two-tailed Student’s t-test or one-way ANOVA.\u003c/p\u003e","description":"","filename":"fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4282785/v1/6b107b12be25628ebbd1652d.png"},{"id":55190797,"identity":"f583b559-a7af-4c18-8fa9-25fb3ce292f3","added_by":"auto","created_at":"2024-04-23 19:41:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":418644,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth hormone has no effect on ACP cell apoptosis\u003c/p\u003e\n\u003cp\u003e(A) Representative FACS plots of cell apoptosis analysis in ACP cells after treatment with different concentrations of rGH.\u003c/p\u003e\n\u003cp\u003e(B) The expression level of P53 and Bcl-2 protein in ACP cells after treatment with 0.01 μg, 1 μg, and 10 μg rGH. Data are presented as mean ± SEM (n = 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01 by two-tailed Student’s t-test or one-way ANOVA.\u003c/p\u003e","description":"","filename":"fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4282785/v1/d960c09c9f5f59bf46445419.png"},{"id":55266022,"identity":"61c30b76-e0e1-47c0-a734-fe17ec4d188d","added_by":"auto","created_at":"2024-04-25 02:20:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1869134,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4282785/v1/3848c593-3444-4483-9a27-8c5c46a79d5e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of growth hormone on cell proliferation and biological behavior of adamantinomatous craniopharyngioma cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCraniopharyngiomas are benign epithelial tumors primarily found in the intrasellar/suprasellar area. [1] Pituitary hormone deficiency is a common issue linked with craniopharyngioma and/or its treatment. [2, 3]Before surgery, many craniopharyngioma patients show signs of pituitary hormone deficiency, with a significant portion of children exhibiting at least one defect in the hypothalamic-pituitary axis at diagnosis. Growth hormone replacement therapy (GHRT) is frequently used as a supplemental treatment to improve growth and prognosis in pediatric patients with craniopharyngioma. [4, 5] Recent studies have shown that GHRT can aid in the recovery of endocrine functions following hypothalamic damage and restore hypothalamic functionality.[6]\u003c/p\u003e\n\u003cp\u003eThe concern regarding the safety of GHRT in individuals with craniopharyngioma arises from the growth-promoting and anti-apoptotic properties of growth hormone and insulin-like growth factor-1 (IGF-1). [6,7] Research has indicated that growth hormone has the potential to facilitate the advancement of breast cancer, prostate cancer, and other types of cancer cells. [8, 9] Furthermore, a comprehensive longitudinal study involving individuals who received human pituitary growth hormone during childhood and early adulthood revealed a heightened susceptibility to mortality from cancer, notably colorectal cancer and Hodgkin\u0026apos;s disease. The safety implications of GHRT in patients with craniopharyngioma are a matter of particular significance. [10，11] Clinical studies have found that growth hormone does not increase the recurrence rate of craniopharyngioma and is safe for patients. [12, 13] Despite the safety of GHRT demonstrated in clinical studies, the conservative approach to its clinical practice following craniopharyngioma suggests initiating treatment with a low dose. Higher doses of GHRT may result in elevated side effects and a heightened risk of tumor recurrence. [14, 15] It is not clear whether the non-increase in recurrence rate of craniopharyngioma after growth hormone treatment is related to the direct effect of growth hormone on craniopharyngioma. This study employed various concentrations of growth hormone to stimulate and culture the ACP cell line, observing changes in ACP cell growth and examining the influence of growth hormone on ACP cells and its potential mechanisms.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eACP samples designated for primary cell culture were cut into 1 mm\u003csup\u003e3\u003c/sup\u003e fragments after rinsing in phosphate-buffered saline (PBS) solution (Gibco/Thermo Fisher Scientific, Waltham, MA, USA). These pieces were then digested for 30 minutes at 37\u0026deg;C using 0.25% trypsin (Gibco/Thermo Fisher Scientific) before being transferred into Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Gibco/Thermo Fisher Scientific) supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS, Gibco/Thermo Fisher Scientific). After digestion, the cells were centrifuged, resuspended, and incubated at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. The filtered residue was spread into a flask and then placed in an incubator without additional reagents. After 5\u0026ndash;6 passages, CnT-Prime Epithelial Culture Medium was used for continued cultivation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Preparation for High-Throughput RNA-Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells subjected to various concentrations of growth hormone and physiological saline were rapidly washed once with PBS buffer. Add 1ml of TRIzol reagent per 10 cm\u003csup\u003e2\u003c/sup\u003e culture area (equivalent to one well of a six-well plate or a 35mm diameter culture dish). Using a 1ml pipette, pipette the reagent repeatedly to ensure TRIzol contacts all cell surfaces in the culture flasks, thoroughly lysing them; then transfer to an RNase-free 1.5 ml or 2 ml centrifuge tube. RNA purity was verified using a NanoPhotometer\u0026reg; spectrophotometer, and RNA concentration was determined with a Qubit\u0026reg; RNA Assay Kit in a Qubit\u0026reg; 2.0 Fluorometer. RNA integrity was evaluated using the RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLibrary Preparation and Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA samples from three biological replicates were purified using poly-T oligonucleotide-linked magnetic beads. The prepared DNB was loaded onto the microarray chip (Patterned Array) and sequenced using combined probe anchored polymerization technology (cPAS, Combinatorial Probe-Anchor Synthesis), anchoring sequencing primer molecules and fluorescent probes onto the DNA nanospheres. After the polymerization reaction, a high-resolution imaging system collected, read, and identified the light signal to acquire single base sequence information, proceeding to the next cycle to obtain subsequent base sequence information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene Expression Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-seq reads were aligned to the reference genome using Hisat2 v2.0.5. Feature Counts v1.5.0-\\p3 was utilized to tally the reads mapped to each gene. Differential expression analysis was conducted using the DESeq2 method (version 1.10.1). The resulting \u003cem\u003ep\u003c/em\u003e value was adjusted using the Benjamini and Hochberg method to control the false discovery rate (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Genes with an adjusted \u003cem\u003ep\u003c/em\u003e value \u0026lt;0.05, as identified by DESeq2, between different groups were deemed differentially expressed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional Enrichment Analysis of Differentially Expressed Genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe differentially expressed genes (DEGs) between groups (PEL_day 1 vs. control, PEL_day 7 vs. control) with an adjusted \u003cem\u003ep\u003c/em\u003e value \u0026lt;0.05 were analyzed using the Database for Annotation, Visualization and Integrated Discovery. Gene ontology (GO) terms and KEGG pathways were identified based on cluster analysis with Benjamini multiple test correction (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). The gene set enrichment analysis (GSEA), GO analysis, and KEGG pathway analysis results were visualized using the R package clusterProfiler.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGSVA analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene set variation analysis (GSVA), a non-parametric, unsupervised approach to gene set enrichment analysis, evaluates the gene set enrichment outcomes for each transcriptome sample. The process involves two main steps: First, the expression matrix of genes across different samples is transformed into a matrix of gene set expressions across different samples using the GSVA package. Then, based on the official recommendations, the limma package is employed to determine if significant differences exist in the enrichment scores of each functional pathway between different groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCCK-8 assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess ACP cell proliferation, a CCK-8 kit (Dojindo) was used. A total of 1,000 cells were cultured in 100 \u0026mu;L per well in five replicate wells within a 96-well plate using CnT medium. The CCK-8 reagent (10 \u0026mu;L) was then mixed with 90 \u0026mu;L DMEM to create a working solution. This working solution (100 \u0026mu;L) was added to each well, and the plate was incubated for 1.5 h at various time points: 0 h, 24 h, 48 h, and 72 h. The 50% inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) value was calculated using GraphPad (San Diego, CA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cycle detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells in the logarithmic growth phase were dissociated with 0.25% trypsin, suspended in PBS, and fixed in ice-cold 70% ethanol overnight at 4\u0026deg;C. After fixation, the cells were centrifuged at 1,000 rpm for 5 min, resuspended in 50 \u0026mu;L of RNase A, and incubated at 37\u0026deg;C for 30 min. Then, 400 \u0026mu;L of propidium iodide (PI) was added to the cell suspension for an additional 30 min, and the mixture was analyzed using ModFit LT (Verity Software House, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApoptosis was evaluated using the Annexin V FITC kit (Keygentec, Nanjing, China) and analyzed with the Cellometer (Nexcelom, Lawrence, MA). Briefly, 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well were seeded into each well of 6-well plates, incubated overnight, and then treated with SIM at IC\u003csub\u003e50\u003c/sub\u003e concentrations for 24h and 48h. Afterward, the cells were collected, washed with PBS, and resuspended in 100 \u0026mu;L binding buffer. Then, 1 \u0026mu;L of Annexin V-FITC (100 \u0026mu;g/mL) and 0.5 \u0026mu;L of propidium iodide (2 mg/mL) were added to the binding buffer, and the mixture was incubated in the dark for 30 min. The samples were immediately analyzed using the Cellometer. To ensure consistency in responses, all experiments were conducted in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCellular and fresh tissue lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. These membranes were then blocked with 5% fat-free milk and incubated overnight at 4\u0026deg;C with primary antibodies, as specified in Supplemental Table 2. Immunoreactivity was detected using an enhanced chemiluminescence kit (Millipore), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the loading control. Relative protein expression levels were normalized to the expression level of \u0026beta;-Tubulin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical and immunofluorescence staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecimens were fixed in 10% formalin immediately post-surgery and embedded in paraffin 24 hours later. Sections 4 \u0026mu;m thick were placed on poly-L-lysine-coated slides. The slides underwent deparaffinization, rehydration, and soaking in 10 mm sodium citrate buffer, pH 6.0, before pretreatment in a microwave oven for 15 minutes. After blocking with 3% hydrogen peroxide for 10 minutes at room temperature, the slides were incubated with primary antibodies against GHR overnight at 4\u0026deg;C. Subsequently, a 2-step application of Poly-HRP Anti-Rabbit IgG detection system (ZSGB-Bio, Beijing, China) was employed. For immunofluorescence staining of cells, cells were plated on confocal dishes at a density of 5w/ml, allowed to adhere overnight, and fixed with 4% formaldehyde for 10 minutes. Post-fixation, the cells were washed with PBST three times for 5 minutes each, blocked with goat serum for one hour, and incubated with GHR antibody overnight at 4\u0026deg;C. After washing with PBST three times for 5 minutes each, the cells were incubated with goat anti-rabbit 488 secondary antibody at 37\u0026deg;C for one hour, followed by three more PBST washes. Stained samples were imaged using an Olympus BX63 fluorescence microscope.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Effects of different concentrations of growth hormone stimulation on the growth of craniopharyngioma cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe tested various concentrations and durations of growth hormone to study its impact on ACP cell growth. ACP cells were stimulated with various concentrations of growth hormone for 24h, 48h, and 72h[16] (Fig. 1 A-C). Compared to the control group, growth hormone did not promote ACP cell growth. High concentrations of growth hormone inhibited ACP cell growth at 48 hours, but this inhibitory effect disappeared after 72 hours. Our findings indicate that growth hormone does not impact ACP cell proliferation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. The differential gene enrichment pathways in ACP cells after growth hormone stimulation are not related to tumor progression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter observing no effect in the CCK8 experiment, we aimed to study the transcriptional changes in ACP cells following growth hormone stimulation.RNA sequencing was conducted on both the growth hormone-treated group and the saline-treated group. The sequencing results indicated that 129 genes were up-regulated and 391 genes were down-regulated following treatment with 10ug of growth hormone (Fig. 2A). GO and KEGG enrichment analyses revealed that the top 15 enriched pathways were primarily involved in inflammatory responses and cell adhesion (Fig. 2B-C). After treatment with 0.01ug of growth hormone, 104 genes were up-regulated and 254 genes were down-regulated (Fig. 2A). The GO and KEGG enrichment analyses indicated that the top 15 enriched pathways were mainly related to inflammatory response and cell-to-cell signaling (Fig. 2B-C). The pathways significantly enriched in these analyses are not associated with Wnt, cell cycle, apoptosis, or other pathways that promote malignant transformation or proliferation of craniopharyngioma cells. Therefore, growth hormone does not facilitate the proliferation of craniopharyngioma cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. There is no change in the transcriptome levels of ACP cell proliferation and apoptosis after growth hormone stimulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe WNT-\u0026beta;-catenin pathway, implicated in the malignant phenotypic transformation of craniopharyngioma, showed reduced expression of Wnt pathway activator (WNT4) and no significant change or a slight reduction in the expression of inhibitors (SFRP1, DKK3, AXIN1, and APC). The gene expression of activator (TCF7), target genes (WISP2 and CDH1), and Wnt regulator (TP53) was reduced (Fig. 3A). GSEA enrichment indicated that ACP cells are enriched in the WNT-\u0026beta;-catenin pathway after growth hormone treatment, but with no significant difference (Fig. 3B). This suggests that growth hormone does not activate the WNT-\u0026beta;-catenin pathway in ACP cells. Furthermore, GSVA analysis indicated that growth hormone stimulation does not affect the proliferation and apoptosis of ACP cells, but it reduces the inflammatory response in ACP cells (Fig. 3C-D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Growth hormone does not stimulate the growth of craniopharyngioma cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining was performed on craniopharyngioma specimens, revealing that growth hormone receptors (GHR) are highly expressed in craniopharyngioma (Fig. 4A). Similarly, when culturing the ACP cell line, it was observed that GHR is also highly expressed in ACP cells (Fig. 4B). However, upon treating the ACP cell line with growth hormone, it was found that concentrations of 10ug, 1ug, and 0.01ug/ml of growth hormone could promote an increase in STAT5 expression in craniopharyngioma cells (Fig. 4C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Growth hormone has no effect on ACP cell cycle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStimulation of craniopharyngioma with various concentrations of growth hormone showed that growth hormone has no impact on the cell cycle of craniopharyngioma (Fig. 5A). Furthermore, no effect was observed on CD44 expression after stimulation with different concentrations of growth hormone (Fig. 5B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Growth hormone has no effect on ACP cell apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUpon stimulation of craniopharyngioma with different concentrations of growth hormone, it was found that growth hormone had no effect on the apoptosis of craniopharyngioma cells (Fig. 6A). Apoptosis-related proteins BCL-2 and P53 were also tested, and no differences in their expression levels were found, confirming that growth hormone does not influence the apoptosis of craniopharyngioma cells (Fig. 6B).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe increased clinical focus on growth hormone deficiency (GHD) in both pediatric and adult populations has led to widespread discussion about the safety of GHRT. [4] Guidelines from the European Society of Endocrinology and Pediatric Endocrinology, as well as the American Society of Pediatric Endocrinology, support the long-term use of GHRT in GHD patients with a history of childhood cancer and intracranial tumors, considering it safe based on extensive clinical evidence. Currently, there is a paucity of foundational experimental research on the potential link between growth hormone and the proliferation activity of craniopharyngioma cells. In this study, various concentrations of growth hormone were used to stimulate and culture the ACP cell line. Results showed that growth hormone stimulation did not increase the proliferation of ACP cells. Moreover, sequencing results suggested that growth hormone might reduce the inflammatory response of ACP cells. These findings indicate that GHRT (GH-RT) may be administered safely to growth hormone deficient (GHD) patients with craniopharyngioma regarding tumor progression.\u003c/p\u003e\n\u003cp\u003eGrowth hormone influences proliferation, angiogenesis, and anti-apoptosis [17], with GH and GHR expressed in both tumor cells and the tumor microenvironment [18]. GHR expression has been observed in craniopharyngioma cells, and increased GHR expression may lead to more invasive craniopharyngioma [19]. Limited research has been conducted on the stimulation of ACP cells by growth hormone, with some researchers using growth hormone to stimulate primary craniopharyngioma ACP cells and noting growth stimulation in craniopharyngioma cells as a result [20]. Instead of the keratinocyte medium used in this study, we used CnT-Prime Epithelial Proliferation Medium to grow primary ACP cells. Our culture method can maintain the characteristics of ACP cells during the passage process, ensuring the reliability of our experiment. [21] Culturing primary craniopharyngioma cells is challenging, and different culturing methods may yield ACP cells with diverse characteristics. Our cultured primary cells closely resemble tumor tissue and effectively mimic tumors in vivo. At the same time, although craniopharyngioma is a benign tumor, there are still differences between tumor specimens of different individuals, and differences between specimens may lead to different experimental results.\u003c/p\u003e\n\u003cp\u003eThe mechanism of direct stimulation of growth hormone on ACP cells remains unclear. Activation of GHR leads to tyrosine phosphorylation of Janus kinase-2 and other substrates like STAT5, involved in genomic GH actions and potentially oncogenesis. [22]GH also activates MAPK/ERK, Ras-like GTPases, INS receptor substrate/PI3K/Akt, and interacts with focal adhesion kinases to propagate the signal through various intracellular pathways.[23]\u0026nbsp;Our findings indicate that growth hormone can activate the STAT5 pathway in ACP cells, but further research is needed to fully understand the mechanism. sequencing of craniopharyngioma tissue has revealed that the CTNNB1 mutation in adamantinomatous craniopharyngioma (ACP) activates the Wnt signaling pathway, thereby facilitating the proliferation of craniopharyngioma cells [24-26]. Our sequencing analysis revealed enrichment of the Wnt signaling pathway in ACP cells, while growth hormone stimulation did not result in the upregulation of related pathways. Previous research has shown that elevated expression of IGF1R can exacerbate ACP inflammation, leading to compromised pituitary function and prognosis [27]. Additionally, our enrichment analysis demonstrated a downregulation of the inflammatory response pathway in craniopharyngioma cells following growth hormone stimulation, despite the common association of craniopharyngioma with severe inflammatory response. Our findings provide additional evidence supporting the safety and efficacy of GHRT for postoperative patients with craniopharyngioma. Previous clinical research has shown that the serum concentration of growth hormone exceeds 10ng/ml following a single dose [28], aligning with the dosages administered in our study. In contrast to a conservative approach involving low-dose initiation of recombinant human GHRT, personalized dosing regimens may more effectively address the individualized needs of patients. Timely and appropriate administration of growth hormone in pediatric populations has been shown to significantly enhance short-term height gain and ultimate adult stature in both growth hormone deficient (GHD) and non-GHD individuals. [29] Furthermore, numerous clinical studies have demonstrated that GHRT does not increase the risk of craniopharyngioma recurrence, consistent with the outcomes of our investigation [30-31].\u003c/p\u003e\n\u003cp\u003eWhile \u003cem\u003ein vitro\u003c/em\u003e cell experiments suggest that growth hormone treatment does not stimulate the growth of craniopharyngioma cells, there is a lack of appropriate \u003cem\u003ein vivo\u003c/em\u003e experiments to confirm the impact of growth hormone. However, the available animal models for adamantinomatous craniopharyngioma (ACP) primarily consist of the Hesx1Cre/+/Ctnnb1+/lox(ex3) embryonic ACP model [32] and the Sox2++CreERT2/+; Ctnnb1+/lox(ex3) adult ACP model [33]. These models have led to the development of tumors resembling human ACP through the targeting of CTNNB1 mutations. Nevertheless, these tumors lacked key characteristics of human ACP, including calcification and wet keratin, and were confined to the sellar region without infiltration into the hypothalamus. Thus, the establishment of \u003cem\u003ein vivo\u003c/em\u003e models that accurately replicate human ACP is imperative for a more comprehensive investigation into growth hormone\u0026apos;s mechanism of action.\u003c/p\u003e\n\u003cp\u003eIn conclusion, the \u003cem\u003ein vitro\u003c/em\u003e cell experiments conducted in this study suggest that growth hormone treatment does not stimulate the growth of craniopharyngioma cells. These results align with previous research findings and offer additional support for the notion that GHRT does not increase the risk of tumor progression in craniopharyngioma patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eStatement of Ethics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll materials were approved by the Ethics Committee of Nanfang Hospital (No. NFEC201096), and written informed consent was obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work; there is no professional or other personal interest of any nature or kind in any product, service, and/or company that could be construed as influencing the position presented in, or the review of, this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is financially supported by the Startup fund from the Natural Science Foundation of Guangdong Province (Grant No. 2019A1515012140 and No. 2021A1515011371).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJunxiang Peng, Danling Li provided the concept and designed the study. Zhiwei Xiong, Kai Li, and Milai Yu conducted the analyses. Zijing Wang and Yihan Wang wrote the manuscript. ,Rongjun Chen, Weizhao Li,Keying Zhang, Yucong Lin and Zhixuan Zhang participated in data analysis. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or used during the study appear in the submitted article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOrtiz, T.M., I. Shafiq and F.B. Mesfin, Craniopharyngioma. 2024.\u003c/li\u003e\n\u003cli\u003eHietamaki, J., et al., Presentation and diagnosis of childhood-onset combined pituitary hormone deficiency: A single center experience from over 30 years. EClinicalMedicine, 2022. 51: p. 101556.\u003c/li\u003e\n\u003cli\u003eBaldelli, R., et al., Characteristics of adult patients with growth hormone deficiency who underwent neurosurgery for functioning and non-functioning pituitary adenomas and craniopharyngiomas. J Endocrinol Invest, 2005. 28(2): p. 157-61.\u003c/li\u003e\n\u003cli\u003eMaghnie, M., et al., Safety and Efficacy of Pediatric Growth Hormone Therapy: Results From the Full KIGS Cohort. J Clin Endocrinol Metab, 2022. 107(12): p. 3287-3301.\u003c/li\u003e\n\u003cli\u003eVerhelst, J., et al., Baseline characteristics and response to 2 years of growth hormone (GH) replacement of hypopituitary patients with GH deficiency due to adult-onset craniopharyngioma in comparison with patients with nonfunctioning pituitary adenoma: data from KIMS (Pfizer International Metabolic Database). J Clin Endocrinol Metab, 2005. 90(8): p. 4636-43.\u003c/li\u003e\n\u003cli\u003eLi, K., et al., Growth hormone promotes the reconstruction of injured axons in the hypothalamo-neurohypophyseal system. Neural Regen Res, 2024. 19(10): p. 2249-2258.\u003c/li\u003e\n\u003cli\u003eDonato, J.J. and J.J. Kopchick, New findings on brain actions of growth hormone and potential clinical implications. Rev Endocr Metab Disord, 2023.\u003c/li\u003e\n\u003cli\u003eKleinberg, D.L., et al., Growth hormone and insulin-like growth factor-I in the transition from normal mammary development to preneoplastic mammary lesions. Endocr Rev, 2009. 30(1): p. 51-74.\u003c/li\u003e\n\u003cli\u003eZhu, M.L. and N. Kyprianou, Androgen receptor and growth factor signaling cross-talk in prostate cancer cells. Endocr Relat Cancer, 2008. 15(4): p. 841-9.\u003c/li\u003e\n\u003cli\u003eSwerdlow, A.J., et al., Risk of cancer in patients treated with human pituitary growth hormone in the UK, 1959-85: a cohort study. Lancet, 2002. 360(9329): p. 273-7.\u003c/li\u003e\n\u003cli\u003eNguyen, Q.A., et al., GH and Childhood-onset Craniopharyngioma: When to Initiate GH Replacement Therapy? J Clin Endocrinol Metab, 2023. 108(8): p. 1929-1936.\u003c/li\u003e\n\u003cli\u003eBoguszewski, M., et al., Safety of growth hormone replacement in survivors of cancer and intracranial and pituitary tumours: a consensus statement. Eur J Endocrinol, 2022. 186(6): p. P35-P52.\u003c/li\u003e\n\u003cli\u003eOlsson, D.S., et al., Tumour recurrence and enlargement in patients with craniopharyngioma with and without GH replacement therapy during more than 10 years of follow-up. Eur J Endocrinol, 2012. 166(6): p. 1061-8.\u003c/li\u003e\n\u003cli\u003eThomas-Teinturier, C., et al., Influence of growth hormone therapy on the occurrence of a second neoplasm in survivors of childhood cancer. Eur J Endocrinol, 2020. 183(4): p. 471-480.\u003c/li\u003e\n\u003cli\u003eFleseriu, M., et al., Hormonal Replacement in Hypopituitarism in Adults: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab, 2016. 101(11): p. 3888-3921.\u003c/li\u003e\n\u003cli\u003eUnterberger, C.J., et al., GH Action in Prostate Cancer Cells Promotes Proliferation, Limits Apoptosis, and Regulates Cancer-related Gene Expression. Endocrinology, 2022. 163(5).\u003c/li\u003e\n\u003cli\u003ePerry, J.K., et al., Tumour-Derived Human Growth Hormone As a Therapeutic Target in Oncology. Trends Endocrinol Metab, 2017. 28(8): p. 587-596.\u003c/li\u003e\n\u003cli\u003eChesnokova, V. and S. Melmed, Growth hormone in the tumor microenvironment. Arch Endocrinol Metab, 2019. 63(6): p. 568-575.\u003c/li\u003e\n\u003cli\u003eHofmann, B.M., et al., Hormone receptor expression in craniopharyngiomas: a clinicopathological correlation. Neurosurgery, 2010. 67(3): p. 617-25; discussion 625.\u003c/li\u003e\n\u003cli\u003eLi, Q., et al., Craniopharyngioma cell growth is promoted by growth hormone (GH) and is inhibited by tamoxifen: involvement of growth hormone receptor (GHR) and IGF-1 receptor (IGF-1R). J Clin Neurosci, 2013. 20(1): p. 153-7.\u003c/li\u003e\n\u003cli\u003eZhang, P.D., et al., Feasibility of primary human cell cultures as a model for adamantinomatous craniopharyngioma research: Evidence from RNA-Seq analysis. Oncol Lett, 2020. 19(3): p. 2346-2354.\u003c/li\u003e\n\u003cli\u003eWaters, M.J., The growth hormone receptor. Growth Horm IGF Res, 2016. 28: p. 6-10.\u003c/li\u003e\n\u003cli\u003eBergan-Roller, H.E. and M.A. Sheridan, The growth hormone signaling system: Insights into coordinating the anabolic and catabolic actions of growth hormone. Gen Comp Endocrinol, 2018. 258: p. 119-133.\u003c/li\u003e\n\u003cli\u003eHe, J., et al., Characterization of novel CTNNB1 mutation in Craniopharyngioma by whole-genome sequencing. Mol Cancer, 2021. 20(1): p. 168.\u003c/li\u003e\n\u003cli\u003eMota, J., et al., Telomere length and Wnt/beta-catenin pathway in adamantinomatous craniopharyngiomas. Eur J Endocrinol, 2022. 187(2): p. 219-230.\u003c/li\u003e\n\u003cli\u003eApps, J.R., et al., Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target. Acta Neuropathol, 2018. 135(5): p. 757-777.\u003c/li\u003e\n\u003cli\u003eYang, L., et al., Expression of Insulin-Like Growth Factor Type 1 Receptor Is Linked to Inflammation in Adamantinomatous Craniopharyngioma. Neuroendocrinology, 2022. 112(9): p. 917-926.\u003c/li\u003e\n\u003cli\u003eUnterberger, C.J., et al., GH Action in Prostate Cancer Cells Promotes Proliferation, Limits Apoptosis, and Regulates Cancer-related Gene Expression. Endocrinology, 2022. 163(5).\u003c/li\u003e\n\u003cli\u003eRanke, M.B., et al., Final height in children with medulloblastoma treated with growth hormone. Horm Res, 2005. 64(1): p. 28-34.\u003c/li\u003e\n\u003cli\u003eRichmond, E. and A.D. Rogol, Treatment of growth hormone deficiency in children, adolescents and at the transitional age. Best Pract Res Clin Endocrinol Metab, 2016. 30(6): p. 749-755.\u003c/li\u003e\n\u003cli\u003eLosa, M., et al., Growth Hormone Therapy Does Not Increase the Risk of Craniopharyngioma and Nonfunctioning Pituitary Adenoma Recurrence. J Clin Endocrinol Metab, 2020. 105(5).\u003c/li\u003e\n\u003cli\u003eGaston-Massuet, C., et al., Increased Wingless (Wnt) signaling in pituitary progenitor/stem cells gives rise to pituitary tumors in mice and humans. Proc Natl Acad Sci U S A, 2011. 108(28): p. 11482-7.\u003c/li\u003e\n\u003cli\u003eAndoniadou, C.L., et al., Sox2(+) stem/progenitor cells in the adult mouse pituitary support organ homeostasis and have tumor-inducing potential. Cell Stem Cell, 2013. 13(4): p. 433-45.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplemental Table","content":"\u003cp\u003eSupplemental Table 2 is not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"craniopharyngioma, growth hormone, cell proliferation","lastPublishedDoi":"10.21203/rs.3.rs-4282785/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4282785/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePurpose: \u003c/strong\u003e\u003c/em\u003eThis study aims to investigate the impact of growth hormone therapy on the postoperative management of craniopharyngioma and to evaluate the direct influence of GH on craniopharyngioma.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/em\u003e The research involved the application of varying concentrations of growth hormone to stimulate and cultivate the ACP cell line. It focused on assessing the influence of growth hormone on the growth, proliferation, and apoptosis of ACP cells, and explored changes in transcription levels following growth hormone treatment of ACP cells through transcriptome sequencing. The results shed light on the effects and underlying mechanisms through which growth hormone impacts ACP cells.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/em\u003e The CCK8 cell viability assay's results indicate that growth hormone stimulation does not significantly increase ACP cell proliferation. Furthermore, sequencing data analysis suggests that growth hormone does not promote the activation of pathways associated with craniopharyngioma proliferation. Immunofluorescence analysis confirmed the presence of GHR receptors on ACP cells. Moreover, flow cytometry experiments focusing on cell cycle and apoptosis in ACP cells show that growth hormone does not affect cell cycle progression or apoptosis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/em\u003e The findings suggest that growth hormone can impact craniopharyngioma directly but it didn’t promote the growth of ACP..\u003c/p\u003e","manuscriptTitle":"Effect of growth hormone on cell proliferation and biological behavior of adamantinomatous craniopharyngioma cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-23 19:40:26","doi":"10.21203/rs.3.rs-4282785/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"175a2a11-08e6-490f-b72b-39f379d3b53c","owner":[],"postedDate":"April 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-26T08:47:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-23 19:40:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4282785","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4282785","identity":"rs-4282785","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}