{"paper_id":"2d8307c6-2aa8-4d8d-a44d-a929e182a75b","body_text":"Relationship between angiogenesis and hypoxia-inducible factor-1α and erythropoietin in superficial esophageal carcinoma | 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 Relationship between angiogenesis and hypoxia-inducible factor-1α and erythropoietin in superficial esophageal carcinoma Eisuke Yamamoto, Youichi Kumagai, Tetsuhiko Tachikawa, Morihiro Higashi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4452751/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 Hypoxia-inducible factor (HIF-1α) is the initial switch in angiogenesis; however, its expression in superficial esophageal carcinoma is unclear. We determined the timing of the hypoxia-induced angiogenesis switch by investigating the expression of HIF-1α and the erythropoietin (Epo) it activates in superficial esophageal carcinoma. We used 53 lesions of superficial esophageal carcinoma and conducted immunohistochemistry of HIF-1α and Epo. Cases were divided into three groups: T1a-EP/LPM (22 lesions), T1a-MM/T1b-SM1 (15 lesions), and T1b-SM2,3 (16 lesions). We compared HIF-1α and Epo expression in the deepest area and examined the correlation between HIF-1α and Epo scores. In 24 cases of T1a-MM or deeper carcinomas with intraepithelial spread, we compared HIF-1α and Epo scores in the intraepithelial spread and deepest areas in the same cases. Both HIF-1α and Epo were most strongly expressed in T1a-EP/LPM, with significant attenuation at T1a-MM and deeper. Both HIF-1α and Epo had significantly higher scores in the T1a-EP/LPM group than in the T1a-MM/T1b-SM1 and T1b-SM2,3 groups. HIF-1α and Epo were strongly correlated, and were significantly higher in intraepithelial spread areas than in invaded areas. HIF-1α and Epo are strongly expressed from an early stage of esophageal carcinoma; as the carcinoma invades the muscularis mucosae and deeper, expression decreases. Biological sciences/Cancer Health sciences/Gastroenterology esophageal carcinoma hypoxia-inducible factor-1α erythropoietin angiogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cancer is a population of cells that proliferate in a disorderly manner, causing proliferation, migration, and invasion into the surrounding tissue. Cancer invasion occurs when cancer cells acquire the ability to proliferate and migrate, and a factor that promotes these abilities is the supply of nutrients to the cancer cells via angiogenesis [ 1 ]. Recently, observations of superficial esophageal carcinoma using magnifying endoscopy revealed that the superficial capillary network is correlated with the depth of cancer invasion [ 2 ]. This finding allows real-time in vivo observation of the angiogenesis process in the early stages of esophageal cancer progression. We have previously examined the expression status of angiogenic and angiogenesis inhibition factors in superficial esophageal carcinoma cases to elucidate the mechanism of angiogenesis in superficial esophageal squamous cell carcinoma [ 3 – 6 ]. In the tumor microenvironment, comprising the malignant tumor and its surrounding tumor stroma, increased oxygen demand due to excessive cell proliferation and insufficient oxygen supply as a result of a longer distance from blood vessels results in a hypoxic state. When in this state, the hypoxia-inducible factor (HIF), which is involved in angiogenesis, is expressed [ 6 ]. HIF-1α is the first switch for inducing tumor blood vessels and supporting growth and extension [ 7 , 8 ]. Several studies have examined the status of HIF1 expression in esophageal carcinomas [ 9 – 13 ], whereas a few studies have examined the status of HIF-1α expression in superficial esophageal carcinomas. HIF-1α activates erythropoietin (Epo), the expression of which is the most correlated with HIF-1α. The expression status of HIF-1α and Epo in cervical cancer has been examined; both are strongly expressed from the early stages of carcinogenesis and substantially correlated [ 14 ]. However, to the best of our knowledge, the expression status of HIF-1α and Epo and their correlation in esophageal carcinomas, including advanced carcinomas, have not been investigated. Here, we aimed to conduct an immunohistochemical examination of the expression status of HIF-1α and Epo in superficial esophageal carcinoma to examine the hypoxic state in the early stages of esophageal cancer cell growth and development. Materials and Methods Materials We used 61 samples obtained from 50 consecutive patients whose esophageal carcinoma (squamous cell carcinoma) was removed by surgery or endoscopic treatment at Saitama Medical Center from 2007 to 2016 and underwent histopathological examination. Patients who received radiotherapy or chemotherapy, or both, before treatment were excluded. The median age was 69 years (54–80), with 45 males and 5 females. The macroscopic types of the tumors were as follows: superficial and protruding type (0–I), 14 lesions; superficial and flat type (0–II), 37 lesions (0–IIa: 4 lesions, 0–IIb, 11 lesions, 0–IIc: 22 lesions); and superficial and excavated type (0–III), 2 lesions [ 15 ]. The samples included 8 normal esophagus samples and 53 esophageal squamous cell carcinoma samples (T1a-EP: 9 lesions, T1a-LPM: 13 lesions, T1a-MM: 7 lesions, T1b-SM1: 8 lesions, T1b-SM2: 2 lesions, T1b-SM3: 14 lesions) that were obtained from 9 endoscopic resection and 46 esophagectomy cases. Normal esophagus samples were obtained from normal squamous epithelium positioned away from the cancerous lesion in the esophagectomy specimens. Pathological diagnosis was conducted according to the Japanese Classification of Esophageal Cancer (11th Edition) [ 15 ]. This study was conducted under a protocol approved by the ethics committee of our institution (1968-II). All experiments were performed in accordance with relevant guidelines and regulations. Opt-out informed consent was obtained from all patients. Immunohistochemistry (IHC) We established formalin-fixed, paraffin-embedded sections with a thickness of 4 µm obtained from endoscopically or surgically resected esophagus cases, and we conducted IHC for HIF-1α and Epo. For HIF-1α and Epo, we used Leica IHC NovoLink kits (Leica Biosystems, Nußloch, Germany), and the primary antibodies used were an anti-HIF-1 alpha antibody (ab82832; Abcam, Cambridge, UK) and an EPO Rabbit polyclonal antibody (17908-1-AP; Proteintech, Rosemont, IL, USA), respectively, both at 50-fold dilution. Examination items The cases were divided into three groups: T1a-EP/LPM (24 lesions), T1a-MM/T1b-SM1 (15 lesions), and T1b-SM2,3 (16 lesions). We used a 200× field of view to observe three hot spots each for HIF-1α and Epo in the carcinoma epithelium for T1a-EP/LPM and deepest area for T1a-MM and deeper, which were then scored using the H score. We examined the differences in the HIF-1α and Epo scores among the T1a-EP/LPM, T1a-MM/T1b-SM1, and T1b-SM2,3 groups. In addition, we examined the correlation between HIF-1α and Epo score. Furthermore, in 24 cases of carcinoma that invaded T1a-MM or deeper with an intraepithelial spread, we compared the HIF-1α and Epo scores between the intraepithelial spread and deepest areas. H score The criteria for expression intensity were based on the expression intensity in the nucleus for HIF-1α in the renal tubule and that in the nucleus and cytoplasm for Epo. A score of 1 was given for those with weaker expression than that in the renal tubule, a score of 2 for expression similar to that in the renal tubule, and a score of 3 for expression greater than that in the renal tubule. A score of 0 was given when no expression was confirmed (Fig. 1 ). The positive cell count/total cell count (%) was calculated for each score, after which each percentage was multiplied by the score and added for scoring. The average value calculated by two clinicians was used as the final score, with a separate pathologist confirming the value. Statistics Continuous variables in the evaluations are expressed as median values. The difference between the scores of each group was analyzed using Kruskal–Wallis tests, followed by post-hoc Dunn’s tests. Correlations between variables were examined using Spearman’s rank correlation test. Comparisons between related groups were also analyzed using the Wilcoxon test. Statistical significance was considered at P < 0.05. All statistical analyses were conducted using Statflex (version 7.0, Artec Co., Osaka, Japan). Results IHC Regarding the expression status of HIF-1α in the normal esophagus, the basal and prickle cell layers showed very slight expression, and the muscularis mucosae showed diffuse expression. HIF-1α in esophageal carcinoma cells was expressed in the nucleus, but the expression intensity varied even within the same case. Regarding the expression status of Epo in the normal esophagus, the basal cell layer showed very slight expression in the nucleus, and the vascular endothelium showed uniform expression. Moreover, Epo expression in the carcinoma cells was observed in both the nucleus and cytoplasm. The degree of expression intensity also varied within the same case (Fig. 2 ). Expression status of HIF-1α and Epo in each group and correlation between HIF-1α and Epo The median scores for HIF-1α were as follows: normal esophageal mucosa, 0.75 (0.5–1.5) points; T1a-EP/LPM group, 222.5 (50–294) points; T1a-MM/T1b-SM1 group, 0 (0–157) points; and T1b-SM2,3 group, 0 (0–150) points. Expression was strongest in the T1a-EP/LPM group, with significant changes observed ( P < 0.001, Kruskal–Wallis test). The post-hoc Dunn’s test showed that the T1a-EP/LPM group had a significantly higher score than the T1a-MM/T1b-SM1 ( P < 0.001) and T1b-SM2,3 ( P < 0.001) groups (Fig. 3 a). The median scores for Epo were as follows: normal esophageal mucosa, 0.5 (0–5) points; T1a-EP/LPM group, 239 (95–300) points; T1a-MM/T1b-SM1 group, 63 (0–195) points; and T1b-SM2,3 group, 68 (0–295) points. Expression was strongest in the T1a-EP/LPM group, with significant changes observed ( P < 0.001, Kruskal–Wallis test). The post-hoc Dunn’s test showed that the T1a-EP/LPM group had a significantly higher score than the T1a-MM/T1b-SM1 ( P < 0.001) and T1b-SM2,3 ( P < 0.001) groups (Fig. 3 b). HIF-1α and Epo scores were strongly positively correlated in esophageal carcinoma cases (Spearman’s rank correlation test: rS = 0.74, P < 0.001) (Fig. 3 c). Changes in HIF-1α and Epo scores depending on invasion depth in the same cases In the 24 cases of T1a-MM or deeper with an intraepithelial spread, we compared the scores for the intraepithelial spread and invaded areas in the same cases. The HIF-1α scores significantly differed at 219 (1–297) points for the intraepithelial spread area and 0 (0–157) points for the invaded area (Wilcoxon test, P < 0.001) (Fig. 4 a). Similarly, the Epo scores significantly differed at 236 (2–300) points for the intraepithelial spread area and 52 (0–196) points for the invaded area ( P < 0.001, Wilcoxon test) (Fig. 4 b and Fig. 5 ). Discussion In this study, we obtained two important results. First, in T1a-EP/LPM carcinomas, the expression status of HIF1α and Epo indicated that the tumors were in a hypoxic state. Using magnifying endoscopy, the vascular morphology of T1a-EP/LPM carcinomas was shown to be dilated, and the number of the subepithelial and intrapapillary capillaries was shown to be increased, which was also observed in the normal squamous epithelium. At this stage, the tumor was likely to be hypoxic owing to increased oxygen demand. Second, as the tumor invaded deeper than the muscularis mucosa, the hypoxic state was resolved at the invasive front. Tumor vessels were observed by magnifying endoscopy from this stage, at which neovascularization was induced by various angiogenic factors, and the hypoxic state was thought to be resolved. The expression status of HIF-1 in the early stages of cancer growth and development varies depending on the organ. Various reports have shown early gastric cancer expression rates of 15, 61, and 100%, with no consensus on the topic [ 16 – 18 ]. Only one report demonstrated that HIF-1α is expressed in 51% of early esophageal carcinomas [ 9 ], and no studies have scored and compared the expression status of HIF-1α in superficial esophageal carcinoma cases according to invasion depth, as in the present study. HIF-1 is composed of HIF-1α and HIF-1β, and the protein expression level of HIF-1α regulates the hypoxic response. This is because HIF-1β is a constant nuclear protein, whereas HIF-1α is degraded by proteasomes under normal oxygen concentration and partial pressure. This degradation occurs because the von Hippel Lindau gene binds to the oxygen-dependent degradation domain of HIF-1α through the ubiquitin E3 ligase complex, which hydroxylates proline residues. Under a hypoxic state, degradation by ubiquitination is suppressed, and HIF-1α accumulates in the cytoplasm. HIF-1α is then translocated into the nucleus and forms a dimer with the β subunit, after which it forms a complex with CBP/p300 and binds to hypoxia-responsive elements, which are target genes that induce the transcription of various growth factors. Genes that are reportedly induced include EPO , vascular endothelial growth factor ( VEGF ) and its receptor endothelin 1, plasminogen activators, platelet-derived growth factor, glucose transporters, and tyrosine hydroxylase [ 19 – 21 ]. In a previous study, we showed that VEGF-A, which is induced by HIF-1α, is expressed in esophageal mucosal carcinoma and similarly strongly expressed even when invading deeper than T1a-MM [ 4 ]. This result is inconsistent with the expression status of HIF-1α reported in the present study. However, VEGF-A is also induced by other cytokines, such as cyclooxygenase (COX)-2. We previously reported that COX-2 is strongly expressed at T1a-MM and deeper [ 22 ]. Therefore, VEGF-A expression is thought to be induced by the action of multiple angiogenesis factors. Epo, which is mainly produced in the tubulointerstitial cells of the kidney, is a hematopoietic growth factor that promotes the differentiation and proliferation of erythropoietic cells by binding to the erythropoietin receptor (EPOR) present on erythroid progenitor cells in bone marrow. Recently, Epo has also been reported to have angiogenesis-promoting effects [ 23 , 24 ]. The presence of Epo and EPOR has been reported in malignant tumor cells, such as neuroblastoma and uterine, ovarian, and breast cancers, and it has been suggested that they affect tumor growth and angiogenesis [ 25 ]. The present study showed that the expression status of Epo is very similar to that of HIF1, and Epo is speculated to affect angiogenesis in the early stages of esophageal carcinoma growth and development. In the present study, we used the H score obtained via IHC for scoring. Using this method enables the scoring of the expression intensity determined by IHC across a wide range from 0 to 300 points. The current standard is for investigators to conduct manual evaluations, but reproducibility when examining multiple cases is a challenge. Image analysis using software has been attempted to solve this problem, and a significant correlation with visual examination results has been observed [ 26 ]. This study had some limitations. First, the number of cases was limited, and the cases were from a single institution; further studies with more cases are needed in the future. Second, the present study only included superficial esophageal carcinoma cases. HIF-1α is reportedly strongly expressed in advanced esophageal carcinoma cases and associated with prognosis [ 11 ], with similar results obtained in cervical cancer cases [ 27 ]. In the present study, HIF-1α was positive from T1a-EP/LPM, with decreased expression from T1a-MM and deeper. In the future, there is a need to examine advanced esophageal carcinoma cases as well. Third, our study targeted only carcinoma cells, and stromal cells, such as macrophages, were not investigated. Examining the roles of HIF-1α in cancer progression, as well as its association with cells in the tumor stroma, such as macrophages, is required. Recently, whole-genome analysis using next-generation sequencers has advanced, and new genomic abnormalities have been discovered in somatic cells related to cancer growth and development. Previous research has shown that carcinoma invasion beyond the muscularis mucosae in esophageal carcinoma cases causes several changes in the expression status of angiogenic factors and angiogenesis inhibition factors. [ 5 ]A comprehensive analysis of how genetic mutations occur in the process of progression from mucosal to invasive cancer will lead to an elucidation of the mechanisms of cancer growth and development. In conclusion, we showed that, in superficial esophageal carcinoma cases, both HIF-1α and Epo were expressed from T1a-EP/LPM, with expression tending to decrease from T1a-MM and deeper. Moreover, carcinoma cells were in a hypoxic state in the early stages of proliferation, and the hypoxic state was surmised to be resolved in carcinomas at T1a-MM and deeper, where angiogenesis is active. Declarations Acknowledgments: This manuscript is supported by MEXT Kakenhi (grant number 19K08477). We would like to thank Editage (www.editage.com) for English language editing. Author contributions: E.Y. and Y.K., study concept and design, analysis and interpretation of data, and writing of the report. T.T., analysis and interpretation of data. M.H., A.T., and M.O., acquisition of data. H.I., critical revision of the manuscript for important intellectual content and material support. All authors agreed to submit this manuscript for publication. Data availability The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Conflict of interest: All authors declare that they have no conflict of interest. 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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-4452751\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":318180721,\"identity\":\"33dc0932-9108-4f57-921d-01a0ae85e96f\",\"order_by\":0,\"name\":\"Eisuke Yamamoto\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYFACNjApByIOPCBBi4ExWEsCKVoSG0AUUVr4+Y+lSd3M+ZM+P+zwQ6AtdnK6DQS0SM5IOyadu80gd+PtNAOglmRjswMEtBjcYG+DaJmdANJyIHEbIS3254+DtaQbzk7/QJwWAwaIwxLkpXOItEXiRlqyde42Y8MN0jkFBxIMiPALf/8xw9u52+Tk5Wenb/7wocJOjqAWIGCRALvwAMSdRAHmDyBSvoE41aNgFIyCUTACAQBB80T3ch/q3AAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"Saitama Medical Center, Saitama Medical University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Eisuke\",\"middleName\":\"\",\"lastName\":\"Yamamoto\",\"suffix\":\"\"},{\"id\":318180724,\"identity\":\"3975cdeb-17ce-4b99-8496-552bf73664db\",\"order_by\":1,\"name\":\"Youichi Kumagai\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Saitama Medical Center, Saitama Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Youichi\",\"middleName\":\"\",\"lastName\":\"Kumagai\",\"suffix\":\"\"},{\"id\":318180725,\"identity\":\"27af6d08-406f-4f70-9254-8cc32d6b21b0\",\"order_by\":2,\"name\":\"Tetsuhiko Tachikawa\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Saitama Cancer Center\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Tetsuhiko\",\"middleName\":\"\",\"lastName\":\"Tachikawa\",\"suffix\":\"\"},{\"id\":318180727,\"identity\":\"0c206758-0f85-4665-9f82-780220104723\",\"order_by\":3,\"name\":\"Morihiro Higashi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Saitama Medical Center, Saitama Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Morihiro\",\"middleName\":\"\",\"lastName\":\"Higashi\",\"suffix\":\"\"},{\"id\":318180728,\"identity\":\"fa79bd5c-e526-43ff-9a6f-7c447a3d2fbf\",\"order_by\":4,\"name\":\"Masashi Oka\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Saitama Medical Center, Saitama Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Masashi\",\"middleName\":\"\",\"lastName\":\"Oka\",\"suffix\":\"\"},{\"id\":318180729,\"identity\":\"64f2e90e-8447-412d-841d-0714d294dfef\",\"order_by\":5,\"name\":\"Hideyuki Ishida\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Saitama Medical Center, Saitama Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hideyuki\",\"middleName\":\"\",\"lastName\":\"Ishida\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-05-21 07:00:15\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4452751/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4452751/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":59871096,\"identity\":\"4236dca3-43ea-4aa1-92fd-e7e4f31fbb75\",\"added_by\":\"auto\",\"created_at\":\"2024-07-08 17:03:20\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":413451,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImmunohistochemistry images of HIF-1α and Epo in normal kidney and esophageal carcinoma (200×). \\u003cstrong\\u003ea\\u003c/strong\\u003e HIF-1α image of the normal renal tubule. Uniform expression was observed in the nucleus. \\u003cstrong\\u003eb\\u003c/strong\\u003e Epo image of the normal renal tubule. Expression was observed in the nucleus and cytoplasm. \\u003cstrong\\u003ec\\u003c/strong\\u003e HIF-1α image where score 0 cell count was over 99%. \\u003cstrong\\u003ed\\u003c/strong\\u003e Epo image where score 0 cell count was over 99%. \\u003cstrong\\u003ee\\u003c/strong\\u003e HIF-1α image where score 1 cell count was 90%. \\u003cstrong\\u003ef\\u003c/strong\\u003e Epo image where score 1 cell count was 92%. \\u003cstrong\\u003eg\\u003c/strong\\u003e HIF-1α image where score 2 cell count was 86%. \\u003cstrong\\u003eh\\u003c/strong\\u003eEpo image where score 2 cell count was 91%. \\u003cstrong\\u003ei\\u003c/strong\\u003e HIF-1α image where score 3 cell count was 88%. \\u003cstrong\\u003ej\\u003c/strong\\u003e Epo image where score 3 cell count was 86%.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4452751/v1/d3f294df169d9a5c7505dda2.jpg\"},{\"id\":59872171,\"identity\":\"37ca95db-e06b-4129-896a-e5f7787b22f8\",\"added_by\":\"auto\",\"created_at\":\"2024-07-08 17:11:20\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":774514,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImmunohistochemistry images of normal esophageal mucosa, mucosal carcinoma, and submucosal invasive carcinoma considering HIF-1α and Epo. \\u003cstrong\\u003ea\\u003c/strong\\u003e HIF-1α image of normal mucosa. The normal mucosal epithelial cell layer showed hardly any HIF1 expression, and expression was observed in the nuclei of 1% of basal cells (100×). \\u003cstrong\\u003eb\\u003c/strong\\u003e HIF-1α image of a T1a-EP case. Expression was observed in cancer cells, with particularly high expression in the nucleus. (score: 288) (100×, black square: 200×). \\u003cstrong\\u003ec\\u003c/strong\\u003e HIF-1α image of a T1b-SM1 case (score: 3) (100×, black square: 200×). \\u003cstrong\\u003ed\\u003c/strong\\u003eHIF-1α image of a T1b-SM3 case. (score: 0). (100×, black square: 200×). \\u003cstrong\\u003ee\\u003c/strong\\u003e Epo image of normal mucosa. The cytoplasm of the basal cells from the upper endothelium did not exhibit Epo expression, but the nucleus showed slight expression (1%) in the basal layer (100×). \\u003cstrong\\u003ef\\u003c/strong\\u003e Epo image of a T1a-EP case. Epo expression was observed in the cytoplasm and nucleus of mucosal epithelial atypical cells (score: 255) (100×, black square: 200×). \\u003cstrong\\u003eg\\u003c/strong\\u003e Epo image of a T1b-SM1 case (score: 9). (100×, black square: 200×). \\u003cstrong\\u003eh\\u003c/strong\\u003eEpo image of a T1b-SM3 case (score: 87). (100×, black square: 200×).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4452751/v1/e84f634f9e462af3db86b09a.jpg\"},{\"id\":59871093,\"identity\":\"de8dffd1-81e5-4704-9a84-f28d73b80268\",\"added_by\":\"auto\",\"created_at\":\"2024-07-08 17:03:20\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":116631,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eHIF-1α and Epo scores according to invasion depth. \\u003cstrong\\u003ea\\u003c/strong\\u003e HIF-1α score. The T1a-EP/LPM group had the highest score, with significant changes exhibited (\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.01, Kruskal–Wallis test). The post-hoc Dunn’s test showed significantly higher scores for the T1a-EP/LPM group compared with those of the T1a-MM/T1b-SM1 (\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.01) and T1b-SM2,3 groups (\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.01). \\u003cstrong\\u003eb\\u003c/strong\\u003e Epo score. The T1a-EP/LPM group had the highest score, with significant changes exhibited (\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.01, Kruskal–Wallis test). Additionally, the post-hoc Dunn’s test showed significantly higher scores for the T1a-EP/LPM group compared with those of the T1a-MM/T1b-SM1 (\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.01) and T1b-SM2,3 groups (\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.01). \\u003cstrong\\u003ec\\u003c/strong\\u003e HIF-1α and Epo scores were significantly correlated (Spearman’s rank correlation test rS = 0.74, \\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.001).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4452751/v1/c3465c7a377998847c650535.jpg\"},{\"id\":59872172,\"identity\":\"ec50e6f3-4317-47ec-85aa-5d31ffa938b9\",\"added_by\":\"auto\",\"created_at\":\"2024-07-08 17:11:20\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":88196,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eChanges in HIF-1α and Epo scores in the intraepithelial spread and invaded areas within the same case. \\u003cstrong\\u003ea\\u003c/strong\\u003e The HIF-1α score of the intraepithelial spread area was significantly higher compared with that in the invaded area, even within the same case (\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.01, Wilcoxon test). \\u003cstrong\\u003eb\\u003c/strong\\u003eThe Epo score of the intraepithelial spread area was significantly higher compared with that in the invaded area, even within the same case (\\u003cem\\u003eP \\u003c/em\\u003e\\u0026lt; 0.01, Wilcoxon test).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4452751/v1/65744aa2cbe778313d319769.jpg\"},{\"id\":59871095,\"identity\":\"02c2d151-c125-4c81-a6f1-549fa470c1af\",\"added_by\":\"auto\",\"created_at\":\"2024-07-08 17:03:20\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1513091,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eImmunohistochemistry image of HIF-1α and Epo in an SM1 case with intraepithelial spread (40×, black square: 200×). \\u003cstrong\\u003ea\\u003c/strong\\u003e HIF-1α expression was observed in cancer cells in the intraepithelial spread area, with particularly high expression in the nucleus (score: 277). \\u003cstrong\\u003eb\\u003c/strong\\u003eHIF-1α image of the invaded area of the submucosal layer. No expression was observed in cancer cells (score: 0). \\u003cstrong\\u003ec\\u003c/strong\\u003eEpo expression was observed in cancer cells in the intraepithelial spread area (score: 267). \\u003cstrong\\u003ed\\u003c/strong\\u003e Epo image of the invaded area of the submucosal layer (score: 81).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4452751/v1/ea770c79e2f677db33695943.jpg\"},{\"id\":85197765,\"identity\":\"955177b8-6702-42d5-a09e-bddd733ab5cd\",\"added_by\":\"auto\",\"created_at\":\"2025-06-23 09:47:18\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3495495,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4452751/v1/566c37ed-2e40-4d55-9b50-d0906f27d81c.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Relationship between angiogenesis and hypoxia-inducible factor-1α and erythropoietin in superficial esophageal carcinoma\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eCancer is a population of cells that proliferate in a disorderly manner, causing proliferation, migration, and invasion into the surrounding tissue. Cancer invasion occurs when cancer cells acquire the ability to proliferate and migrate, and a factor that promotes these abilities is the supply of nutrients to the cancer cells via angiogenesis [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eRecently, observations of superficial esophageal carcinoma using magnifying endoscopy revealed that the superficial capillary network is correlated with the depth of cancer invasion [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. This finding allows real-time in vivo observation of the angiogenesis process in the early stages of esophageal cancer progression. We have previously examined the expression status of angiogenic and angiogenesis inhibition factors in superficial esophageal carcinoma cases to elucidate the mechanism of angiogenesis in superficial esophageal squamous cell carcinoma [\\u003cspan additionalcitationids=\\\"CR4 CR5\\\" citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn the tumor microenvironment, comprising the malignant tumor and its surrounding tumor stroma, increased oxygen demand due to excessive cell proliferation and insufficient oxygen supply as a result of a longer distance from blood vessels results in a hypoxic state. When in this state, the hypoxia-inducible factor (HIF), which is involved in angiogenesis, is expressed [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. HIF-1α is the first switch for inducing tumor blood vessels and supporting growth and extension [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. Several studies have examined the status of HIF1 expression in esophageal carcinomas [\\u003cspan additionalcitationids=\\\"CR10 CR11 CR12\\\" citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e], whereas a few studies have examined the status of HIF-1α expression in superficial esophageal carcinomas.\\u003c/p\\u003e \\u003cp\\u003eHIF-1α activates erythropoietin (Epo), the expression of which is the most correlated with HIF-1α. The expression status of HIF-1α and Epo in cervical cancer has been examined; both are strongly expressed from the early stages of carcinogenesis and substantially correlated [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. However, to the best of our knowledge, the expression status of HIF-1α and Epo and their correlation in esophageal carcinomas, including advanced carcinomas, have not been investigated.\\u003c/p\\u003e \\u003cp\\u003eHere, we aimed to conduct an immunohistochemical examination of the expression status of HIF-1α and Epo in superficial esophageal carcinoma to examine the hypoxic state in the early stages of esophageal cancer cell growth and development.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eMaterials\\u003c/h2\\u003e \\u003cp\\u003eWe used 61 samples obtained from 50 consecutive patients whose esophageal carcinoma (squamous cell carcinoma) was removed by surgery or endoscopic treatment at Saitama Medical Center from 2007 to 2016 and underwent histopathological examination. Patients who received radiotherapy or chemotherapy, or both, before treatment were excluded. The median age was 69 years (54\\u0026ndash;80), with 45 males and 5 females. The macroscopic types of the tumors were as follows: superficial and protruding type (0\\u0026ndash;I), 14 lesions; superficial and flat type (0\\u0026ndash;II), 37 lesions (0\\u0026ndash;IIa: 4 lesions, 0\\u0026ndash;IIb, 11 lesions, 0\\u0026ndash;IIc: 22 lesions); and superficial and excavated type (0\\u0026ndash;III), 2 lesions [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eThe samples included 8 normal esophagus samples and 53 esophageal squamous cell carcinoma samples (T1a-EP: 9 lesions, T1a-LPM: 13 lesions, T1a-MM: 7 lesions, T1b-SM1: 8 lesions, T1b-SM2: 2 lesions, T1b-SM3: 14 lesions) that were obtained from 9 endoscopic resection and 46 esophagectomy cases. Normal esophagus samples were obtained from normal squamous epithelium positioned away from the cancerous lesion in the esophagectomy specimens. Pathological diagnosis was conducted according to the Japanese Classification of Esophageal Cancer (11th Edition) [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. This study was conducted under a protocol approved by the ethics committee of our institution (1968-II). All experiments were performed in accordance with relevant guidelines and regulations. Opt-out informed consent was obtained from all patients.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eImmunohistochemistry (IHC)\\u003c/h2\\u003e \\u003cp\\u003e We established formalin-fixed, paraffin-embedded sections with a thickness of 4 \\u0026micro;m obtained from endoscopically or surgically resected esophagus cases, and we conducted IHC for HIF-1α and Epo. For HIF-1α and Epo, we used Leica IHC NovoLink kits (Leica Biosystems, Nu\\u0026szlig;loch, Germany), and the primary antibodies used were an anti-HIF-1 alpha antibody (ab82832; Abcam, Cambridge, UK) and an EPO Rabbit polyclonal antibody (17908-1-AP; Proteintech, Rosemont, IL, USA), respectively, both at 50-fold dilution.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eExamination items\\u003c/h2\\u003e \\u003cp\\u003eThe cases were divided into three groups: T1a-EP/LPM (24 lesions), T1a-MM/T1b-SM1 (15 lesions), and T1b-SM2,3 (16 lesions). We used a 200\\u0026times; field of view to observe three hot spots each for HIF-1α and Epo in the carcinoma epithelium for T1a-EP/LPM and deepest area for T1a-MM and deeper, which were then scored using the H score. We examined the differences in the HIF-1α and Epo scores among the T1a-EP/LPM, T1a-MM/T1b-SM1, and T1b-SM2,3 groups. In addition, we examined the correlation between HIF-1α and Epo score. Furthermore, in 24 cases of carcinoma that invaded T1a-MM or deeper with an intraepithelial spread, we compared the HIF-1α and Epo scores between the intraepithelial spread and deepest areas.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eH score\\u003c/h2\\u003e \\u003cp\\u003eThe criteria for expression intensity were based on the expression intensity in the nucleus for HIF-1α in the renal tubule and that in the nucleus and cytoplasm for Epo. A score of 1 was given for those with weaker expression than that in the renal tubule, a score of 2 for expression similar to that in the renal tubule, and a score of 3 for expression greater than that in the renal tubule. A score of 0 was given when no expression was confirmed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The positive cell count/total cell count (%) was calculated for each score, after which each percentage was multiplied by the score and added for scoring. The average value calculated by two clinicians was used as the final score, with a separate pathologist confirming the value.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistics\\u003c/h2\\u003e \\u003cp\\u003eContinuous variables in the evaluations are expressed as median values. The difference between the scores of each group was analyzed using Kruskal\\u0026ndash;Wallis tests, followed by post-hoc Dunn\\u0026rsquo;s tests. Correlations between variables were examined using Spearman\\u0026rsquo;s rank correlation test. Comparisons between related groups were also analyzed using the Wilcoxon test. Statistical significance was considered at \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05. All statistical analyses were conducted using Statflex (version 7.0, Artec Co., Osaka, Japan).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIHC\\u003c/h2\\u003e \\u003cp\\u003eRegarding the expression status of HIF-1α in the normal esophagus, the basal and prickle cell layers showed very slight expression, and the muscularis mucosae showed diffuse expression. HIF-1α in esophageal carcinoma cells was expressed in the nucleus, but the expression intensity varied even within the same case. Regarding the expression status of Epo in the normal esophagus, the basal cell layer showed very slight expression in the nucleus, and the vascular endothelium showed uniform expression. Moreover, Epo expression in the carcinoma cells was observed in both the nucleus and cytoplasm. The degree of expression intensity also varied within the same case (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eExpression status of HIF-1α and Epo in each group and correlation between HIF-1α and Epo\\u003c/h2\\u003e \\u003cp\\u003eThe median scores for HIF-1α were as follows: normal esophageal mucosa, 0.75 (0.5\\u0026ndash;1.5) points; T1a-EP/LPM group, 222.5 (50\\u0026ndash;294) points; T1a-MM/T1b-SM1 group, 0 (0\\u0026ndash;157) points; and T1b-SM2,3 group, 0 (0\\u0026ndash;150) points. Expression was strongest in the T1a-EP/LPM group, with significant changes observed (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001, Kruskal\\u0026ndash;Wallis test). The post-hoc Dunn\\u0026rsquo;s test showed that the T1a-EP/LPM group had a significantly higher score than the T1a-MM/T1b-SM1 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) and T1b-SM2,3 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe median scores for Epo were as follows: normal esophageal mucosa, 0.5 (0\\u0026ndash;5) points; T1a-EP/LPM group, 239 (95\\u0026ndash;300) points; T1a-MM/T1b-SM1 group, 63 (0\\u0026ndash;195) points; and T1b-SM2,3 group, 68 (0\\u0026ndash;295) points. Expression was strongest in the T1a-EP/LPM group, with significant changes observed (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001, Kruskal\\u0026ndash;Wallis test). The post-hoc Dunn\\u0026rsquo;s test showed that the T1a-EP/LPM group had a significantly higher score than the T1a-MM/T1b-SM1 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) and T1b-SM2,3 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb). HIF-1α and Epo scores were strongly positively correlated in esophageal carcinoma cases (Spearman\\u0026rsquo;s rank correlation test: rS\\u0026thinsp;=\\u0026thinsp;0.74, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ec).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eChanges in HIF-1α and Epo scores depending on invasion depth in the same cases\\u003c/h2\\u003e \\u003cp\\u003eIn the 24 cases of T1a-MM or deeper with an intraepithelial spread, we compared the scores for the intraepithelial spread and invaded areas in the same cases. The HIF-1α scores significantly differed at 219 (1\\u0026ndash;297) points for the intraepithelial spread area and 0 (0\\u0026ndash;157) points for the invaded area (Wilcoxon test, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea). Similarly, the Epo scores significantly differed at 236 (2\\u0026ndash;300) points for the intraepithelial spread area and 52 (0\\u0026ndash;196) points for the invaded area (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001, Wilcoxon test) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb and Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eIn this study, we obtained two important results. First, in T1a-EP/LPM carcinomas, the expression status of HIF1α and Epo indicated that the tumors were in a hypoxic state. Using magnifying endoscopy, the vascular morphology of T1a-EP/LPM carcinomas was shown to be dilated, and the number of the subepithelial and intrapapillary capillaries was shown to be increased, which was also observed in the normal squamous epithelium. At this stage, the tumor was likely to be hypoxic owing to increased oxygen demand. Second, as the tumor invaded deeper than the muscularis mucosa, the hypoxic state was resolved at the invasive front. Tumor vessels were observed by magnifying endoscopy from this stage, at which neovascularization was induced by various angiogenic factors, and the hypoxic state was thought to be resolved.\\u003c/p\\u003e \\u003cp\\u003eThe expression status of HIF-1 in the early stages of cancer growth and development varies depending on the organ. Various reports have shown early gastric cancer expression rates of 15, 61, and 100%, with no consensus on the topic [\\u003cspan additionalcitationids=\\\"CR17\\\" citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e]. Only one report demonstrated that HIF-1α is expressed in 51% of early esophageal carcinomas [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e], and no studies have scored and compared the expression status of HIF-1α in superficial esophageal carcinoma cases according to invasion depth, as in the present study.\\u003c/p\\u003e \\u003cp\\u003eHIF-1 is composed of HIF-1α and HIF-1β, and the protein expression level of HIF-1α regulates the hypoxic response. This is because HIF-1β is a constant nuclear protein, whereas HIF-1α is degraded by proteasomes under normal oxygen concentration and partial pressure. This degradation occurs because the von Hippel Lindau gene binds to the oxygen-dependent degradation domain of HIF-1α through the ubiquitin E3 ligase complex, which hydroxylates proline residues. Under a hypoxic state, degradation by ubiquitination is suppressed, and HIF-1α accumulates in the cytoplasm. HIF-1α is then translocated into the nucleus and forms a dimer with the β subunit, after which it forms a complex with CBP/p300 and binds to hypoxia-responsive elements, which are target genes that induce the transcription of various growth factors. Genes that are reportedly induced include \\u003cem\\u003eEPO\\u003c/em\\u003e, vascular endothelial growth factor (\\u003cem\\u003eVEGF\\u003c/em\\u003e) and its receptor endothelin 1, plasminogen activators, platelet-derived growth factor, glucose transporters, and tyrosine hydroxylase [\\u003cspan additionalcitationids=\\\"CR20\\\" citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn a previous study, we showed that VEGF-A, which is induced by HIF-1α, is expressed in esophageal mucosal carcinoma and similarly strongly expressed even when invading deeper than T1a-MM [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. This result is inconsistent with the expression status of HIF-1α reported in the present study. However, VEGF-A is also induced by other cytokines, such as cyclooxygenase (COX)-2. We previously reported that COX-2 is strongly expressed at T1a-MM and deeper [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. Therefore, VEGF-A expression is thought to be induced by the action of multiple angiogenesis factors.\\u003c/p\\u003e \\u003cp\\u003eEpo, which is mainly produced in the tubulointerstitial cells of the kidney, is a hematopoietic growth factor that promotes the differentiation and proliferation of erythropoietic cells by binding to the erythropoietin receptor (EPOR) present on erythroid progenitor cells in bone marrow. Recently, Epo has also been reported to have angiogenesis-promoting effects [\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. The presence of Epo and EPOR has been reported in malignant tumor cells, such as neuroblastoma and uterine, ovarian, and breast cancers, and it has been suggested that they affect tumor growth and angiogenesis [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. The present study showed that the expression status of Epo is very similar to that of HIF1, and Epo is speculated to affect angiogenesis in the early stages of esophageal carcinoma growth and development.\\u003c/p\\u003e \\u003cp\\u003e In the present study, we used the H score obtained via IHC for scoring. Using this method enables the scoring of the expression intensity determined by IHC across a wide range from 0 to 300 points. The current standard is for investigators to conduct manual evaluations, but reproducibility when examining multiple cases is a challenge. Image analysis using software has been attempted to solve this problem, and a significant correlation with visual examination results has been observed [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eThis study had some limitations. First, the number of cases was limited, and the cases were from a single institution; further studies with more cases are needed in the future. Second, the present study only included superficial esophageal carcinoma cases. HIF-1α is reportedly strongly expressed in advanced esophageal carcinoma cases and associated with prognosis [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e], with similar results obtained in cervical cancer cases [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. In the present study, HIF-1α was positive from T1a-EP/LPM, with decreased expression from T1a-MM and deeper. In the future, there is a need to examine advanced esophageal carcinoma cases as well. Third, our study targeted only carcinoma cells, and stromal cells, such as macrophages, were not investigated. Examining the roles of HIF-1α in cancer progression, as well as its association with cells in the tumor stroma, such as macrophages, is required.\\u003c/p\\u003e \\u003cp\\u003eRecently, whole-genome analysis using next-generation sequencers has advanced, and new genomic abnormalities have been discovered in somatic cells related to cancer growth and development. Previous research has shown that carcinoma invasion beyond the muscularis mucosae in esophageal carcinoma cases causes several changes in the expression status of angiogenic factors and angiogenesis inhibition factors. [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]A comprehensive analysis of how genetic mutations occur in the process of progression from mucosal to invasive cancer will lead to an elucidation of the mechanisms of cancer growth and development.\\u003c/p\\u003e \\u003cp\\u003eIn conclusion, we showed that, in superficial esophageal carcinoma cases, both HIF-1α and Epo were expressed from T1a-EP/LPM, with expression tending to decrease from T1a-MM and deeper. Moreover, carcinoma cells were in a hypoxic state in the early stages of proliferation, and the hypoxic state was surmised to be resolved in carcinomas at T1a-MM and deeper, where angiogenesis is active.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments:\\u0026nbsp;\\u003c/strong\\u003eThis manuscript is supported by MEXT Kakenhi (grant number 19K08477).\\u0026nbsp;We would like to thank Editage (www.editage.com) for English language editing.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions:\\u003c/strong\\u003e E.Y. and Y.K., study concept and design, analysis and interpretation of data, and writing of the report. T.T., analysis and interpretation of data. M.H., A.T., and M.O., acquisition of data. H.I., critical revision of the manuscript for important intellectual content and material support. All authors agreed to submit this manuscript for publication.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest:\\u0026nbsp;\\u003c/strong\\u003eAll authors declare that they have no conflict of interest.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis study was conducted under a protocol approved by the ethics committee of Saitama Medical Center, Saitama Medical University (1968-II). All experiments were performed in accordance with relevant guidelines and regulations. Opt-out informed consent was obtained from all patients.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eCarmeliet, P. \\u0026amp; Jain, R. K. Angiogenesis in cancer and other diseases. \\u003cem\\u003eNature\\u003c/em\\u003e \\u003cstrong\\u003e407\\u003c/strong\\u003e, 249\\u0026ndash;257; 10.1038/35025220 (2000).\\u003c/li\\u003e\\n\\u003cli\\u003eKumagai, Y., Inoue, H., Nagai, K., Kawano, T. \\u0026amp; Iwai, T. Magnifying endoscopy, stereoscopic microscopy, and the microvascular architecture of superficial esophageal carcinoma. \\u003cem\\u003eEndoscopy\\u003c/em\\u003e \\u003cstrong\\u003e34\\u003c/strong\\u003e, 369\\u0026ndash;375; 10.1055/s-2002-25285 (2002).\\u003c/li\\u003e\\n\\u003cli\\u003eKumagai, Y. et al. Angiogenesis in superficial esophageal squamous cell carcinoma: assessment of microvessel density based on immunostaining for CD34 and CD105. \\u003cem\\u003eJpn. J. Clin. 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Critical role for PI 3-kinase in the control of erythropoietin-induced erythroid progenitor proliferation. \\u003cem\\u003eBlood\\u003c/em\\u003e \\u003cstrong\\u003e101\\u003c/strong\\u003e, 3436\\u0026ndash;3443; 10.1182/blood-2002-07-2332 (2003).\\u003c/li\\u003e\\n\\u003cli\\u003eAnnese, T., Tamma, R., Ruggieri, S. \\u0026amp; Ribatti, D. Erythropoietin in tumor angiogenesis. \\u003cem\\u003eExp. Cell Res\\u003c/em\\u003e. \\u003cstrong\\u003e374\\u003c/strong\\u003e, 266\\u0026ndash;273; 10.1016/j.yexcr.2018.12.013 (2019).\\u003c/li\\u003e\\n\\u003cli\\u003eHardee, M. E., Arcasoy, M. O., Blackwell, K. L., Kirkpatrick, J. P. \\u0026amp; Dewhirst, M. W. Erythropoietin biology in cancer. \\u003cem\\u003eClin. Cancer Res\\u003c/em\\u003e. \\u003cstrong\\u003e12\\u003c/strong\\u003e, 332\\u0026ndash;339; 10.1158/1078-0432.CCR-05-1771 (2006).\\u003c/li\\u003e\\n\\u003cli\\u003eRam, S. et al. Pixelwise H-score: A novel digital image analysis-based metric to quantify membrane biomarker expression from immunohistochemistry images. \\u003cem\\u003ePLOS ONE\\u003c/em\\u003e. \\u003cstrong\\u003e16\\u003c/strong\\u003e, e0245638; 10.1371/journal.pone.0245638 (2021).\\u003c/li\\u003e\\n\\u003cli\\u003eBirner, P., Schindl, M., Obermair, A., Plank, C., Breitenecker, G. \\u0026amp; Oberhuber, G. Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer. \\u003cem\\u003eCancer Res\\u003c/em\\u003e. \\u003cstrong\\u003e60\\u003c/strong\\u003e, 4693\\u0026ndash;4696 (2000).\\u003c/li\\u003e\\n\\u003c/ol\\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\":\"info@researchsquare.com\",\"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\":\"esophageal carcinoma, hypoxia-inducible factor-1α, erythropoietin, angiogenesis\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4452751/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4452751/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eHypoxia-inducible factor (HIF-1α) is the initial switch in angiogenesis; however, its expression in superficial esophageal carcinoma is unclear. We determined the timing of the hypoxia-induced angiogenesis switch by investigating the expression of HIF-1α and the erythropoietin (Epo) it activates in superficial esophageal carcinoma. We used 53 lesions of superficial esophageal carcinoma and conducted immunohistochemistry of HIF-1α and Epo. Cases were divided into three groups: T1a-EP/LPM (22 lesions), T1a-MM/T1b-SM1 (15 lesions), and T1b-SM2,3 (16 lesions). We compared HIF-1α and Epo expression in the deepest area and examined the correlation between HIF-1α and Epo scores. In 24 cases of T1a-MM or deeper carcinomas with intraepithelial spread, we compared HIF-1α and Epo scores in the intraepithelial spread and deepest areas in the same cases. Both HIF-1α and Epo were most strongly expressed in T1a-EP/LPM, with significant attenuation at T1a-MM and deeper. Both HIF-1α and Epo had significantly higher scores in the T1a-EP/LPM group than in the T1a-MM/T1b-SM1 and T1b-SM2,3 groups. HIF-1α and Epo were strongly correlated, and were significantly higher in intraepithelial spread areas than in invaded areas. HIF-1α and Epo are strongly expressed from an early stage of esophageal carcinoma; as the carcinoma invades the muscularis mucosae and deeper, expression decreases.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Relationship between angiogenesis and hypoxia-inducible factor-1α and erythropoietin in superficial esophageal carcinoma\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-07-08 17:03:13\",\"doi\":\"10.21203/rs.3.rs-4452751/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"e92ce74b-af78-4cd2-9c9b-58f303c0d4e9\",\"owner\":[],\"postedDate\":\"July 8th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":33639406,\"name\":\"Biological sciences/Cancer\"},{\"id\":33639407,\"name\":\"Health sciences/Gastroenterology\"}],\"tags\":[],\"updatedAt\":\"2025-06-23T09:39:11+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-07-08 17:03:13\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4452751\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4452751\",\"identity\":\"rs-4452751\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}