The prognostic significance of co-expression of α-SMA and Trichrome in the tumor microenvironment of poorly differentiated endometrial carcinoma

The prognostic significance of co-expression of α-SMA and Trichrome in the tumor microenvironment of poorly differentiated endometrial 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 Research Article The prognostic significance of co-expression of α-SMA and Trichrome in the tumor microenvironment of poorly differentiated endometrial carcinoma Xi Chen, Yuan Cheng, Na Zhao, Fangfang Liu, Jianliu Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2479907/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 Backgroud: In recent years, the relationship between tumor microenvironment (TME) and adjuvant therapy has become a research hotspot. α-SMA is heterogeneous in different tumors. Type I collagen deposition influenced the access of immune cells to cancer cells. This study aims to describe the prognostic significance of co-expression of α-SMA and Trichrome in the tumor microenvironment of poorly differentiated endometrial carcinoma. Methods A total of 45 cases of poorly differentiated endometrial carcinoma (EC) from March 2013 to December 2020 was collected in the study. There were 17 cases of grade 3 endometrioid endometrial carcinomas (EECs), 16 cases of endometrial serous carcinomas, and 12 cases of endometrial clear cell carcinomas. The expression of α-SMA, CD10, and desmin was detected in the tumor microenvironment (TME) by the immunohistochemical method. Trichrome was detected to identify collagen in TME by a special dyeing method. Between-group comparison in categorical variables was assessed via using Fisher’s exact test. The overall survival was analyzed by a Kaplan-Meier curve. Results The total expression rate of α-SMA in endometrial carcinoma was 71.1%. The expression rate of α-SMA was 82.4%, 62.5%, and 66.7% positively expressed in EEC, serous carcinoma, and clear cell carcinoma, respectively. CD10 and desmin were negative in all EC TME. Both α-SMA and Trichrome positive groups showed significantly higher mortality rates ( p = 0.042) and poorer prognosis ( p = 0.041) than both α-SMA and Trichrome negative groups. Conclusions The co-expression of α-SMA and Trichrome is of great significance in predicting the poor prognosis of endometrial cancer. endometrial carcinoma immunohistochemistry α-SMA Trichrome prognosis Figures Figure 1 Figure 2 Introduction Endometrial carcinoma (EC) is a common malignant tumor of the female reproductive system. It ranks first in incidence and second in mortality rate among gynecologic cancers. 1 Jan V. Bokhman first proposed a dualistic view of ECs in 1983. ECs could be classified into 2 broad categories: “Type I” (mainly endometrioid endometrial carcinoma, EEC) and “Type II” (Non-endometrioid tumors). The most common ones of the latter are endometrial serous carcinoma (ESC) and endometrial clear cell carcinoma (ECCC). 2 ESC, ECCC, and grade 3 EEC are highly malignant and are the cause of death in most patients. 3 Surgery plus postoperative adjuvant chemoradiotherapy is the classic treatment for grade 3 EEC and type II EC. However, the role of these treatment options in improving prognosis, overall survival (OS), and relapse-free survival (RFS) of these poorly differentiated ECs is controversial. 4 , 5 In recent years, the relationship between tumor microenvironment (TME) and adjuvant therapy including targeted therapy and immunotherapy has become a research hotspot. Some researchers think that the host’s robust response to evolving cancer cells results in the formation of TME or tumor stroma, which includes cancer-associated fibroblasts (CAFs), immune cells, capillaries, basement membrane, and extracellular matrix (ECM). 6 CAFs, deriving from activation of normal resident tissue quiescent fibroblasts or transdifferentiation of non-fibroblastic cells, are the most abundant and main component of TME. 7 Many different biomarkers can identify CAFs, such as α-smooth muscle actin (α-SMA), fibroblasts specific protein 1 (FSP1/S100A4), vimentin, fibroblast activation protein (FAP), platelet-derived growth factor receptor-α (PDGFRα), PDGFRβ, desmin, discoidin domain-containing receptor 2 (DDR2) and so on. 6 As the first discovered and most common CAFs biomarker, α-SMA is heterogeneous in different tumors. 8 Studies of some malignant tumors such as gastric adenocarcinomas, retroperitoneal leiomyosarcomas, and ovarian epithelial carcinomas have verified that α-SMA + CAFs could promote tumorigenesis, tumor proliferation, and metastasis, impede drug delivery and mediate immunosuppressive. 9 – 12 Studies showed that α-SMA + CAFs could produce type I collagen. 12 , 13 As a part of ECM remodeling, type I collagen deposition influenced the access of immune cells to cancer cells. 14 However, other studies showed that α-SMA + CAFs might be tumor-restraining because of the improved prognosis and overall survival they found among pancreatic ductal adenocarcinoma (PDAC), breast and lung cancer patients with high desmoplasia. 13 , 15 There is no research report on the prognostic significance of co-expression of α-SMA and Trichrome in the tumor microenvironment of poorly differentiated endometrial cancer after reviewing the kinds of literature. Materials And Methods Patients 122 cases with grade 3 endometrioid endometrial carcinoma (EECG3), ESC, and ECCC in Peking University People’s Hospital from March 2013 to December 2020 were collected in the retrospective study. The main exclusion criteria were cases with preoperative chemotherapy, radiotherapy, or endocrine therapy, mixed carcinomas, cases with incomplete clinicopathological data or inability to obtain tissue wax blocks, and cases with tumors showing invasive growth into the myometrium. Finally, forty-five patients who have complete medical records and follow-up data were included in the study. Immunohistochemistry and special dyeing methods The paraffin-embedded sections were cut for Hematoxylin-Eosin (HE) staining and Trichrome staining. Immunohistochemistry was performed concerning our previous study. 16 Incubation with mouse antibodies against human α-SMA (GA-0106, China), CD10 (GC-0371, China), desmin (ZA-0610, China) antibody was performed at 4℃ overnight and followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (GB23301 for goat anti-mouse, China). Positive was defined when Trichrome staining showed blue. α-SMA was judged to be negative when it showed no expression or was expressed only in a few proliferating or aggregated fibroblasts and diffusely positive when it was expressed in a large number of fibroblasts that aggregated into bundles. Statistical analysis Statistical analyses were performed via using SPSS software (version 26.0, IBM, Armonk, New York, USA). Categorical variables are expressed as counts and percentages. Between-group comparison in categorical variables was tested with Fisher’s exact test. A two-sided P value < 0.05 was defined as statistically significant. Kaplan-Meier product-limit method was applied for the survival analysis. The difference of survival between different groups was estimated by the 2-side log-rank test. The level of significance was set as a P value < 0.05. The Kaplan-Meier curve was drawn by GraphPad Prism (version 8.0). Results A total of 45 cases of poor differentiated endometrial carcinoma (EC) were included in the study. There were 17 grade 3 endometrioid endometrial carcinomas (EECs), 16 endometrial serous carcinomas (ESCs), and 12 endometrial clear cell carcinomas (ECCCs). Microscopically, we found the disappearance of the normal endometrial stroma in the TME of poorly differentiated ECs, replaced by the accumulation of fibroblasts. In some cases, fibroblasts aggregated into bundles. The expression of CD10, SMA, desmin, and Trichrome was detected in all 45 cases by the IHC method in order to make clear the property of the tumor stroma. The total expression of α-SMA was 71.1% (32/45) in three types of cancer. The expression of α-SMA was 17.6% (3/17) negative and 82.4% (14/17) positive in EEC cases. The expression of α-SMA was 37.5% (6/16) negative and 62.5% (10/16) positive in ESC cases. The expression of α-SMA was 33.3% (4/12) negative and 66.7% (8/12) positive in ECCC cases. All α-SMA diffusely positive cases showed Trichrome positive simultaneously, and Trichrome expressed negative when α-SMA negative. CD10 and desmin showed negative in ECs tumor stroma. (Fig. 1 ) To further explore the relationship between α-SMA expression and clinicopathologic features, we divided 45 EC cases into two groups, α-SMA negative and α-SMA positive group. Statistical analysis showed that the expression of α-SMA was not associated with age, FIGO stage, histopathologic type, tumor size, myometrium infiltration, LVSI, and lymph nodes metastasis of these 45 EC patients ( p > 0.05). However, the mortality rate of the α-SMA positive group was significantly higher than that in the α-SMA negative group ( p = 0.042) (Table 1 ). Then we performed a Kaplan-Meier survival analysis between these two groups. The overall survival of the positive group was significantly worse than the negative group ( p = 0.041) (Fig. 2 ). Table 1 The relationship between α-SMA expression and clinicopathologic factors in ECs Total No. α-SMA expression in TME P Negative (N = 13) Positive (N = 32) Age (y) ༜60 19 7 (36.8) 12 (63.2) 0.341 ≥ 60 26 6 (23.1) 20 (76.9) FIGO stage I 29 10 (34.5) 19 (65.5) 0.816 II 3 0 (0.0) 3 (100.0) III 11 3 (27.3) 8 (72.7) IV 2 0 (0.0) 2 (100.0) Histopathologic type Endometrioid 17 3 (17.6) 14 (82.4) 0.421 Serous 16 6 (37.5) 10 (62.5) Clear cell 12 4 (32.3) 8 (66.7) Tumor size (cm) ༜3 25 7 (28.0) 18 (72.0) > 0.999 ≥ 3 20 6 (30.0) 14 (70.0) Myometrium infiltration ༜50% 26 8 (30.8) 18 (69.2) > 0.999 ≥ 50% 19 5 (26.3) 14 (73.7) LVSI No 24 8 (33.3) 16 (66.7) 0.528 Yes 21 5 (23.8) 16 (76.2) Lymph nodes metastasis No 35 10 (28.6) 25 (71.4) > 0.999 Yes 10 3 (30.0) 7 (70.0) Death No 36 13 (36.1) 23 (63.9) 0.042* Yes 9 0 (0.0) 9 (100.0) α-SMA: α-smooth muscle actin, TME: tumor microenvironment, LVSI: lymph-vascular space invasion. P* : P < 0.05. Discussion Endometrial cancer is a common malignant tumor of the female reproductive system, and its incidence ranks second in China. Our study found that postoperative adjuvant chemoradiotherapy for endometrial cancer, including EEC G3 and type II endometrial cancer, had no significant effect on improving overall survival, disease-free survival, and recurrence-free survival. The tumor microenvironment plays an important role in the treatment of tumors. The tumor microenvironment (TME) and the ecosystem formed by the tumor itself have an important impact on the treatment of tumors. cancer-associated fibroblasts (CAFs) were identified in many TME studies of malignant tumors, but their biomarkers and functions upon different types of tumors were heterogeneous. 9 – 14 The heterogeneity of CAFs reflects in their origin and function. Most of CAFs are activated from normal resident tissue quiescent fibroblasts. They could also derive from transdifferentiation of non-fibroblastic cells such as pericytes, adipocytes, chondrocytes, bone marrow-derived precursors, mesenchymal stem cells (MSCs), endothelial cells, epithelial cells, liver, and pancreas stellate cells and so on. 6 – 8 Kalluri proposed that CAFs could be divided into five subtypes on the basis of function, tumor-restraining (F1 subtype), tumor-promoting (F2 subtype), secretory (F3 subtype), ECM-remodeling (F4 subtype) and other functions (F5 subtype) CAFs. 6 In fact, if merely focused on their effect on tumors, CAFs can be simply classified into two subtypes, tumor-promoting CAFs and tumor-restraining CAFs. On one hand, CAFs may participate in the proliferation, metastasis, immunosuppressive, and drug resistance of cancer cells through ECM remodeling, immune-modulating, metabolic reprogramming, and secretion of growth factors, cytokines and chemokines. 6 , 17 , 18 On the other hand, CAFs may arrest tumor growth and metastasis by influencing host defense through the modulation of innate and adaptive tumor immunity. 19 As the first identified and most frequent CAFs biomarker, α-SMA has proved to be heterogeneous on the basis of its functions upon different tumors. Zhan observed that high expression of α-SMA was associated with poor prognosis in 268 gastric adenocarcinomas. 9 Ma also found that α-SMA was an indicator of poor prognosis in 50 cases of retroperitoneal leiomyosarcoma. 10 While Ozdemir showed that direct depletion of α-SMA + CAFs led to increased tumor invasion associated with decreased survival of mice with pancreatic ductal adenocarcinoma (PDAC) by inducing immunosuppression. 13 In our study, although the relationship between α-SMA expression and FIGO stage, myometrium infiltration, LVSI, and lymph nodes metastasis were of no statistically significance, probably for the reason that all the EC cases included were poorly differentiated. However, the mortality rate of the α-SMA positive group was significantly higher than that in the α-SMA negative group ( p = 0.042). This result suggested that α-SMA + CAFs in the ECs stroma may protect and promote tumor growth. CD10 is a marker of the normal endometrial stroma. CD10 showed negative in all ECs tumor stroma which indicated that all normal endometrial stroma has disappeared. α-SMA + CAFs could produce type I collagen. type I collagen crosslinking leads to the formation of collagen fibers. α-SMA + myofibroblasts depletion reduced type I collagen content. 12 , 13 All α-SMA positive cases showed Trichrome positive, which further confirmed that collagen fibers had formed in the tumor stroma of ECs. It was consistent with a study made by Ozdemir. 13 We also observed that Trichrome expressed negative when α-SMA negative. This means that collagen fibers cannot be formed when only a few α-SMA + fibroblasts focally proliferated and gathered in the tumor stroma. The appearance of collagen fibers might be a hint of further deterioration of the tumor. Therefore, we considered that the tumor stroma in ECs first undergoes the disappearance of normal endometrial stroma, the aggregation and proliferation of fibroblasts, and finally aggregates into fibrous bundles and produces type I collagen, which maintains tumor growth and defends against the attack of therapeutic drugs. Probably this is the process of CAFs formation in ECs. Both α-SMA and desmin can be expressed in myofibroblasts and smooth muscle cells. But the desmin showed negative in ECs tumor stroma in this study, which demonstrated that the α-SMA + CAFs did not show the characteristics of muscle fiber tissue and smooth muscle tissue. Here, the number of cases included was limited due to our strict exclusion criteria. Next, we will increase the number of cases and further study the mechanism of the interaction of α-SMA + CAF, chemotherapy, and immunotherapy. Our research will provide ideas for the precision treatment of poorly differentiated ECs. Conclusion The co-expression of α-SMA and Trichrome in TME proves the formation of CAFs and suggests poor prognosis. Abbreviations α-SMA: α-smooth muscle actin; CAFs: cancer-associated fibroblasts; DDR2: discoidin domain-containing receptor 2; EC: endometrial carcinoma; ECCC: endometrial clear cell carcinoma; ECM: extracellular matrix; EEC: endometrioid endometrial carcinoma; ESC: endometrial serous carcinoma; FAP: fibroblast activation protein; FSP1/S100A4: fibroblasts specific protein 1; HE: Hematoxylin-Eosin; HRP: horseradish peroxidase; LVSI: lymph-vascular space invasion; MSCs: mesenchymal stem cells; OS: overall survival; PDAC: pancreatic ductal adenocarcinoma; PDGFRα: platelet-derived growth factor receptor-α; RFS: relapse-free survival; TME: tumor microenvironment. Declarations Author contribution Both Fangfang Liu and Jianliu Wang was responsible for the conception and design of the research and the revision of the manuscript. Fangfang Liu carried out the assessment of IHC staining. Xi Chen and Yuan Cheng carried out the statistical analysis. Na Zhao made the chart. Xi Chen contributed to the preparation of the manuscript. Funding This study was supported by National Natural Science Foundation of China (Grant Nos: 82072861, 81672571, and 81874108). Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Sample collection was approved by the Hospital Ethics Review. Consent for publication Not applicable. Conflict of interest All authors declare no conflicts of interests. Author details 1 Department of Obstetrics and Gynecology, Peking University People’s Hospital, Beijing, China 2 Department of Pathology, Peking University First Hospital, Beijing, China Acknowledgments The authors thank Laboratory of Gynecology and Department of Pathology to support our study. References Siegel RL, Miller KD, Fuchs HE, et al . Cancer Statistics. CA Cancer J Clin 2021; 71(1): 7-33. SuarezAA, Felix AS, Cohn DE. Bokhman Redux: Endometrial cancer "types" in the 21st century. Gynecol Oncol 2017; 144(2): 243–249. Gulec UK, Bagir EK, Paydas S, et al . Programmed death‑1 (PD‑1) and programmed death‑ligand 1 (PD‑L1) expressions in type 2 endometrial cancer. Gynecology and Obstetrics 2019; 300: 377–382. Cook A, Khalil R, Burmeister C, et al .The Impact of Adjuvant Management Strategies on Outcomes in Women With Early Stage Uterine Serous Carcinoma. Cureus 2021; 13(2): 1-9. Han Y & Liu C. Clinicopathological characteristics and prognosis of uterine serous carcinoma: A SEER program analysis of 1016 cases. J. Obstet. Gynaecol. Res 2021; 47(7): 2460-2472. Kalluri. The biology and function of fibroblasts in cancer. Nat Rev Cancer 2016; 16(9): 582-98. Han C, Liu T, Yin R. Biomarkers for cancer-associated fibroblasts. Biomarker Research 2020; 8: 64. Park D, Sahai E, Rullan A. SnapShot: Cancer-Associated Fibroblasts. Cell 2020; 181(2): 486-486. Zhan S, Liu Z, Zhang M, et al . Overexpression of B7-H3 in α-SMA-Positive Fibroblasts Is Associated With Cancer Progression and Survival in Gastric Adenocarcinomas. Front Oncol 2020; 9: 1466. Ma C, Li P, Zhang N, et al . Prognostic factors analysis of Ki-67, α-SMA expression in retroperitoneal leiomyosarcoma. Zhonghua Zhong Liu Za Zhi 2018; 40(4): 258-263. Cui Y, Wang D, Xie M. Tumor-Derived Extracellular Vesicles Promote Activation of Carcinoma-Associated Fibroblasts and Facilitate Invasion and Metastasis of Ovarian Cancer by Carrying miR-630. Front Cell Dev Biol 2021; 9: 652322. Anggorowati N, Kurniasari CR, Damayanti K, et al . Histochemical and Immunohistochemical Study of α-SMA, Collagen, and PCNA in Epithelial Ovarian Neoplasm. Asian Pac J Cancer Prev 18 (3): 667-671. Ozdemir BC, Pentcheva-Hoang T, Carstens JL, et al . Depletion of carcinoma associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell 2014; 25: 719–34. Chakravarthy A, Khan L, Bensle NP, et al . TGF-β-associated extracellular matrix genes link cancer-associated fibroblasts to immune evasion and immunotherapy failure. Nat Commun 2018; 9(1): 4692. Wang WQ, Liu L, Xu HX, et al . Intratumoral alpha-SMA enhances the prognostic potency of CD34 associated with maintenance of microvessel integrity in hepatocellular carcinoma and pancreatic cancer. PLoS One 2013; 8(8). Fangfang Liu, Danhua Shen,Xiaoping Kang,et al. New tumor antigen PLAC1/CP1, a potentially useful prognostic marker and immunotherapy target for gastric adenocarcinoma. J Clin Pathol 2015; 68(11):913-916. Liu FF, Dong XY, Pang XW, et al. The specific immune response to tumor antigen CP1 and its correlation with improved survival in colon cancer patients. Gastroenterology 2008;134 (4): 998-1006. Lau EY, Lo J, Cheng BY, et al . Cancer-associated fibroblasts regulate tumor-initiating cell plasticity in hepatocellular carcinoma through c-met/FRA1/HEY1 signaling. Cell Rep 2016; 15: 1175–89. Huang M, Li Y, Zhang H, et al . Breast cancer stromal fibroblasts promote the generation of CD44+CD24- cells through SDF-1/CXCR4 interaction. J Exp Clin Cancer Res 2010; 29:80. 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-2479907","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":168343047,"identity":"d0a7f10d-cade-42c6-a73a-f33c28f21450","order_by":0,"name":"Xi Chen","email":"","orcid":"","institution":"Peking University People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Chen","suffix":""},{"id":168343048,"identity":"6fef1c54-b5ec-46ae-8954-fad18f89a48b","order_by":1,"name":"Yuan Cheng","email":"","orcid":"","institution":"Peking University People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Cheng","suffix":""},{"id":168343049,"identity":"653db46d-9f7e-4477-80ef-3d1d0fadd693","order_by":2,"name":"Na Zhao","email":"","orcid":"","institution":"Peking University People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Zhao","suffix":""},{"id":168343050,"identity":"dc2d703a-0c5a-4be8-aa2c-fd1b35c8e8cb","order_by":3,"name":"Fangfang Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYHACAwkwycB8gOGBAWla2BIYEkjUwmPAkECMevP25o03PrbZMJjzr/n4IaHgcOJ2BuaHj27g0SJz5lix5cy2NAbLGW83SyQYHE7c2cBmbJyDR4uERI6ZNM+ZwwwGN85uAGvZcICHTRqvFvk3IC3/gVrOPP5BnBYJHqCWigMMBud72Ii0hSet2HJGRTLQFjYziwSDdOMNhwn5hf3wxhsfDOyAthx+fOPDH2vZDcebHz7GpwUG6hskEkB0MwMDMxHKIYD/AIisI1r9KBgFo2AUjBwAAHfgTvyJ6jIEAAAAAElFTkSuQmCC","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Fangfang","middleName":"","lastName":"Liu","suffix":""},{"id":168343051,"identity":"ec544ba0-7c9b-46d6-b776-5575de39c3dc","order_by":4,"name":"Jianliu Wang","email":"","orcid":"","institution":"Peking University People’s Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianliu","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2023-01-15 08:14:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2479907/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2479907/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":31758466,"identity":"6c68fc40-881c-4de9-bb01-0cca18d15ad2","added_by":"auto","created_at":"2023-01-18 15:58:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":743845,"visible":true,"origin":"","legend":"\u003cp\u003eA. Grade 3 endometrioid endometrial carcinomas, HE, 100x. B. The expression of α-SMA in tumor stroma, SP, 100x. C. The expression of Trichrome in tumor stroma, SD, 100x.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2479907/v1/4fa6102e6b6f3ca3e6f242da.png"},{"id":31759423,"identity":"525fae0e-2e42-4921-b48d-dac71398a5c6","added_by":"auto","created_at":"2023-01-18 16:06:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":133108,"visible":true,"origin":"","legend":"\u003cp\u003eKeplan-Meier curve of overall survival according to the expression of α-SMA (α-SMA\u003csup\u003e-\u003c/sup\u003e vs α-SMA\u003csup\u003e+\u003c/sup\u003e,\u003cem\u003e p\u003c/em\u003e=0.041).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2479907/v1/2752655ac93f2dd1b5a24a1f.png"},{"id":31961190,"identity":"e4eae556-545b-46d2-ad01-4b57049b3b3b","added_by":"auto","created_at":"2023-01-23 18:29:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1136960,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2479907/v1/7d3a3345-5f3e-4616-a1b8-3f59ef4fccbc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The prognostic significance of co-expression of α-SMA and Trichrome in the tumor microenvironment of poorly differentiated endometrial carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometrial carcinoma (EC) is a common malignant tumor of the female reproductive system. It ranks first in incidence and second in mortality rate among gynecologic cancers.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Jan V. Bokhman first proposed a dualistic view of ECs in 1983. ECs could be classified into 2 broad categories: \u0026ldquo;Type I\u0026rdquo; (mainly endometrioid endometrial carcinoma, EEC) and \u0026ldquo;Type II\u0026rdquo; (Non-endometrioid tumors). The most common ones of the latter are endometrial serous carcinoma (ESC) and endometrial clear cell carcinoma (ECCC).\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e ESC, ECCC, and grade 3 EEC are highly malignant and are the cause of death in most patients.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Surgery plus postoperative adjuvant chemoradiotherapy is the classic treatment for grade 3 EEC and type II EC. However, the role of these treatment options in improving prognosis, overall survival (OS), and relapse-free survival (RFS) of these poorly differentiated ECs is controversial.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e In recent years, the relationship between tumor microenvironment (TME) and adjuvant therapy including targeted therapy and immunotherapy has become a research hotspot.\u003c/p\u003e \u003cp\u003eSome researchers think that the host\u0026rsquo;s robust response to evolving cancer cells results in the formation of TME or tumor stroma, which includes cancer-associated fibroblasts (CAFs), immune cells, capillaries, basement membrane, and extracellular matrix (ECM).\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e CAFs, deriving from activation of normal resident tissue quiescent fibroblasts or transdifferentiation of non-fibroblastic cells, are the most abundant and main component of TME.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Many different biomarkers can identify CAFs, such as α-smooth muscle actin (α-SMA), fibroblasts specific protein 1 (FSP1/S100A4), vimentin, fibroblast activation protein (FAP), platelet-derived growth factor receptor-α (PDGFRα), PDGFRβ, desmin, discoidin domain-containing receptor 2 (DDR2) and so on.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e As the first discovered and most common CAFs biomarker, α-SMA is heterogeneous in different tumors.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Studies of some malignant tumors such as gastric adenocarcinomas, retroperitoneal leiomyosarcomas, and ovarian epithelial carcinomas have verified that α-SMA\u003csup\u003e+\u003c/sup\u003e CAFs could promote tumorigenesis, tumor proliferation, and metastasis, impede drug delivery and mediate immunosuppressive.\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Studies showed that α-SMA\u003csup\u003e+\u003c/sup\u003e CAFs could produce type I collagen.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e As a part of ECM remodeling, type I collagen deposition influenced the access of immune cells to cancer cells.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e However, other studies showed that α-SMA\u003csup\u003e+\u003c/sup\u003e CAFs might be tumor-restraining because of the improved prognosis and overall survival they found among pancreatic ductal adenocarcinoma (PDAC), breast and lung cancer patients with high desmoplasia.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e There is no research report on the prognostic significance of co-expression of α-SMA and Trichrome in the tumor microenvironment of poorly differentiated endometrial cancer after reviewing the kinds of literature.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003e122 cases with grade 3 endometrioid endometrial carcinoma (EECG3), ESC, and ECCC in Peking University People\u0026rsquo;s Hospital from March 2013 to December 2020 were collected in the retrospective study. The main exclusion criteria were cases with preoperative chemotherapy, radiotherapy, or endocrine therapy, mixed carcinomas, cases with incomplete clinicopathological data or inability to obtain tissue wax blocks, and cases with tumors showing invasive growth into the myometrium. Finally, forty-five patients who have complete medical records and follow-up data were included in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry and special dyeing methods\u003c/h2\u003e \u003cp\u003eThe paraffin-embedded sections were cut for Hematoxylin-Eosin (HE) staining and Trichrome staining. Immunohistochemistry was performed concerning our previous study.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Incubation with mouse antibodies against human α-SMA (GA-0106, China), CD10 (GC-0371, China), desmin (ZA-0610, China) antibody was performed at 4℃ overnight and followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (GB23301 for goat anti-mouse, China). Positive was defined when Trichrome staining showed blue. α-SMA was judged to be negative when it showed no expression or was expressed only in a few proliferating or aggregated fibroblasts and diffusely positive when it was expressed in a large number of fibroblasts that aggregated into bundles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed via using SPSS software (version 26.0, IBM, Armonk, New York, USA). Categorical variables are expressed as counts and percentages. Between-group comparison in categorical variables was tested with Fisher\u0026rsquo;s exact test. A two-sided \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was defined as statistically significant. Kaplan-Meier product-limit method was applied for the survival analysis. The difference of survival between different groups was estimated by the 2-side log-rank test. The level of significance was set as a \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The Kaplan-Meier curve was drawn by GraphPad Prism (version 8.0).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 45 cases of poor differentiated endometrial carcinoma (EC) were included in the study. There were 17 grade 3 endometrioid endometrial carcinomas (EECs), 16 endometrial serous carcinomas (ESCs), and 12 endometrial clear cell carcinomas (ECCCs). Microscopically, we found the disappearance of the normal endometrial stroma in the TME of poorly differentiated ECs, replaced by the accumulation of fibroblasts. In some cases, fibroblasts aggregated into bundles. The expression of CD10, SMA, desmin, and Trichrome was detected in all 45 cases by the IHC method in order to make clear the property of the tumor stroma. The total expression of α-SMA was 71.1% (32/45) in three types of cancer. The expression of α-SMA was 17.6% (3/17) negative and 82.4% (14/17) positive in EEC cases. The expression of α-SMA was 37.5% (6/16) negative and 62.5% (10/16) positive in ESC cases. The expression of α-SMA was 33.3% (4/12) negative and 66.7% (8/12) positive in ECCC cases. All α-SMA diffusely positive cases showed Trichrome positive simultaneously, and Trichrome expressed negative when α-SMA negative. CD10 and desmin showed negative in ECs tumor stroma. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore the relationship between α-SMA expression and clinicopathologic features, we divided 45 EC cases into two groups, α-SMA negative and α-SMA positive group. Statistical analysis showed that the expression of α-SMA was not associated with age, FIGO stage, histopathologic type, tumor size, myometrium infiltration, LVSI, and lymph nodes metastasis of these 45 EC patients (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, the mortality rate of the α-SMA positive group was significantly higher than that in the α-SMA negative group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.042) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Then we performed a Kaplan-Meier survival analysis between these two groups. The overall survival of the positive group was significantly worse than the negative group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.041) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe relationship between α-SMA expression and clinicopathologic factors in ECs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eα-SMA expression in TME\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003cp\u003e(N\u0026thinsp;=\u0026thinsp;32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (y)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e༜60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 (36.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12 (63.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.341\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (23.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20 (76.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFIGO stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10 (34.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19 (65.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.816\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (100.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (27.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (72.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (100.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistopathologic type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEndometrioid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (17.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14 (82.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.421\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (37.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10 (62.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClear cell\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (32.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (66.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor size (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e༜3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 (28.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 (72.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (30.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14 (70.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMyometrium infiltration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e༜50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (30.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 (69.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (26.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14 (73.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLVSI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (33.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16 (66.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.528\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (23.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16 (76.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph nodes metastasis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10 (28.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25 (71.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (30.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7 (70.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDeath\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13 (36.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23 (63.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.042*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9 (100.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eα-SMA: α-smooth muscle actin, TME: tumor microenvironment, LVSI: lymph-vascular space invasion.\u003c/p\u003e \u003cp\u003e \u003cem\u003eP*\u003c/em\u003e: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEndometrial cancer is a common malignant tumor of the female reproductive system, and its incidence ranks second in China. Our study found that postoperative adjuvant chemoradiotherapy for endometrial cancer, including EEC G3 and type II endometrial cancer, had no significant effect on improving overall survival, disease-free survival, and recurrence-free survival. The tumor microenvironment plays an important role in the treatment of tumors. The tumor microenvironment (TME) and the ecosystem formed by the tumor itself have an important impact on the treatment of tumors. cancer-associated fibroblasts (CAFs) were identified in many TME studies of malignant tumors, but their biomarkers and functions upon different types of tumors were heterogeneous.\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e The heterogeneity of CAFs reflects in their origin and function. Most of CAFs are activated from normal resident tissue quiescent fibroblasts. They could also derive from transdifferentiation of non-fibroblastic cells such as pericytes, adipocytes, chondrocytes, bone marrow-derived precursors, mesenchymal stem cells (MSCs), endothelial cells, epithelial cells, liver, and pancreas stellate cells and so on.\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Kalluri proposed that CAFs could be divided into five subtypes on the basis of function, tumor-restraining (F1 subtype), tumor-promoting (F2 subtype), secretory (F3 subtype), ECM-remodeling (F4 subtype) and other functions (F5 subtype) CAFs.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e In fact, if merely focused on their effect on tumors, CAFs can be simply classified into two subtypes, tumor-promoting CAFs and tumor-restraining CAFs. On one hand, CAFs may participate in the proliferation, metastasis, immunosuppressive, and drug resistance of cancer cells through ECM remodeling, immune-modulating, metabolic reprogramming, and secretion of growth factors, cytokines and chemokines.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e On the other hand, CAFs may arrest tumor growth and metastasis by influencing host defense through the modulation of innate and adaptive tumor immunity.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAs the first identified and most frequent CAFs biomarker, α-SMA has proved to be heterogeneous on the basis of its functions upon different tumors. Zhan observed that high expression of α-SMA was associated with poor prognosis in 268 gastric adenocarcinomas.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Ma also found that α-SMA was an indicator of poor prognosis in 50 cases of retroperitoneal leiomyosarcoma.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e While Ozdemir showed that direct depletion of α-SMA\u003csup\u003e+\u003c/sup\u003e CAFs led to increased tumor invasion associated with decreased survival of mice with pancreatic ductal adenocarcinoma (PDAC) by inducing immunosuppression.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e In our study, although the relationship between α-SMA expression and FIGO stage, myometrium infiltration, LVSI, and lymph nodes metastasis were of no statistically significance, probably for the reason that all the EC cases included were poorly differentiated. However, the mortality rate of the α-SMA positive group was significantly higher than that in the α-SMA negative group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.042). This result suggested that α-SMA\u003csup\u003e+\u003c/sup\u003e CAFs in the ECs stroma may protect and promote tumor growth. CD10 is a marker of the normal endometrial stroma. CD10 showed negative in all ECs tumor stroma which indicated that all normal endometrial stroma has disappeared. α-SMA\u003csup\u003e+\u003c/sup\u003e CAFs could produce type I collagen. type I collagen crosslinking leads to the formation of collagen fibers. α-SMA\u003csup\u003e+\u003c/sup\u003e myofibroblasts depletion reduced type I collagen content.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e All α-SMA positive cases showed Trichrome positive, which further confirmed that collagen fibers had formed in the tumor stroma of ECs. It was consistent with a study made by Ozdemir.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e We also observed that Trichrome expressed negative when α-SMA negative. This means that collagen fibers cannot be formed when only a few α-SMA\u003csup\u003e+\u003c/sup\u003e fibroblasts focally proliferated and gathered in the tumor stroma. The appearance of collagen fibers might be a hint of further deterioration of the tumor. Therefore, we considered that the tumor stroma in ECs first undergoes the disappearance of normal endometrial stroma, the aggregation and proliferation of fibroblasts, and finally aggregates into fibrous bundles and produces type I collagen, which maintains tumor growth and defends against the attack of therapeutic drugs. Probably this is the process of CAFs formation in ECs. Both α-SMA and desmin can be expressed in myofibroblasts and smooth muscle cells. But the desmin showed negative in ECs tumor stroma in this study, which demonstrated that the α-SMA\u003csup\u003e+\u003c/sup\u003e CAFs did not show the characteristics of muscle fiber tissue and smooth muscle tissue.\u003c/p\u003e \u003cp\u003eHere, the number of cases included was limited due to our strict exclusion criteria. Next, we will increase the number of cases and further study the mechanism of the interaction of α-SMA\u003csup\u003e+\u003c/sup\u003e CAF, chemotherapy, and immunotherapy. Our research will provide ideas for the precision treatment of poorly differentiated ECs.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe co-expression of α-SMA and Trichrome in TME proves the formation of CAFs and suggests poor prognosis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u0026alpha;-SMA: \u0026alpha;-smooth muscle actin; CAFs: cancer-associated fibroblasts; DDR2: discoidin domain-containing receptor 2; EC: endometrial carcinoma; ECCC: endometrial clear cell carcinoma; ECM: extracellular matrix; EEC: endometrioid endometrial carcinoma; ESC: endometrial serous carcinoma; FAP: fibroblast activation protein; FSP1/S100A4: fibroblasts specific protein 1; HE: Hematoxylin-Eosin; HRP: horseradish peroxidase; LVSI: lymph-vascular space invasion; MSCs: mesenchymal stem cells; OS: overall survival; PDAC: pancreatic ductal adenocarcinoma; PDGFR\u0026alpha;: platelet-derived growth factor receptor-\u0026alpha;; RFS: relapse-free survival; TME: tumor microenvironment.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBoth Fangfang Liu and Jianliu Wang was responsible for the conception and design of the research and the revision of the manuscript. Fangfang Liu carried out the assessment of IHC staining. Xi Chen and Yuan Cheng carried out the statistical analysis. Na Zhao made the chart. Xi Chen contributed to the preparation of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was supported by National Natural Science Foundation of China (Grant Nos: 82072861, 81672571, and 81874108).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u0026nbsp;\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\u0026nbsp;\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSample collection was approved by the Hospital Ethics Review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no conflicts of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Obstetrics and Gynecology, Peking University People\u0026rsquo;s Hospital, Beijing, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eDepartment of Pathology, Peking University First Hospital, Beijing, China\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Laboratory of Gynecology and Department of Pathology to support our study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Miller KD, Fuchs HE, \u003cem\u003eet al\u003c/em\u003e. Cancer Statistics. \u003cem\u003eCA Cancer J Clin\u003c/em\u003e 2021; 71(1): 7-33.\u003c/li\u003e\n\u003cli\u003eSuarezAA, Felix AS, Cohn DE. Bokhman Redux: Endometrial cancer \u0026quot;types\u0026quot; in the 21st century. \u003cem\u003eGynecol Oncol\u003c/em\u003e 2017; 144(2): 243\u0026ndash;249.\u003c/li\u003e\n\u003cli\u003eGulec UK, Bagir EK, Paydas S, \u003cem\u003eet al\u003c/em\u003e. Programmed death‑1 (PD‑1) and programmed death‑ligand 1 (PD‑L1) expressions in type 2 endometrial cancer. \u003cem\u003eGynecology and Obstetrics\u003c/em\u003e 2019; 300: 377\u0026ndash;382.\u003c/li\u003e\n\u003cli\u003eCook A, Khalil R, Burmeister C, \u003cem\u003eet al\u003c/em\u003e.The Impact of Adjuvant Management Strategies on Outcomes in Women With Early Stage Uterine Serous Carcinoma. \u003cem\u003eCureus\u003c/em\u003e 2021; 13(2): 1-9.\u003c/li\u003e\n\u003cli\u003eHan Y \u0026amp; Liu C. 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Type I collagen deposition influenced the access of immune cells to cancer cells. This study aims to describe the prognostic significance of co-expression of α-SMA and Trichrome in the tumor microenvironment of poorly differentiated endometrial carcinoma.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 45 cases of poorly differentiated endometrial carcinoma (EC) from March 2013 to December 2020 was collected in the study. There were 17 cases of grade 3 endometrioid endometrial carcinomas (EECs), 16 cases of endometrial serous carcinomas, and 12 cases of endometrial clear cell carcinomas. The expression of α-SMA, CD10, and desmin was detected in the tumor microenvironment (TME) by the immunohistochemical method. Trichrome was detected to identify collagen in TME by a special dyeing method. Between-group comparison in categorical variables was assessed via using Fisher\u0026rsquo;s exact test. The overall survival was analyzed by a Kaplan-Meier curve.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe total expression rate of α-SMA in endometrial carcinoma was 71.1%. The expression rate of α-SMA was 82.4%, 62.5%, and 66.7% positively expressed in EEC, serous carcinoma, and clear cell carcinoma, respectively. CD10 and desmin were negative in all EC TME. Both α-SMA and Trichrome positive groups showed significantly higher mortality rates (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.042) and poorer prognosis (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.041) than both α-SMA and Trichrome negative groups.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe co-expression of α-SMA and Trichrome is of great significance in predicting the poor prognosis of endometrial cancer.\u003c/p\u003e","manuscriptTitle":"The prognostic significance of co-expression of α-SMA and Trichrome in the tumor microenvironment of poorly differentiated endometrial carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-18 15:58:32","doi":"10.21203/rs.3.rs-2479907/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":"6bb61244-aa04-4276-946b-51f13fdcb3a9","owner":[],"postedDate":"January 18th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-01-23T18:29:31+00:00","versionOfRecord":[],"versionCreatedAt":"2023-01-18 15:58:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2479907","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2479907","identity":"rs-2479907","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}