Lymphatic Markers in Kaposiform Hemangioendothelioma: Implications for Tumor Invasiveness and β-blockers efficacy

Lymphatic Markers in Kaposiform Hemangioendothelioma: Implications for Tumor Invasiveness and β-blockers efficacy | 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 Lymphatic Markers in Kaposiform Hemangioendothelioma: Implications for Tumor Invasiveness and β-blockers efficacy Lu Yu, Li Wei, Lei Qiu, Lin Ma, Li Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4014692/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 Background: Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor that usually expresses lymphatic markers. PROX1 can increase the local invasiveness of KHE in mice. Objectives: To evaluate whether the expression of lymphatic markers in KHE patients increases their invasiveness and decreases the efficacy of β-blockers (propranolol or atenolol). Methods: The tumor tissues of 3 patients with KHE were collected for transcriptomics. The clinical data of 15 patients were collected to analyze the correlation between PROX1, invasiveness and β-blockers’ efficacy. Overexpression PROX1 in EOMAs, cell viability assay, apoptosis, cell cycle, migration, and β-blockers’ efficacy were analyzed in vitro. Tumor invasiveness was analyzed in vivo. Results: PROX1 might increase local invasiveness of the KHE due to impact cell motility, locomotion, and migration. The nearly complete resolution rate (>75%) of β-blockers (propranolol or atenolol) in the treatment of KHE was 40 %. There was no significant correlation between the positive rate of PROX1 and the efficacy of β-blockers’ treatment. Limitations: Due to small numbers, the effect of PROX1 on the efficacy of propranolol needs further study. Conclusion: Our study showed the higher positive rate of PROX1 in the tumor, the more prone the local invasiveness of the KHE patients. There was no significant correlation between the positive rate of PROX1 and the efficacy of 10 patients with β-blockers treatment. Kaposiform hemangioendothelioma PROX1 invasiveness β-blockers Figures Figure 1 Figure 2 Figure 3 Figure 4 Plain language summary Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor that usually expresses lymphatic markers. PROX1 is a specific marker of lymphatic endothelium and can reprogram vascular endothelial cells into lymphatic phenotype. PROX1 can increase the local invasiveness of KHE in mice. However, the role of PROX1 in the pathogenesis of KHE needs to be clarified. Meanwhile, it is not clear whether the expression of lymphatic markers in KHE patients increases their invasiveness and decreases the efficacy of propranolol. Transcriptome revealed that PROX1 was highly expressed in KHE tumor tissues. The clinical data of 15 patients were collected to analyze the correlation between KHE tumors and invasiveness. The higher the positive degree of PROX1, the stronger the local invasiveness of the KHE. The nearly complete resolution rate (>75%) of β-blockers (propranolol or atenolol) in the treatment of KHE was 40 %. There was no significant correlation between the positive rate of PROX1 and the efficacy of 10 patients with propranolol or atenolol treatment. The biological processes and pathways involved in the differentially expressed genes in the PROX1 overexpression group before and after β-blockers treatment were the same as those in the control group. Introduction Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor that is usually diagnosed in early infancy or childhood. KHE resembles an infantile hemangioma (IH) and is a vascular tumor that usually presents as a dilated, erythematous, or purplish soft-tissue mass in infancy 1 . It often occurs in the superficial or deep soft tissue of the limbs and in the trunk, retroperitoneal, head and neck, mediastinum, thymus, and spleen 2 . The tumor is locally invasive and can involve local muscles and bones. It is named because of its morphological characteristics, which are similar to Kaposi 's sarcoma. Kasabach-Merritt phenomenon (KMP) has been used in patients with vascular abnormalities associated with thrombocytopenia and coagulation since 1940. The latest classification of vascular anomalies in 2018 issued by the International Society for the Study of Vascular Anomalies (ISSVA) defines KMP as specifically caused by KHE or tufted angioma (TA). Spindle tumor cells in KHE express vascular endothelial markers CD31 and CD34, and also express specific lymphatic markers Prospero homeobox protein 1 (PROX1), podoplanin (PDPN or D2-40), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), which are considered to be the origin of stem cells that differentiate into vascular endothelium and lymphatic endothelium 3 . PROX1 is specifically expressed in embryonic and postnatal lymphatic vessels and is a critical gene in lymphangiogenesis 4 . PROX1 can increase the local invasiveness of KHE in murine 5 . PROX1 and vascular endothelial growth factor C (VEGFC)/VEGFR3 feedback loops play an important role in lymphatic anomalies 6 . It is not clear whether the expression of lymphatic markers in KHE patients increases their invasiveness. The currently reported KHE regimens include systemic corticosteroids, antiplatelet agents, α-interferon, antifibrinolytic therapy, chemotherapy, surgical resection with or without embolization, rapamycin, and propranolol 7 . The efficacy and safety of β-blockers (propranolol or atenolol) in treating vascular abnormalities, especially infantile hemangioma (IH), have been widely confirmed 8 . However, its efficacy in the treatment of KHE is still controversial. It is unclear whether the difference in the expression of lymphatic marker PROX1 in KHE leads to its insensitivity to propranolol treatment. This study mainly investigates the effect of lymphatic vessel marker PROX1 on the local invasiveness of KHE. To explore the impact of PROX1 on the efficacy of propranolol. Methods Pan-caner analysis of the oncogenic role of PROX1 in human tumors Login to the TIMER database ( http://timer.cistrome.org/ ) and GEPIA website ( http://gepia2.cancer-pku.cn/ ) to obtain the expression of PROX1 mRNA in various tumors, the correlation with clinical stages, and the correlation with overall survival (OS) and disease-free survival (DFS). 50 genes and 99 genes related to PROX1 expression were obtained by using STRING and GEPIA, respectively (supplemental Table 1). Transcriptome Tumor tissue and normal skin of three KHE patients were collected for transcriptome detection. Cells with PROX1 overexpression (OE) EOMA combined normal control (NC) EOMA group, OE and NC EOMA cells were treated with 100µM propranolol were collected for transcriptome detection, respectively. Transcriptomics was performed using Illumina (novaseq 6000) sequencing. Enrichment analysis was conducted in the GO and KEGG databases. Collecting clinical patients with KHE to analyze the expression among lymphatic vessel markers, invasiveness, and efficacy Immunohistochemical staining with antibodies to PROX1 (ab199359, 1:500, abcam), D2-40 (ZM-0465, 1:200, zsbio), LYVE-1 (ab199359, 1:500, abcam), VEGFR-3 (ab27278, 1:500, abcam). The positive rate of immunohistochemistry was evaluated by Image J software. Evaluation of the efficacy of propranolol in the treatment of KHE refers to the evaluation criteria of the efficacy of infantile hemangiomas 9 . Cell culture and transfection EOMAs were acquired from the American type culture collection (ATCC). Dulbecco's modified eagle medium (Vivacell) was applied to culture EOMAs supplemented with 10% fetal bovine serum (FBS) (Zhejiang Tianhang, China) (5%CO 2 at 37°C). Lentiviral constructs stable expressing PROX1 (NM_002763) and GFP with DDDDK-tag (GV492-PROX1-GFP), along with the negative control lentivirus, were purchased from Genechem (Shanghai, China). Cells were infected with lentivirus for 48 h. RNA isolation, qRT-PCR analysis, and Western blot TRIzol Reagent (Invitrogen) was utilized for the extraction of RNA. Then, total RNA was used to generate of complementary DNA via the Revert Aid First-Strand cDNA Synthesis Kit (LABLEAD, China). After that, qRT-PCR was conducted via ABI Prism 7500 Thermal Cycler (Applied Biosystems) with SYBR Green detection reagent (LABLEAD, China). The results were normalized to the Actb gene. The relative expressions were calculated using formula 2 −(△△CT) . All the primer sequences were listed in supplemental table 2 . The primary antibodies of western blot including βactin (Beijing TDY BIOTECH CO.,LTD) (1:10000, TDY051), PROX1 (1:500, ab38692), LYVE1 (1:2000, ab281587), and podoplanin (PDPN) (1:2000, ab256559)A corresponding second antibody (1:5000; ASPEN; AS1107; AS1193) was incubated on the membrane. Cell viability assay, apoptosis, and cell cycle The cell viability and proliferation were evaluated by the CCK8 test. APC Annexin V (Biolegend) and PI (LABLEAD) were used to stain OE and NC cells analyzed apoptosis by flow cytometry. PI were used to stain cells fixed in precooled 70% ethanol for cell cycle by flow cytometry. In vitro monolayer wounding assay and cell migration assay Cell monolayers were wounded with a pipette tip. Identical wounded areas were imaged at time points 0, 6 hours, and 24 hours with a phase contrast microscope. Transwell assay was used to evaluate the ability of cell migration. Animal operation The Animal Care and Experimental Committee of Capital Medical University permitted all the animal procedures in this study. The cell suspension (cell concentration of 5×10 7 /mL) was subcutaneously injected into the abdominal subcutaneous tissue of 5-week-old Balb/c nude mice (female mice, 100ul). The tumor formation of different groups of cells was continuously measured twice a week. Tumor volume (mm 3 ) = long diameter (mm) × short diameter (mm 2 ) × 0.52. Blood samples were collected from the tail tip for blood routine examination. Results A pan-cancer analysis of the oncogenic role of PROX1 in human tumors As shown in Fig. 1 a, the expression level of PROX1 in the tumor tissues COAD (Colon adenocarcinoma), READ (Rectum adenocarcinoma) (P < 0.001), and STAD (Stomach adenocarcinoma) (P < 0.01), is higher than the corresponding control tissues. The expression level of PROX1 in the tumor tissues BLCA (Bladder Urothelial Carcinoma), BRCA (Breast invasive carcinoma), KICH (Kidney chromophobe), KIRC (Kidney renal clear cell carcinoma), KIRP (Kidney renal papillary cell carcinoma), PRAD (Prostate adenocarcinoma), READ (Rectum adenocarcinoma), THCA (Thyroid carcinoma), UCEC (Uterine Corpus Endometrial Carcinoma) (P < 0.001), LUSC (Lung squamous cell carcinoma) (P < 0.01), GBM (Glioblastoma multiforme), LIHC (Liver hepatocellular carcinoma) (P < 0.05), is lower than the corresponding control tissues. The expression level of PROX1 in the tumor tissues SKCM (Skin Cutaneous Melanoma) is lower than in the SKCM metastasis tissues (P < 0.01) (Fig. 1 a). We also used the “Pathological Stage Plot” module of GEPIA2 to observe the correlation between PROX1 expression and the pathological stages of cancers, including SKCM and THCA (Thyroid carcinoma) (Fig. 1 b, P < 0.05). We divided the cancer cases into high-expression and low-expression groups according to the expression levels of PROX1. We investigated the correlation of PROX1 expression with the prognosis of patients with different tumors, mainly using the datasets of TCGA and GEO, respectively. As shown in Fig. 1 c, highly expressed PROX1 was linked to poor prognosis of OS (overall survival) for cancers of KIRC (P = 0.0023) (Fig. 1 c). DFS (disease-free survival) analysis data (Fig. 1 d) showed a correlation between high PROX1 expression and poor prognosis for the TCGA cases of ACC (P = 0.014). Additionally, low expression of the PROX1 gene was related to poor OS prognosis for SKCM (Fig. 1 c, P = 0.0049) and DFS prognosis for PAAD, SKCM, THCA, and UCEC (Fig. 1 d, P = 0.019, 0.033, 0.0012, and 0.029, respectively). To investigate the molecular mechanism of the PROX1 gene in tumorigenesis further, we attempted to screen out the targeting PROX1-binding proteins and the PROX1 expression-correlated genes for a series of pathway enrichment analyses. Fig. s1 a shows the interaction network of 50 proteins based on STRING tool. TCGA and got the top 99 genes that correlated with PROX1 expression. We combined the two datasets to perform KEGG and GO enrichment analyses. The KEGG data of Fig. s1 b suggest that “complement and coagulation cascade” might be involved in the effect of PROX1 on tumor pathogenesis. The GO enrichment analysis data further indicated that most of these genes are linked to the pathways of blood coagulation (Fig. s1 c). High expression of PROX1 in KHE tumor tissues By applying transcriptome analysis of tumor tissues and normal tissues far from tumor tissues, according to the DESeq2 padj =1.0, a total of 2188 differentially expressed genes were screened according to the standard. GO enrichment results showed that the up-regulated biological processes included angiogenesis, positive regulation of cell migration, and positive regulation of cell motility. The differentially expressed genes were involved in the biological pathways PI3K-Akt signaling pathway, Rap1 signaling pathway, Focal adhesion, Ras signaling pathway, and Cell adhesion molecules (CAMs) (Fig.s 2 ). A Volcano map of differentially expressed genes showed that PROX1 was highly expressed in tumor tissues (Fig. 1 e). PROX1 expression increases local invasiveness in KHE The clinical data of 15 patients were collected. All 14 patients had onset within three months after birth, including 11 males (73.33%) and four females (26.67%). Among them, 11 patients involved deep muscles, bone, or deep tissues (such as the parotid gland). Eleven patients (73.33%) were initially treated with β-blockers (propranolol or atenolol)(Table 1 ). Table 1 Clinical information of 15 patients with KHE No. age of onset gender KMP location size(cm) regional invasiveness treatment D2-40 (%) PROX1 (%) LYVE1 (%) 1 at birth M no cervical region 4.7*7.6*7.1 muscle, parotid gland propranolol 9.67 4.64 0.49 2 10 days M no groin 1.8*1.0*4.8 no propranolol 7.70 0.92 0.33 3 at birth M no cervical region 6*2.3*4.8 muscle, parotid gland atenolol 13.43 6.02 2.37 4 2 months M preexistent cervical region and thoracic wall diffuse muscle, bone sirolimus 10.72 1.46 0.24 5 1 month F no face 4.2*1.0*4.3 muscle propranolol 4.56 0.75 0.61 6 at birth M no circumference 5.2*2.0*3.9 parotid gland propranolol 3.24 0.43 1.06 7 1 month F no lower limb 4.5*2.0*7.0 muscle sirolimus 12.38 1.63 0.61 8 1 month M no thoracic wall 3.8*1.3*5.0 muscle surgery 29.91 11.19 2.22 9 at birth F yes shoulder and thoracic wall diffuse muscle, bone corticosteroid 29.36 11.45 7.01 10 at birth M no upper limb 3.7*1.4*3.9 muscle atenolol 10.38 2.52 0.00 11 at birth M no lower limb diffuse no propranolol 1.40 0.61 0.00 12 10 months F no shoulder and thoracic wall 5.3*1*5.1 muscle propranolol 4.21 0.51 0.00 13 at birth M no lower limb 5.6*2.3*5.1 muscle propranolol 12.09 1.69 3.65 14 1 month M no circumference 3.1*0.8*2.9 no propranolol 8.49 0.71 0.29 15 1 month M no lower limb 9.8*2.8*6.0 no propranolol 8.49 1.50 2.39 F: female; M: male; KMP: Kasabach-Merrit Phenomenon The difference in local invasiveness of KHE was correlated with PROX1. The higher the positive degree of PROX1, the stronger the local invasiveness of the tumor. There was no significant correlation between invasiveness and the expression of LYVE1 and D2-40 (Fig. 2 ). Efficacy of propranolol The subjective evaluation of clinicians, combined with B-ultrasound, MRI, or CT examination, was used to evaluate the efficacy one year after treatment. A total of 11 patients with KHE without KMP who were treated with oral β-blockers were followed up for more than six months, and 10 of them were followed up for more than 12 months. Among the 10 cases, 4 cases achieved >75% efficacy at 12 months of treatment, two patients achieved 51%~75% efficacy, 1 case achieved 26%~50% efficacy, and 2 cases achieved ≤ 25% efficacy. There was no significant correlation between the positive rate of PROX1 and the effectiveness of propranolol (P = 0.2569) (Fig. 3 ). Construction of PROX1 stably transfected EOMA cells PROX1 overexpressing cell lines were constructed using lentivirus. The transcription and protein levels of PROX1 in the PROX1 overexpression (OE) group were higher than those in the normal control (NC) group. The transcription and protein expression levels of LYVE1 in the OE group were lower than those in the NC group. There was no significant difference in the two groups transcription and protein levels of PDPN (Fig.). Cell proliferation was not accelerated in the OE group compared with the NC group. There was no significant difference in apoptosis rate, S phase, migration rate, and metastatic cells between the OE and NC groups (Fig. 4 ). The biological processes involved in the differentially expressed genes between the OE group and the NC group included positive regulation of cell motility, positive regulation of locomotion, positive regulation of cell migration, and angiogenesis. The pathways involved in differentially expressed genes include the AGE/RAGE signaling pathway and the PI3K/AKT pathway (Fig.s 3 ). High expression of PROX1 in Balb/c nude increased the invasiveness of EOMA From the 22nd day, the average tumor size in the NC group was larger than in the OE group. PROX1 expression did not reduce platelets and hemoglobin, so there was no significant correlation between PROX1 and coagulation dysfunction in nude mice (Fig. 4 ). Although PROX1 overexpression inhibits tumor growth in vivo, it can increase tumor invasiveness (Table 2 ). Table 2 Local invasion of tumors in OE and NC groups in nude mice OE NC Local invasion 9 6 No local invasion 0 4 Investigate the effect of PROX1 on the efficacy of propranolol in vitro and in vivo The inhibitory effect of propranolol on EOMA in the NC group was more potent than that in the OE group at 100µM and 200µM. At propranolol 500µM, there was no significant difference in the inhibitory effect between the OE and NC groups. After propranolol treatment, the differentially expressed genes in the OE group and the NC group were compared with the control group. The biological processes and pathways involved were the same (Fig.s 4 ). Discussion The pathogenesis of KHE is still unclear. High expression of PROX1 may promote the invasiveness of KHE. In this study, we found that PROX1 may play an essential role in the pathogenesis and prognosis of cutaneous melanoma. In addition, the expression of PROX1 may lead to the disorder of coagulation function. Therefore, we performed transcriptomic analysis of KHE tumor tissues and normal tissues away from tumor tissues and found that PROX1 was highly expressed in KHE tumor tissues. PROX1 is involved in the occurrence and development of cancer and has tumor suppressor and carcinogenic properties in many different cancer types. However, the exact mechanism by which PROX1 regulates cancer cell proliferation, migration, and invasion is unknown 10 . Whether the expression of PROX1 can promote the growth rate of KHE is still controversial 5 , 11 . However, it might can increase the invasion ability of KHE 5 , 11 . The proliferation rate of PROX1-positive tumor cells in high-grade tumors was lower than that of other tumor cells. To further explore the role of PROX1 in the pathogenesis of KHE, we retrospectively analyzed the relationship between lymphatic markers PROX1, LYVE1, and PDPN (D2-40) tumor invasiveness in 15 patients with KHE. The higher the percentage of PROX1 expression, the stronger the local invasion of KHE tumors. We further observed that the invasion ability of the EOMA cell line overexpressing PROX1 was enhanced in nude mice. At the cellular level, it was found that the differentially expressed genes of EOMA overexpressing PROX1 and EOMA in the control group were related to positive regulation of cell motility, positive regulation of locomotion, and positive regulation of cell migration. Although bioinformatics suggested that PROX1 may be associated with abnormal coagulation function, the experimental data of nude mice in this study did not show coagulation dysfunction. The relationship between PROX1 and KMP still needs further investigation. Although sirolimus is effective, it can lead to rare side effects such as interstitial pneumonia and pneumocystis carinii pneumonia due to its immunosuppressive effect, which may endanger life 12 , 13 . Moreover, the patients with KHE are mainly infants and young children, and the blood drug concentration should be closely monitored during the medication process. Prophylactic use of antibiotics is required to prevent serious infections when combined with glucocorticoids. Propranolol is effective for some KHE, and its efficacy may be dose-related 14 – 17 . However, it has also been reported that propranolol is ineffective in treating KHE (with or without KMP) 16 , 18 – 21 . Propranolol monotherapy is not adequate for KHE (with KMP) patients 22 . Propranolol combined with vincristine and glucocorticoid therapy can achieve better efficacy 23 – 25 . At present, the efficacy of propranolol in the treatment of KHE is still controversial, and the factors affecting its effectiveness are still unclear. It was found that the nearly complete resolution (> 75%) rate of propranolol in the treatment of KHE for one year was (4/10, 40%). There are significant individual differences in the efficacy of propranolol in the treatment of KHE. We speculate that PROX1, as a marker in the direction of lymphatic vessels, may reduce the efficacy of propranolol. However, our clinical data show that the expression of lymphatic markers does not reduce the efficacy of propranolol. For younger patients with bone and muscle invasion, sirolimus affects vaccination and chooses a method that does not have immunosuppression. Propranolol has an anti-fibrosis effect as well 26 . It may play a role in reducing the long-term complications of KHE. We found that although the PROX1 positive group decreased the reactivity of propranolol in the low concentration, with the increase of propranolol concentration, the reactivity of propranolol in the PROX1 positive group tended to be consistent with that in the control group. The differentially expressed genes in PROX1 positive and control groups before and after propranolol treatment involved similar biological processes and pathways. So, the effect and mechanism of the expression of lymphatic markers on the efficacy of treatment still needs to be further explored. Limitations Due to the small sample size of this study, the effect of PROX1 on the invasiveness of KHE and efficacy of propranolol needs further study. The mechanism of PROX1 promoting KHE tumor invasion still needs further exploration. The mechanism by which affects propranolol’s treatment of KHE needs further study. Conclusion This study found that PROX1 increased tumor invasiveness in nude mice and KHE patients. PROX1 may play a role by affecting cell motility and migration. The nearly complete resolution rate of propranolol in treating KHE without KMP is 40%. PROX1 expression did not reduce the efficacy of propranolol. Declarations Ethics statement This study protocol was reviewed and approved by Medical Ethics Committee of Beijing Children’s Hospital, Capital Medical University (IEC-C-006-A04-V.06). All the animal procedures in this study got permitted by the Animal Care and Experimental Committee of Capital Medical University (AEEI-2023-049). Consent for publication Not applicable. Informed consent declaration Written informed consent was obtained from the patients for the publication of their anonymized information in this article. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions. Authors’ contributions LL and LM conceptualized, designed the study, and reviewed and revised the manuscript. LY conceptualized and designed the study, drafted the initial manuscript, designed the data collection instruments, collected data, undertook the experiment, carried out the initial analyses, reviewed and revised the manuscript, and critically reviewed the manuscript for important intellectual content. LW and LQ collected data, undertook the experiment, coordinated, contributed to analysis and interpretation of data, supervised data collection, and critically reviewed it for important intellectual content. All authors contributed to the article and approved the submitted version. All authors approved the final manuscript. Competing interests Not applicable. Financial disclosure None of the authors has a financial interest in any of the products, devices, or drugs mentioned in this manuscript. Acknowledgment Thanks for the support of the parents of KHE patients in this article. Thanks to the guidance of Kaihu Yao, Qinghong Meng and Dan Yu from Beijing Pediatric Research Institute. References Wang Y, Kong L, Sun B, Cui J, Shen W. Sirolimus for Kaposiform Hemangioendothelioma With Kasabach-Merritt Phenomenon in Two Infants. J Craniofac Surg . 2020;31(4):1074-1077. Putra J, Gupta A. Kaposiform haemangioendothelioma: a review with emphasis on histological differential diagnosis. Pathology . 2017;49(4):356-362. Eiman A Hasby NEM, Rania Eltatawy C-Kit, CD34 & α-SMA Immunohistochemical Features in Classic Kaposi Sarcoma and Kaposiform Hemangioendothelioma J Microsc Ultrastruct Jan-Mar 2017;5(1):49-57. Meng FW, Jing XN, Song GH, Jie LL, Shen FF. Prox1 induces new lymphatic vessel formation and promotes nerve reconstruction in a mouse model of sciatic nerve crush injury. J Anat . 2020;237(5):933-940. Dadras SS, Skrzypek A, Nguyen L, Shin JW, Schulz MMP, Arbiser J, et al. 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Cancer Manag Res. 2018;10:3325-3331. Uno T, Ito S, Nakazawa A, Miyazaki O, Mori T, Terashima K. Successful treatment of Kaposiform hemangioendothelioma with everolimus. Pediatr Blood Cancer. 2015;62(3):536-538. Wang Z, Li K, Dong K, Xiao X, Zheng S. Variable response to propranolol treatment of kaposiform hemangioendothelioma, tufted angioma, and Kasabach-Merritt phenomenon. Pediatr Blood Cancer. 2014;61(8):1518-1519. Hermans DJJ, Beynum IMv, Vijver RJvd, Kool LJS, Blaauw Id, Vleuten CJMvd. Kaposiform hemangioendothelioma with Kasabach-Merritt syndrome: a new indication for propranolol treatment. J Pediatr Hematol Oncol. 2011;33(4):e171-173. Uppuluri R, Kumar V, Munirathnam D, Doss H, Ramachandrakurup S, Subburaj D, et al. Kaposiform Hemangioendothelioma in a 3 Months Old Infant. Indian J Hematol Blood Transfus. 2016;32(Suppl 1):370-371. Verma S, Dhamija E, Barwad A, Kumar VS, Rastogi S. A case report of Kaposiform haemangioendothelioma; response with propranolol and steroids. Clin Sarcoma Res. 2020;10:12. Ding Q, Li Z, Liu B, Ling L, Tian X, Zhang C. Propranolol prevents liver cirrhosis by inhibiting hepatic stellate cell activation mediated by the PDGFR/Akt pathway. Hum Pathol. 2018;76:37-46. Supplementary Files figures1.tif figures2.tif figures3.tif figures4.tif supplementaltable1.docx supplementaltable2.docx 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4014692","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":282751479,"identity":"840c6504-a90c-4b71-8510-4b5590998c46","order_by":0,"name":"Lu Yu","email":"","orcid":"","institution":"Beijing Children's Hospital Capital Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Yu","suffix":""},{"id":282751480,"identity":"f2e7f37e-fe6a-497c-b4ee-48a5dda5d6ca","order_by":1,"name":"Li Wei","email":"","orcid":"","institution":"Beijing Children's Hospital Capital Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Wei","suffix":""},{"id":282751481,"identity":"964c8d42-57f1-474d-adee-93ab13d73677","order_by":2,"name":"Lei Qiu","email":"","orcid":"","institution":"Beijing Children's Hospital Capital Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Qiu","suffix":""},{"id":282751482,"identity":"6f9b7e3a-f2c3-4cf0-9ecf-28fb6d5a9f67","order_by":3,"name":"Lin Ma","email":"","orcid":"","institution":"Beijing Children's Hospital Capital Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Ma","suffix":""},{"id":282751483,"identity":"aea32369-1226-4fc2-a3d0-734a2ec7059c","order_by":4,"name":"Li Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYBACPgYGZgaGAgkZNvbGxgcfiNHCBtZiIMHDxnO42XAGCVoYeBgk0tukOYjSIpH82JjHwIKHT/JhgzQDg52cbgNBLWnGyTwgh0knNhgXMCQbmx0gqCWH+TBMS/IMhgOJ24jXInmw4TAPsVogDpNgbGwmTgvPM2PDOeBATmxmnGFAhF/42ZMfS7ypqJOTbz/+/MeHCjs5glpAgIkHzjQgQjkIMP4gUuEoGAWjYBSMUAAAX2ozBkC4BIAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8533-1682","institution":"Beijing Children's Hospital Capital Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-03-05 00:59:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4014692/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4014692/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53670491,"identity":"97729c31-ecff-4d58-8eaa-d05d4442f794","added_by":"auto","created_at":"2024-03-28 17:47:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1390797,"visible":true,"origin":"","legend":"\u003cp\u003ePan-cancer analysis of the oncogenic role of PROX1 in human tumors\u003c/p\u003e\n\u003cp\u003e(a) expression status of PROX1 across various cancer types of TCGA; (b) the correlation between PROX1 expression and the pathological stages of cancers (SKCM and THCA); (c) the correlation between expression of PROX1 and OS (overall survival) for cancers; (d) the correlation between expression of PROX1 and DFS (disease-free survival) for cancers; (e) volcano map of differentially expressed genes, in which PROX1 was highly expressed in tumor tissues\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4014692/v1/8c241deabe7a6a8d3581b3e7.png"},{"id":53670490,"identity":"93a17d6b-a583-49f4-a5d0-84e284a8bdcf","added_by":"auto","created_at":"2024-03-28 17:47:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2492781,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship between lymphatic markers and KHE local invasiveness\u003c/p\u003e\n\u003cp\u003e(a)Low expression of PROX1 in KHE (10×); (b) High expression of PROX1 in KHE (10×); (c) There was no significant correlation between invasiveness and the expression of D2-40; (d) The higher the positive degree of PROX1, the stronger the local invasiveness of the tumor; (e) There was no significant correlation between invasiveness and the expression of LYVE1\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4014692/v1/9f0fa832f135054215a6cf8f.png"},{"id":53670493,"identity":"ba39749e-de4e-4598-9751-75b3de91534a","added_by":"auto","created_at":"2024-03-28 17:47:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1298312,"visible":true,"origin":"","legend":"\u003cp\u003eThe efficacy of propranolol and its correlation with PROX1 expression\u003c/p\u003e\n\u003cp\u003e(a) Case 1: neck KHE before propranolol treatment; (b) Case 1: 9 months after propranolol treatment; (c) MRI of the neck before treatment in case 1; (d) Case 1: neck MRI after one year of propranolol treatment; (e) The relationship between the positive expression of PROX1 and the efficacy of propranolol\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4014692/v1/bf0111be614be0adf7fe03b0.png"},{"id":53670480,"identity":"ee541b68-f5c0-4074-a1c3-d5a96117a355","added_by":"auto","created_at":"2024-03-28 17:47:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3367858,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of human PROX1 in EOMA cell clones, in vivo and in vitro tumorigenesis\u003c/p\u003e\n\u003cp\u003e(a)Western blot analysis confirmed high amounts of PROX1, LYVE1, PDPN protein in cell lysates obtained from EOMA cells stably transfected with lentivirus, as compared with control transfectants; (b) qPCR analysis confirmed high amounts of PROX1, LYVE1, PDPN mRNA obtained from EOMA cells stably transfected with lentivirus, as compared with control transfectants; (c)cell proliferation was not accelerated in the OE group; (d) There was no significant difference in apoptosis rate between the OE group and the NC group; (e) Compared with the NC group, the number of cells in the G1 phase and the G2/M phase in OE group increased; (f) and (g) There was no significant difference in the migration rate between the OE group and the NC group after 6 hours and 24 hours, bar=20μm; (h) and (i) There was no significant difference in the number of metastatic cells detected by Transwell in the OE combined NC group, bar=30μm (100×); (j) Tumors on day 43 in OE group and NC group; (k) Tumor proliferation curve in nude mice; (l) Platelet count on day 32 and day 43 in OE group and NC group; (m) Propranolol in different concentrations treated OE and NC EOMA cells; (n) Sirolimus in different concentrations treated OE and NC EOMA cells\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4014692/v1/b538cb52b58fb08e435d1038.png"},{"id":61035135,"identity":"75632593-f962-488c-95b1-99aaa76482cf","added_by":"auto","created_at":"2024-07-24 21:20:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11525717,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4014692/v1/2c1a15f2-4bd2-4d8d-a23e-b1928d2fc357.pdf"},{"id":53670485,"identity":"f77bae0d-8bd9-47c1-a49d-0ea74f4bec3b","added_by":"auto","created_at":"2024-03-28 17:47:14","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":523028,"visible":true,"origin":"","legend":"","description":"","filename":"figures1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4014692/v1/6321a568cd48b2a9e9442c3e.tif"},{"id":53670492,"identity":"3a2b855c-6cb1-49d6-a63b-3ecd14dd23f4","added_by":"auto","created_at":"2024-03-28 17:47:16","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":920330,"visible":true,"origin":"","legend":"","description":"","filename":"figures2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4014692/v1/9aa596dc53e23c9e5b3863cc.tif"},{"id":53670482,"identity":"450e09e5-372e-4868-b5eb-26303747c7e8","added_by":"auto","created_at":"2024-03-28 17:47:11","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":349492,"visible":true,"origin":"","legend":"","description":"","filename":"figures3.tif","url":"https://assets-eu.researchsquare.com/files/rs-4014692/v1/ab838e7b271f6dab8b7284b6.tif"},{"id":53670481,"identity":"477647c1-5145-4242-867c-d0fc86081c98","added_by":"auto","created_at":"2024-03-28 17:47:11","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":645578,"visible":true,"origin":"","legend":"","description":"","filename":"figures4.tif","url":"https://assets-eu.researchsquare.com/files/rs-4014692/v1/49f15c8971bebde102c31e22.tif"},{"id":53670487,"identity":"9ccc6290-4b50-40a9-acc5-e31d7a18cf2b","added_by":"auto","created_at":"2024-03-28 17:47:14","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":19525,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaltable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4014692/v1/322916ba0bf981055d8b14ba.docx"},{"id":53670484,"identity":"2a642507-51b1-4044-93a7-7424f5fd681c","added_by":"auto","created_at":"2024-03-28 17:47:12","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14720,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaltable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4014692/v1/45f5c0eeb97e5ece3f112728.docx"}],"financialInterests":"","formattedTitle":"Lymphatic Markers in Kaposiform Hemangioendothelioma: Implications for Tumor Invasiveness and β-blockers efficacy","fulltext":[{"header":"Plain language summary ","content":"\u003cp\u003eKaposiform hemangioendothelioma (KHE) is a rare vascular tumor that usually expresses lymphatic markers. PROX1 is a specific marker of lymphatic endothelium and can reprogram vascular endothelial cells into lymphatic phenotype. PROX1 can increase the local invasiveness of KHE in mice. However, the role of PROX1 in the pathogenesis of KHE needs to be clarified. Meanwhile, it is not clear whether the expression of lymphatic markers in KHE patients increases their invasiveness and decreases the efficacy of propranolol. Transcriptome revealed that PROX1 was highly expressed in KHE tumor tissues. The clinical data of 15 patients were collected to analyze the correlation between KHE tumors and invasiveness. The higher the positive degree of PROX1, the stronger the local invasiveness of the KHE. The nearly complete resolution rate (\u0026gt;75%) of \u0026beta;-blockers (propranolol or atenolol) in the treatment of KHE was 40 %. There was no significant correlation between the positive rate of PROX1 and the efficacy of 10 patients with propranolol or atenolol treatment. The biological processes and pathways involved in the differentially expressed genes in the PROX1 overexpression group before and after \u0026beta;-blockers treatment were the same as those in the control group.\u003c/p\u003e\n"},{"header":"Introduction","content":"\u003cp\u003eKaposiform hemangioendothelioma (KHE) is a rare vascular tumor that is usually diagnosed in early infancy or childhood. KHE resembles an infantile hemangioma (IH) and is a vascular tumor that usually presents as a dilated, erythematous, or purplish soft-tissue mass in infancy\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. It often occurs in the superficial or deep soft tissue of the limbs and in the trunk, retroperitoneal, head and neck, mediastinum, thymus, and spleen\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The tumor is locally invasive and can involve local muscles and bones. It is named because of its morphological characteristics, which are similar to Kaposi 's sarcoma. Kasabach-Merritt phenomenon (KMP) has been used in patients with vascular abnormalities associated with thrombocytopenia and coagulation since 1940. The latest classification of vascular anomalies in 2018 issued by the International Society for the Study of Vascular Anomalies (ISSVA) defines KMP as specifically caused by KHE or tufted angioma (TA).\u003c/p\u003e \u003cp\u003eSpindle tumor cells in KHE express vascular endothelial markers CD31 and CD34, and also express specific lymphatic markers Prospero homeobox protein 1 (PROX1), podoplanin (PDPN or D2-40), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), which are considered to be the origin of stem cells that differentiate into vascular endothelium and lymphatic endothelium\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePROX1 is specifically expressed in embryonic and postnatal lymphatic vessels and is a critical gene in lymphangiogenesis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. PROX1 can increase the local invasiveness of KHE in murine\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. PROX1 and vascular endothelial growth factor C (VEGFC)/VEGFR3 feedback loops play an important role in lymphatic anomalies\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. It is not clear whether the expression of lymphatic markers in KHE patients increases their invasiveness.\u003c/p\u003e \u003cp\u003eThe currently reported KHE regimens include systemic corticosteroids, antiplatelet agents, α-interferon, antifibrinolytic therapy, chemotherapy, surgical resection with or without embolization, rapamycin, and propranolol\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The efficacy and safety of β-blockers (propranolol or atenolol) in treating vascular abnormalities, especially infantile hemangioma (IH), have been widely confirmed\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, its efficacy in the treatment of KHE is still controversial. It is unclear whether the difference in the expression of lymphatic marker PROX1 in KHE leads to its insensitivity to propranolol treatment. This study mainly investigates the effect of lymphatic vessel marker PROX1 on the local invasiveness of KHE. To explore the impact of PROX1 on the efficacy of propranolol.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePan-caner analysis of the oncogenic role of PROX1 in human tumors\u003c/h2\u003e \u003cp\u003eLogin to the TIMER database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://timer.cistrome.org/\u003c/span\u003e\u003cspan address=\"http://timer.cistrome.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and GEPIA website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia2.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia2.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to obtain the expression of PROX1 mRNA in various tumors, the correlation with clinical stages, and the correlation with overall survival (OS) and disease-free survival (DFS). 50 genes and 99 genes related to PROX1 expression were obtained by using STRING and GEPIA, respectively (supplemental Table\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome\u003c/h2\u003e \u003cp\u003eTumor tissue and normal skin of three KHE patients were collected for transcriptome detection. Cells with PROX1 overexpression (OE) EOMA combined normal control (NC) EOMA group, OE and NC EOMA cells were treated with 100\u0026micro;M propranolol were collected for transcriptome detection, respectively. Transcriptomics was performed using Illumina (novaseq 6000) sequencing. Enrichment analysis was conducted in the GO and KEGG databases.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCollecting clinical patients with KHE to analyze the expression among lymphatic vessel markers, invasiveness, and efficacy\u003c/b\u003e \u003c/p\u003e \u003cp\u003eImmunohistochemical staining with antibodies to PROX1 (ab199359, 1:500, abcam), D2-40 (ZM-0465, 1:200, zsbio), LYVE-1 (ab199359, 1:500, abcam), VEGFR-3 (ab27278, 1:500, abcam). The positive rate of immunohistochemistry was evaluated by Image J software. Evaluation of the efficacy of propranolol in the treatment of KHE refers to the evaluation criteria of the efficacy of infantile hemangiomas\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and transfection\u003c/h2\u003e \u003cp\u003eEOMAs were acquired from the American type culture collection (ATCC). Dulbecco's modified eagle medium (Vivacell) was applied to culture EOMAs supplemented with 10% fetal bovine serum (FBS) (Zhejiang Tianhang, China) (5%CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C). Lentiviral constructs stable expressing PROX1 (NM_002763) and GFP with DDDDK-tag (GV492-PROX1-GFP), along with the negative control lentivirus, were purchased from Genechem (Shanghai, China). Cells were infected with lentivirus for 48 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation, qRT-PCR analysis, and Western blot\u003c/h2\u003e \u003cp\u003eTRIzol Reagent (Invitrogen) was utilized for the extraction of RNA. Then, total RNA was used to generate of complementary DNA via the Revert Aid First-Strand cDNA Synthesis Kit (LABLEAD, China). After that, qRT-PCR was conducted via ABI Prism 7500 Thermal Cycler (Applied Biosystems) with SYBR Green detection reagent (LABLEAD, China). The results were normalized to the Actb gene. The relative expressions were calculated using formula 2\u003csup\u003e\u0026minus;(△△CT)\u003c/sup\u003e. All the primer sequences were listed in supplemental table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe primary antibodies of western blot including βactin (Beijing TDY BIOTECH CO.,LTD) (1:10000, TDY051), PROX1 (1:500, ab38692), LYVE1 (1:2000, ab281587), and podoplanin (PDPN) (1:2000, ab256559)A corresponding second antibody (1:5000; ASPEN; AS1107; AS1193) was incubated on the membrane.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay, apoptosis, and cell cycle\u003c/h2\u003e \u003cp\u003eThe cell viability and proliferation were evaluated by the CCK8 test. APC Annexin V (Biolegend) and PI (LABLEAD) were used to stain OE and NC cells analyzed apoptosis by flow cytometry. PI were used to stain cells fixed in precooled 70% ethanol for cell cycle by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro monolayer wounding assay and cell migration assay\u003c/h2\u003e \u003cp\u003eCell monolayers were wounded with a pipette tip. Identical wounded areas were imaged at time points 0, 6 hours, and 24 hours with a phase contrast microscope. Transwell assay was used to evaluate the ability of cell migration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnimal operation\u003c/h2\u003e \u003cp\u003eThe Animal Care and Experimental Committee of Capital Medical University permitted all the animal procedures in this study. The cell suspension (cell concentration of 5\u0026times;10\u003csup\u003e7\u003c/sup\u003e/mL) was subcutaneously injected into the abdominal subcutaneous tissue of 5-week-old Balb/c nude mice (female mice, 100ul). The tumor formation of different groups of cells was continuously measured twice a week. Tumor volume (mm\u003csup\u003e3\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;long diameter (mm) \u0026times; short diameter (mm\u003csup\u003e2\u003c/sup\u003e) \u0026times; 0.52. Blood samples were collected from the tail tip for blood routine examination.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eA pan-cancer analysis of the oncogenic role of PROX1 in human tumors\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the expression level of PROX1 in the tumor tissues COAD (Colon adenocarcinoma), READ (Rectum adenocarcinoma) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and STAD (Stomach adenocarcinoma) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), is higher than the corresponding control tissues. The expression level of PROX1 in the tumor tissues BLCA (Bladder Urothelial Carcinoma), BRCA (Breast invasive carcinoma), KICH (Kidney chromophobe), KIRC (Kidney renal clear cell carcinoma), KIRP (Kidney renal papillary cell carcinoma), PRAD (Prostate adenocarcinoma), READ (Rectum adenocarcinoma), THCA (Thyroid carcinoma), UCEC (Uterine Corpus Endometrial Carcinoma) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), LUSC (Lung squamous cell carcinoma) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), GBM (Glioblastoma multiforme), LIHC (Liver hepatocellular carcinoma) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), is lower than the corresponding control tissues. The expression level of PROX1 in the tumor tissues SKCM (Skin Cutaneous Melanoma) is lower than in the SKCM metastasis tissues (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also used the \u0026ldquo;Pathological Stage Plot\u0026rdquo; module of GEPIA2 to observe the correlation between PROX1 expression and the pathological stages of cancers, including SKCM and THCA (Thyroid carcinoma) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eWe divided the cancer cases into high-expression and low-expression groups according to the expression levels of PROX1. We investigated the correlation of PROX1 expression with the prognosis of patients with different tumors, mainly using the datasets of TCGA and GEO, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, highly expressed PROX1 was linked to poor prognosis of OS (overall survival) for cancers of KIRC (P\u0026thinsp;=\u0026thinsp;0.0023) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). DFS (disease-free survival) analysis data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) showed a correlation between high PROX1 expression and poor prognosis for the TCGA cases of ACC (P\u0026thinsp;=\u0026thinsp;0.014). Additionally, low expression of the PROX1 gene was related to poor OS prognosis for SKCM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, P\u0026thinsp;=\u0026thinsp;0.0049) and DFS prognosis for PAAD, SKCM, THCA, and UCEC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, P\u0026thinsp;=\u0026thinsp;0.019, 0.033, 0.0012, and 0.029, respectively).\u003c/p\u003e \u003cp\u003eTo investigate the molecular mechanism of the PROX1 gene in tumorigenesis further, we attempted to screen out the targeting PROX1-binding proteins and the PROX1 expression-correlated genes for a series of pathway enrichment analyses. Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003es1\u003c/span\u003ea shows the interaction network of 50 proteins based on STRING tool. TCGA and got the top 99 genes that correlated with PROX1 expression. We combined the two datasets to perform KEGG and GO enrichment analyses. The KEGG data of Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003es1\u003c/span\u003eb suggest that \u0026ldquo;complement and coagulation cascade\u0026rdquo; might be involved in the effect of PROX1 on tumor pathogenesis. The GO enrichment analysis data further indicated that most of these genes are linked to the pathways of blood coagulation (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003es1\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHigh expression of PROX1 in KHE tumor tissues\u003c/h2\u003e \u003cp\u003eBy applying transcriptome analysis of tumor tissues and normal tissues far from tumor tissues, according to the DESeq2 padj\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05 and |log2FoldChange|\u0026gt;=1.0, a total of 2188 differentially expressed genes were screened according to the standard. GO enrichment results showed that the up-regulated biological processes included angiogenesis, positive regulation of cell migration, and positive regulation of cell motility. The differentially expressed genes were involved in the biological pathways PI3K-Akt signaling pathway, Rap1 signaling pathway, Focal adhesion, Ras signaling pathway, and Cell adhesion molecules (CAMs) (Fig.s\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A Volcano map of differentially expressed genes showed that PROX1 was highly expressed in tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePROX1 expression increases local invasiveness in KHE\u003c/h2\u003e \u003cp\u003eThe clinical data of 15 patients were collected. All 14 patients had onset within three months after birth, including 11 males (73.33%) and four females (26.67%). Among them, 11 patients involved deep muscles, bone, or deep tissues (such as the parotid gland). Eleven patients (73.33%) were initially treated with β-blockers (propranolol or atenolol)(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\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\u003eClinical information of 15 patients with KHE\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\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=\"left\" 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=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eage of onset\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003egender\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKMP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003elocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003esize(cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eregional invasiveness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003etreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eD2-40\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003ePROX1\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eLYVE1\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eat birth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ecervical region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.7*7.6*7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emuscle, parotid gland\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003epropranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e9.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e4.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003egroin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.8*1.0*4.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003epropranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e7.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eat birth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ecervical region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6*2.3*4.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emuscle, parotid gland\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eatenolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e13.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e6.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e2.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003epreexistent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ecervical region and thoracic wall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ediffuse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emuscle, bone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esirolimus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e10.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 month\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eface\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.2*1.0*4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emuscle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003epropranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eat birth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ecircumference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.2*2.0*3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eparotid gland\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003epropranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e3.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 month\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003elower limb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.5*2.0*7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emuscle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esirolimus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e12.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 month\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ethoracic wall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.8*1.3*5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emuscle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esurgery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e29.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e11.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e2.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eat birth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eyes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eshoulder and thoracic wall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ediffuse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emuscle, bone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ecorticosteroid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e29.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e11.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e7.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eat birth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eupper limb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.7*1.4*3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emuscle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eatenolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e10.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e2.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eat birth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003elower limb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ediffuse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003epropranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eshoulder and thoracic wall\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.3*1*5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emuscle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003epropranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eat birth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003elower limb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.6*2.3*5.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emuscle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003epropranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e12.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 month\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ecircumference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.1*0.8*2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003epropranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 month\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003elower limb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.8*2.8*6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eno\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003epropranolol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e2.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003eF: female; M: male; KMP: Kasabach-Merrit Phenomenon\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe difference in local invasiveness of KHE was correlated with PROX1. The higher the positive degree of PROX1, the stronger the local invasiveness of the tumor. There was no significant correlation between invasiveness and the expression of LYVE1 and D2-40 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEfficacy of propranolol\u003c/h2\u003e \u003cp\u003eThe subjective evaluation of clinicians, combined with B-ultrasound, MRI, or CT examination, was used to evaluate the efficacy one year after treatment. A total of 11 patients with KHE without KMP who were treated with oral β-blockers were followed up for more than six months, and 10 of them were followed up for more than 12 months. Among the 10 cases, 4 cases achieved \u0026gt;75% efficacy at 12 months of treatment, two patients achieved 51%~75% efficacy, 1 case achieved 26%~50% efficacy, and 2 cases achieved\u0026thinsp;\u0026le;\u0026thinsp;25% efficacy. There was no significant correlation between the positive rate of PROX1 and the effectiveness of propranolol (P\u0026thinsp;=\u0026thinsp;0.2569) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of PROX1 stably transfected EOMA cells\u003c/h2\u003e \u003cp\u003ePROX1 overexpressing cell lines were constructed using lentivirus. The transcription and protein levels of PROX1 in the PROX1 overexpression (OE) group were higher than those in the normal control (NC) group. The transcription and protein expression levels of LYVE1 in the OE group were lower than those in the NC group. There was no significant difference in the two groups transcription and protein levels of PDPN (Fig.).\u003c/p\u003e \u003cp\u003eCell proliferation was not accelerated in the OE group compared with the NC group. There was no significant difference in apoptosis rate, S phase, migration rate, and metastatic cells between the OE and NC groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe biological processes involved in the differentially expressed genes between the OE group and the NC group included positive regulation of cell motility, positive regulation of locomotion, positive regulation of cell migration, and angiogenesis. The pathways involved in differentially expressed genes include the AGE/RAGE signaling pathway and the PI3K/AKT pathway (Fig.s\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHigh expression of PROX1 in Balb/c nude increased the invasiveness of EOMA\u003c/h2\u003e \u003cp\u003eFrom the 22nd day, the average tumor size in the NC group was larger than in the OE group. PROX1 expression did not reduce platelets and hemoglobin, so there was no significant correlation between PROX1 and coagulation dysfunction in nude mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Although PROX1 overexpression inhibits tumor growth in vivo, it can increase tumor invasiveness (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLocal invasion of tumors in OE and NC groups in nude mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocal invasion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo local invasion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eInvestigate the effect of PROX1 on the efficacy of propranolol in vitro and in vivo\u003c/h2\u003e \u003cp\u003eThe inhibitory effect of propranolol on EOMA in the NC group was more potent than that in the OE group at 100\u0026micro;M and 200\u0026micro;M. At propranolol 500\u0026micro;M, there was no significant difference in the inhibitory effect between the OE and NC groups. After propranolol treatment, the differentially expressed genes in the OE group and the NC group were compared with the control group. The biological processes and pathways involved were the same (Fig.s\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe pathogenesis of KHE is still unclear. High expression of PROX1 may promote the invasiveness of KHE. In this study, we found that PROX1 may play an essential role in the pathogenesis and prognosis of cutaneous melanoma. In addition, the expression of PROX1 may lead to the disorder of coagulation function. Therefore, we performed transcriptomic analysis of KHE tumor tissues and normal tissues away from tumor tissues and found that PROX1 was highly expressed in KHE tumor tissues. PROX1 is involved in the occurrence and development of cancer and has tumor suppressor and carcinogenic properties in many different cancer types. However, the exact mechanism by which PROX1 regulates cancer cell proliferation, migration, and invasion is unknown\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhether the expression of PROX1 can promote the growth rate of KHE is still controversial\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, it might can increase the invasion ability of KHE\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The proliferation rate of PROX1-positive tumor cells in high-grade tumors was lower than that of other tumor cells.\u003c/p\u003e \u003cp\u003eTo further explore the role of PROX1 in the pathogenesis of KHE, we retrospectively analyzed the relationship between lymphatic markers PROX1, LYVE1, and PDPN (D2-40) tumor invasiveness in 15 patients with KHE. The higher the percentage of PROX1 expression, the stronger the local invasion of KHE tumors.\u003c/p\u003e \u003cp\u003eWe further observed that the invasion ability of the EOMA cell line overexpressing PROX1 was enhanced in nude mice. At the cellular level, it was found that the differentially expressed genes of EOMA overexpressing PROX1 and EOMA in the control group were related to positive regulation of cell motility, positive regulation of locomotion, and positive regulation of cell migration. Although bioinformatics suggested that PROX1 may be associated with abnormal coagulation function, the experimental data of nude mice in this study did not show coagulation dysfunction. The relationship between PROX1 and KMP still needs further investigation.\u003c/p\u003e \u003cp\u003eAlthough sirolimus is effective, it can lead to rare side effects such as interstitial pneumonia and pneumocystis carinii pneumonia due to its immunosuppressive effect, which may endanger life\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Moreover, the patients with KHE are mainly infants and young children, and the blood drug concentration should be closely monitored during the medication process. Prophylactic use of antibiotics is required to prevent serious infections when combined with glucocorticoids.\u003c/p\u003e \u003cp\u003ePropranolol is effective for some KHE, and its efficacy may be dose-related\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, it has also been reported that propranolol is ineffective in treating KHE (with or without KMP)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Propranolol monotherapy is not adequate for KHE (with KMP) patients \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Propranolol combined with vincristine and glucocorticoid therapy can achieve better efficacy\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. At present, the efficacy of propranolol in the treatment of KHE is still controversial, and the factors affecting its effectiveness are still unclear.\u003c/p\u003e \u003cp\u003eIt was found that the nearly complete resolution (\u0026gt;\u0026thinsp;75%) rate of propranolol in the treatment of KHE for one year was (4/10, 40%). There are significant individual differences in the efficacy of propranolol in the treatment of KHE. We speculate that PROX1, as a marker in the direction of lymphatic vessels, may reduce the efficacy of propranolol. However, our clinical data show that the expression of lymphatic markers does not reduce the efficacy of propranolol.\u003c/p\u003e \u003cp\u003eFor younger patients with bone and muscle invasion, sirolimus affects vaccination and chooses a method that does not have immunosuppression. Propranolol has an anti-fibrosis effect as well\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. It may play a role in reducing the long-term complications of KHE.\u003c/p\u003e \u003cp\u003eWe found that although the PROX1 positive group decreased the reactivity of propranolol in the low concentration, with the increase of propranolol concentration, the reactivity of propranolol in the PROX1 positive group tended to be consistent with that in the control group. The differentially expressed genes in PROX1 positive and control groups before and after propranolol treatment involved similar biological processes and pathways. So, the effect and mechanism of the expression of lymphatic markers on the efficacy of treatment still needs to be further explored.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eDue to the small sample size of this study, the effect of PROX1 on the invasiveness of KHE and efficacy of propranolol needs further study. The mechanism of PROX1 promoting KHE tumor invasion still needs further exploration. The mechanism by which affects propranolol\u0026rsquo;s treatment of KHE needs further study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study found that PROX1 increased tumor invasiveness in nude mice and KHE patients. PROX1 may play a role by affecting cell motility and migration. The nearly complete resolution rate of propranolol in treating KHE without KMP is 40%. PROX1 expression did not reduce the efficacy of propranolol.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study protocol was reviewed and approved by Medical Ethics Committee of Beijing Children\u0026rsquo;s Hospital, Capital Medical University (IEC-C-006-A04-V.06). All the animal procedures in this study got permitted by the Animal Care and Experimental Committee of Capital Medical University (AEEI-2023-049).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the patients for the publication of their anonymized information in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLL and LM conceptualized, designed the study, and reviewed and revised the manuscript. LY conceptualized and designed the study, drafted the initial manuscript, designed the data collection instruments, collected data, undertook the experiment, carried out the initial analyses, reviewed and revised the manuscript, and critically reviewed the manuscript for important intellectual content. LW and LQ collected data, undertook the experiment, coordinated, contributed to analysis and interpretation of data, supervised data collection, and critically reviewed it for important intellectual content. All authors contributed to the article and approved the submitted version. All authors approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial disclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone of the authors has a financial interest in any of the products, devices, or drugs mentioned in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks for the support of the parents of KHE patients in this article. Thanks to the guidance of Kaihu Yao, Qinghong Meng and Dan Yu from Beijing Pediatric Research Institute.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang Y, Kong L, Sun B, Cui J, Shen W. 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Comparison of Effectiveness of Two Different Doses of Propranolol on Kaposiform Hemangioendothelioma. \u003cem\u003eFront Pediatr.\u003c/em\u003e 2022;10:760401. \u003c/li\u003e\n\u003cli\u003eFilippi L, Tamburini A, Berti E, Perrone A, Defilippi C, Favre C, et al. Successful Propranolol Treatment of a Kaposiform Hemangioendothelioma Apparently Resistant to Propranolol. \u003cem\u003ePediatr Blood Cancer\u003c/em\u003e 2016;63(7):1290-1292. \u003c/li\u003e\n\u003cli\u003eChiu YE, Drolet BA, Blei F, Carcao M, Fangusaro J, Kelly ME, et al. Variable response to propranolol treatment of kaposiform hemangioendothelioma, tufted angioma, and Kasabach-Merritt phenomenon. \u003cem\u003ePediatr Blood Cancer\u003c/em\u003e 2012;59(5):934-938. \u003c/li\u003e\n\u003cli\u003eChoeyprasert W, Natesirinilkul R, Charoenkwan P. Successful treatment of mild pediatric kasabach-merritt phenomenon with propranolol monotherapy. \u003cem\u003eCase Rep Hematol.\u003c/em\u003e 2014;2014:364693. \u003c/li\u003e\n\u003cli\u003eJi Y, Chen S, Li L, Yang K, Xia C, Li L, et al. Kaposiform hemangioendothelioma without cutaneous involvement. \u003cem\u003eJ Cancer Res Clin Oncol.\u003c/em\u003e 2018;144(12):2475-2484. \u003c/li\u003e\n\u003cli\u003eTribolet S, Hoyoux C, Boon LM, Cheruy C, Demarche M, Jamblin P, et al. A not so harmless mass: Kaposiform hemangioendothelioma complicated by a Kasabach-Merritt phenomenon. \u003cem\u003eArch Pediatr.\u003c/em\u003e 2019;26(6):365-369. \u003c/li\u003e\n\u003cli\u003eJi Y, Yang K, Chen S, Peng S, Lu G, Liu X. Musculoskeletal complication in kaposiform hemangioendothelioma without Kasabach-Merritt phenomenon: clinical characteristics and management. \u003cem\u003eCancer Manag Res. \u003c/em\u003e2018;10:3325-3331. \u003c/li\u003e\n\u003cli\u003eUno T, Ito S, Nakazawa A, Miyazaki O, Mori T, Terashima K. Successful treatment of Kaposiform hemangioendothelioma with everolimus. \u003cem\u003ePediatr Blood Cancer.\u003c/em\u003e 2015;62(3):536-538. \u003c/li\u003e\n\u003cli\u003eWang Z, Li K, Dong K, Xiao X, Zheng S. Variable response to propranolol treatment of kaposiform hemangioendothelioma, tufted angioma, and Kasabach-Merritt phenomenon. \u003cem\u003ePediatr Blood Cancer.\u003c/em\u003e 2014;61(8):1518-1519. \u003c/li\u003e\n\u003cli\u003eHermans DJJ, Beynum IMv, Vijver RJvd, Kool LJS, Blaauw Id, Vleuten CJMvd. Kaposiform hemangioendothelioma with Kasabach-Merritt syndrome: a new indication for propranolol treatment. \u003cem\u003eJ Pediatr Hematol Oncol.\u003c/em\u003e 2011;33(4):e171-173. \u003c/li\u003e\n\u003cli\u003eUppuluri R, Kumar V, Munirathnam D, Doss H, Ramachandrakurup S, Subburaj D, et al. Kaposiform Hemangioendothelioma in a 3 Months Old Infant. \u003cem\u003eIndian J Hematol Blood Transfus.\u003c/em\u003e 2016;32(Suppl 1):370-371. \u003c/li\u003e\n\u003cli\u003eVerma S, Dhamija E, Barwad A, Kumar VS, Rastogi S. A case report of Kaposiform haemangioendothelioma; response with propranolol and steroids. \u003cem\u003eClin Sarcoma Res.\u003c/em\u003e 2020;10:12. \u003c/li\u003e\n\u003cli\u003eDing Q, Li Z, Liu B, Ling L, Tian X, Zhang C. Propranolol prevents liver cirrhosis by inhibiting hepatic stellate cell activation mediated by the PDGFR/Akt pathway. \u003cem\u003eHum Pathol.\u003c/em\u003e 2018;76:37-46. \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":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Kaposiform hemangioendothelioma, PROX1, invasiveness, β-blockers","lastPublishedDoi":"10.21203/rs.3.rs-4014692/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4014692/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor that usually expresses lymphatic markers. PROX1 can increase the local invasiveness of KHE in mice.\u003c/p\u003e\n\u003cp\u003eObjectives: To evaluate whether the expression of lymphatic markers in KHE patients increases their invasiveness and decreases the efficacy of β-blockers (propranolol or atenolol).\u003c/p\u003e\n\u003cp\u003eMethods: The tumor tissues of 3 patients with KHE were collected for transcriptomics. The clinical data of 15 patients were collected to analyze the correlation between PROX1, invasiveness and β-blockers’ efficacy. Overexpression PROX1 in EOMAs, cell viability assay, apoptosis, cell cycle, migration, and β-blockers’ efficacy were analyzed in vitro. Tumor invasiveness was analyzed in vivo.\u003c/p\u003e\n\u003cp\u003eResults: PROX1 might increase local invasiveness of the KHE due to impact cell motility, locomotion, and migration. The nearly complete resolution rate (\u0026gt;75%) of β-blockers (propranolol or atenolol) in the treatment of KHE was 40 %. There was no significant correlation between the positive rate of PROX1 and the efficacy of β-blockers’ treatment.\u003c/p\u003e\n\u003cp\u003eLimitations: Due to small numbers, the effect of PROX1 on the efficacy of propranolol needs further study.\u003c/p\u003e\n\u003cp\u003eConclusion: Our study showed the higher positive rate of PROX1 in the tumor, the more prone the local invasiveness of the KHE patients. There was no significant correlation between the positive rate of PROX1 and the efficacy of 10 patients with β-blockers treatment.\u003c/p\u003e","manuscriptTitle":"Lymphatic Markers in Kaposiform Hemangioendothelioma: Implications for Tumor Invasiveness and β-blockers efficacy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-28 17:46:43","doi":"10.21203/rs.3.rs-4014692/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":"e2855fe5-ba19-47fb-85c1-dc0d5639a3a3","owner":[],"postedDate":"March 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-24T21:11:51+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-28 17:46:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4014692","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4014692","identity":"rs-4014692","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}