Lymphatic Microvessel Density Research Articles

ZNF468-mediated epigenetic upregulation of VEGF-C facilitates lymphangiogenesis and lymphatic metastasis in ESCC via PI3K/Akt and ERK1/2 signaling pathways.

Dysfunctional lymphangiogenesis is pivotal for various pathological processes including tumor lymph node metastasis which is a crucial cause of therapeutic failure for ESCC. In this study, we aim to elucidate the molecular mechanisms and clinical relevance of Zinc-finger protein ZNF468 in lymphangiogenesis and lymphatic metastasis in ESCC. Immunohistochemistry, Western blot, Kaplan-Meier plotter analysis and Gene Set Enrichment Analysis were preformed to detect the association of ZNF468 with lymphangiogenesis and poor prognosis in ESCC patients. Foot-pads lymph node metastasis model, tube formation assay, 3D-culture assay and invasion assay were preformed to verify the effect of ZNF468 on lymphangiogenesis and lymph node metastasis. CUT&Tag analysis, immunoprecipitation and mass spectrometry analysis and ChIP-PCR assay were preformed to study the molecular mechanisms of ZNF468 in lymphangiogenesis. We found that ectopic expression of ZNF468 was correlated with higher microlymphatic vessel density in ESCC tissues, leading to poorer prognosis of ESCC patients. ZNF468 enhanced the capacity of lymphangiogenesis and promoted lymphatic metastasis in ESCC both in vitro and in vivo. However, silencing ZNF468 reversed these phenotypes in ESCC. Mechanically, we demonstrated that ZNF468 recruits the histone modification factors (PRMT1/HAT1) to increase the levels of H4R2me2a and H3K9ac, which then leads to the recruitment of the transcription initiation complex on the VEGF-C promoter, ultimately promoting the upregulation of VEGF-C transcription. Strikingly, the promoting effect of lymphatic metastasis induced by ZNF468 in ESCC was abrogated by targeting PRMT1 using Arginine methyltransferase inhibitor-1 or silencing VEGF-C. Furthermore, we found that the activation of PI3K/AKT and ERK1/2 signaling is required for ZNF468-medicated lymphatic metastasis in ESCC. Importantly, the clinical relevance between ZNF468 and VEGF-C were confirmed not only in ESCC samples and but also in multiple cancer types. Our results identified a precise mechanism underlying ZNF468-induced epigenetic upregulation of VEGF-C in facilitating lymphangiogenesis and lymph node metastasis of ESCC, which might provide a novel prognostic biomarker and potential therapeutic for ESCC patients.

Downregulation of miR-100-5p in cancer-associated fibroblast-derived exosomes facilitates lymphangiogenesis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC), an aggressive gastrointestinal tumor, often has high early lymphatic metastatic potential. Cancer-associated fibroblasts (CAFs) are primary components in tumor microenvironment (TME), and the impact of CAFs and its derived exosomes on lymphangiogenesis remains elusive. CAFs and the microlymphatic vessel density (MLVD) in ESCC was examined. Exosomes were extracted from primary normal fibroblast (NFs) and CAFs. Subsequently, tumor-associated lymphatic endothelial cells (TLECs) were treated with these exosomes, and the effect on their biological behavior was examined. miR-100-5p was selected as the target miRNA, and its effect on TLECs was examined. The target of miR-100-5p was predicted and confirmed. Subsequently, IGF1R, PI3K, AKT, and p-AKT expression in TLECs and tumors treated with exosomes and miR-100-5p were examined. A large number of CAFs and microlymphatic vessels were present in ESCC, leading to a poor prognosis. CAF-derived exosomes promoted proliferation, migration, invasion, and tube formation in TLECs. Further, they also enhanced lymphangiogenesis in ESCC xenografts. miR-100-5p levels were significantly lower in CAF-derived exosomes than in NF-derived exosomes. miR-100-5p inhibited proliferation, migration, invasion, and tube formation in TLECs. Further, miR-100-5p inhibited lymphangiogenesis in ESCC xenografts. Mechanistic studies revealed that this inhibition was mediated by the miR-100-5p-induced inhibition of IGF1R/PI3K/AKT axis. Taken together, our study demonstrates that CAF-derived exosomes with decreased miR-100-5p levels exhibit pro-lymphangiogenesis capacity, suggesting a possibility of targeting IGF1R/PI3K/AKT axis as a strategy to inhibit lymphatic metastasis in ESCC.

BANCR positively regulates the HIF-1α/VEGF-C/VEGFR-3 pathway in a hypoxic microenvironment to promote lymphangiogenesis in pancreatic cancer cells.

The aim of the present study was to explore the effects of BRAF-activated non-protein coding RNA (BANCR) on pancreatic microlymphangiogenesis in pancreatic cancer (PC) and its molecular mechanism under hypoxic conditions. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect the expression of BANCR in SW1990 and PANC-1 PC cell lines under normoxic and hypoxic conditions. Subsequently, the expression of BANCR in the PC cells was knocked down using small interfering RNAs (siRNAs). Western blotting and RT-qPCR analyses were performed to detect the expression of hypoxia-inducible factor (HIF-1α), VEGF-C and VEGFR-3 in the transfected cells. In addition, the transfected PC cells were co-cultured with human lymphatic endothelial cells and the lymphatic microvessel density (MLVD) was detected under normal and hypoxic conditions. Furthermore, HIF-1α expression in the PC cells was knocked down using siRNAs, and VEGF-C and VEGFR-3 mRNA expression in the HIF-1α knockdown cells was detected using RT-qPCR. The results showed that the expression of BANCR in the SW1990 and PANC-1 PC cell lines was significantly higher than that in human pancreatic duct endothelial cells. Additionally, the expression of BANCR was significantly increased in PC cells under hypoxic conditions compared with normoxic conditions. The MLVD of PC cells under hypoxic conditions was significantly higher compared with that under normoxic conditions, and the MLVD in the si-BANCR group was lower than that in the si-NC group, indicating that si-BANCR downregulated MLVD. These results indicate that BANCR positively regulated the expression of HIF-1α in PC cells at the transcriptional and translational levels. Finally, the expression levels of VEGF-C and VEGFR-3 in PC cells were significantly reduced when BANCR or HIF-1α expression was knocked down. In conclusion, the results demonstrate that the expression of BANCR in PC cells was significantly increased under hypoxic conditions and suggest that BANCR promoted tumor cell lymphangiogenesis by upregulating the HIF-1α/VEGF-C/VEGFR-3 pathway, which plays an important role in the process of PC lymph node metastasis.

Integrin α6 overexpression promotes lymphangiogenesis and lymphatic metastasis via activating the NF-κB signaling pathway in lung adenocarcinoma.

It has been reported that tumor-associated lymphangiogenesis plays an important role in lymph node metastasis and contributes to the poor survival of lung adenocarcinoma (LUAD) patients. As yet, however, the molecular mechanism underlying LUAD-associated lymphangiogenesis has remained elusive. Immunohistochemistry (IHC) was used to determine the expression of integrin subunit alpha 6 (ITGA6) and the lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) in clinicopathologically characterized LUAD specimens. The effect of ITGA6 overexpression on lymphangiogenesis and lymphatic metastasis was examined by tube formation, scratch wound-healing, and cell migration assays in vitro and a popliteal lymph node metastasis model in vivo. Mechanistically, overexpression of ITGA6 and activation of NF-κB signaling were examined by real-time PCR, ubiquitination and dual-luciferase reporter assays. Finally, high ITGA6 expression in LUAD tissue samples was related to copy number variation (CNV) using the TCGA database. We found that ITGA6 overexpression correlated with microlymphatic vessel density in LUAD specimens (p < 0.01). Importantly, by using a popliteal lymph node metastasis model, we found that ITGA6 upregulation significantly enhanced lymphangiogenesis and lymphatic metastasis in vivo (p < 0.05). In addition, we found that ITGA6 overexpression enhanced the capability of A549 and H1299 LUAD cells to induce tube formation and migration in human lymphatic endothelial cells (HLECs). Mechanistically, we found that ITGA6 sustained NF-κB activity via binding and promoting K63 polyubiquitination of TNF receptor-associated factor 2 (TRAF2). Finally, CNV analysis revealed ITGA6 amplification of 27.5% in the LUAD tissue samples in the TCGA database. Taken together, our results uncover a plausible role for ITGA6 in mediating lymphangiogenesis and lymphatic metastasis and may provide a basis for targeting ITGA6 to treat LUAD lymphatic metastasis.

BRAFV600E mutant colorectal cancer cells mediate local immunosuppressive microenvironment through exosomal long noncoding RNAs.

BACKGROUNDBRAFV600E mutated colorectal cancer (CRC) is prone to peritoneal and distant lymph node metastasis and this correlates with a poor prognosis. The BRAFV600E mutation is closely related to the formation of an immunosuppressive microenvironment. However, the correlation between BRAFV600E mutation and changes in local immune microenvironment of CRC is not clear.AIMTo explore the effect and mechanism of BRAFV600E mutant on the immune microenvironment of CRC.METHODSThirty patients with CRC were included in this study: 20 in a control group and 10 in a treatment group. The density of microvessels and microlymphatic vessels, and M2 subtype macrophages in tumor tissues were detected by immunohistochemistry. Screening and functional analysis of exosomal long noncoding RNAs (lncRNAs) were performed by transcriptomics. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (HLECs) were detected by CCK-8 assay and scratch test, respectively. The tube-forming ability of endothelial cells was detected by tube formation assay. The macrophage subtypes were obtained by flow cytometry. The expression of vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β1, VEGF-C, claudin-5, occludin, zonula occludens (ZO)-1, fibroblast activation protein, and α-smooth muscle actin was assessed by western blot analysis. The levels of cytokines interleukin (IL)-6, TGF-β1, and VEGF were assessed by enzyme-linked immunosorbent assay.RESULTSBRAFV600E mutation was positively correlated with the increase of preoperative serum carbohydrate antigen 19-9 (P < 0.05), and with poor tumor tissue differentiation in CRC (P < 0.01). Microvascular density and microlymphatic vessel density in BRAFV600E mutant CRC tissues were higher than those in BRAF wild-type CRC (P < 0.05). The number of CD163+ M2 macrophages in BRAFV600E mutant CRC tumor tissue was markedly increased (P < 0.05). Compared with exosomes from CRC cells with BRAF gene silencing, the expression of 13 lncRNAs and 192 mRNAs in the exosomes from BRAFV600E mutant CRC cells was upregulated, and the expression of 22 lncRNAs and 236 mRNAs was downregulated (P < 0.05). The biological functions and signaling pathways predicted by differential lncRNA target genes and differential mRNAs were closely related to angiogenesis, tumor cell proliferation, differentiation, metabolism, and changes in the microenvironment. The proliferation, migration, and tube formation ability of HUVECs and HLECs induced by exosomes in the 1627 cell group (HT29 cells with BRAF gene silencing) was greatly reduced compared with the HT29 cell group (P < 0.05). Compared with the HT29 cell group, the expression levels of VEGF-A, bFGF, TGF-β1, and VEGF-C in the exosomes derived from 1627 cells were reduced. The expression of ZO-1 in HUVECs, and claudin-5, occludin, and ZO-1 in HLECs of the 1627 cell group was higher. Compared with the 1627 cell group, the exosomes of the HT29 cell group promoted the expression of CD163 in macrophages (P < 0.05). IL-6 secretion by macrophages in the HT29 cell group was markedly elevated (P < 0.05), whereas TGF-β1 was decreased (P < 0.05). The levels of IL-6, TGF-β1, and VEGF secreted by fibroblasts in the 1627 cell group decreased, compared with the HT29 cell group (P < 0.05).CONCLUSIONBRAFV600E mutant CRC cells can reach the tumor microenvironment by releasing exosomal lncRNAs, and induce the formation of an immunosuppressive microenvironment.

Correlation Analysis Among the Level of IL-35, Microvessel Density, Lymphatic Vessel Density, and Prognosis in Non-Small Cell Lung Cancer.

The aim of this study was to determine the expression of IL‐35 and the lymphatic vessel density (LVD) and microvessel density (MVD) in the pathological tissues from patients with non‐small cell lung cancer (NSCLC) and to analyze their correlation with other common clinical prognostic factors, as well as patients’ overall survival and progression‐free survival. We analyzed the pathological characteristics of 130 patients with NSCLC and determined the IL‐35 expression, MVD, and LVD changes in the pathological tissues by immunohistochemistry. The results showed that IL‐35 expression was significantly correlated with tumor differentiation, lymph node metastasis, T staging, LVD, and MVD (P < 0.05) but was not associated with age, sex, smoking, and other factors. Univariate analysis of risk models showed that age, lymph node metastasis, T stage, and high IL‐35 expression, LVD, and MVD were significantly associated with NSCLC prognosis (P < 0.05), whereas sex, smoking, and high differentiation were not correlated with prognosis. Multivariate analysis of the proportional risk model showed that the IL‐35 expression, lymph node metastasis, high LVD, and high MVD were significantly correlated with NSCLC prognosis (P < 0.05). In conclusion, IL‐35, MVD, and LVD may be independent prognostic markers. In addition, IL‐35 might represent a promising clinical drug target for the treatment of NSCLC.

Maspin suppresses cell invasion and migration in gastric cancer through inhibiting EMT and angiogenesis via ITGB1/FAK pathway.

This study aims to investigate how Maspin affects the EMT and angiogenesis of gastric cancer (GC) cells via ITGB1/FAK pathway. Immunohistochemistry was used to evaluate the expressions of Maspin, ITGB1, FAK, E-cadherin, Vimentin, D2-40, and CD34 in GC and adjacent normal tissues from 160 patients. Then, the human GC cells with different degree of differentiation were transfected with Maspin CRISPR activation plasmid, ITGB1 siRNA and/or Maspin siRNA, followed by the following experiments, including qRT-PCR, western blotting, tube formation assay, Transwell assay and wound healing. GC tumor tissues manifested decreased Maspin with the activated ITGB1/FAK pathway. In tumor tissues, Maspin was negatively correlated with the expressions of ITGB1 and FAK, as well as Lauren's classification, differentiation degree, and TNM stage. Besides, Maspin was negatively related with lymphatic vessel density (LVD) and microvessel density (MVD), Vimentin and VEGF, but was positive correlated with E-cadherin. Maspin expression decreased, but ITGB1 and p-FAK expressions increased gradually in MKN-28 (well differentiated), SGC-7901 (moderate differentiated), and MKN-45 (poorly differentiated). Maspin CRISPR and ITGB1 siRNA increased E-cadherin with the decreased Vimentin, VEGF and bFGF, and the reductions of tube length. In comparison with the ITGB1 siRNA group, cells in the Maspin siRNA + ITGB1 siRNA group presented the more evident EMT and angiogenesis. Furthermore, ITGB1 siRNA reduced the malignancies of GC cells, which could be restored by Maspin siRNA. Maspin was downregulated in GC tissues, which could inhibit the EMT and angiogenesis by blocking the ITGB1/FAK pathway, thereby decreasing cell invasion and migration of GC.

Podoplanin: a Prognostic Marker and a Potential Target for Therapy in Bladder Cancer

Invasive bladder cancer is a frequent neoplastic diseases, and despite progresses made in early diagnosis and surgical procedures, the outcome of patients is characterized by high rate of mortality. This is mainly due to the lack of response to chemotherapy and radiotherapy, and other new resources are limited. In the present work we analyzed 50 consecutive cases of invasive bladder cancer treated by open surgery. Specimens were processed using standard histological procedures, including establishment of the histological diagnosis and grading. Additional slides were stained for podoplanin expression, clone D2-40 to investigate the structure and number of lymphatic vessels. We found lymphatic vessels in both intra- and peritumor areas, showing significant differences in morphology and number. Lymphatic invasion by tumor cells was higher in peritumor than in intratumor vessels. Lymphatic microvessel density correlated with stage and grade of the tumor. Of interest is the expression of podoplanin by tumor cells of urothelial carcinoma in about 14% of the cases, with strong correlation with grading. Based on our results, expression of podoplanin in invasive bladder cancer might indicate three potential targets: lymphatics, myofibroblasts, and tumor cells in selected cases. Keywords: bladder cancer, immunohistochemistry, podoplanin, therapeutic target

Comparison of the Roles of Lymphatic Vessels and Blood Vessels in the Recurrence of Pterygia: A Retrospective Study

Purpose: Increased conjunctival arteries and lymphangions accelerate the cellular immune response in recurrent pterygium, however, which plays a more important role warrants further investigation. The aim of the study is to compare the roles of lymphatic and blood vessels in pterygium recurrence. Methods: Histological sections from 48 excised recurrent pterygia (including 14 Grade 1, 20 Grade 2, and 14 Grade 3 tissues) were examined. Histological sections from seven nasal epibulbar conjunctival segments served as normal controls. Blood and lymphatic vessels were evaluated and compared according to blood microvessel density (BMD), blood vascular area (BVA), lymphatic microvessel density (LMD), and lymph-vascular area (LVA). Furthermore, the following relationships were analyzed: LMD and pterygium recurrence time (RT), LVA and RT, BMD and RT, BVA and RT. Results: Compared to LVA, LMD, BVA, and BVD values in normal control tissues, these values were markedly enhanced in recurrent pterygia tissues. The LMD/BMD and LVA/BVA ratios were significant increased in Grade 2 and 3 recurrent pterygia, suggesting that development of conjunctival lymphangions was not proportional to blood vessel growth. LMD, LVA and BVA were significantly correlated with RT for all grades of pterygia, while BMD was the only factor correlated with RT in Grade 1 pterygia. Moreover, no significant correlation was found between BMD and RT in Grade 2 and 3 pterygia. Conclusion: Compared to blood vessels, lymphangions might have a greater impact on pterygium recurrence.

Development of a biomarker of efficacy in second-line treatment for lymphangioma of the tongue: a pilot study.

Lymphangioma of the tongue is a rare lymphatic malformation, and various authors have reported the successful use of sirolimus for its treatment. However, the safety of sirolimus in children needs further evaluation so that those who do not respond are not necessarily exposed to its potential adverse effects. We hypothesised that assessment of lymphangiogenesis can be used to predict whether the patient will respond to sirolimus, so we organised a prospective study after ethics committee approval had been given. After clinical and histological diagnoses of lymphangioma of the tongue had been confirmed, 16 patients were given sirolimus 0.8mg/day in three divided doses. Clinical response was assessed and compared with lymphatic microvessel density (LMVD), which was calculated immunohistochemically using the monoclonal antibody D2-40 as the lymphatic endothelial marker. Nine patients responded well, five partially, and two failed to respond. Mean (SD) LVD among the good responders was 21.00 (3.74), whereas among non-responders it was 8.00 (4.24). There was a significant difference in mean LVD between good responders, partial responders, and non-responders (p=0.04). Sirolimus is effective in treating children with lymphangioma of the tongue, and lymphangiogenesis is a useful therapeutic predictive marker.

Histological features of skin and subcutaneous tissue in patients with breast cancer who have received neoadjuvant chemotherapy and their relationship to post-treatment edema.

Lymphedema is a major complication of treatment for breast cancer. Although chemotherapy can cause lymphedema, there have been few reports about histological changes in skin and subcutaneous tissue after chemotherapy. The aim of our study was to determine whether chemotherapy affects blood and lymphatic vessels in the skin and subcutaneous fat and to investigate the relationship between these changes and extent of post-chemotherapy edema. We compared histological findings in skin and subcutaneous fat of mastectomy specimens from 38 patients who had received NAC (neoadjuvant chemotherapy) and 56 who had not (non-NAC) attending our institution from 2007 to 2016. Patients whose tumor may have affected the area examined were excluded. Blood and lymphatic vessels were identified by CD31 and D2-40, respectively. We assessed microvessel density (MVD), lymphatic microvessel density (MLVD), lumen cross-sectional area (LA), and amount of endothelium (AE) in blood and lymphatic vessels. To minimize surgical effects, we measured edema, defined as ≥ 15% thicker dorsal subcutaneous tissue than baseline, on the contralateral side. MVD, LA, and AE of blood vessels were greater and MLVD not significantly different in the skin of NAC patients than in that of non-NAC patients. MVD was greater and AE of blood vessels less in subcutaneous fat of NAC patients than in that of non-NAC patients. Patients with edema had significantly less AE of blood vessels in skin than did those without it. These pathological findings can help to identify patients who will develop edema and improve their treatment.

Relationship between one-stop CT spectral perfusion imaging parameters and expression of lymphatic microvessel density and vascular endothelial growth factor-C in axillary lymph nodes of rabbit VX2 breast cancer

Objective: To analyze the correlation between quantitative parameters of one-stop spectral perfusion computed tomography imaging and the expression level of vascular endothelial growth factor-C(VEGF-C) and MLVD in metastatic lymph nodes of rabbit VX2 breast cancer. Methods: Thirty New Zealand purebred female rabbits were used to establish the lymph node metastasis model of rabbit VX2 breast cancer, one-stop spectral and perfusion CT imaging protocol was performed.The axillary lymph nodes were selected for corresponding image markers. The observed morphology of conventional HE staining and the EnVision method were used to quantitatively analyze VEGF-C expression and calculate MLVD.Pearson linear correlation was used to analyze the perfusion parameters of metastatic lymph node energy spectrum and the correlation of MLVD and VEGF-C expression. Results: Twenty-four experimental rabbits were successfully modeled and performed a one-stop CT scan on the 28th day. A total of 39 metastatic lymph nodes were included. The VEGF-C of metastatic lymph node was 20.0%±2.8%,and the MLVD was 12.5±3.5. There was a positive correlation between BF, AP(λHU), IC(VP), NIC(VP), VP(λHU) of metastatic lymph node and VEGF-C and MLVD (P<0.05). There was a positive correlation between lymph node IC(AP) and MLVD (P=0.027) and no correlation with VEGF-C expression (P=0.386).There was no correlation between BV, NIC(A)P and VEGF-C, MLVD(P>0.05). The correlation between VP(λHU) and MLVD was higher (r=0.448, P=0.001). Conclusions: One-stop CT spectral perfusion imaging quantitative parameters and pathological indicators have a good correlation, and it can reflect lymphatic vessel metastasis in lymph nodes.

Pursuit of an optimal surgical margin in pulmonary metastasectomy

Pulmonary metastasectomy is an accepted treatment modality for stage IV disease in various primary carcinomas and sarcomas. In addition to tumor removal, its objectives are preservation of normal lung parenchyma and maintaining optimal length of surgical margins. We analyzed the impact of risk factors and local recurrence on disease course, effect of surgical techniques and devices on local recurrence, relationship between molecular constellations and microscopic tumor spread in lung with local recurrence, and the role played by the type of resection and lymphadenectomy. Local recurrence at resection margin and in lymph nodes is attributable to resection site inadequacy and the extent of lymphadenectomy respectively, with lower incidence when surgical margins are wider and metastases are smaller. The incidence of local recurrence is comparable between minor and major resections, moreover when employing different resection techniques. Laser resection (Nd:YAG) has lower recurrence by causing alveolar coagulation and occlusion as well as leading to coagulation and necrosis of parenchyma, capillaries and lymphatics in the resection margin. The evaporation and coagulation zones on both sides of the resection line can be added to the pathologic measurement in order to calculate the size of the resection margin. Spread through air spaces (STAS), lymphatic microvessel density (LMVD), lymphovascular invasion (LVI), tumor type and metastasis growth patterns, driver mutations are all major factors impacting locoregional recurrence. Safe resection margin during metastasectomy prevents local recurrence and wider resection margins are required when aggressive patterns of local spread are present. A safe resection margin should be at least either half the diameter of the metastasis. Microscopic tumor dissemination can only be identified postoperatively. Studies are required to additionally describe the radiomorphology and microscopic growth patterns of metastases, to facilitate surgeons to plan better and adequate resection margins.

Tumor-associated lymphangiogenesis predicts unfavorable prognosis of intrahepatic cholangiocarcinoma

BackgroundTumor-associated lymphangiogenesis is considered significant in number of solid malignancies. However, its impact on prognosis of intrahepatic cholangiocarcinoma (ICC) after resection remains further confirmation. Herein, we conducted this study to evaluate prognostic impact of tumor-associated lymphangiogenesis in patients with ICC.MethodsExtent of tumor-associated lymphangiogenesis of ICC was evaluated by quantifying microlymphatic vessel density (MLVD) from immunohistochemical staining of a lymphatic endothelial-specific antibody (podoplanin). Clinicopathological characteristics were comprehensively analyzed to identify MLVD-associated factors. The patients were stratified into high and low MLVD groups according to the distinctive correlation between the MLVD and overall survival using the Spearman’s correlation test. Kaplan-Meier estimation was performed to confirm prognostic impact of MLVD in patients with ICC. Univariate and multivariate analyses were performed using the Cox proportional hazard model.ResultsThe MLVD between 4 to 12 counts showed inverse proportion to the overall survival (Spearman’s r = − 0.66; 95% confidence interval [CI], − 0.82 to − 0.39; p < 0.0001), which was set as a cut-off for the high MLVD group, whereas the MLVD between 13 to 25 showed no correlation to the overall survival (r = − 0.11; 95% CI, − 0.38 to 0.19; p = 0.4791). The high MLVD group showed more frequent lymph node metastasis (p < 0.001) and were more likely to suffer from recurrence of the tumor compared to the low MLVD group (p < 0.001). The high MLVD was found to be independently associated with reduced overall and recurrence-free survival. The 5-year overall survival of the patients with high MLVD was significantly lower compared to those with low MLVD (0% vs 48%).ConclusionsOur study reveals that tumor-associated lymphangiogenesis is significantly associated with increased lymphatic metastasis, recurrence of the tumor, and reduced overall survival in patients with ICC, thus providing guidance when estimating postresection prognosis.

Aberrant Cyclin E and Hepatocyte Growth Factor Expression, Microvascular Density, and Micro-Lymphatic Vessel Density in Esophageal Squamous Cell Carcinoma.

Cyclin E and hepatocyte growth factor (HGF) have been observed as a multifaceted factor in many cancers, and the assessment of microvascular density (MVD) and micro-lymphatic vessel density (MLVD) has been used to quantify tumor angiogenesis and lymphangiogenesis. The aim of this study was to explore the association between expression of cyclin E, HGF, MVD, and MLVD, and clinicopathologic parameters in esophageal squamous cell carcinoma (ESCC). The expression of cyclin E, HGF, MVD, and MLVD were detected using immunohistochemically anticyclin E, HGF, CD34, and lymphatic vessel endothelial hyaluronan receptor 1 in 168 surgically resected ESCC cases and 30 normal esophageal mucosal samples. The expression levels of cyclin E, HGF, MVD, and MLVD were higher compared to controls. High cyclin E and HGF expression was found more frequently in the tumors larger than 5 cm (P < .001), with poorer differentiation (P = .034) and higher tumor node metastasis (TNM) staging (P = .009) compared to their counterparts. Both MVD and MLVD values were found to be higher in the tumors larger than 5 cm (P < .001), with poorer differentiation (P < .001) and higher TNM staging (P < .001) compared to their counterparts. Furthermore, the expression of MVD and MLVD in both the high cyclin E and high HGF expression groups was significantly higher compared to the low cyclin E and HGF expression groups (P < .001). This study demonstrated that high cyclin E and HGF expression is closely correlated with tumor size, tumor differentiation degree, and TNM stage in patients with ESCC. These findings proposed that cyclin E and HGF could serve as novel molecular markers for preoperational evaluation of ESCC.

MicroRNA-488 inhibits progression of colorectal cancer via inhibition of the mitogen-activated protein kinase pathway by targeting claudin-2.

Colorectal cancer (CRC) affects people globally, and lymph node metastasis (LNM) is an important indicator of poor clinical outcome in CRC. The current study aims to evaluate the role of microRNA-448 (miR-488) and claudin-2 (CLDN2) in epithelial-mesenchymal transition (EMT) and LNM of CRC through the MAPK signaling pathway. First, microarray analysis indicated that miR-488 was poorly expressed in CRC, whereas CLDN2 was highly expressed. Additionally, the bioinformatics website MicroRNA.org and the dual luciferase reporter gene assay found that CLDN2 was a target gene of miR-488. Next, the results for the correlations between expression of miR-488 and clinicopathological characteristics of CRC indicated that the expression of miR-488 was closely associated with differentiation degree, LNM, and Dukes stages in CRC patients. Moreover, overexpression of miR-488 inhibited the activation of the MAPK signal transduction pathway. Notably, loss- and gain-of-function experiments demonstrated that upregulation of miR-488 suppressed SW480 cell viability, invasion, and migration and promoted apoptosis in SW480 cells. Finally, overexpression of miR-488 inhibited LNM, microlymphatic vessel density, and tumor growth in nude mice. We conclude that overexpression of miR-488 could suppress the cell proliferation, EMT, and LNM of CRC cells via inhibition of the CLDN2-mediated MAPK signaling pathway, which could be a new molecular therapy target for CRC.

Laringeal skuamöz hücreli karsinomlarda lenfatik damar yoğunluğu ve mikrodamar yoğunluğunun değerlendirilmesi

Amaç: Larinks karsinomlarında boyun lenf nodlarına metastaz, sağ kalımı önemli oranda azaltan bir etkendir. Bu çalışmanın amacı; larinksin skuamöz hücreli tümörlerinde tümör içi ve tümör dışı alanlarda lenfatik damar yoğunluğu ve mikrodamar yoğunluğunun lenf nodu metastazı ile ilişkisini ortaya koymaktır.Yöntemler: Çalışmamızda 2000-2006 yılları arasında Erciyes Üniversitesi Tıp Fakültesi Patoloji Ana Bilim Dalı’nda larinks skuamöz hücreli karsinom tanısı almış 86 olgu incelemeye alındı. Tümör içi ve tümör dışı alanlarda immünhistokimyasal bir belirteç olan D2-40 ile lenfatik damar yoğunluğu ve CD34 ile mikrodamar damar yoğunluğu değerlendirildi. Sonuçlar tümör derecesi, tümör yerleşim yeri, lenf nodu metastazı ile karşılaştırıldı. Bulgular: Olguların tümör içi ve tümör dışı lenfatik damar yoğunluğu sırası ile 8,93±12,5 ve 24,1±20,1 idi (p=0,001). Tümör içi mikrodamar yoğunluğu değeri 217,53±89,8 ve tümör dışı mikrodamar yoğunluğu değeri 330,43±92,4 olarak sayıldı (p=0,001). İyi diferansiye tümörlerde tümör içi lenfatik damar yoğunluğu değeri, iyi diferansiye olmayan tümörlere göre yüksekti, fakat anlamlı sonuç elde edilemedi (p=0,100). Kötü diferansiye tümörlerde tümör dışı mikrodamar yoğunluğu değeri anlamlı olarak yüksek bulundu (p=0,050). Tümör içi mikrodamar yoğunluğu değeri lenf nodu metastazı olan tümörlerde anlamlı olarak düşüktü (p=0,028). Lenf nodu metastazı olan ve olmayan her iki grupta da tümör dışı lenfatik damar yoğunluğu değeri yüksekti, istatiksel olarak anlamlı bir sonuç elde edilemedi (p=0,084). Sonuç: Bu çalışmada iyi diferansiye tümörlerde tümör içi mikrodamar yoğunluğu ve kötü diferansiye olgularda tümör dışı mikrodamar yoğunluğu daha yüksek idi. Lenfatik damar yoğunluğu değeri ile diferansiasyon arasında bir ilişki saptanmadı. Metastaz yapmayan grupta tümör içi ve tümör dışı mikrodamar yoğunluğu değeri yüksek bulundu. Metastaz ile lenfatik damar yoğunluğu değeri arasında bir ilişki izlenmedi. D2-40 ve CD34 ‘ün birlikte kullanımı ile lenfatik damar yoğunluğunun değerlendirilmesinin, metastazın erken belirlenmesinde daha önemli olabileceğini düşünmekteyiz.

Oxyresveratrol prevents murine H22 hepatocellular carcinoma growth and lymph node metastasis via inhibiting tumor angiogenesis and lymphangiogenesis.

The purpose of this study was to investigate the effects and mechanisms of oxyresveratrol (Oxyres) on hepatocellular carcinoma (HCC) in vitro and in vivo. The MTT and Transwell assays were performed to investigate the effects of Oxyres on cell proliferation and migration of two HCC cell lines, QGY-7701 and SMMC-7721 cells. H22 cells were subcutaneously injected into hind foot pads of 70 male mice to establish a lymph node metastasis model. These mice were randomly divided into seven groups as follows, control group, HCC group, Oxyres 20mg/kg group, Oxyres 40mg/kg group, Oxyres 60mg/kg group, Resveratrol (Res) group, and Adriamycin (ADM) group. Oxyres, Res, and ADM were intraperitoneally injected daily for consecutive 21days. Tumors and popliteal lymph node were isolated and embedded for histology analysis. Expressions of CD31 and vascular endothelial growth factor receptor-3 (VEGFR3) in tumors were detected by immunohistocehmistry. Expressions of vascular endothelial growth factor C (VEGF-C) were measured by Western blot. Oxyres significantly inhibited the proliferation and migration of QGY-7701 and SMMC-7721 cells. Oxyres significantly inhibited tumor growth (p<0.001) and metastasis to sentinel lymph nodes (70%) in a dose-dependent manner. Oxyres showed a similar inhibition rate as Res. Oxyres also significantly decreased micro-blood vessel density and micro-lymphatic vessel density in tumors (p<0.05). Expressions of CD31, VEGFR3, and VEGF-C of tumors were also inhibited by Oxyres (p<0.05). Oxyres exerts anti-tumor effects against HCC through inhibiting both angiogenesis and lymph node metastasis, which suggests Oxyres be a potential therapeutic agent.

Expression of VEGFR-3 in intrahepatic cholangiocarcinoma correlates with unfavorable prognosis through lymphangiogenesis.

Background & aims: VEGFR-3 has been shown of great significance in lymph node metastasis and some malignancies, however, its expression in tumors and impact on outcome of intrahepatic cholangiocarcinoma (iCCA) remains unknown. The aim of this study was to assess the role of VEGFR-3 positive tumors for prognosis of iCCA and tumor-associated lymphangiogenesis.Methods: Clinicopathological features, prognostic factors and survival rate were analyzed to evaluate the influence of VEGFR-3 positive expression on prognosis of iCCA. In addition, tumor-associated lymphangiogenesis quantified as micro-lymphatic vessel density (MLVD) was assessed to explore the correlation between VEGFR-3 expression and lymph node metastasis for iCCA.Results: Patients with VEGFR-3 positive tumors had increased lymph node metastasis (p=0.025) and were more likely to suffer from tumor recurrence compared with VEGFR-3 negative tumors (p<0.001). VEGFR-3 expression in tumors was identified as an independent prognostic factor for both overall and recurrence-free survival in surgical resected patients with iCCA. In addition, higher MLVD was significantly associated with VEGFR-3 positive expression in tumors (p<0.001), which facilitate lymph node metastasis and significantly worse survival rates.Conclusions: Our study reveals that VEGFR-3 positive expression in tumors represents an independent prognostic factor for both overall and recurrence-free survival in hepatic resected patients with iCCA. VEGFR-3 positive tumors favor lymph node metastasis, tumor recurrence and worse outcomes through tumor-associated lymphangiogenesis.

A meta-analysis of the lymphatic microvessel density and survival in gastric cancer with 1809 cases.

Lymph node metastasis commonly occurs in gastric cancer. Previous studies have demonstrated that the overexpression of lymphatic microvessel density (LVD) is correlated with various malignancies. To evaluate the potential role of LVD in various malignancies, we conducted a systematic review and meta-analysis to thoroughly investigate the association of LVD expression with tumor progression and survival in gastric cancer. We performed a comprehensive search of common databases and selected studies demonstrating the relationship between LVD expression and gastric cancer prognosis. Hazard ratios (HR) were used to determine the value of LVD for predicting gastric cancer metastasis and prognosis. The data were extracted from the included studies and pooled with the appropriate effects model using STATA 12.0. The results showed that high LVD expression obviously impacted the prognosis of gastric cancer, based on an overall survival (OS) HR of 2.58 (95% CI: 1.91–3.48, P < 0.001) and a disease-free survival (DFS) HR of 2.51 (95% CI: 1.35–4.68, P = 0.004) in the univariate analysis. In addition, the results of the multivariate analysis indicated a remarkable relationship between high LVD expression and gastric neoplasm prognosis. The pooled OS HR was 4.12 (95% CI: 3.45–4.91, P < 0.001). The current meta-analysis shows that high LVD is closely related to tumor metastasis and poor prognosis in gastric malignancy. LVD could be a key factor in tumor lymphatic metastasis. Moreover, LVD is likely a potential index and an effective biomarker for the prediction of patient prognosis.