High Lymphatic Metastasis Research Articles

Effective Combination of 3D-DenseNet's Artificial Intelligence Technology and Gallbladder Cancer Diagnosis Model

Gallbladder cancer is the most common malignant tumor in the biliary system. It has the characteristics of low early diagnosis rate, strong invasiveness and high lymphatic metastasis rate. In recent years, with the rapid development of artificial intelligence technology, relevant technologies based on machine learning and deep learning algorithms have been applied to the diagnosis and treatment of malignant tumors, prognosis assessment and medical image processing, bringing revolutionary changes to the diagnosis and treatment mode of malignant tumors. At present, artificial intelligence technology has been preliminarily studied in the early screening and diagnosis of gallbladder cancer, preoperative lymph node status assessment, intraoperative lymph node dissection, surgical treatment and prognosis assessment, showing certain clinical value. In this paper, in order to assist clinical diagnosis of gallbladder cancer, an improved 3D-DenseNet was used to establish an assisted diagnosis model of gallbladder cancer based on enhanced CT images of patients. Firstly, multiple arterial CT images of patients were converted into 3D images, and the regions of interest were cut out using the gallbladder area marked by doctors. Then, the traditional Dense Net network is optimized, the Dropout mechanism and Soft max loss function are improved, and the cross-entropy function is replaced by Focal loss in the output part for imbalance correction, so as to establish the auxiliary diagnosis model of gallbladder cancer.

Patterns of lymph node metastasis in level IIB and contralateral level VI for papillary thyroid carcinoma with pN1b and safety of low collar extended incision for neck dissection in level II

ObjectiveTo explore relevant clinical factors of level IIB and contralateral level VI lymph node metastasis and evaluate the safety of low-collar extended incision (LCEI) for lymph node dissection in level II for papillary thyroid carcinoma (PTC) with pN1b.MethodA retrospective analysis was performed on 218 patients with PTC with pN1b who were treated surgically in the Head and Neck Surgery Center of Sichuan Cancer Hospital from September 2021 to May 2022. Data on age, sex, body mass index (BMI), tumor location, maximum tumor diameter, multifocality, Braf gene, T staging, surgical incision style, and lymph node metastasis in each cervical subregion were collected. The chi-square test was used for comparative analysis of relevant factors. All statistical analyses were completed by SPSS 24 software.ResultEach subgroup on sex, age, BMI, multifocality, tumor location, extrathyroidal extension, Braf gene, and lymphatic metastasis in level III, level IV, and level V had no significant difference in the positive rate of lymph node metastasis in level IIB (P > 0.05). In contrast, patients with bilateral lateral cervical lymphatic metastasis were more likely to have level IIB lymphatic metastasis than those with unilateral lateral cervical lymphatic metastasis, with a statistically significant difference (P = 0.000). In addition, lymph node metastasis in level IIA was significantly associated with lymph node metastasis in level IIB (P = 0.001). After multivariate analysis, lymph node metastasis in level IIA was independently associated with lymph node metastasis in level IIB (P = 0.010). The LCEI group had a similar lymphatic metastasis number and lymphatic metastasis rate in both level IIA and level IIB as the L-shaped incision group (P > 0.05). There were 86 patients with ipsilateral central lymphatic metastasis (78.2%). Patients with contralateral central lymphatic metastasis accounted for 56.4%. The contralateral central lymphatic metastasis rate was not correlated with age, BMI, multifocality, tumor invasion, or ipsilateral central lymphatic metastasis, and there was no significant difference (P > 0.05). The contralateral central lymphatic metastasis in males was slightly higher than that in females, and the difference was statistically significant (68.2% vs. 48.5%, P = 0.041).ConclusionLymphatic metastasis in level IIA was an independent predictor of lymphatic metastasis in level IIB. When bilateral lateral cervical lymphatic metastasis or lymph node metastasis of level IIA is found, lymph node dissection in level IIB is strongly recommended. When unilateral lateral cervical lymphatic metastasis and lymphatic metastasis in level IIA are negative, lymph node dissection in level IIB may be performed as appropriate on the premise of no damage to the accessory nerve. LCEI is safe and effective for lymph node dissection in level II. When the tumor is located in the unilateral lobe, attention should be given to contralateral central lymph node dissection because of the high lymphatic metastasis rate.

LncRNA HULC as a potential predictor of prognosis and clinicopathological features in patients with digestive system tumors: a meta-analysis.

Objective: This meta-analysis aimed to evaluate the correlation between lncRNA HULC, prognosis and clinicopathological characteristics in patients with digestive system tumors.Methods: The relevant literatures were collected through PubMed, Web of Science and Embase up to February 2021. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated to assess the prognostic value of HULC in patients with digestive system tumors. The clinicopathological characteristics of HULC in patients were estimated by odds ratios (ORs).Results: A total of 14 studies involving 1312 patients were included. The up-regulated expression level of HULC was associated with poorer overall survival (OS) in patients with digestive system tumors (HR = 1.83, 95% CI: 1.05-3.19, P = 0.033). Subgroup analysis showed that cancer type (pancreatic cancer or gastric cancer), residence region (China, Japan or Korea), and specimen (serum) significantly associated between HULC and OS. In addition, high HULC expression significantly increased the risk of high TNM stage (OR = 2.51, 95%CI: 1.36-4.62, P < 0.05), poor differentiation (OR = 1.38, 95%CI: 1.02-1.87, P < 0.05) and lymphatic node metastasis (LNM, OR = 4.93, 95% CI: 3.47-6.99, P < 0.05).Conclusions: High expression level of HULC is related to OS, TNM stage, differentiation and LNM. Therefore, HULC can be used as a new potential predictor for prognosis and clinicopathological features of patients with digestive system tumors.

Effect of ultrasound contrast agent targeting gelatin on uptake of mouse ascites hepatocellular carcinoma cell lines with high lymphatic metastasis

Objective: To analyze the effect of ultrasound contrast agent targeting gelatin on uptake of high lymphatic metastasis cell lines of mouse hepatocellular carcinoma with peritoneal effusion. Methods: The modified double emulsifying solvent evaporation method was used to construct the macromolecule contrast agent PLGA-Cooh. The carbodiimide was used to connect the monoclonal antibody of gelatin with the contrast agent PLGA-Cooh, and the targeted ultrasound contrast agent Gsn-PLGA was established. The particle size and Zeta potential of the targeted ultrasound contrast agent were measured by laser particle size analyzer. The surface binding of the contrast agent to the gelatin monoclonal antibody was evaluated by immunofluorescence. Hca-F cells with high lymphatic metastasis were cultured in mice with peritoneal effusion hepatocellular carcinoma. Target-seeking ability in vitro was evaluated by in vitro uptake test, and the imaging effect of the contrast agent in vitro was evaluated by in vitro developing test. Results: The contrast agent is white powder with good water solubility. The average particle size and surface potential were (569.68±6.96) nm and (-10.95±2.43) mV, respectively. The fluorescent antibody binding rate of non-targeted and targeted ultrasound contrast agent labeled with DiI were 0.84% and 95.89%, respectively. The results showed that the targeted ultrasound contrast agent Gsn-PLGA had a better of developing effect in vitro. Hca-F cells with high expression of gelsolin protein had stronger uptake ability of targeted ultrasound contrast agent and stronger green fluorescence in vitro than those with low expression of gelsolin protein (P<0.05). Moreover, targeted ultrasound contrast agent Gsn-PLGA had stronger targeting to the gelsolin protein. The echo of the targeted ultrasound contrast agent Gsn-PLGA was uniform and fine, without attenuating echo of the back. Simultaneously, the development effect was more obvious with the increase of contrast agent concentration (P<0.05). Conclusion: Ultrasound contrast agent Gsn-PLGA targeting gelatin can bind Hca-F cells with high expression of gelatin and display a good imaging effect in vitro.

&lt;p&gt;High Tiam1 expression predicts positive lymphatic metastasis and worse survival in patients with malignant solid tumors: a systematic review and meta-analysis&lt;/p&gt;

BackgroundMany studies have explored the prognostic value of T-cell lymphoma invasion and metastasis inducing factor 1 (Tiam1) and its association with lymphatic metastasis in malignant solid tumors, but the conclusions remain controversial. Therefore, we performed a meta-analysis to systematically assess the prognostic value of Tiam1 expression and its association with lymphatic metastasis in malignant solid tumors.MethodsWe searched eligible studies in PubMed, Web of Science and EMBASE databases (from inception up to October 2018). The combined HR with 95% CI was used to estimate the prognostic value of Tiam1 expression. The correlation between Tiam1 expression and lymphatic metastasis was assessed using the combined odds ratio (OR) with 95% CI.ResultsA total of 17 studies with 2,228 patients with solid tumors were included in this meta-analysis. The overall estimated results showed that high Tiam1 expression was significantly associated with shorter overall survival (HR= 2.08, 95% CI: 1.62–2.68, P<0.01), and disease-free survival (HR = 1.86, 95% CI: 1.49–2.32, P<0.01). Besides, we also found that there was a close relationship between high Tiam1 expression and positive lymphatic metastasis (OR=2.63; 95% CI: 1.79–3.84, P<0.01).ConclusionHigh Tiam1 expression was significantly associated with shorter survival and positive lymphatic metastasis in patients with malignant solid tumors. Therefore, Tiam1 may be a promising prognostic biomarker and an effective therapeutic target for malignant solid tumors.

Genome-wide analysis of long non-coding RNAs in esophageal squamous cell carcinoma reveals their potential role in invasion and metastasis.

BackgroundA high lymphatic metastasis rate and strong local invasive ability are the key characteristics of esophageal squamous cell carcinoma (ESCC) that affect patient survival, and long non‐coding RNAs (lncRNAs) may play a crucial role. We performed genome‐wide analysis of lncRNAs to identify novel biomarkers associated with local invasion and lymphatic metastasis in ESCC.MethodsSix pairs of ESCC tumor and para‐tumor tissues were subjected to microarray analysis to identify differentially expressed lncRNAs, and 25 pairs of tissues samples were used to verify the effectiveness of screened lncRNAs using quantitative reverse transcription PCR. The correlations between verified lncRNAs and clinicopathological characteristics were analyzed to confirm specific lncRNAs associated with the local invasion and lymphatic metastasis of ESCC, and gene co‐expression analysis was used to predict potential mechanisms.ResultsMicroarray analysis identified 1850 lncRNAs with significant differential expression in ESCC. Of 22 lncRNAs selected for quantitative reverse transcription PCR verification, four were significantly upregulated and one was significantly downregulated in ESCC cancer compared to para‐cancer tissues. ENST00000508406.1 was significantly associated with T, N, and tumor node metastasis stages, and NR_037652.1 was significantly associated with N stage. Moreover, 49 lncRNA‐messenger RNA pairs were significantly associated with the two dysregulated lncRNAs and possibly involved in the regulation of local invasion and lymphatic metastasis of ESCC.ConclusionThe present genome‐wide analysis identified two novel and tumor‐specific lncRNAs for predicting ESCC local invasion and lymphatic metastasis, providing insight into the potential underlying mechanism, which warrants further investigation.

Prognostic and clinicopathological role of long non-coding RNA taurine upregulated 1 in various human malignancies: A systemic review and meta-analysis.

The aberrant dysregulation of taurine upregulated 1, a novel discovered long non-coding RNA, was ubiquitous in different human solid tumors. Accumulating researches have indicated that taurine upregulated 1 is an independent prognostic indicator in cancer patients. This investigation aimed to further explore the prognostic and clinical significance of taurine upregulated 1 in various types of cancers. Eligible studies were systematically searched in PubMed, Embase, Medline, and Web of Science databases. A total of 12/14 studies with 1303/1228 individuals were included to evaluate the association of taurine upregulated 1 with overall survival and clinicopathological features by pooled hazard ratio and odds ratio in malignancies. The meta-analysis suggested overexpression of taurine upregulated 1 was significantly correlated with unfavorable overall survival in patients with cancer (pooled hazard ratio = 1.63, 95% confidence interval: 1.29-2.06). There was also a significantly positive correlation between high level of taurine upregulated 1 and high pathological grade carcinoma (pooled odds ratio = 4.41, 95% confidence interval: 3.07-6.43) and positive lymphatic metastasis (pooled odds ratio = 2.00, 95% confidence interval: 1.31-3.06). In summary, upregulated taurine upregulated 1 is correlated with more advanced clinicopathological characteristics and poor prognosis, suggesting that taurine upregulated 1 may serve as a novel predictive biomarker of patients with numerous tumors.

Ezrin expression is altered in mice lymphatic metastatic hepatocellular carcinoma and subcellular fractions upon Annexin 7 modulation in-vitro

Ezrin and Annexin seven (A7) have been suggested to be involved in several roles in cancers metastasis. However, the role of Ezrin and the effect of A7 on Ezrin expression in lymphatic metastatic hepatocellular carcinoma (LNM-HCC) have not been extensively explored yet.This study reports expression of Ezrin in high lymphatic metastasis (Hca-F >70%) and low metastatic metastasis (Hca-P <30%) HCC cell lines, and the effect of A7 on Ezrin expression.Real-Time PCR, Western blot, Subcellular fractionation, Immunocytochemistry and Immunofluorescence were used to investigate Ezrin expression in addition to migration and invasion behaviors of A7 up-regulated Hca-F cells, A7 down-regulated Hca-P and in their respective negative control (NC) cells.Ezrin expression was higher in high LNM-HCC than low LNM-HCC (p=0.0046). Cell fractionation analysis reveals that Ezrin was highly present in the cytoplasm, nucleus and cytoskeleton of NC-Hca-F cells. However, Ezrin was highly observed in the cell membrane, nucleus and cytoskeleton of NC-Hca-P cells. A7 up-regulation in Hca-F suppressed Ezrin expression (p=0.0248), but increase the migration and invasion, whereas Ezrin was mainly located in the cytoplasm and nucleus fractions. Down-regulation of A7 in Hca-P cells, enhanced Ezrin expression (p<0.0001) in the cytoplasm and nucleus fractions, and suppressed migration and invasion.In conclusion, Ezrin may play a role in LNM-HCC and might be inversely associated with A7 expression. The subcellular localization of Ezrin and A7 was varied according to the metastatic levels. Ezrin may thus be a potential diagnostic and/or prognostic biomarker for HCC.

DCLK1 is up-regulated and associated with metastasis and prognosis in colorectal cancer.

Metastasis is a primary cause of colorectal cancer (CRC)-related death, and cancer stem cells (CSCs) are thought to be majorly responsible for initiating metastatic behaviors. Doublecortin-like kinase 1 (DCLK1) was recently discovered to be a marker for gastrointestinal CSCs. Here, we aimed to explore whether DCLK1 is associated with CRC metastasis through clinical and in vitro investigations. The expression levels of DCLK1 mRNA and protein in human CRC tissues were analyzed through quantitative RT-PCR and immunohistochemistry staining, respectively. Human CRC cell line SW480 was selected to explore the effect of DCLK1 overexpression on cell migration and invasion. Besides, the associations between DCLK1 and epithelial-mesenchymal transition (EMT) were determined. Compared to normal colorectal tissues, DCLK1 expression was significantly up-regulated in human CRC tissues and correlated well with high lymphatic metastasis and poor prognosis in patients. DCLK1 expression was inversely associated with overall survival in CRC patients. Overexpression of DCLK1 in SW480 cells markedly promoted cell migration and invasion. Furthermore, we validated that DCLK1 could facilitate EMT in cancer cells by up-regulation of the mesenchymal markers Vimentin and ZEB1 and down-regulation of the epithelial marker E-cadherin in SW480 cells. DCLK1 up-regulation may play a contributory role in CRC metastasis and poor prognosis via activation of EMT. DCLK1 may serve as an independent predictor for CRC prognosis.

Human lymphatic endothelial cells contribute to epithelial ovarian carcinoma metastasis by promoting lymphangiogenesis and tumour cell invasion.

The microenvironment of a tumour is an important factor in ovarian cancer metastasis. The present study aimed to simulate the in vivo microenvironment of an ovarian carcinoma using a co-culture system consisting of human lymphatic endothelial cells (HLECs) and human ovarian carcinoma cells with directional high lymphatic metastasis (SKOV3-PM4s) in order to investigate the role of both cell types in ovarian carcinoma metastasis. The SKOV3-PM4s cultured in the HLEC-conditioned medium exhibited increased numbers of pseudopodia and mitotic figures, proliferated at a faster rate and exhibited enhanced invasion and migratory abilities. Furthermore, the HLECs cultured in SKOV3-PM4-conditioned medium exhibited significant morphological alterations and vacuolisation of the cytoplasm, as well as increased invasion, migratory and tube forming abilities. In addition, spontaneous fusion of the SKOV3-PM4s and HLECs was observed in the co-culture system using laser confocal microscopy. The gelatin zymography assay demonstrated that matrix metalloproteinase-2, which was downregulated in the SKOV3-PM4s, was upregulated in the co-culture system. The results of the present study suggested that the invasion ability of the SKOV3-PM4s was increased in the in vitro co-culture system of SKOV3-PM4 and HLECs. Therefore, alterations in the cell microenvironment may represent a novel strategy for ovarian cancer therapy.

Clinicopathological Differences and Prognostic Value of Hypoxia-Inducible Factor-2α Expression for Gastric Cancer: Evidence From Meta-Analysis.

Published literatures have reported the relationship between hypoxic-inducible factor-2α (HIF-2α) expression and clinicopathological features in gastric cancer (GC), but the evaluated conclusions remain controversial.A meta-analysis was carried to examine the clinicopathological features and prognostic values of HIF-2α in patients with GC. Systematic detailed searches were performed to Pub Med, Cochrane Library, and EBSCO until to August 2015.Six studies (508 specimens) were included in this meta-analysis. HIF-2α-positive expression indicates an unfavorable prognosis value and advanced clinicopathological differences for the available patient dates with GC. Further multivariate meta-analysis revealed that HIF-2α-positive expression in gastric cancer associated with deeper tumor infiltration (OR = 3.08; 95%CI: 1.18–8.04), higher rates of lymphatic metastasis (OR = 3.26; 95%CI: 1.10–9.63), higher TNM stage (III+IV) (OR = 2.61; 95%CI: 1.40–4.84), and much lower 5-year overall survival (OR = 2.08; 95%CI: 1.21–3.58). Nevertheless, there is no association between HIF-2α-positive expression and worse tumor differentiation (OR = 2.03; 95%CI: 0.73–5.64). In addition, by this subgroup analysis, HIF-2α-positive expression associated with deeper tumor infiltration (OR = 3.81; 95%CI: 1.03–14.08), higher lymphatic metastasis (OR = 4.71; 95%CI: 1.08–20.50), higher TNM stage (OR = 3.21; 95%CI: 1.57–6.57), worse tumor differentiation (OR = 3.08; 95%CI: 1.51–6.31), and lower 5-year overall survival (OR = 2.34; 95%CI: 1.15–4.79).Our results indicate that HIF-2α overexpression can potently predict the poor prognosis and may be a potential therapeutic target for gastric carcinoma, according to the limited evidence. Meanwhile, further studies are needed to elucidate the accuracy of these results.

Expression of SRSF3 is Correlated with Carcinogenesis and Progression of Oral Squamous Cell Carcinoma.

Objective: Oral squamous cell carcinoma (OSCC) is the most common malignancy of head and neck with high mortality rates. The mechanisms of initiation and development of OSCC remain largely unknown. Dysregulated alternative splicing of pre-mRNA has been associated with OSCC. Splicing factor SRSF3 is a proto-oncogene and overexpressed in multiple cancers. The aim of this study was to uncover the relationship between SRSF3 and carcinogenesis and progression of oral squamous cell carcinoma.Design and Methods: The expression of SRSF3 in oral normal, dysplasia, or carcinoma tissues was analyzed by immunohistochemistry. The expression levels of EMT-related genes were quantified by real-time quantitative RT-PCR. The expression of SRSF3 in DMBA treated primary cultured oral epithelial cells were analyzed by western blot.Result: SRSF3 is overexpressed in oral cancer and moderate or severe dysplasia tissues. Patients with high grade cancer or lymphatic metastasis showed up-regulated expression of SRSF3. Knockdown of SRSF3 repressed the expression of Snail and N-cadherin in vitro. Carcinogen DMBA treated primary cultured oral epithelial cells showed significantly increased SRSF3 level than in control cells.Conclusion: Our results suggested that SRSF3 is associated with the initiation and development of OSCC and may be a biomarker and therapeutic target of OSCC.

Anti-metastasis effect of fucoidan from Undaria pinnatifida sporophylls in mouse hepatocarcinoma Hca-F cells.

Metastasis is one of the major causes of cancer-related death. It is a complex biological process involving multiple genes, steps, and phases. It is also closely connected to many biological activities of cancer cells, such as growth, invasion, adhesion, hematogenous metastasis, and lymphatic metastasis. Fucoidan derived from Undaria pinnatifida sporophylls (Ups-fucoidan) is a sulfated polysaccharide with more biological activities than other fucoidans. However, there is no information on the effects of Ups-fucoidan on tumor invasion and metastasis. We used the mouse hepatocarcinoma Hca-F cell line, which has high invasive and lymphatic metastasis potential in vitro and in vivo, to examine the effect of Ups-fucoidan on cancer cell invasion and metastasis. Ups-fucoidan exerted a concentration- and time-dependent inhibitory effect on tumor metastasis in vivo and inhibited Hca-F cell growth, migration, invasion, and adhesion capabilities in vitro. Ups-fucoidan inhibited growth and metastasis by downregulating vascular endothelial growth factor (VEGF) C/VEGF receptor 3, hepatocyte growth factor/c-MET, cyclin D1, cyclin-dependent kinase 4, phosphorylated (p) phosphoinositide 3-kinase, p-Akt, p-extracellular signal regulated kinase (ERK) 1/2, and nuclear transcription factor-κB (NF-κB), and suppressed adhesion and invasion by downregulating L-Selectin, and upregulating protein levels of tissue inhibitor of metalloproteinases (TIMPs). The results suggest that Ups-fucoidan suppresses Hca-F cell growth, adhesion, invasion, and metastasis capabilities and that these functions are mediated through the mechanism involving inactivation of the NF-κB pathway mediated by PI3K/Akt and ERK signaling pathways.

Clinicopathological and Prognostic Characteristics of Hepatoid Adenocarcinoma of the Stomach

The present study was undertaken to clarify the association of the clinicopathological features of hepatoid adenocarcinoma (HAC) in the stomach, a special kind of carcinoma that histologically resembled hepatocellular carcinoma (HCC) and is characterized by large amounts of α-fetoprotein (AFP) in serum, with the clinical prognosis. We collected the data of the clinicopathological features and the follow-up information from a total of 31 HACs from January 2005 to December 2012 in our hospital. High lymphatic (54.8%) and distant (25.8%) metastasis rates before surgery, large proportion of advanced HACs (71.0%) at admission, short median overall survival time (6 months), and low three-year survival rate (22.6%) suggested that HAC in the stomach was an aggressive disease, resulting in a poor prognosis. And pTNM stages, immunohistochemical staining of AFP, CEA, CK7, and CK20 had statistically relation with the survival as the independent risk factors, P < 0.05. Therefore, early and clear differentiation of HAC from cancerous or noncancerous conditions with AFP elevation and assessment of high risk patients by histopathology may improve the clinical prognosis.

Prognostic significance of XB130 expression in surgically resected pancreatic ductal adenocarcinoma

BackgroundXB130 is a newly discovered adaptor protein for intracellular signal transduction; it is involved in gene regulation, cell proliferation, cell survival, cell migration, and tumorigenesis. However, its expression and role in pancreatic ductal adenocarcinoma (PDAC) have not been investigated. The present study was designed to clarify the prognostic significance of XB130 expression in PDAC.MethodsA total of 76 consecutive patients with surgically resected PDAC were retrospectively reviewed. XB130 expression was detected by immunohistochemical analysis on the paraffin-embedded tumour sections. Correlation between the expression of XB130 and clinicopathological parameters was analyzed.ResultsXB130 expression was significantly upregulated in PDAC(56.5%, 43/76) compared to normal pancreas (0%, 0/15; P < 0.05). Increased XB130 expression was correlated with lymph node metastasis (P = 0.017), distant metastasis (P = 0.0024), high tumour-node-metastasis (TNM) stage (P =0.001), and high tumour grade (P = 0.013). The survival of 43 patients with high XB130 expression was significantly worse than that of the 33 patients with low XB130 expression (P = 0.001). Univariate analysis showed that high XB130 expression (P = 0.0045), tumour size (P = 0.024), distant metastasis (P = 0.003), TNM stage (P = 0.002) and lymphatic metastasis (P = 0.016) were independent prognostic factors of postoperative survival. Multivariate analysis using the Cox proportional hazards model showed that high XB130 expression and distant metastasis (P = 0.0239) were significant independent risk factors.ConclusionsXB130 was overexpressed in the PDAC. XB130 is a promising pathological marker for the prediction of outcome in patients with PDAC.

Optimization of human hepatocellular cell clones with high lymphatic metastasis potential and the establishment of nude mouse model

Objective To isolate hepatocellular carcinoma (HCC) cell clones with high metastatic potentials from lymphatic metastatic HCC cell line and establish specific metastasis model in nude mice.Methods Cell cloning was conducted on the source cell line HCCLYM-H using limited dilution method,and 25 cell clones were obtained (No.1-25).On the first selection round,9 cell clones with potentials of continuous proliferation were seeded into the food pad off mice.The lymph node metastatic rate of 4 cell clones was 100％,and these cell clones were used for liver orthotopic transplantation for further selection.Metastasis in lungs and lymph nodes in mice was detected by series pathological examination,then cell clones with various metastatic potentials were screened.Results HCC cell clone with high lymphatic metastatic and low lung metastatic potential (HCCLYM-H2) was cstablished.The lymph node metastatic rate was 100％,while the pulmonary metastatic rate was 20％.Conclusion We established the specific lymphatic metastasis models,and the results confirmed the heterogeneity of tumor cells. Key words: Carcinoma,hepatocellular; Cell clone ; Metastasis ; Nude mouse model

Highly lymphatic metastatic pancreatic cancer cells possess stem cell-like properties

Cancer stem cells are thought to be the origin of tumor metastasis. However, evidence of cancer stem cells as the source of lymphatic metastasis in pancreatic cancer is not clear. In this study, we examined the stem cell-like properties of the highly lymphatic metastatic pancreatic cancer cells BxPC-3-LN. Compared with the parental BxPC-3 cells, the BxPC-3-LN cells showed stem cell-like properties, including high lymphatic metastasis potential, self-renewal ability and chemoresistance. In addition, the BxPC-3-LN cells also expressed higher levels of sonic hedgehog and migrating cancer stem cell surface markers (CD133 and CXCR4) compared to the parental BxPC-3 cells. The growth of BxPC-3-LN cells was significantly inhibited by gemcitabine combined with the sonic hedgehog inhibitor cyclopamine. The BxPC-3-LN cells expressed lower levels of let-7, miR-34, miR-107, miR-125, miR-128, miR-130, miR-132 and miR-141 than the parental BxPC-3 cells detected by microRNA PCR array, which were reported to have close relation to stem cell factors. This study provides evidence that cancer stem cells are the major sources of pancreatic cancer lymphatic metastasis, and microRNAs may regulate lymphatic metastasis in pancreatic cancer through modulating cancer stem cells.

Development of a unique mouse model for pancreatic cancer lymphatic metastasis

Lymphatic metastasis of pancreatic cancer is a predictor of poor prognosis. However, the molecular mechanisms are largely unknown, thus, making the development of appropriate cell lines and experimental models critically important for future investigations. The purpose of the present study was to establish a 'pancreatic cancer cell and mouse model with high lymphatic metastasis potential' for in-depth study of the underlying mechanisms. The BxPC-3-LN subline, derived from the BxPC-3 human pancreatic cancer cell line, was established through serial passages in nude mice via footpad injections. The subline was able to develop notable lymphatic metastases in 100% of the recipient mice 8 weeks after tumor cell implantation. Compared with the parental BxPC-3 cells, BxPC-3-LN cells were more aggressive, displaying invasive ultrastructure, increased migration and invasion ability, and chemoresistance. Metastasis-related gene alteration including upregulation of MMP14, MMP24, MIF and ADRM1, and downregulation of TGFB2 and ROBO1 were also observed in BxPC-3-LN cells by cDNA microarrays. Thus, the newly selected BxPC-3-LN subline can serve as a unique model for further study of lymphatic metastasis of pancreatic cancer.

Subcellular proteomics: Determination of specific location and expression levels of lymphatic metastasis associated proteins in hepatocellular carcinoma by subcellular fractionation

Background Subcellular fractionation and proteomics form an ideal partnership when it comes to specific location and analysis of intracellular organelles and expression levels of multiprotein complexes. Lymphatic metastasis is the major complicated system involving multiple factors. However, to date lymphatic metastatic mechanism is poorly understood. Aim To specifically locate expression site by subcellular fractionation, based on expression levels, interpret the involvement of different lymphatic metastasis associated proteins in hepatocellular carcinoma cell lines with different lymphatic metastasis potential. Method Mouse hepatocellular cell lines Hca-F and Hca-P are used to evaluate the location and expression levels of some lymphatic metastasis-associated proteins in the cell by using subcellular fractionation kit and Western blot analysis. The proteins under studies were Gelsolin, JNK and Annexin 7. Results Gelsolin was sequestered in cytoplasm, membrane and cytoskeleton in F-cells but in P-cells, it was found in cytoplasm and cytoskeleton .JNK was located in nuclear fraction and cytoskeleton in F and P cells, Annexin7 was in cytoplasm with its two isoforms only at this location, cell membrane and cytoskeleton in F and P cells. With the high expression level of Gelsolin, JNK and Annexin 7 in Hca-F cell line than Hca-P cell line. Conclusion With subcellular fractionation specific location of Gelsolin, JNK and Annexin 7 at various cell sites during lymphatic metastasis were determined. High expression levels were found in high lymphatic metastasis potential cell lines which indicate their roles according to different expression sites in the disease.

Differential proteomic analysis and function study of human prostate carcinoma cells with different osseous metastatic tendency

To investigate the differential protein expression profiles of human prostatic carcinoma cells with different metastatic tendency and to screen the osseous metastasis associated proteins and investigate their function. Proteomics and Western blotting were applied to screen and identify the differentially expressed proteins in the prostatic carcinoma cells of different lines: line T3B with high osseous metastasis potential, line P2-4 with high lymphatic metastasis potential, and their common parent cell line PC-3. The eukaryotic expression vector carrying human Pgenesil-1/HMGB1 siRNA was constructed and transfected into T3B cells by Lipofectamine 2000 and the positive clones was screened by G418. Pgenesil-1/HMGB1 siRNA/T3B, Pgenesil-1/T3B, and T3B cells were inoculated into the left ventricles of nude mice. Twelve weeks later the mice were killed. The number of osseous metastatic nodules and osseous metastasis inhibition rate were calculated. The mice metastatic tumor cells were identified by immunohistochemistry. Six differential expressed proteins, correlated with cytoskeleton, transcriptional control, cellular metabolism, and phosphorylation were identified by proteomics and Western blotting. The recombinant plasmid Pgenesil-1/HMGB1 siRNA was successfully constructed. The HMGB1 expression of the T3B cells transfected with Pgenesil-1/HMGB1 siRNA was significantly lower than those of the other 2 groups (both P <0.05). The number of osseous metastatic nodules of the mice inoculated with Pgenesil-1/HMGB1 siRNA/T3B was significantly less than those of the other 2 groups (both P < 0.05). The metastatic osseous tumor cells were identified as the human prostatic carcinoma cells. Osseous metastasis associated proteins exist in prostatic carcinoma cells of the line with high osseous metastasis potential. HMGB1 is closely related to the osseous metastasis of human prostatic carcinoma cells. siRNA targeting HMGB1 specifically suppresses the expression of HMGB1 gene in the human prostatic carcinoma cells with high osseous metastasis potential and effectively inhibits the osseous metastasis.