Single-cell RNA-sequencing atlas reveals an MDK-dependent immunosuppressive environment in ErbB pathway-mutated gallbladder cancer.

BACKGROUND & AIMS: Our previous genomic whole-exome sequencing (WES) data identified the key ErbB pathway mutations that play an essential role in regulating the malignancy of gallbladder cancer (GBC). Herein, we tested the hypothesis that individual cellular components of the tumor microenvironment (TME) in GBC function differentially to participate in ErbB pathway mutation-dependent tumor progression. METHODS: We engaged single-cell RNA-sequencing to reveal transcriptomic heterogeneity and intercellular crosstalk from 13 human GBCs and adjacent normal tissues. In addition, we performed WES analysis to reveal the genomic variations related to tumor malignancy. A variety of bulk RNA-sequencing, immunohistochemical staining, immunofluorescence staining and functional experiments were employed to study the difference between tissues with or without ErbB pathway mutations. RESULTS: We identified 16 cell types from a total of 114,927 cells, in which epithelial cells, M2 macrophages, and regulatory T cells were predominant in tumors with ErbB pathway mutations. Furthermore, epithelial cell subtype 1, 2 and 3 were mainly found in adenocarcinoma and subtype 4 was present in adenosquamous carcinoma. The tumors with ErbB pathway mutations harbored larger populations of epithelial cell subtype 1 and 2, and expressed higher levels of secreted midkine (MDK) than tumors without ErbB pathway mutations. Increased MDK resulted in an interaction with its receptor LRP1, which is expressed by tumor-infiltrating macrophages, and promoted immunosuppressive macrophage differentiation. Moreover, the crosstalk between macrophage-secreted CXCL10 and its receptor CXCR3 on regulatory T cells was induced in GBC with ErbB pathway mutations. Elevated MDK was correlated with poor overall survival in patients with GBC. CONCLUSIONS: This study has provided valuable insights into transcriptomic heterogeneity and the global cellular network in the TME, which coordinately functions to promote the progression of GBC with ErbB pathway mutations; thus, unveiling novel cellular and molecular targets for cancer therapy. LAY SUMMARY: We employed single-cell RNA-sequencing and functional assays to uncover the transcriptomic heterogeneity and intercellular crosstalk present in gallbladder cancer. We found that ErbB pathway mutations reduced anti-cancer immunity and led to cancer development. ErbB pathway mutations resulted in immunosuppressive macrophage differentiation and regulatory T cell activation, explaining the reduced anti-cancer immunity and worse overall survival observed in patients with these mutations.

Hepatic hypertrophy and hemodynamics of portal venous flow after percutaneous transhepatic portal embolization.

BACKGROUND: Percutaneous transhepatic portal embolization (PTPE) is useful for safe major hepatectomy. This study investigated the correlation between hepatic hypertrophy and hemodynamics of portal venous flow by ultrasound sonography after PTPE. METHODS: We analyzed 58 patients with PTPE, excluding those who underwent recanalization (n = 10). Using CT volumetry results 2 weeks after PTPE, the patients were stratified into a considerable hypertrophy group (CH; n = 15) with an increase rate of remnant liver volume (IR-RLV) ≥ 40% and a minimal hypertrophy group (MH; n = 33) with an IR-RLV < 40%. We investigated the hemodynamics of portal venous flow after PTPE and the favorable factors for hepatic hypertrophy. RESULTS: Univariate and multivariate analysis identified the indocyanine green retention rate at 15 min (ICGR15) and increase rate of portal venous flow volume (IR-pFV) at the non-embolized lobe on day 3 after PTPE as independent favorable factors of IR-RLV. Patients with IR-pFV on day 3 after PTPE ≥100% and ICGR15 ≤ 15% (n = 13) exhibited significantly increased IR-RLV compared with others (n = 35). CONCLUSIONS: Cases with high IR-pFV on day 3 after PTPE exhibited better hepatic hypertrophy. Preserved liver function and increased portal venous flow on day 3 were important.

Bedside ultrasound-guided celiac plexus neurolysis in upper abdominal cancer patients: a randomized, prospective study for comparison of percutaneous bilateral paramedian vs. unilateral paramedian needle-insertion technique.

INTRODUCTION: Percutaneous anterior abdominal ultrasound guidance for performing celiac plexus neurolysis is a relatively new but more economical, less time-consuming, more comfortable bedside technique for interventional pain management. Paucity of studies evaluating the efficacy of single-site vs. double-site injections at celiac trunk for ultrasound-guided celiac plexus neurolysis (USCPN) prompted us to conduct a prospective, randomized, single-blind clinical trial to compare USCPN using bilateral paramedian (double needle) technique with unilateral paramedian (single needle) technique. METHODS: Sixty patients aged 18 years or older with unresectable upper abdominal cancers were randomized into two groups to receive USCPN. A 20-mL mixture of 50% ethanol with 0.25% bupivacaine was injected either unilaterally (20 mL×1 site) or bilaterally (10 mL×2 sites) depending on the randomization group. Subjects were assessed for the pain relief using Numerical rating scale (NRS) to assess their pain relief. RESULTS: Baseline parameters being comparable (P > 0.05), the site of drug injections (single or double needle) had no bearing on the onset of pain relief and patient satisfaction scores (P > 0.05). Pain relief during follow-up visits was comparable between the two groups (P > 0.05). The discomfort score correlated well with the pain relief scoring without any significant difference between the two groups except in the last visit (at 3 month). Incidences of the complications were comparable in the two groups (P > 0.05). CONCLUSION: Ultrasound-guided celiac plexus neurolysis using unilateral paramedian (single needle) needle-insertion technique is comparable with bilateral paramedian (double needle) needle-insertion technique with regard to pain relief and side effects.

Gallbladder cancer: lessons from a rare tumour.

Gallbladder cancer is a relatively rare form of malignancy about which our knowledge is scant. However, a unique combination of predisposing factors - including genetic predisposition, geographic distribution, female gender bias, chronic inflammation and congenital developmental abnormalities - makes this type of cancer unique and offers potential for understanding cancer pathogenesis in general. An understanding of how these risk factors contribute to the molecular basis of the disease is essential for understanding the origins of this unusual cancer.

[Change of coagulation in patients with gallbladder cancer and its clinical significance].

OBJECTIVE: To study the relationship between the change of coagulation and the clinicopathologic characteristics in patients with gallbladder cancer. METHODS: The 64 gallbladder cancer patients (GBC group) and 60 cholecystitis patients (control group) had been reviewed from January 2007 to June 2013. The prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fib), and thrombin time (TT) had been measured and compared between patients of GBC group and control group. The relationship of coagulation function and prognosis were analyzed. RESULTS: Compared with control group, APTT in GBC group ((29.0 ± 4.2) s) was significantly shortened (t = -4.265, P = 0.000) and PT ((11.5 ± 1.4) s), TT ((15.3 ± 3.5) s), Fib ((4.1 ± 0.9) g/L) were significantly increased in GBC group (t = 2.521, 4.147 and 4.365, all P < 0.05). The level of Fib was higher in patients with medium or poor-differentiated tumor cells (F = 4.069, P = 0.022), lymph metastasis (t = 2.640, P = 0.010) and advanced staging (II-IV) (t = 3.003, P < 0.01) than those of well-differentiated, non-lymph metastasis and early staging (0-I). The ratio of gallbladder cancer with hyperfibrinogenemia (32/64) was significantly higher than control group (11/60, χ(2) = 13.709, P < 0.01). In GBC group, compared with normal Fib patients, hyperfibrinogenemia patients showed significantly difference in clinicopathologic characteristics (χ(2) = 5.851-10.573, P < 0.05). The average survival period of hyperfibrinogenemia patients and normal Fib patients were 8.63 months and 16.73 months. The 1-, 3-year survival rate of patients with hyperfibrinogenemia were significantly lower than those with normal Fib (64.7%, 14.9% vs. 74.9%, 21.1%, P < 0.05). CONCLUSION: Preoperative plasma level of Fib might be a new promising biomarker in patients with gallbladder cancer for evaluating disease progression and prognosis.

Association and functional significance of genetic variants present in regulatory elements of SERPINB5 gene in gallbladder cancer.

SERPINB5 is a mammary serine protease inhibitor, which is involved in various cellular functions. The aberrant expression of SERPINB5 is reported in many cancers along with GBC but limited information is available about its role in genetic predisposition for GBC. We carried out case-control study in 206 cases and 219 controls. Promoter SNPs were genotyped by Sanger's sequencing. In-silico promoter analysis and luciferase reporter assay were done to elucidate the role of promoter variants in regulation of SERPINB5 expression. Out of four SNPs, three SERPINB5 promoter variants showed association with GBC in different models. The 'C' allele of variant rs17071138 was found to be significantly associated with GBC (p = 0.017). The 'T' allele of rs3744940 significantly increased the risk for GBC in dominant (p = 0.035) and additive models (p = 0.005). Also, rs3744941 'T' allele increased the risk for GBC by dominant (p = 0.042) as well as additive models (p = 0.016). In-silico promoter analysis and luciferase reporter assay revealed the probable regulatory role of the SERPINB5 promoter variant rs17071138 on the expression. Overall, our study reveals the genetic association of SERPINB5 promoter variants with GBC and possible role of rs17071138 in the regulation of expression.

Predictive factors for pain relief after endoscopic ultrasound-guided celiac plexus neurolysis.

BACKGROUND: Celiac plexus neurolysis (CPN) is an established treatment for upper abdominal cancer pain. Recently, endoscopic ultrasound-guided CPN (EUS-CPN) was introduced and has enabled the performance of CPN under real-time imaging guidance, thereby making this technique much safer and easier. However, this procedure is not always efficacious, and a limited number of patients benefit from it. It should not be recommended for patients suspected of having unfavorable outcomes. We determined the predictive factors for response to EUS-CPN in order to enable rational selection of the therapeutic strategy. PATIENTS AND METHODS: Forty-seven consecutive patients who underwent EUS-CPN at our institutions were eligible for this study. Absolute ethanol containing a contrast medium was injected just above the origin of the celiac trunk from the aorta under real-time EUS guidance, and abdominal computed tomography was performed immediately after the procedure to evaluate the distribution of the injected ethanol. The efficacy in pain relief was evaluated based on the pain score at day 7 after EUS-CPN. RESULTS: Pain relief was obtained in 32 patients (68.1%). Multivariate analysis using a multiple logistic regression model revealed that direct invasion of the celiac plexus and distribution of ethanol only on the left side of the celiac artery were significant factors for a negative response to EUS-CPN (odds ratio = 4.82 and 8.67, P = 0.0387 and 0.0224, respectively). CONCLUSION: EUS-CPN seems to be less effective in patients with direct invasion of the celiac plexus. Ethanol should be injected on both sides of the celiac axis to obtain greater pain relief.

Gallbladder cancer with ascites in a child with metachromatic leukodystrophy.

INTRODUCTION: Metachromatic leukodystrophy (MLD) refers to leukodystrophy caused by the accumulation of sulfatide from arylsulfatase A (ARSA) gene mutations. Sulfatide also accumulates in various organs, including the peripheral nerves, kidney, and gallbladder. Proliferative changes in the gallbladder have been reported in several patients, while gallbladder cancer is reported in only two adult MLD cases. We report what is likely the first pediatric case of MLD with gallbladder cancer. CASE REPORT: The patient was a 5-year-old girl diagnosed with MLD using head magnetic resonance imaging and detecting a homozygous mutation of c.302G>A (p.Gly101Asp) in ARSA. Abdominal bloating was observed at the age of 4 years; CT revealed a giant tumor in the gallbladder and massive ascites. Cholecystectomy was performed and pathological examination revealed adenocarcinoma. Measurement of serum sulfatide revealed increased levels compared to the average healthy range. DISCUSSION: Rapidly increased ascites and large polyps which are reported as risk factors for cancer were characteristic in our MLD case. When such lesions are detected, they should be removed immediately because of the possibility of cancer, even in a pediatric patient.

Screening and identification of haptoglobin showing its important role in pathophysiological process of gallbladder carcinoma.

Gallbladder cancer (GBC) with poor prognosis has been a major cause of cancer-related deaths worldwide. In this study, we aimed to screen and identify crucial genes in GBC through integrative analysis of multiple datasets and further experimental validation. A candidate crucial gene, up-regulated haptoglobin (HP), was firstly screened, and then further analysis and validation mainly focused on whether higher enrichment level of HP was responsible for pathophysiological process of GBC. HP was found with diverse expression patterns in various cancer types, and the dynamic expression patterns indicated its spatiotemporal characteristics in different tissues and disease stages, implicating its role in multiple biological processes. Further experimental validation showed that HP could promote the GBC-SD cell proliferation, migration and invasion, implying its role in pathophysiological process of GBC. HP may have a crucial role in occurrence and development of GBC, and it provides possibility as a potential biomarker or target in cancer prognosis and treatment.

Expression of VEGF-A, HER2/neu, and KRAS in gall bladder carcinoma and their correlation with clinico-pathological parameters.

BACKGROUND: Gall bladder carcinoma (GBC) is a multi-factorial disease, involving multiple genetic alterations. The present pilot study aims to explore some of the molecular pathways, by studying immunohistochemical (IHC) expression of biomarkers (HER2/neu, KRAS, and VEGF) in GBC with their correlation with various clinicopathological parameters. AIM OF THE STUDY: To study the expression of prognostic biomarkers (HER2/neu, KRAS and VEGF-A) in GBC and their correlation with clinico-morphological parameters. Materials and. METHODS: This prospective study was conducted over a period of 2 years. The study group included tissue of GBC (29) reported as malignant on histopathology and cholecystitis as a control group (29) for histopathological evaluation and IHC expression of above markers. RESULTS: HER2/neu was expressed in 27.5% cases, and KRAS in 51.6%; however, both showed no association with tumor type, stage and grade. No association was found in KRAS expression and dysplasia. Vascular Endothelial Growth Factor - A (VEGF-A) was expressed in 86.1% cases, of which strong positivity was seen in 48.27%; it showed significant association with tumor stage (P value-0.027, Fishers' exact test), hence possibly suggesting its role in tumor progression; though no association was found in VEGF expression with tumor type and grade. No significant association was seen with vascular and tumor invasion also. CONCLUSION: The results suggest that the VEGF-A expression may be used as a potential prognostic biomarker in GBC.

Abdominal wall nodule in a cholecystectomy scar : A rare marker of occult carcinoma gallbladder.

Gallbladder carcinoma (GBC) is a rare pathology. We reviewed our hospital database for prognosticating the patients with post-cholecystectomy abdominal wall nodule. On reviewing the database we could find 7 patients who were diagnosed with GBC after simple cholecystectomy. Three of those patients were diagnosed after evaluation of a scar site nodule. Two patients were females and one patient was male. The mean age of the patients was 55 years. Two patients underwent laparoscopic cholecystectomy and one patient underwent open cholecystectomy. The average time of detection of malignancy was 10.6 months. The gallbladder was not subjected to histopathological examination in all three patients. The patients had the unresectable disease on restaging workup. Two patients had adenocarcinoma while one patient had a neuroendocrine tumor. abdominal wall nodule is a rare marker of occult gallbladder carcinoma. Subjecting every gallbladder specimen to histopathology should help in improving the survival in these patients.

Clinicopathological correlation of cancer stem cell markers Oct-4 and CD133 expression as prognostic factor in malignant lesions of gallbladder: An immunohistochemical study.

BACKGROUND: Gallbladder cancer (GBC) is the most frequent biliary tract cancer, with high morbidity and poor prognosis, and shows early metastasis and invasiveness. No reliable biomarkers are available for detection of GBC progression. AIM: To investigate the immunohistochemical expression of Oct-4 and CD133 in malignant and nonneoplastic lesions of gallbladder and to analyze the clinical significance of the expressions related to clinicopathological parameters. SETTINGS AND DESIGN: This is a prospective case control study, conducted in medical college background. MATERIALS AND METHODS: A total of 103 cases of gallbladder were grouped into malignant lesions (n = 48) and nonneoplastic lesions (simple epithelial hyperplasia; n = 35 and chronic cholecystitis; n = 20). All tissue samples were evaluated for expression of Oct-4 and CD133 using immunohistochemistry in an effort to elucidate the correlation between their expressions with clinicopathological parameters. STATISTICAL ANALYSIS: The final score was calculated by multiplying the intensity to the percentage of positive cells. The scores ≥2 were considered as positive. RESULTS: Significant positive correlation of higher expression levels of Oct-4 and CD133 were observed in malignant as compared to nonneoplastic lesions of gallbladder (P < 0.0001). High expression of Oct-4 and CD133 were significantly associated with tumor grading (Oct-4, P = 0.04; CD133, P = 0.02), staging (Oct-4, P = 0.03; CD133, P = 0.02), and liver metastasis (Oct-4, P = 0.01; CD133, P = 0.007). Significantly reduced survival was observed with high expression of Oct-4 (P = 0.002). No significant correction was observed between CD 133 and survival. CONCLUSION: This study revealed that high expression level of Oct-4 may provide a new insight for the prognosis of the disease in terms of clinical staging and grade.

Cholesterol gallstones and cancer of gallbladder (CAGB): molecular links.

There is a known association between cholesterol gallstones and cancer of gall bladder (CAGB). However, the exact relation is not clear. It is proposed they are linked at molecular level by the activity of the orphan nuclear receptors (ONRs) and ABC transporter pumps involved in cholesterol and xenobiotic efflux from the liver into bile. There is evidence that these two pathways are closely interlinked and influence each other. Genetic and environmental factors that upregulate these systems can lead to the simultaneous pumping of cholesterol (which precipitate as gallstones) and a food carcinogen into the bile in gall bladder; the latter causes malignant transformation. Aflatoxin B, a potent hepatocarcinogen, could be the culprit in endemic regions such as South America and North India.

MicroRNA-30a-5p inhibits gallbladder cancer cell proliferation, migration and metastasis by targeting E2F7.

Gallbladder carcinoma (GBC), the most common malignant tumour of the bile duct, is highly aggressive and has a poor prognosis. MicroRNA-30a-5p (miR-30a-5p) is an important tumour suppressor that participates in many aspects of carcinogenesis and cancer development. However, the role of miR-30a-5p in GBC development remains to be determined, as do the mechanisms underlying its effects in GBC. Using samples collected from 42 subjects with gallbladder carcinoma (GBC), we showed decreased miR-30a-5p expression in the primary lesions vs. non-tumour adjacent tissues (NATs). Decreased miR-30a-5p was associated with shorter disease-free survival (DFS) and overall survival (OS). Inhibiting miR-30a-5p expression in 2 representative GBC cell lines (GBC-SD and NOZ) increased cell proliferation, migration, invasiveness, as well as ß-catenin nuclear translocation, vice versa. In nude mice, NOZ cells transfected with miR-30a-5p mimics grew slower (vs. miR-NC) upon subcutaneous inoculation, and had lower rate of hepatic metastasis upon spleen inoculation. Dual luciferase assay confirmed that E2F transcription factor 7 (E2F7) was a direct target of miR-30a-5p and antagonized the effects induced by miR-30a-5p downregulation in GBC cells. MiR-30a-5p attenuates the EMT and metastasis in GBC cells by targeting E2F7, suggesting miR-30a-5p is a tumour suppressor that may serve as a novel potential prognostic biomarker or molecular therapeutic target for GBC.

Cytokeratin 17 Expression is Associated With Poor Prognosis in Gallbladder Adenocarcinoma.

Cytokeratin 17 (CK17), a basal/myoepithelial cell keratin, is a poor prognostic marker for cancers of organs such as the stomach, ovary, and breast as well as a useful diagnostic marker for pancreatobiliary adenocarcinoma. However, its expression pattern and prognostic significance have not been studied in gallbladder adenocarcinoma. We constructed a tissue microarray from samples from 82 consecutive patients with gallbladder adenocarcinoma treated by cholecystectomy at the Kangbuk Samsung Hospital from 2000 to 2011. CK17 expression was examined by immunohistochemistry and correlated with clinicopathologic prognostic factors. CK17 stained the cytoplasm of tumor cells and immunohistochemical interpretation was possible in 77 cases. Among these, 41 (53.2%) were considered positive using a 5% cutoff determined by a receiver operating characteristic curve (area under the curve=0.656, P=0.021). CK17 expression was associated with poor tumor differentiation (P<0.001), high pT stage (P<0.001), presence of distant metastasis (P=0.036), and low disease-specific survival rate (P<0.001). These results indicate that CK17 can be used as a marker for poor prognosis for gallbladder adenocarcinoma.

Expression of cyclooxygenase-2 is associated with p53 accumulation in premalignant and malignant gallbladder lesions.

AIM: To examine the relationship between cyclooxygenase-2 (COX-2) overexpression and p53 accumulation in gallbladder carcinoma and its precursor lesions. METHODS: Sixty-eight gallbladder tissue samples comprising 14 cases of normal gallblader epithelium, 27 cases of dysplasia (11 low-grade dyplasia and 16 high-grade dysplasia) and 27 adenocarcinomas were evaluated by immunohistochemistry for COX-2 expression and p53 accumulation. The relationship among COX-2 expression, p53 accumulation and clinicopathological characteristics was analysed. RESULTS: COX-2 was expressed in 14.3% of normal gallbladder epithelium, 70.3% of dysplastic epite hlium, and 59.2% of adenocarcinomas. When divided into low- and high-grade dysplasia, COX-2 was positive in 5 (45.4%) cases of low-grade and 14 (87.5%) of high-grade dysplasia (P = 0.019). Accumulation of p53 was detected in 5 (31.2%) cases of high-grade dysplasia and in 13 (48.1%) of carcinomas. No p53 accumulation was found in normal epithelium or low-grade dysplasia. COX-2 overexpression was observed in 17 of 18 (94.4%) cases with p53-accumulation in comparison with 20 (40.0%) out of 50 cases without p53 accumulation (P < 0.001). CONCLUSION: The significant differences in COX-2 expression among normal epithelium, low-grade dysplasia and high-grade dysplasia suggest that overexpression of COX-2 enzyme is an early event in gallbladder carcinogenensis. Furthermore, since accumulation of p53 correlates with COX-2 expression, COX-2 overexpression observed in gallbladder high-grade dysplasia and carcinoma might be partly due to the dysfunction of p53.

Does laparoscopy worsen the prognosis for incidental gallbladder cancer?

BACKGROUND: More than 75% of cholecystectomies are done laparoscopically and less than one-third of gallbladder carcinomas are known presurgically. It is supposed that the laparoscopic technique could adversely affect the prognosis of gallbladder cancer. METHODS: The C-A-E has started a register of all cases of cholecystectomy with a postoperative incidental finding of gallbladder carcinoma. The aim is to compare the prospectively collected follow-up data on the outcome of these patients and to answer the question of whether laparoscopic cholecystectomy affects the prognosis of incidental gallbladder cancer. RESULTS: A total of 377 cases have been recorded so far. These include 201 patients treated by the laparoscopic procedure, 119 by an open procedure, and 57 by an intraoperative conversion. The survival shows a significantly better life expectancy for the patients treated laparoscopically. CONCLUSION: The life expectancy is higher for the laparoscopically treated patients and this cannot be explained by the fact that the laparoscopic technique is used to treat the earlier stages of cancer. The access technique does not seem to influence the prognosis for gallbladder carcinomas.

Gastroduodenal dysmotility in patients with gallbladder carcinoma: frequency of occurrence and clinical importance.

BACKGROUND: Gastric stasis, common in patients with gall-bladder carcinoma (GBC), results from anatomical obstruction or motor abnormalities. We studied patients with GBC for antroduodenal motor dysfunction using manometry. METHODS: Forty-one patients with GBC without endoscopic gastric outlet obstruction and 10 healthy controls were evaluated using a symptom scoring system for gastric stasis, saline load test and water perfusion antroduodenal manometry. Fasting, post-prandial and post-octreotide motility were recorded and analysed on a computer using GiPC manometry software. RESULTS: Sixteen of 41 patients (39%) with GBC reported recurrent vomiting; patients with vomiting had a higher symptom score (13 [11-17] v. 6 [4-10], p<0.0001] and higher volume of aspirate on the saline load test (460 ml [210-650] v. 160 ml [70-260], p<0.0001) as compared with those without vomiting. Healthy subjects more often had spontaneous fasting migratory motor complex than patients with GBC (9/10 v. 13/41, p=0.002). The amplitudes of contractions in the antrum and duodenum were significantly lower in patients with GBC than in healthy subjects. Patients with GBC had lower fasting (157 [68-284] v. 190.5 [150-284], p=0.01) and post-prandial (200 [96-395] v. 284 [178-395], p<0.0001) antral motor indices than healthy subjects. Patients with GBC and vomiting had significantly lower contraction amplitude and motility indices than those without vomiting. Motility indices correlated inversely with the symptom score and volume of aspirate on the saline load test (Spearman correlation, p = 0.01 for all). CONCLUSION: Antroduodenal motor abnormalities are common in patients with GBC. These may explain the symptoms of gastric stasis and abnormal results of the saline load test in the absence of anatomical obstruction in such patients.

Inhibition of tumor growth and invasion by a four-kringle antagonist (HGF/NK4) for hepatocyte growth factor.

Invasion of various carcinoma cells follows their interaction with stromal cells. Hepatocyte growth factor (HGF), four-kringle-containing growth factor, is a mesenchymal or stromal-derived mediator which affects the growth and the invasiveness of carcinoma cells. We now have evidence that a four-kringle-containing antagonist for HGF, HGF/NK4 inhibits invasion of tumors in vivo, as well as in vitro. HGF/NK4 competitively inhibited the binding of HGF to Met/ HGF receptors on GB-d1 human gallbladder carcinoma cells. HGF induced invasion of the cells through Matrigel basement membrane components and into collagen gels, but HGF-induced invasion was inhibited by HGF/NK4. Invasion of GB-d1 cells was induced by co-cultivation with stromal fibroblasts, which mimics tumor-stromal interaction, but it was almost completely suppressed by HGF/NK4. Likewise, invasive growth induced by HGF in collagen gels in GB-dl cells, HuCC-T1 human cholangiocarcinoma cells, and ME-180 human uterus cervical carcinoma cells was also strongly inhibited by HGF/NK4. When GB-d1 cells were implanted subcutaneously into nude mouse, tumor cells invaded muscular tissue, but the infusion of HGF/NK4 inhibited this invasion. Furthermore, HGF/NK4 increased apoptotic cell death of GB-d1 cells and inhibited tumor growth in vivo. These results indicate that HGF/ NK4 may inhibit growth and invasion of carcinoma cells, as mediated by HGF during tumor-stromal interactions. We propose that there is a unique therapeutic potential for HGF/NK4 to prevent tumor invasion and perhaps even metastasis.