Gain-of-function mutant p53 promotes cell growth and cancer cell metabolism via inhibition of AMPK activation.

Many mutant p53 proteins (mutp53s) exert oncogenic gain-of-function (GOF) properties, but the mechanisms mediating these functions remain poorly defined. We show here that GOF mutp53s inhibit AMP-activated protein kinase (AMPK) signaling in head and neck cancer cells. Conversely, downregulation of GOF mutp53s enhances AMPK activation under energy stress, decreasing the activity of the anabolic factors acetyl-CoA carboxylase and ribosomal protein S6 and inhibiting aerobic glycolytic potential and invasive cell growth. Under conditions of energy stress, GOF mutp53s, but not wild-type p53, preferentially bind to the AMPKα subunit and inhibit AMPK activation. Given the importance of AMPK as an energy sensor and tumor suppressor that inhibits anabolic metabolism, our findings reveal that direct inhibition of AMPK activation is an important mechanism through which mutp53s can gain oncogenic function.

A metabolomics-based approach for predicting stages of chronic kidney disease.

Chronic kidney disease (CKD) is a major epidemiologic problem and a risk factor for cardiovascular events and cerebrovascular accidents. Because CKD shows irreversible progression, early diagnosis is desirable. Renal function can be evaluated by measuring creatinine-based estimated glomerular filtration rate (eGFR). This method, however, has low sensitivity during early phases of CKD. Cystatin C (CysC) may be a more sensitive predictor. Using a metabolomic method, we previously identified metabolites in CKD and hemodialysis patients. To develop a new index of renal hypofunction, plasma samples were collected from volunteers with and without CKD and metabolite concentrations were assayed by quantitative liquid chromatography/mass spectrometry. These results were used to construct a multivariate regression equation for an inverse of CysC-based eGFR, with eGFR and CKD stage calculated from concentrations of blood metabolites. This equation was able to predict CKD stages with 81.3% accuracy (range, 73.9-87.0% during 20 repeats). This procedure may become a novel method of identifying patients with early-stage CKD.

Impact of CYP3A5*3 on plasma exposure and urinary excretion of fentanyl and norfentanyl in the early postsurgical period.

BACKGROUND: The pharmacokinetic characteristics of intravenous fentanyl have not been fully clarified in the early postsurgical period. The aim of this study was to evaluate the plasma exposure and urinary excretion of fentanyl and norfentanyl according to cytochrome P450 (CYP) 3A5 genetic polymorphism. METHODS: Fifty-two adult Japanese postoperative patients receiving a continuous intravenous fentanyl infusion were enrolled. Plasma concentrations of fentanyl and norfentanyl were determined at 24 hours after the operation, and their urinary excretion from 12 to 36 hours was evaluated. RESULTS: Plasma concentrations of fentanyl normalized for infusion rate were significantly higher in the *3/*3 group than in the *1 carrier group. The plasma concentration ratio of norfentanyl to fentanyl was significantly lower in the *3/*3 group than in the *1 carrier group. Urinary excretion rates of fentanyl and norfentanyl were 4.4% and 71%, respectively, and no significant differences were observed between the CYP3A5 genotypes. Renal clearance ratios of fentanyl and norfentanyl to creatinine were 0.34 and 3.4, respectively. There were no significant differences in the renal clearance ratios between the genotypes. Free fractions of fentanyl and norfentanyl in human plasma were 4.9% and 95%, respectively. Total and nonrenal clearance of fentanyl were significantly lower in the *3/*3 group than in the *1 carrier group. CONCLUSIONS: CYP3A5*3 affected the plasma exposure of fentanyl but not urinary excretion in our postoperative patients. The renal clearance ratios of fentanyl and norfentanyl to creatinine were much higher than their free fractions in plasma. These findings suggest a slight contribution of renal tubular secretion of fentanyl and norfentanyl to their plasma exposures.

Exploration of novel predictive markers in rat plasma of the early stages of chronic renal failure.

To identify blood markers for early stages of chronic kidney disease (CKD), blood samples were collected from rats with adenine-induced CKD over 28 days. Plasma samples were subjected to metabolomic profiling by liquid chromatography-mass spectrometry, followed by multivariate analyses. In addition to already-identified uremic toxins, we found that plasma concentrations of N6-succinyl adenosine, lysophosphatidylethanolamine 20:4, and glycocholic acid were altered, and that these changes during early CKD were more sensitive markers than creatinine concentration, a universal indicator of renal dysfunction. Moreover, the increase in plasma indoxyl sulfate concentration occurred earlier than increases in phenyl sulfate and p-cresol sulfate. These novel metabolites may serve as biomarkers in identifying early stage CKD.

In vivo imaging of lymph node metastasis with telomerase-specific replication-selective adenovirus.

Currently available methods for detection of tumors in vivo such as computed tomography and magnetic resonance imaging are not specific for tumors. Here we describe a new approach for visualizing tumors whose fluorescence can be detected using telomerase-specific replication-competent adenovirus expressing green fluorescent protein (GFP) (OBP-401). OBP-401 contains the replication cassette, in which the human telomerase reverse transcriptase (hTERT) promoter drives expression of E1 genes, and the GFP gene for monitoring viral replication. When OBP-401 was intratumorally injected into HT29 tumors orthotopically implanted into the rectum in BALB/c nu/nu mice, para-aortic lymph node metastasis could be visualized at laparotomy under a three-chip color cooled charged-coupled device camera. Our results indicate that OBP-401 causes viral spread into the regional lymphatic area and selectively replicates in neoplastic lesions, resulting in GFP expression in metastatic lymph nodes. This technology is adaptable to detect lymph node metastasis in vivo as a preclinical model of surgical navigation.

Inferior alveolar nerve regeneration after bifocal distraction osteogenesis in dogs.

PURPOSE: Bifocal distraction osteogenesis has been shown to be a reliable method for reconstructing missing bone segments. However, no reports have been published regarding inferior alveolar nerve regeneration during this procedure. We assumed that the nerve could regenerate with the bone regeneration during bifocal distraction, if the nerve had been saved at a mesial site of the transport disc. In the present study, we investigated that possibility in dogs. MATERIALS AND METHODS: Using a bifocal distraction osteogenesis method, we produced a 10-mm mandibular defect, including the nerve defect, and distracted the transport disc at a rate of 1 mm/day in 12 dogs. The nerve was saved at the mesial site of the transport disc. The regenerated nerve was evaluated by a jaw opening reflex examination performed once daily. Histologic examinations with hematoxylin-eosin and immunohistochemical staining with neurofilament and S-100 antibody were also performed on all dogs after death at 3, 6, and 12 months after the first operation. RESULTS: The jaw opening reflex had recovered in all dogs. The average period of recovery was 109.5 ± 24.7 days. On histologic examination, although consecutive nerves were observed in all areas, cellular nerve fascicles were seen, consistent with wallerian degeneration at 3 and 6 months in the nerve connection area on the distal side of the transport disc. CONCLUSIONS: Our results have indicated that inferior alveolar nerve regeneration after bifocal distraction osteogenesis is successful in dogs. Although our research is still at the stage of animal experiments, future application in humans can be considered to be possible.

Steroid-free living donor liver transplantation for HCV--a multicenter prospective cohort study in Japan.

This prospective, non-randomized, multicenter cohort study analyzed the safety and efficacy of a steroid-free immunosuppressive (IS) protocol for hepatitis C virus (HCV)-positive living donor liver transplant (LDLT) recipients in Japan. Of 68 patients enrolled from 13 transplant centers, 56 fulfilled the inclusion/exclusion criteria; 27 were assigned the steroid-free IS protocol (Fr group) and 29 the traditional steroid-containing IS protocol (St group). Serum HCV RNA levels increased over time and were higher in the St group until postoperative day 90 (POD 14, p=0.013). Preemptive anti-HCV therapy was started in a higher percentage of recipients (59.3%) in the Fr group than in the St group (31.0%, p=0.031), mainly due to early HCV recurrence. The incidence of HCV recurrence at one yr was lower in the Fr group (22.2%) than in the St group (41.4%; p=0.066). The incidence of acute cellular rejection was similar between groups. New onset diabetes after transplant, cytomegalovirus infection, and renal dysfunction were significantly less frequent in the Fr group than in the St group (p=0.022, p<0.0001, p=0.012, respectively). The steroid-free IS protocol safely reduced postoperative morbidity and effectively suppressed both the HCV viral load in the early post-transplant period and HCV recurrence in HCV-positive LDLT recipients.

In vivo internal tumor illumination by telomerase-dependent adenoviral GFP for precise surgical navigation.

Cancer surgery requires the complete and precise identification of malignant tissue margins including the smallest disseminated lesions. Internal green fluorescent protein (GFP) fluorescence can intensely illuminate even single cells but requires GFP sequence transcription within the cell. Introducing and selectively activating the GFP gene in malignant tissue in vivo is made possible by the development of OBP-401, a telomerase-dependent, replication-competent adenovirus expressing GFP. This potentially powerful adjunct to surgical navigation was demonstrated in 2 nude mouse models that represent difficult surgical challenges--the resection of widely disseminated cancer. HCT-116, a model of intraperitoneal disseminated human colon cancer, was labeled by virus injection into the peritoneal cavity. A549, a model of pleural dissemination of human lung cancer, was labeled by virus administered into the pleural cavity. Only the malignant tissue fluoresced brightly in both models. In the intraperitoneal model of disseminated cancer, fluorescence-guided surgery enabled resection of all tumor nodules labeled with GFP by OBP-401. The data in this report suggest that adenoviral-GFP labeling tumors in patients can enable fluorescence-guided surgical navigation.

[A case of resected rectal cancer with hepatic node and multiple liver metastases effectively treated by preoperative modified FOLFOX6 and sLV5FU2 chemotherapy].

A 55-year-old male had complained of melena.Colonoscopy revealed a type 2 tumor at the rectum.CT demonstrated hepatic lymph nodes and multiple liver metastases(stage IV).Low anterior resection was performed(tub2, RsRa, circ, type 2, pSS, pN1, sH3, cHN1, sP0, cM0: fstage IV).The patient was treated with mFOLFOX6 and sLV5FU2 after operation.CT revealed a partial response after 14 courses of systemic chemotherapy.sLV5 FU2 therapy was converted to capecitabine because he experienced bone marrow suppression.CT showed that the liver metastases had enlarged but the hepatic lymph nodes disappeared.Right portal vein embolization was performed.After 4 weeks, right hepatectomy and hepatic lymph node dissection were performed.Preoperative chemotherapy with mFOLFOX6 seems beneficial as a neoadjuvant chemotherapy for hepatic lymph node-positive advanced colorectal cancer.

[A case of primary adenocarcinoma of small intestine responding to XELOX chemotherapy and leading to a partial metabolic response].

We report a case of adenocarcinoma of the small intestine responding to XELOX chemotherapy, leading to a partial metabolic response(PMR). The patient was a 58-year-old male with multiple peritoneal dissemination of adenocarcinoma of the small intestine. Chemotherapy with XELOX(L-OHP 130 mg/m² on day 1 , and capecitabine 1,000 mg/m2 on days 1-14)was performed. After 4 courses, a significant tumor reduction was obtained. This case suggests that chemotherapy with XELOX is a potential regimen for small intestinal adenocarcinoma.

Inhibition of mTOR by temsirolimus contributes to prolonged survival of mice with pleural dissemination of non-small-cell lung cancer cells.

Temsirolimus (CCI-779), a recently synthesized analogue of rapamycin, specifically inhibits mTOR and has been approved for clinical use in renal cell carcinoma. Recent reports have indicated the growth inhibitory effect of temsirolimus in some cancers including non-small-cell lung carcinoma (NSCLC). In this study, we aimed to explore the potential therapeutic use of temsirolimus as a treatment for NSCLC. Using cultured NSCLC cells (A549, H1299, and H358), we determined the effect of temsirolimus on cell proliferation and its antitumor effects on subcutaneous tumors, as well as its contribution to the survival of mice having pleural dissemination of cancer cells, mimicking advanced NSCLC. Temsirolimus suppressed proliferation of NSCLC cells in a dose-dependent manner, with an IC(50) of <1 nM. Western blot analysis revealed that temsirolimus treatment specifically inhibited the phosphorylation of mTOR and its downstream effectors in 1 h, accompanied by an increased cell population in the G(0) /G(1) phase, but according to flow cytometry, the cell population did not increase in the sub-G(0) phase. When NSCLC subcutaneous tumor-bearing mice were treated with temsirolimus, tumor volume was significantly reduced (tumor volume on day 35: vehicle vs temsirolimus = 1239 vs 698 cm(3) ; P < 0.05). Furthermore, prolonged survival was observed in pleural disseminated tumor-bearing mice with temsirolimus treatment (median survival: vehicle vs temsirolimus = 53.5 vs 72.5 days; P < 0.05). These results suggest that temsirolimus could be useful for NSCLC treatment, due to its antiproliferative effect, and could be a potential treatment for advanced NSCLC, giving prolonged survival.

Metabolomic analysis of human plasma from haemodialysis patients.

BACKGROUND: Urea and creatinine are widely used as biomarkers for disease. However, these parameters have been criticized as markers for several reasons. Thus, we conducted this study to identify novel biomarkers that can be used as alternatives to urea and creatinine to estimate the adequate dialysis dose by metabolomic analyses of plasma samples from patients undergoing haemodialysis. MATERIAL AND METHODS: Liquid chromatography-electrospray ionization (ESI)-time-of-flight mass spectrometry (MS) was used to analyse low molecular weight molecules present in the plasma samples of 10 patients with end-stage renal disease (ESRD) who were being treated with haemodialysis, and in 16 healthy subjects. RESULTS: In plasma samples obtained after haemodialysis, the relative quantities of 54 peaks were significantly (P < 0·05) decreased when compared with those in the plasma before haemodialysis. The candidate biomarkers were allocated to three groups. Molecules in Group A improved completely with a large variance, molecules in Group B improved partially but with a large variance, and molecules in Group C improved partially with low variance after haemodialysis. Small cohort validation study consisting of the patients with ESRD undergoing haemodialysis indicates that three candidate biomarkers in Group C would be a very useful marker to estimate adequate haemodialysis dose. CONCLUSIONS: 1-Methylinosine and two unknown molecules whose m/z at ESI-positive mode are 257·1033 and 413·1359 were found as effective candidate biomarkers to estimate adequate haemodialysis dose, which has to be confirmed in prospective studies.

A novel antiangiogenic effect for telomerase-specific virotherapy through host immune system.

Soluble factors in the tumor microenvironment may influence the process of angiogenesis; a process essential for the growth and progression of malignant tumors. In this study, we describe a novel antiangiogenic effect of conditional replication-selective adenovirus through the stimulation of host immune reaction. An attenuated adenovirus (OBP-301, Telomelysin), in which the human telomerase reverse transcriptase promoter element drives expression of E1 genes, could replicate in and cause selective lysis of cancer cells. Mixed lymphocyte-tumor cell culture demonstrated that OBP-301-infected cancer cells stimulated PBMC to produce IFN-gamma into the supernatants. When the supernatants were subjected to the assay of in vitro angiogenesis, the tube formation of HUVECs was inhibited more efficiently than recombinant IFN-gamma. Moreover, in vivo angiogenic assay using a membrane-diffusion chamber system s.c. transplanted in nu/nu mice showed that tumor cell-induced neovascularization was markedly reduced when the chambers contained the mixed lymphocyte-tumor cell culture supernatants. The growth of s.c. murine colon tumors in syngenic mice was significantly inhibited due to the reduced vascularity by intratumoral injection of OBP-301. The antitumor as well as antiangiogenic effects, however, were less apparent in SCID mice due to the lack of host immune responses. Our data suggest that OBP-301 seems to have antiangiogenic properties through the stimulation of host immune cells to produce endogenous antiangiogenic factors such as IFN-gamma.

A new cell-permeable peptide allows successful allogeneic islet transplantation in mice.

Calcineurin inhibitors such as cyclosporine A and FK506 have been used for transplant therapy and treatment of autoimmune diseases. However, the inhibition of calcineurin outside the immune system has a number of side effects, including hyperglycemia. In the search for safer drugs, we developed a cell-permeable inhibitor of NFAT (nuclear factor of activated T cells) using the polyarginine peptide delivery system. This peptide provided immunosuppression for fully mismatched islet allografts in mice. In addition, it did not affect insulin secretion, whereas FK506 caused a dose-dependent decrease in insulin secretion. Cell-permeable peptides can thus provide a new strategy for drug development and may eventually be useful clinically.

Nationwide survey for bisphosphonate-related osteonecrosis of the jaws in Japan.

PURPOSE: A nationwide retrospective cohort study was conducted by the Japanese Society of Oral and Maxillofacial Surgeons to assess the occurrence of bisphosphonate (BP)-related osteonecrosis of the jaws (BRONJ) during 2006 to 2008 and to elucidate the outcome and factors associated with remission of BRONJ. MATERIALS AND METHODS: A written questionnaire, including the clinical characteristics, management, and outcome of patients with BRONJ, was sent to 248 institutions certified as training facilities by the Japanese Society of Oral and Maxillofacial Surgeons in 2008. RESULTS: A total of 568 patients with BRONJ, including suspicious cases, were registered. Of these 568 patients, 263, including the maxilla in 81, the mandible in 160, and both in 22, met the working definition of BRONJ proposed by the American Association of Oral and Maxillofacial Surgeons. The patients included 219 women (83.3%) and 44 men (16.7%). Of these patients, 152 (57.8%) had received intravenous BPs, 104 (39.5%) had received oral BPs, and 7 (2.7%) had received both. The mean duration of administration until onset of BRONJ was 23.6 months for intravenous BPs and 33.2 months for oral BPs. BRONJ was stage 1 in 42 patients (16.0%), stage 2 in 187 (71.1%), stage 3 in 32 (12.2%), and unknown in 2. Of these patients, 34.2% had remission of BRONJ, 46.0% had persistent or progressive disease, and 19.7% died of malignancy or were lost to follow-up. Statistical analysis revealed that surgical treatment, including tooth extraction, sequestrectomy, and segmental mandibulectomy, contributed to the remission of BRONJ. In contrast, conservative treatment, concurrent anticancer drugs, poor oral hygiene, and the use of intravenous BPs did not. CONCLUSIONS: The relative ratio of BRONJ related to the use of oral BPs was greater in Japan than in the United States and European Union. Surgical treatment contributed to remission of BRONJ, and conservative treatment, concurrent anticancer drugs, poor oral hygiene, and intravenous BPs did not.

Combined Inhibition of Rad51 and Wee1 Enhances Cell Killing in HNSCC Through Induction of Apoptosis Associated With Excessive DNA Damage and Replication Stress.

Despite advances in surgery, chemotherapy, and radiation, there are limited treatment options for advanced head and neck squamous cell carcinoma (HNSCC) and survival remains very poor. Therefore, effective therapies are desperately needed. Recently, selective exploitation of DNA damage and replication stress responses has become a novel approach for cancer treatment. Wee1 kinase and Rad51 recombinase are two proteins involved in regulating replication stress and homologous recombination repair in cancer cells. In this study, we investigated the combined effect of Rad51 inhibitor (B02) and Wee1 inhibitor (AZD1775) in vitro and in vivo in various HNSCC cell lines. Clonogenic survival assays demonstrated that B02 synergized with AZD1775 in vitro in all HNSCC cell lines tested. The synergy between these drugs was associated with forced CDK1 activation and reduced Chk1 phosphorylation leading to induction of excessive DNA damage and replication stress, culminating in aberrant mitosis and apoptosis. Our results showed that elevated Rad51 mRNA expression correlated with worse survival in HNSCC patients with HPV-positive tumors. The combination of B02 and AZD1775 significantly inhibited tumor growth in vivo in mice bearing HPV-positive HNSCC tumors as compared to HPV-negative HNSCC. This differential sensitivity appears to be linked to HPV-positive tumors having more in vivo endogenous replication stress owing to transformation by E6 and E7 oncogenes. Furthermore, addition of B02 radiosensitized the HPV-negative HNSCC tumors in vitro and in vivo In conclusion, our data implicate that a novel rational combination with Rad51 and Wee1 inhibitors holds promise as synthetic lethal therapy, particularly in high-risk HPV-positive HNSCC.

Two distinct pathways mediated by PA28 and hsp90 in major histocompatibility complex class I antigen processing.

Major histocompatibility complex (MHC) class I ligands are mainly produced by the proteasome. Herein, we show that the processing of antigens is regulated by two distinct pathways, one requiring PA28 and the other hsp90. Both hsp90 and PA28 enhanced the antigen processing of ovalbumin (OVA). Geldanamycin, an inhibitor of hsp90, almost completely suppressed OVA antigen presentation in PA28alpha(-/-)/beta(-/-) lipopolysaccharide blasts, but not in wild-type cells, indicating that hsp90 compensates for the loss of PA28 and is essential in the PA28-independent pathway. In contrast, treatment of cells with interferon (IFN)-gamma, which induces PA28 expression, abrogated the requirement of hsp90, suggesting that IFN-gamma enhances the PA28-dependent pathway, whereas it diminishes hsp90-dependent pathway. Importantly, IFN-gamma did not induce MHC class I expressions in PA28-deficient cells, indicating a prominent role for PA28 in IFN-gamma-stimulated peptide supply. Thus, these two pathways operate either redundantly or specifically, depending on antigen species and cell type.

Intramuscular transplantation of engineered hepatic tissue constructs corrects acute and chronic liver failure in mice.

BACKGROUND & AIMS: Transplantation of isolated hepatocytes holds great promise as an alternative to whole organ liver transplantation. For treatment of liver failure, access to the portal circulation has significant risks and intrahepatic hepatocyte engraftment is poor. In advanced cirrhosis, transplantation into an extrahepatic site is necessary and intrasplenic engraftment is short-lived. Strategies that allow repeated extrahepatic infusion of hepatocytes could improve the efficacy and safety of hepatocyte transplantation for the treatment of liver failure. METHODS: A non-immunogenic self-assembling peptide nanofiber (SAPNF) was developed as a three-dimensional scaffold and combined with growth factors derived from a conditionally immortalized human hepatocyte cell line to engineer a hepatic tissue graft that would allow hepatocyte engraftment outside the liver. RESULTS: The hepatic tissue constructs maintained hepatocyte-specific gene expression and functionality in vitro. When transplanted into skeletal muscle as an extrahepatic site for engraftment, the engineered hepatic grafts provided life-saving support in models of acute, fulminant, and chronic liver failure that recapitulates these clinical diseases. CONCLUSIONS: SAPNF-engineered hepatic constructs engrafted and functioned as hepatic tissues within the muscle to provide life-sustaining liver support. These engineered tissue constructs contained no animal products that would limit their development as a therapeutic approach.

Advanced glycation end products enhance monocyte activation during human mixed lymphocyte reaction.

Posttransplant diabetes mellitus (PTDM) is a frequent complication among transplant recipients. Ligation of advanced glycation end products (AGEs) with their receptor (RAGE) on monocytes/macrophages plays roles in the diabetes complications. The enhancement of adhesion molecule expression on monocytes/macrophages activates T-cells, leading to reduced allograft survival. We investigated the effect of four distinct AGE subtypes (AGE-2/AGE-3/AGE-4/AGE-5) on the expressions of intracellular adhesion molecule (ICAM)-1, B7.1, B7.2 and CD40 on monocytes, the production of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha and the proliferation of T-cells during human mixed lymphocyte reaction (MLR). AGE-2 and AGE-3 selectively induced the adhesion molecule expression, cytokine production and T-cell proliferation. The AGE-induced up-regulation of adhesion molecule expression was involved in the cytokine production and T-cell proliferation. AGE-2 and AGE-3 up-regulated the expression of RAGE on monocytes; therefore, the AGEs may activate monocytes, leading to the up-regulation of adhesion molecule expression, cytokine production and T-cell proliferation.

In vivo biological purging for lymph node metastasis of human colorectal cancer by telomerase-specific oncolytic virotherapy.

BACKGROUND/OBJECTIVE: The aim of this study was to develop a less invasive way of targeting lymph node metastasis for the treatment of human gastrointestinal cancer. Lymphatic invasion is a major route for cancer cell dissemination, and adequate treatment of locoregional lymph nodes is required for curative treatment in patients with malignancies. METHODS: Human telomerase reverse transcription (hTERT) is the catalytic subunit of telomerase, which is highly active in cancer cells but quiescent in most normal somatic cells. OBP-301 (Telomelysin) is an attenuated adenovirus with oncolytic potency that contains the hTERT promoter element to regulate viral replication. We examined whether OBP-301 injected into the primary tumor might be useful for purging micrometastasis from regional lymph nodes in an orthotopic colorectal cancer model. RESULTS: OBP-301 was intratumorally injected into HT29 tumors orthotopically implanted into the rectum in BALB/c nu/nu mice. By using a highly sensitive quantitative PCR analysis that targets the human-specific Alu sequence, we showed that OBP-301 caused viral spread into the regional lymphatic area and selectively replicated in neoplastic lesions, resulting in tumor-cell-specific death in metastatic lymph nodes. Moreover, although the surgical removal of primary tumors increased the tendency of lymph node metastasis, preoperative intratumoral injection of virus significantly reduced lymph node metastasis. CONCLUSIONS: Our results indicate that intratumoral injection of OBP-301 mediates effective in vivo purging of metastatic tumor cells from regional lymph nodes, which may help optimize treatment of human cancer, especially gastrointestinal malignancies.