Mouse Model Of Peritoneal Metastasis Research Articles

Exosomal TRIM3 is a novel marker and therapy target for gastric cancer

BackgroundExosomes are critically involved in cancer development and progression. The exosomal contents have been suggested as ideal cancer biomarkers. In this study, we investigated the expression of exosomal proteins in the serum of gastric cancer patients and their roles in gastric cancer.MethodsThe proteomic profile of exosomes from the serum of gastric cancer patients was detected by using LC-MS/MS. The expression of TRIM3 in exosomes from the serum of gastric cancer patients and healthy controls was assessed by ELISA and western blot. Immunohistochemistry was used to detect TRIM3 expression in gastric cancer tissues and their matching adjacent tissues. The growth and migration abilities of gastric cancer cells with TRIM3 overexpression or knockdown in vitro were evaluated by colony formation assay and transwell migration assay. The effects of TRIM3 overexpression or knockdown on gastric cancer growth and metastasis in vivo were investigated by using subcutaneous xenograft tumor and peritoneal metastasis mouse model. The effects of TRIM3-overexpressing exosomes on gastric cancer growth and metastasis in vitro and in vivo were also evaluated.ResultsWe found that the expression levels of TRIM3 mRNA and protein were decreased in gastric cancer tissues compared to the matched control tissues. In addition, the levels of TRIM3 protein in the serum exosomes of gastric cancer patients were lower than that in healthy controls. We demonstrated that TRIM3 overexpression reduced while TRIM3 knockdown promoted the growth and metastasis of gastric cancer in vitro and in vivo through the regulation of stem cell factors and EMT regulators. Moreover, exosomes-mediated delivery of TRIM3 protein could suppress gastric cancer growth and metastasis in vitro and in vivo.ConclusionsTaken together, our findings suggest that exosomal TRIM3 may serve as a biomarker for gastric cancer diagnosis and the delivery of TRIM3 by exosomes may provide a new avenue for gastric cancer therapy.

Experimental pharmacokinetics evaluation of chemotherapy delivery by PIPAC for colon cancer: first evidence for efficacy.

Pressurised intraperitoneal aerosol chemotherapy (PIPAC) is a novel technique of intraperitoneal chemotherapy devoted to unresectable peritoneal metastasis (PM). The first results obtained with PIPAC in preclinical models of colon cancer are presented here. In vitro, PIPAC (normotherm oxaliplatin at 0.028 mg/mL for 10 min at 1.6 bars) and HIPEC (hyperthermic oxaliplatin at 0.14 mg/mL for 30 min) were compared using the apoptosis and proliferation assay on two colon cancer cell lines (LS 174 and CT 26); ex vivo tumours from an orthotopic mouse model of PM and non-tumour peritoneum from a patient treated according to the two modalities were assessed, investigating the percentage of penetration of oxaliplatin in the tumour and oxaliplatin concentration below the peritoneum. In vivo, a mouse model of colon (CT 26) PM was used to create a PIPAC model (same modalities) for the comparison of IV oxaliplatin (at 5 mg/mL). In vitro, the rate of apoptotic and proliferative cells as well as the level of oxaliplatin penetration in tumour nodes was higher in PIPAC groups with less systemic passage through the peritoneum. In vivo, in the colon PM mouse model, the peritoneal cancer index (PCI) was decreased to the same level using PIPAC or IV oxaliplatin. Systemic passage was lower in the PIPAC group. PIPAC with low-dose oxaliplatin is efficient in both in vitro and in vivo models of colon PM. Lower concentrations of chemotherapy are needed in PIPAC to achieve the same effect as IV chemotherapy on PCI. With a very low systemic oxaliplatin passage, this technique of drug delivery seems to be as effective as IV delivery for PM control.

Intraperitoneal Administration of Plasma-Activated Medium: Proposal of a Novel Treatment Option for Peritoneal Metastasis From Gastric Cancer.

The administration of fluid irradiated with non-equilibrium atmospheric pressure plasma (NEAPP) has attracted much interest as a novel therapeutic method for cancer. The authors previously reported on the efficacy of plasma-activated medium (PAM) for treating cancer cell lines through the induction of apoptosis. In this study, the therapeutic effect of PAM was evaluated in vivo using a peritoneal metastasis mouse model. Two gastric cancer cell lines were used in proliferation assays performed to optimize the production of PAM by changing the distance between the plasma source and the medium surface and by altering the volume of irradiated medium. Wound-healing and adhesion assays were conducted to determine the effect of PAM therapy on cell migration and adhesion capacity in vitro. Finally, a mouse model established by the intraperitoneal injection of enhanced green fluorescent protein-tagged gastric cancer cells was used to explore the efficacy of PAM administered intraperitoneally in inhibiting peritoneal metastasis formation. Shorter distances between the plasma source and the medium surface and smaller volumes of treated medium increased the anti-tumor effect of PAM. The PAM treatment attenuated gastric cancer cell migration and adhesion in vitro. The intraperitoneal administration of PAM decreased the formation of peritoneal metastatic nodules by 60% in the mouse model, and no adverse events were observed. Plasma-activated liquids may represent a novel therapeutic method for the treatment of peritoneal metastases in gastric cancer.

A Strong B-cell Response Is Part of the Immune Landscape in Human High-Grade Serous Ovarian Metastases.

In high-grade serous ovarian cancer (HGSOC), higher densities of both B cells and the CD8+ T-cell infiltrate were associated with a better prognosis. However, the precise role of B cells in the antitumor response remains unknown. As peritoneal metastases are often responsible for relapse, our aim was to characterize the role of B cells in the antitumor immune response in HGSOC metastases. Unmatched pre and post-chemotherapy HGSOC metastases were studied. B-cell localization was assessed by immunostaining. Their cytokines and chemokines were measured by a multiplex assay, and their phenotype was assessed by flow cytometry. Further in vitro and in vivo assays highlighted the role of B cells and plasma cell IgGs in the development of cytotoxic responses and dendritic cell activation. B cells mainly infiltrated lymphoid structures in the stroma of HGSOC metastases. There was a strong B-cell memory response directed at a restricted repertoire of antigens and production of tumor-specific IgGs by plasma cells. These responses were enhanced by chemotherapy. Interestingly, transcript levels of CD20 correlated with markers of immune cytolytic responses and immune complexes with tumor-derived IgGs stimulated the expression of the costimulatory molecule CD86 on antigen-presenting cells. A positive role for B cells in the antitumor response was also supported by B-cell depletion in a syngeneic mouse model of peritoneal metastasis. Our data showed that B cells infiltrating HGSOC omental metastases support the development of an antitumor response. Clin Cancer Res; 23(1); 250-62. ©2016 AACR.

T-Type Ca2+ Channel Inhibition Sensitizes Ovarian Cancer to Carboplatin.

Ovarian cancer is the deadliest gynecologic cancer, due in large part to the diagnosis of advanced stage disease, the development of platinum resistance, and inadequate treatment alternatives. Recent studies by our group and others have shown that T-type calcium (Ca(2+)) channels play a reinforcing role in cancer cell proliferation, cell-cycle progression, and apoptosis evasion. Therefore, we investigated whether T-type Ca(2+) channels affect ovarian tumor growth and response to platinum agents. Inhibition of T-type Ca(2+) channels with mibefradil or by silencing expression resulted in growth suppression in ovarian cancer cells with a simultaneous increase in apoptosis, which was accompanied by decreased expression of the antiapoptotic gene survivin (BIRC5). Analysis of intracellular signaling revealed mibefradil reduced AKT phosphorylation, increased the levels and nuclear retention of FOXO transcription factors that repress BIRC5 expression, and decreased the expression of FOXM1, which promotes BIRC5 expression. Combining carboplatin with mibefradil synergistically increased apoptosis in vitro. Importantly, mibefradil rendered platinum-resistant ovarian tumors sensitive to carboplatin in a mouse model of peritoneal metastasis. Together, the data provide rationale for future use of T-type channel antagonists together with platinum agents for the treatment of ovarian cancer.

The effect of SCF and ouabain on small intestinal motility dysfunction induced by gastric cancer peritoneal metastasis.

The interstitial cells of Cajal (ICCs) play an important role in maintaining the normal function of gastrointestinal dynamics. In our previous study, we reported that, in advanced gastric cancer, the frequency of bowel movement is always reduced, due in part to the decreased number of ICCs. To investigate the impact of ICCs in gastric cancer, we established a mouse model of gastric cancer peritoneal metastasis using SGC-7901 gastric adenocarcinoma cells and their supernatant. Then, stem cell factor (SCF) and ouabain were used as therapeutic agents to improve gut dynamics. Our data showed that, compared with the normal mice, treatment with SGC-7901 cells and their supernatant led to a significant reduction of the muscle layer thickness, a decreased number of ICCs, broadened gaps between ICCs and surrounding cells, degeneration and necrosis of smooth muscle cells (SMCs), and infiltration of inflammatory cells. In contrast to SGC-7901 cell and supernatant treatment, SCF intervention caused mild submucosal edema and mitochondrial proliferation in the ICCs and SMCs. Additionally, ouabain treatment led to inflammatory cells infiltration into the submucosa and a decreased volume of ICCs. In conclusion, our data illustrated that, under the condition of gastric cancer peritoneal metastasis, the dysfunction of intestinal peristalsis may be related to pathological changes in ICCs. Moreover, we demonstrated that SCF treatment may help to improve intestinal dynamics by regulating the number and function of ICCs.

Epigenetic modulation and repression of miR-200b by cancer-associated fibroblasts contribute to cancer invasion and peritoneal dissemination in gastric cancer.

Cancer-associated fibroblasts (CAFs) have recently been linked to the invasion and metastasis of gastric cancer. In addition, the microRNA (miR)-200 family plays a central role in the regulation of the epithelial-mesenchymal transition process during cancer metastasis, and aberrant DNA methylation is one of the key mechanisms underlying regulation of the miR-200 family. In this study, we clarified whether epigenetic changes of miR-200b by CAFs stimulate cancer invasion and peritoneal dissemination in gastric cancer. We evaluated the relationship between miR-200b and CAFs using a coculture model. In addition, we established a peritoneal metastasis mouse model and investigated the expression and methylation status of miR-200b. We also investigated the expression and methylation status of miR-200b and CAFs expression in primary gastric cancer samples. CAFs (CAF-37 and CAF-50) contributed to epigenetic changes of miR-200b, reduced miR-200b expression and promoted tumor invasion and migration in NUGC3 and OCUM-2M cells in coculture. In the model mice, epigenetic changes of miR-200b were observed in the inoculated high-frequency peritoneal dissemination cells. In the 173 gastric cancer samples, the low miR-200b expression group demonstrated a significantly poorer prognosis compared with the high miR-200b expression group and was associated with peritoneal metastasis. In addition, downregulation of miR-200b in cancer cells was significantly correlated with alpha-smooth muscle actin expression. Our data provide evidence that CAFs reduce miR-200b expression and promote tumor invasion through epigenetic changes of miR-200b in gastric cancer. Thus, CAFs might be a therapeutic target for inhibition of gastric cancer.

Inhibition of α4β1 integrin increases ovarian cancer response to carboplatin

ObjectiveThe inability to successfully treat women with ovarian cancer is due to the presence of metastatic disease at diagnosis and the development of platinum resistance. Ovarian cancer metastasizes throughout the peritoneal cavity by attaching to and invading through the mesothelium lining the peritoneum using a mechanism that involves α4β1 integrin and its ligand (vascular cell adhesion molecule) VCAM-1. Integrin α4β1 expression on tumor cells is known to confer protection from therapy in other cancers, notably multiple myeloma. We evaluated the role of α4β1 integrin in response to platinum-based therapy in a mouse model of peritoneal ovarian cancer metastasis by treatment with a humanized anti-α4β1 integrin function-blocking antibody. MethodsIntegrin α4β1 expression on primary human ovarian cancer cells, fallopian tube and ovarian surface epithelia and fresh tumor was assessed by flow-cytometry. The therapeutic impact of anti-α4β1 treatment was assessed in murine models of platinum-resistant peritoneal disease and in vitro using the platinum resistant ovarian cancer cell lines. ResultsTreatment of tumor-bearing mice with human-specific α4β1 integrin function-blocking antibodies, anti-VCAM-1 antibody or carboplatin alone had no effect on tumor burden compared to the IgG control group. However, the combined treatment of anti-α4β1 integrin or anti-VCAM-1 with carboplatin significantly reduced tumor burden. In vitro, the combination of carboplatin and anti-α4β1 integrin antibodies resulted in increased cell death and doubling time. ConclusionsOur findings support a role for α4β1 integrin in regulating treatment response to carboplatin, implicating α4β1 integrin as a potential therapeutic target to influence platinum responsiveness in otherwise resistant disease.

Abstract A62: Imaging the tumor micro-environment to monitor ovarian cancer metastasis

Abstract The inability to successfully treat women with ovarian cancer is due in large part to the advanced stage of disease at diagnosis, the development of platinum resistance, and the lack of sensitive methods to monitor tumor progression and response to treatment. Vascular cell adhesion molecule-1 (VCAM-1) is expressed on the mesothelium of ovarian cancer patients. We investigated VCAM-1 expression as a marker of peritoneal metastasis and tumor response to platinum-based chemotherapy. Immunohistochemical staining of peritoneal or omental biopsies obtained from women diagnosed with Stage I, Stage II or Stage III/IV ovarian cancer demonstrated that VCAM-1 expression correlated with tumor stage. All specimens from Stage I patients were negative, while 29% of Stage II patients and the 73% of Stage III/IV patients were positive. While the majority of women with advanced stage disease expressed VCAM-1, the incidence of expression was reduced among women who received neoadjuvant chemotherapy, suggesting a role for chemotherapy in regulating VCAM-1 expression. The effects of carboplatin on mesothelial VCAM-1 expression were determined in a mouse model of ovarian cancer peritoneal metastasis using radiolabeled VCAM-1-specific peptide imaging probes and single photon emission computed tomography (SPECT). This approach showed that 1) VCAM-1 is a viable imaging target in ovarian cancer metastasis, 2) maximal expression was detected with microscopic tumor burden, and 3) VCAM-1 expression reflected the effect of platinum-based treatment on tumor burden. Specifically, carboplatin treatment of mice with platinum-sensitive tumors showed reduced VCAM-1 expression, which correlated with reduced tumor burden; mice with platinum-resistant tumors retained elevated VCAM-1 expression and tumor burden following treatment. These observations support testing the utility of VCAM-1 imaging probes to monitor treatment response in ovarian cancer patients, thus providing the potential to improve management of women with this disease. Citation Format: Jill K. Slack-Davis, Jennifer M. Scalici, Stephanie Thomas, Christine Harrer, Timothy A. Raines, Joanna Curran, Kristen A. Atkins, Mark R. Conaway, Linda Duska, Kimberly A. Kelly. Imaging the tumor micro-environment to monitor ovarian cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A62.

Imaging VCAM-1 as an Indicator of Treatment Efficacy in Metastatic Ovarian Cancer

The inability to successfully treat women with ovarian cancer is due in large part to the advanced stage of disease at diagnosis, the development of platinum resistance, and the lack of sensitive methods to monitor tumor progression and response to treatment. Vascular cell adhesion molecule-1 (VCAM-1) is expressed on the mesothelium of ovarian cancer patients. We investigated VCAM-1 expression as a marker of peritoneal metastasis and tumor response to platinum-based chemotherapy. Peritoneal or omental biopsies obtained from women diagnosed with stage I, stage II, or stage III/IV ovarian cancer were evaluated by immunohistochemistry. The effects of carboplatin on mesothelial VCAM-1 expression were determined in cultured cells by Western blot. Radiolabeled VCAM-1-specific peptide imaging probes and SPECT were used in a mouse model of ovarian cancer peritoneal metastasis to identify VCAM-1 as a viable imaging target. VCAM-1 expression correlated with tumor stage. All specimens from stage I patients were negative, whereas 29% of stage II patients and 73% of stage III/IV patients were positive. Although most women with advanced stage disease expressed VCAM-1, the incidence of expression was reduced among women who received neoadjuvant chemotherapy, suggesting a role for chemotherapy in regulating VCAM-1 expression. Treatment of mesothelial cells in culture with carboplatin resulted in a transient decrease in VCAM-1 expression 4 h after treatment that returned to baseline within 16-24 h. In vivo imaging of VCAM-1 also demonstrated an acute decrease in expression 4 h after carboplatin administration that recovered within 48 h in mice harboring platinum-resistant tumors. Chronic VCAM-1 expression reflected the effect of platinum-based treatment on tumor burden. Specifically, carboplatin treatment of mice with platinum-sensitive tumors showed reduced VCAM-1 expression, which correlated with reduced tumor burden; mice with platinum-resistant tumors retained elevated VCAM-1 expression and tumor burden after treatment. Clinically relevant VCAM-1-specific imaging probes identify VCAM-1 expression as an indicator of ovarian cancer peritoneal metastasis and therapeutic response to platinum-based agents. These observations support testing the utility of VCAM-1 imaging probes to monitor treatment response in ovarian cancer patients, thus providing the potential to improve management of women with this disease.

EEF1A2 promotes cell migration, invasion and metastasis in pancreatic cancer by upregulating MMP-9 expression through Akt activation

eEF1A2 is a protein translation factor involved in protein synthesis that is overexpressed in various cancers, with important functions in tumor genesis and progression. We have previously showed that the ectopic expression of eEF1A2 is correlated with lymph node metastasis and perineural invasion in pancreatic cancer. In this study, we investigated the functional role of eEF1A2 in the regulation of cell migration, invasion, and metastasis in pancreatic cancer. Furthermore, we investigated the potential molecular mechanisms involved. By evaluating the invasive ability of a panel of pancreatic cancer cell lines with different metastatic potentials, eEF1A2 expression in cells was positively associated with their invasive ability. The knockdown of eEF1A2 by siRNA decreased the migration and invasion of PANC-1 cells. By contrast, the ectopic expression of exogenous eEF1A2 significantly promoted the migration and invasion of SW1990 cells. Stable eEF1A2 overexpression in a nude mouse model of peritoneal metastasis likewise dramatically enhanced the intraperitoneal metastatic ability of SW1990 cells. In addition, eEF1A2 overexpression could upregulate MMP-9 expression and activity. A significant positive correlation between the overexpression of both eEF1A2 and MMP-9 was observed in pancreatic cancer tissues. The inhibition of MMP-9 activity reduced the promoting effect of eEF1A2 on cell migration and invasion. Furthermore, eEF1A2-mediated cell migration and invasion, as well as MMP-9 expression and upregulation, were largely dependent on the eEF1A2-induced Akt activation. The findings suggested the potentially important role of eEF1A2 in pancreatic cancer migration, invasion, and metastasis. Thus, the results provide evidence of eEF1A2 as a potential therapeutic target in the treatment of aggressive pancreatic cancer.

Abstract 945: Inhibition of T-type calcium channels sensitizes ovarian cancer cell growth and metastasis to platinum-based chemotherapy.

Abstract The inability to successfully treat women with ovarian cancer is due in large part to the advanced stage of disease at diagnosis and the development of platinum resistance. T-type calcium channels have recently garnered interest as potential targets for the treatment of many cancers. We investigated the hypothesis that inhibiting T-type calcium channels would sensitize ovarian cancer cells to platinum based chemotherapy using cell culture and mouse models. Sequential treatment of cultured ovarian cancer cell lines or primary cells obtained from patients with mibefradil followed by carboplatin showed a dose-dependent decrease in cell number that was greater than observed with either drug alone. Importantly, treatment of platinum-resistant tumor cells with mibefradil followed by carboplatin also decreased cell number. Moreover, either simultaneous administration of both drugs or treatment with carboplatin followed by mibefradil did not alter cellular response to carboplatin. Based on these findings, we tested the effect of mibefradil and carboplatin in a mouse model of peritoneal ovarian cancer metastasis with platinum-resistant ovarian cancer cells. Tumor-bearing mice were subjected to 3 cycles of Interlaced TherapyTM, which consists of 5 days of mibefradil treatment followed by one dose of carboplatin and 2 days off. We found that while neither drug alone significantly affected tumor growth compared to control, the mice that received the dual treatment had a dramatic reduction in tumor burden. Together, the data provide pre-clinical evidence supporting the use of T-type calcium channel blockers with platinum-based chemotherapy to treat ovarian cancer. Citation Format: Eli V. Casarez, Lloyd Gray, Amir A. Jazaeri, Jill K. Slack-Davis. Inhibition of T-type calcium channels sensitizes ovarian cancer cell growth and metastasis to platinum-based chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 945. doi:10.1158/1538-7445.AM2013-945

JS1-3 - Rapid in vivo Cancer Imaging by Topically Spraying a Newly Designed Activatable Fluorescence Probe

ABSTRACT Fluorescence imaging is one of the most powerful techniques currently available for continuous observation of dynamic responses in living cells and animals. For highly efficient development of novel fluorescence probes, we have established several rational strategies for controlling the properties of visible light to NIR-excitable fluorophores based on the intramolecular photoinduced electron transfer. These strategies are quite powerful and versatile and, indeed, based on these strategies, we have succeeded to develop a series of fluorescence probes for reactive oxygen species, ions, and various enzymatic activities. Recently, we have established another strategy to control the fluorescent properties of various fluorophores based on intramolecular spirocyclization. For example, if one of the amino groups of hydroxymethyl rhodaminegreen (HMRG), a new rhodaminegreen derivative bearing a hydroxymethyl group instead of the original carboxy group, is amidated with an amino acid, the resultant product is colorless in neutral pH buffer due to the preferred spirocyclization. This compound can be a highly sensitive fluorescence probe for the target aminopeptidase, because it is selectively reactive towards the target enzyme to yield highly fluorescent HMRG. Based on these HMRG-based fluorescence probes, we have succeeded to establish a novel strategy for in vivo tumor imaging by utilizing upregulated enzymatic activities in cancer cells. Gamma-glutamyltranspeptidase (GGT) is well known to be significantly overexpressed in several human tumors, and we have developed a novel fluorescence probe (gGlu-HMRG) for GGT based on HMRG scaffold, which is quickly activated by GGT and yields an over 350-fold increase in fluorescence signal compared with the quenched state. gGlu-HMRG showed a large fluorescence increase in several cancer cell lines, but not in a normal cell line. Indeed, tumor cells in a mouse model of peritoneal metastases were successfully visualized with high signal contrast between the tumor and background. The activation occurred within 1 min of spraying the probe, and the signal was strong enough to be detected even with our naked eyes. This probe is believed to be practical for clinical application during surgical or endoscopic procedures.

Staging laparoscopy using ALA‐mediated photodynamic diagnosis improves the detection of peritoneal metastases in advanced gastric cancer

This study evaluated the usefulness of photodynamic diagnosis (PDD) using oral 5-aminolevulinic acid (ALA) for the detection of peritoneal metastases in advanced gastric cancer. First, the numbers of peritoneal metastatic nodules that were visible under conventional white light (WL) and ALA-induced fluorescence (ALA-F) were quantified in a mouse model of peritoneal metastasis to compare the tumor detection rate. Next, staging laparoscopy (SL) using ALA-PDD was performed in 13 advanced gastric cancer patients with serosa-invading tumors, and the detection sensitivity of ALA-PDD was compared to the observations using WL. The tumor detection rate using ALA-F was significantly higher than the detection rate using WL (72% vs. 39%, respectively, P < 0.0001). Peritoneal metastases were detected in five patients using SL with ALA-PDD, and liver metastases were detected in one patient. These metastases were confirmed using histological examination. Three metastatic lesions that were invisible under WL were detected under ALA-F. This study demonstrated that SL with ALA-PDD improved the detection sensitivity for peritoneal metastases. ALA-PDD may be an important technique for the preoperative staging of advanced gastric cancer, and ALA-PDD will provide useful information for the selection of therapeutic modality.

Suppressive effect of bevacizumab on peritoneal dissemination from gastric cancer in a peritoneal metastasis model

Vascular endothelial growth factor (VEGF) has been reported to enhance vascular permeability and angiogenesis in the abdominal wall, thereby contributing to peritoneal dissemination with malignant ascites. We conducted this experimental study to find out if bevacizumab, a humanized monoclonal antibody against VEGF, had a suppressive effect on peritoneal dissemination from gastric cancer, in an experimental nude mouse model of peritoneal metastasis. Each mouse was treated with a single intraperitoneal (i.p.) injection of bevacizumab. Five mice were killed, and we measured their body weight, the mean number of tumor nodules, and the volume of ascites. We also extracted retroperitoneal tissues for histological examination, to count the frequency of mitosis, and to calculate the mitotic index. Another five mice were monitored until death, and their mean survival duration was calculated. The volume of ascites and the mitotic index were significantly lower in the therapy group than in the nontherapy group (P = 0.042 and P < 0.01, respectively). The survival curve of the therapy group was significantly higher than that of the nontherapy group (P = 0.005). Bevacizumab may suppress peritoneal dissemination from gastric cancer.

T1993 Inhibition of NF-κB in Colon Cancer Cells Significantly Decreases Tumour Burden and Increases Survival Time in a Mouse Model of Peritoneal Metastasis

G A A b st ra ct s cell migration was reduced in TSC2(-/-) rat embryonic fibroblast (REF) cells. In addition, an increase of actin stress fiber formation was observed in TSC2(-/-) REF cells upon stimulation with IGF-1. Furthermore, we showed that the activation of Rac-GTPase was largely impaired in both TSC2(-/-) REF and TSC2 knockdown cancer cells, suggesting that maintaining TSC2 expression is required for Rac activation and cell migration. Our results are consistent with the fact that patients with TSC2 mutations only develop benign and nonmetastatic tumors. Collectively, we identified a novel role of TSC2 in controlling cell motility and cytoskeleton rearrangement by regulating Rac activation.

Activatable Fluorescent Molecular Imaging of Peritoneal Metastases following Pretargeting with a Biotinylated Monoclonal Antibody

Optical probes that yield high target-to-background ratios are necessary to detect microfoci of cancer that would otherwise escape detection with white light imaging. Target-specific activation of the optical signal at tumor foci is one mechanism by which high target and low background signal can be achieved. Here, we describe a two-step activation process in which the tumors are first pretargeted with a nonfluorescent biotinylated monoclonal antibody [cetuximab (Erbitux) targeting human epidermal growth factor receptor type 1 (HER1)]. Following this, a second agent, neutravidin-BODIPY-FL fluorescent conjugate, is given and binds to the previously targeted antibody, resulting in an approximately 10-fold amplification of the optical fluorescence signal, leading to high tumor-to-background ratios. Spectral fluorescence imaging was done in a mouse model of peritoneal metastasis using a HER1-overexpressing cell line (A431) after pretargeting with biotinylated cetuximab and 3 h after administration of neutravidin-conjugated BODIPY-FL. Both aggregated tumors as well as small cancer implants were clearly visualized in vivo. For lesions approximately 0.8 mm or greater in diameter, the spectral fluorescence imaging had a sensitivity of 96% (178 of 185) and a specificity of 98% (188 of 191). This two-step activation paradigm (pretargeting followed by neutravidin-biotin binding with an attached activatable fluorophore) could be useful in tumor-specific molecular imaging of various targets to guide surgical resections.

A mouse model of early-stage peritoneal metastasis: Optimal RT-PCR-based method for detection of peritoneal micrometastases

The presence of peritoneal micrometastasis in the abdomen is a poor prognostic factor in advanced gastric cancer. However, there is no standardized method for detection of peritoneal micrometastases. The aim of this study was to establish an animal model mimicking early phase peritoneal dissemination of advanced gastric cancer and then to use this model to compare the sensitivity and specificity of reverse transcription-polymerase chain reaction (RT-PCR) with peritoneal lavage fluid, randomly collected omentum with or without milky spots stained with activated carbon particles for detection of disseminated cancer cells. MKN-45-EGFP gastric cancer cells (1 x 10(3), 5 x 10(3) and 1 x 10(4)) were injected intraperitoneally into 4-week-old female BALBc nu/nu mice on day 0. Mice were sacrificed at 7 or 14 days postinjection. Peritoneal seeding was assessed by fluorescence stereomicroscopy. The sensitivities of RT-PCR with peritoneal lavage fluid, omentum with or without milky spots, were compared. Peritoneal seeding was confirmed in 8 of 10 mice injected with 1 x 10(4) MKN-45-EGFP cells by fluorescence stereomicroscopy. On days 7 and 14, the rate of the detection of CEA mRNA was 0, 50.0, and 87.5% in the omentum, omentum with milky spots and peritoneal lavage fluid, respectively. On days 7 and 14, the average level of CEA mRNA was 0, 51113+/-28225, and 3556+/-2842 in the omentum, omentum with milky spots and peritoneal lavage fluid, respectively. Molecular diagnosis using peritoneal lavage fluid is more sensitive than that using the omentum.