Liver Metastasis Of Pancreatic Cancer Research Articles

MiR-29c suppresses pancreatic cancer liver metastasis in an orthotopic implantation model in nude mice and affects survival in pancreatic cancer patients

We investigated mechanisms of pancreatic cancer metastasis and defined the biological role of miR-29c in pancreatic cancer metastasis. After two rounds of cell selection in vivo, pancreatic cancer cells with various metastatic potentials derived from spontaneous liver metastases were used as a model of pancreatic cancer to determine the role of miR-29c in pancreatic cancer metastasis. Pancreatic cancer samples were analyzed for miRNA-29c expression, and these levels were associated with survival between groups. miR-29c suppresses cell migration and invasion by targeting the MMP2 3'UTR. Overexpression of miR-29c suppresses pancreatic cancer liver metastasis in a nude mouse orthotopic implantation model. miR-29c expression was associated with metastasis and pancreatic cancer patient survival. miR-29c plays an important role in mediating pancreatic cancer metastasis to the liver by targeting MMP2. Therefore, miR-29c may serve as a novel marker of pancreatic cancer metastasis and possibly as a therapeutic target to treat pancreatic cancer liver metastasis.

Risk factors of liver metastasis in patients after radical resection of pancreatic cancer

To analyze the risk factors of liver metastasis in patients after radical resection of pancreatic cancer. One hundred and twenty-four patients with non-metastatic, resectable pancreatic cancer treated in our department between 2006 and 2012 were included in this study. All of these patients underwent resection of the primary tumor combined with extensive lymph node dissection. The development of postoperative liver metastases was carefully followed up, and the clinicopathological factors and molecular characteristics were evaluated by univariate analysis and multivariate logistic regression using SPSS 16.0 software. Forty-eight cases of liver metastases were found among the 124 cases of pancreatic cancer after radical surgery (38.7%). The rate of liver metastasis of pancreatic cancer after radical surgery in the age groups < 40, 40-60, and > 60 were 68.8%, 33.3% and 35.1%, respectively. The rate of liver metastasis in the body mass index (BMI) group < 20 kg/m2, 20-25 kg/m2, and > 25 kg/m2 were 21.6%, 44.1% and 52.6%, and the rate of liver metastasis in the time between the onset and diagnosis groups ≥ 3 months and < 3 months were 59.4% and 31.5%, respectively. The rate of liver metastasis in patients with preoperative fatty liver was 14.3% and it was 43.7% in patients without preoperative fatty liver. The rate of liver metastasis in patients of histological high, medium and low grade was 10.0%, 35.4% and 49.0%, respectively. The rate of liver metastasis in patients with venous tumor thrombus was 68.8% and it was 34.3% in patients without venous tumor embolus. The rate of liver metastasis in patients with postoperative chemotherapy was 31.2% and it was 51.1% in patients without postoperative chemotherapy. All those differences had statistical significance (P < 0.05). Univariate analysis revealed that age, body mass index (BMI), time between the onset and diagnosis, preoperative fatty liver, histological grading, tumor invasion depth, venous tumor embolus, and postoperative chemotherapy were significantly related to postoperative liver metastasis. Multivariate analysis revealed five statistically independent risk factors for postoperative liver metastasis: BMI, time between onset and diagnosis, preoperative fatty liver, histological grading, and venous tumor embolus. Our data suggest that patient's BMI, time between onset and diagnosis, histological grade, and venous tumor embolus are significantly correlated with postoperative liver metastases in patients with pancreatic cancer. Pancreatic cancer patients with preoperative fatty liver have less postoperative liver metastasis.

Neurotensin receptors in pancreatic ductal carcinomas.

BackgroundThe frequent expression of neurotensin receptors (NT-R) in primaries of pancreatic ductal carcinomas has triggered the development of radioactive neurotensin analogs for possible in vivo targeting of these tumors. However, the complete lack of information regarding NT-R in liver metastases of pancreatic cancer and pancreatic intraepithelial neoplasia (PanIN) makes an in vitro study of NT-R in these tissues indispensable.MethodsUsing in vitro receptor autoradiography with 125I-[Tyr3]-neurotensin, NT-R were investigated in 18 primaries and 23 liver metastases of pancreatic ductal carcinomas as well as in 19 PanIN lesions.ResultsWe report here that 13 of 18 ductal carcinoma primaries and 14 of 23 liver metastases expressed NT-R. Moreover, none of the six PanIN 1B cases expressed NT-R, while two of six PanIN 2 and five of seven PanIN 3 expressed NT-R. Binding was fully displaced by the type 1 NT-R-selective antagonist SR48692, indicating that the NT-R in the tumors are of the type 1 NT-R subtype.ConclusionsThese in vitro data extend the currently available information on NT-R in invasive and non-invasive pancreatic ductal tumors. They suggest that type 1 NT-R may be a novel, specific marker of PanIN of higher degree. The high expression of NT-R in primaries and metastases of invasive cancer strongly support the need to develop radioactive neurotensin analogs for the diagnosis and therapy of this tumor type.

Riproximin’s activity depends on gene expression and sensitizes PDAC cells to TRAIL

Riproximin (Rpx) is a type II ribosome inactivating protein, which was investigated for its activity in pancreatic ductal adenocarcinoma (PDAC) in a panel of 17 human and rat PDAC cell lines and in rat pancreatic cancer liver metastasis. Cytotoxicity in response to Rpx was determined by MTT assay, apoptosis by flow cytometry and qRT-PCR for apoptosis related genes, and the modulation of the transcriptome was monitored by micro array analysis. The combination effect of Rpx and TRAIL was assessed by MTT assay. Rpx showed high but varying cytotoxicity in PDAC cells. Based on overall gene expression, the sensitivity of these cells was linked to genes involved in apoptosis. Furthermore, based on the affinity of Rpx for CEA, the expression of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) genes was significantly related to Rpx’s cytotoxicity in cells with CEACAM gene expression. Exposure of Suit2–007 cells to Rpx induced the mRNA expression of members of signaling pathways initiating from most death receptors, and down modulation of TRAIL. Apoptosis was increased as shown by FACS analysis. Combination of Rpx with TRAIL resulted in a synergistic cytotoxic effect in human Suit2–007 and rat ASML cells, as evidenced by a 6-fold lower tumor cell survival than expected from an additive combination effect. Treatment of BDX rats bearing intra-portally implanted Suit2–007 cells showed a highly significant anticancer effect and indicated an application of Rpx against pancreatic cancer metastasis to the liver. These data favor further evaluation of Rpx as anticancer agent in PDAC.

Integrin αvβ6–Targeted SPECT Imaging for Pancreatic Cancer Detection

Integrin αvβ6, a member of the integrin family, is specifically expressed in many malignancies but not in normal organs. Overexpression of integrin αvβ6 is usually correlated with malignant potential and poor prognosis. In this study, we describe the synthesis and evaluation of a (99m)Tc-labeled integrin αvβ6-targeting peptide as a SPECT radiotracer for the in vivo imaging of integrin αvβ6 expression. An integrin αvβ6-targeting peptide (denoted as the HK peptide) was conjugated with 6-hydrazinonicotinyl (HYNIC) and radiolabeled with (99m)Tc using tricine and TPPTS (trisodium triphenylphosphine-3,3',3″-trisulfonate) as coligands. The in vitro and in vivo characteristics of (99m)Tc-HYNIC(tricine)(TPPTS)-HK ((99m)Tc-HHK) were investigated in BxPC-3 (integrin αvβ6-positive) and HEK293 (integrin αvβ6-negative) models. The ability of (99m)Tc-HHK to detect liver metastasis of pancreatic cancer was evaluated using small-animal SPECT/CT. (99m)Tc-HHK showed high integrin αvβ6-binding specificity both in vitro and in vivo. (99m)Tc-HHK was cleared rapidly from the blood and normal organs except for the kidneys. The highest uptake (0.88 ± 0.12 percentage injected dose per gram) of (99m)Tc-HHK in BxPC-3 tumors was observed at 0.5 h after injection. High-contrast images of integrin αvβ6-positive tumors were obtained using (99m)Tc-HHK. The minimum nonspecific activity accumulation in normal liver tissues rendered high-quality SPECT/CT images of metastatic lesions. (99m)Tc-HHK is a promising SPECT radiotracer for the noninvasive imaging of integrin αvβ6 expression in vivo. SPECT/CT with (99m)Tc-HHK could provide an effective approach for the noninvasive detection of primary and metastatic lesions of integrin αvβ6-positive tumors.

Liver Metastases of Pancreatic Cancer: Role of Repetitive Transarterial Chemoembolization (TACE) on Tumor Response and Survival

Background: The study was conducted to evaluate the effect of chemoembolization on pancreatic cancer liver metastases. Patients and Methods: Thirty-two patients with pancreatic cancer liver metastases who had underwent chemoembolization (48 weeks intervals) were retrospectively enrolled into this study. Size based evaluation (RECIST) and survival indexes were assessed in general and for gender and number of lesions. Results: 71.87% of patients showed stable disease (SD), 9.37% partial response (PR) and 18.75% progressive disease (PD). Survival rate for 1-, 3-, and 5-years from first TACE was 60%, 25%, and 11% respectively. Median survival time was 16 months and for SD group was 20 months. Progression-free survival for 6 month, 1-, 3- and 5-years was 84%, 57.3%, 20%, and 10%, respectively. There was statistically significant difference between male and female patients in their response. Survival rate for 1- and 5-years for males was 80% and 14% and for females was 47% and 0%. There was no significant difference between oligonodular liver lesion (less than 5) and multinodular (more than 5) groups. Survival rate for 1-and 5-years for oligonodular was 84% and 14% and for multinodular was 50% and 0% respectively. Conclusions: Repetitive TACE resulted in a relevant response for the control of liver metastases of pancreatic cancer with considerable median survival time. Interestingly, this study was limited by the low number of subjects to produce significant results.

Abstract 264: Mesenchymal stem cells mediate epithelial-to-mesenchymal transition (EMT) and stemness regulation in pancreatic cancer via Notch signal.

Abstract Cancer associate fibroblasts (CAFs), one of the major components of the cancer stroma, are prominently involved in tumor progression including epithelial to mesenchymal transition (EMT). Recently, bone marrow derived mesenchymal stem cells (MSCs) have been suggested as candidate for the source of tumor-promoting CAFs. We previously demonstrated that side population (SP) cells, which are enriched with so-called tumor initiating stem cell like cells (TISCs), play a pivotal role of local invasion and liver metastasis in pancreatic cancer (Int-J-Cancer 124:2771, 2009). Therefore, we formulated the hypothesis that MSCs regulate SP cells EMT and TISC-like properties such as tumor formation/progression. The AIM of the present study was to examine whether bone marrow-derived MSCs have ability to mediate EMT and/or tumor progression in pancreatic cancer SP cells. METHODS: MSCs were pre-treated with TGF-beta for 3 days. SP or non-SP cells were co-cultured in the presence or absence of TGF-beta-pretreated MSCs (Tb-MSCs) for 3 days. Real-time PCR experiments were performed to quantitate mRNA levels of EMT associated genes as well as so-called stemness related genes. PANC-1 derived SP or non-SP cells were co-injected with Tb-MSCs into NOD/SCID mice, and their tumorigenesity was evaluated. RESULTS: Co-culturing with Tb-MSCs dramatically induced EMT-associated mRNA alterations including suppression of E-cadherin and induction of slug in PANC-1 SP cells. In SP cells but not in non-SP cells, levels of stemness genes including CD133, Oct4, LGR5, were enhanced by co-culturing with Tb-MSCs. Tb-MSCs enhanced in vitro sphere forming activity as well as in vivo tumor formation in SP cells. These alterations including EMT-associated gene modifications and enhanced sphere formation were suppressed by Notch signal inhibition, suggested MSCs-associated regulation of EMT and TISC-like properties are mediated by Notch-signaling. In CONCLUSION, MSCs regulate EMT and they maintain TISC-like properties in pancreatic cancer SP cells via Notch signal-dependent mechanisms. Targeting Notch signaling would appear to be attractive to prevent MSCs and/or stromal cell-associated cancer progression. Citation Format: Ayano Kabashima-Niibe, Hajime Higuchi, Yumi Matsuzaki, Yo Mabuchi, Shinsuke Funakoshi, Masayuki Adachi, Yasuo Hamamoto, Hiromasa Takaishi, Toshifumi Hibi. Mesenchymal stem cells mediate epithelial-to-mesenchymal transition (EMT) and stemness regulation in pancreatic cancer via Notch signal. [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 264. doi:10.1158/1538-7445.AM2013-264

Combined effect of dehydroxymethylepoxyquinomicin and gemcitabine in a mouse model of liver metastasis of pancreatic cancer

Activation of nuclear factor-κB (NF-κB) has been implicated in metastasis of pancreatic cancer. We investigated the effects of the novel NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) on the inhibition of liver metastasis of pancreatic cancer in a mouse model of clinical liver metastasis. Nude mice were xenografted by intra-portal-vein injection with the human pancreatic adenocarcinomas cell line AsPC-1 via small laparotomy. Mice were treated with DHMEQ and gemcitabine (GEM), alone or in combination. The combination of GEM+DHMEQ showed a stronger antitumor effect than either monotherapy. Apoptosis induction in the metastatic foci was greatest in the DHMEQ+GEM group. Significant reductions in the numbers of neovessels were also seen in the DHMEQ and/or GEM groups. Cell growth inhibition assays revealed no synergistic effect of combination therapy, although each monotherapy had an individual cytotoxic effect. Combination therapy produced the greatest inhibition of tumor cell invasiveness in chemoinvasion assay. In addition, combination therapy significantly down-regulated the expression level of matrix metalloproteinase (MMP)-9 mRNA in AsPC-1 cells. DHMEQ also markedly down-regulated interleukin-8 and MMP-9, while GEM caused moderate down-regulation of vascular endothelial growth factor in metastatic foci, demonstrated by quantitative reverse transcription-polymerase chain reaction. These results demonstrate that DHMEQ can exert anti-tumor effects by inhibiting angiogenesis and tumor cell invasion, and by inducing apoptosis. Combination therapy with DHMEQ and GEM also showed potential efficacy. DHMEQ is a promising drug for the treatment of advanced pancreatic cancer.

Few genes are associated with the capability of pancreatic ductal adenocarcinoma cells to grow in the liver of nude rats

Owing to aggressiveness and chemoresistance, pancreatic ductal adenocarcinoma (PDAC) is characterised by a poor prognosis. To address this disease-spe-cific dilemma we aimed to establish animal models, which can be used for identifying new specific tumor markers, as well as serving as tools for potential therapeutic approaches. From a panel of sixteen pancreatic cancer cell lines, two human (Suit2-007 and Suit2-013) and a rat (ASML) cell line were selected for their properties to grow in the liver of male RNU rats and mimic liver metastasis of PDAC. For better monitoring of metastatic tumor growth invivo, all three pancreatic cancer cell lines were stably transfected with eGFP and luciferase marker genes. In addition, the mRNA expression profile of 13 human PDAC cell lines was analyzed by BeadChip array analysis. Only 33 genes and 5 signaling pathways were identified as significantly associated with the ability of the cell lines to grow initially and/or consistently in rat liver. Only a minority of these genes (osteopontin, matrix metalloproteinase-1 and insulin-like growth factor 1) has been intensively studied and shown to be closely related to cancer progression. The function of the remaining 30 genes ranges from moderate to poorly investigated, and their function in cancer progression is still unclear. The ensuing three pancreatic cancer liver metastasis models vary in their aggressiveness and macroscopic growth. They will be used for preclinical evaluation of new therapeutic approaches aiming at the genes identified.

Collapsin Response Mediator Protein 4 Expression is Associated with Liver Metastasis and Poor Survival in Pancreatic Cancer

Pancreatic cancer is an aggressive malignancy with one of the worst mortality rates of all cancers. Recently, collapsin response mediator proteins (CRMPs) were reported to be associated with proliferation, apoptosis, differentiation, and invasion in several cancers. However, CRMP expression and their role in pancreatic cancer have not been investigated. This study aimed to clarify the clinical significance of CRMPs in pancreatic cancer. Expression of crmp genes in 11 pairs of pancreatic cancer and corresponding noncancerous pancreas tissues were examined by real-time RT-PCR. Knockdown of CRMP4 expression using siRNA was examined in pancreatic cancer cell lines to determine whether CRMP4 regulates cell proliferation and invasion in vitro. Furthermore, CRMP4 protein levels in primary tumors of pancreatic cancer (n = 53) were examined by immunohistochemistry and compared with the clinicopathological features of the tumors. Of all the CRMPs, only CRMP4 was differentially expressed in pancreatic cancer tissues (p = 0.008). CRMP4 knockdown using siRNA reduced cellular invasion, but did not affect proliferation. The expression of CRMP4 was detected immunohistochemically in 34 (64.2 %) of the 53 pancreatic cancer samples, and CRMP4 expression was correlated with severe venous invasion (p = 0.044), stage (p = 0.019), and liver metastasis (p = 0.021). Multivariate analyses suggested that venous invasion and CRMP4 overexpression were prognostic factors for survival. Our results suggested that CRMP4 is significantly associated with poor prognosis by promoting liver metastasis and can serve as a novel therapeutic target for pancreatic cancer.

Increased E-selectin in hepatic ischemia-reperfusion injury mediates liver metastasis of pancreatic cancer

Several recent studies have reported that selectins are produced during ischemia-reperfusion injury, and that selectin ligands play an important role in cell binding to the endothelium and in liver metastasis. Portal clamping during pancreaticoduodenectomy with vessel resection for pancreatic head cancer causes hepatic ischemia-reperfusion injury, which might promote liver metastasis. We investigated the liver colonization of pancreatic cancer cells under hepatic ischemia-reperfusion and examined the involvement of E-selectin and its ligands. A human pancreatic cancer cell line (Capan-1) was injected into the spleen of mice after hepatic ischemia-reperfusion (I/R group). In addition, to investigate the effect of an anti-E-selectin antibody on liver colonization in the IR group, mice received an intraperitoneal injection of the anti-E-selectin antibody following hepatic ischemia-reperfusion and tumor inoculation (IR+Ab group). Four weeks later, mice were sacrificed and the number of tumor nodules on the liver was compared to mice without hepatic ischemia-reperfusion (control group). The incidence of liver metastasis in the I/R group was significantly higher (16 of 20, 80%) than that in the control group (6 of 20, 30%) (P<0.01). Moreover, mice in the I/R group had significantly more tumor nodules compared to those in the control group (median, 9.9 vs. 2.7 nodules) (P<0.01). In the I/R+Ab group, only 2 of 5 (40%) mice developed liver metastases. RT-PCR and southern blotting of the liver extracts showed that the expression of IL-1 and E-selectin mRNA after hepatic ischemia-reperfusion was significantly higher than the basal levels. Hepatic ischemia-reperfusion increases liver metastases and E-selectin expression in pancreatic cancer. These results suggest that E-selectin produced due to hepatic ischemia-reperfusion is involved in liver metastasis.

Abstract 3840: Evaluation of the synergetic effect of a combination of dehydroxymethylepoxyquinomicin and gemcitabine on liver metastasis of pancreatic cancer

Abstract Activation of the nuclear transcription factor-kappaB (NF-κB) has been implicated in the progression and metastasis of pancreatic cancer. This study was designed to evaluate the effects of a novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) in inhibition of liver metastasis of pancreatic cancer in a clinical liver metastasis-mimicking mouse model in which cancer cells were intra-portal injected under small laparotomy. Moreover, the synergistic effect of combination therapy with DHMEQ and gemcitabine (GEM) was investigated. Cytotoxicity of DHMEQ and GEM was evaluated by cell growth inhibition assay. Both DHMEQ and GEM monotherapies inhibited cell growth of the human pancreas cell line AsPC-1. However, there was no synergistic effect in the combination therapy. For the in vivo study, male BALB/c nude mice were xenografted with intra-portal vein injection of AsPC-1. Mice were divided into four groups: 1) control (without any treatment), 2) GEM-treated group, 3) DHMEQ-treated group, and 4) combined treatment group (GEM+DHMEQ). Mice were fed for four weeks, and the number of liver metastases, induction of apoptosis in metastasis focuses and angiogenesis were investigated after sacrifice. The number of liver metastasis was decreased significantly in GEM group, DHMEQ group and GEM+DHMEQ group compared with control group. Significant inductions of apoptosis were revealed in GEM group and DHMEQ group compared with control group, in which apoptosis bodies in metastasis sections were counted by using the TUNEL method. Moreover, combination therapy of DHMEQ+GEM resulted in the strongest inductions of apoptosis. Tumor vessels were identified by immunohistochemical staining of tumor metastasis sections with an antibody against CD31. There was a large reduction in the number of CD31-positive vessels in the tumor of DHMEQ or/and GEM-treated mice as compared with control group. However, there was an inconsistency in the results of cell growth inhibition assay (in vitro) and mice metastasis model (in vivo). Thus we evaluated effect of combination therapy in the invasive potential of tumor cells by using chemoinvasion assay. The invasion ability was significantly inhibited in the GEM group, DHMEQ group and GEM+DHMEQ group. Furthermore, real-time PCR was performed to evaluate expression of mRNA of MMP-9. The expression levels of MMP-9 were inhibited in respective groups in the same order. In conclusion, the anti-tumor effects of combination therapy were significant, as evidenced by angiogenesis inhibition, apoptosis induction and invasiveness inhibition rather than cell growth inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3840. doi:1538-7445.AM2012-3840

Abstract 907: The efficiency of Nek2 siRNA for liver metastasis of pancreatic cancer

Abstract Background: Nek2 (NIMA-related kinase 2) is a member of the serine/threonine kinase family, which related to the essential mitotic regulator NIMA. Nek2 is involved in cell division and mitotic regulation by centrosome splitting. We reported that the efficiency of Nek2 as molecular target for several cancers, such as cholangiocarcinoma, breast cancer and colorectal cancer. Methods: We examined the effect of Nek2 siRNA treatment on the tumor of pancreatic cancer in xenograft mouse model. We also challenged Nek2 siRNA in the liver metastasis model of pancreatic cancer. Results and Discussion: Nek2 siRNA suppressed the growth of tumors in xenograft pancreas cancer model. In liver metastasis model, Nek2 siRNA decreased the number of metastasis and resulted in reduction of metastasis volume. Liver metastasis is one of the major undesirable complications frequently associated with inoperable cases. The Nek2 suppression with siRNA is also effective in the inhibition of liver metastasis of pancreas cancer. Conclusions: Nek2 plays a critical role in tumorigenic growth of various cancers, and inhibition of Nek2 expression with its siRNA causes suppression of cancer growth. Our data provides a strong rationale for the further investigation of Nek2 inhibitors as a new cancer therapy, holding the potential to provide regression of multiple human malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 907. doi:1538-7445.AM2012-907

Abstract 1695: Imaging of the interaction of pancreatic cancer and stellate cells during liver metastasis

Abstract Pancreatic stellate cells are involved in fibrosis of pancreatic cancer. An understanding of pancreatic cancer-cell interactions with stellate cells is critical to our ability to develop effective anti-tumor therapeutics for pancreatic cancer. We report here imaging of the interaction of pancreatic cancer cells and pancreatic stellate cells in liver metastasis. Human pancreatic cancer cell lines (XPA1 and MiaPaCa-2) were engineered to express green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm. Pancreatic stellate cells, engineered to express RFP, were co-injected with the cancer cells into the spleen of transgenic cyan fluorescent protein (CFP) nude mice. Three hours after splenic injection dual-color pancreatic cancer cells and pancreatic stellate cells were found distributed in the host liver. Seven days after cancer cell-stellate cell co-injection, most pancreatic cancer cells and stellate cells were dead. However, by 28 days after injection, liver metastases were observed in the host CFP nude mice. With the high-resolution intravital imaging afforded by the Olympus FV1000 confocal microscope, the interaction of the dual-colored pancreatic cancer cells and the RFP-expressing pancreatic stellate cells could be clearly imaged in the liver metastasis, suggesting that stellate cells participate in metastasis formation. Our hypothesis is that pancreatic stellate cells form a niche for liver metastasis of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1695. doi:1538-7445.AM2012-1695

Abstract 363: Development of computer-aided detection (CAD) tool for liver metastasis micro CT imaging using targeted contrast agent

Abstract Introduction: Preclinical in-vivo micro CT studies of liver metastasis are difficult due to poor inherent soft tissue contrast and the need for highly technical, manual analysis of the data. Research has implicated that Kupffer cells in the liver encapsulate liver metastases providing an opportunity to deliver macrophage-specific contrast agents for the detection of small metastatic lesions. A new, long-acting preclinical CT contrast agent that targets Kupffer cells has been developed that may allow automated detection of liver lesions via CAD software. Method: A pancreatic cancer liver metastasis model was created by surgically implanting human pancreatic cancer cell line (L3.6pl) within the spleen. Mice were injected with the contrast agent and scanned to obtain baseline anatomy of the liver before implantation. The mice were scanned 1 day after implantation and weekly after that for 5 weeks to monitor the liver metastasis progression. The control group followed an identical protocol but with a sham surgery. Liver tissues were harvested and fixed in paraffin blocks after the last scan. Paraffin blocks were scanned using high resolution micro CT before IHC staining. Human Mitochondrial and F4/80 IHC were used to identify L3.6pl and Kupffer cells, respectively. The CT images were compared to the IHC images from the same block to verify that the locations of the contrast agent and the Kupffer cells were related. Once the pattern of contrast agent and metastatic tumors had been identified, CAD software was developed for automatic tumor detection. Results: The contrast agent was evenly distributed throughout the healthy liver tissue within 1 hour post injection. In healthy mice, the homogenous distribution of contrast remained unchanged for at least 6 weeks. In liver metastasis models, the contrast began to concentrate in various areas of the liver within 2 weeks post implantation. As tumors developed and grew, the contrast became highly concentrated on the borders of tumors creating a 3 dimensional outline of the lesion. IHC staining and micro CT imaging of the fixed tissue verified that the tumors are surrounded by Kupffer cells and that the distribution of concentrated contrast agent matched them. Software was able to detect the tumors based on these contrast outlines and compare them over successive weekly scans. Conclusion: Our new imaging method enables automated detection and evaluation of liver metastasis 1 mm or smaller from as early as 2 weeks. In addition to allowing better visualization, it provides new insight into macrophage motility within the liver. CAD software can take advantage of this unique capability to automate data analysis and allow for large scale longitudinal studies. This new imaging method could be a useful tool to facilitate longitudinal imaging of liver metastases in mice and has the potential for translation into clinical practice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 363. doi:1538-7445.AM2012-363

The Efficacy Evaluation of Cryosurgery in Pancreatic Cancer Patients with the Expression of CD44v6, Integrin-β1, CA199, and CEA

Increased expression of cell adhesion molecule CD44v6, integrin-β1, carbohydrate antigen 199 (CA199), and carcinoembryonic antigen (CEA) are closely associated with the progression and metastasis of numerous cancers. In this study, peripheral blood mononuclear cell (PBMC) and serum samples were collected from 37 pancreatic cancer patients and 12 healthy people. A novel triplex TaqMan real-time reverse transcription polymerase chain reaction assay was used to measure the expression levels of CD44v6 and integrin-β1 gene in PBMCs, while chemiluminescence and enzyme-linked immunosorbent assay were used to measure the levels of CA199 and CEA expression in serum. The results showed that both the levels of CD44v6 and integrin-β1 expression had significant correlation with clinical stage, lymph node, and liver metastasis of pancreatic cancer (P < 0.05). Age, tumor size, tumor differentiation, clinical stage, lymph nodes, and liver metastasis were significantly associated with the levels of CA199 and CEA expression (P < 0.05). The levels of CD44v6, integrin-β1, CA199, and CEA expression in the patients prior cryosurgery and chemotherapy were significantly higher than those in the control group (P < 0.05), whereas no significant difference was found between the patients 1 month post cryosurgery and control group (P > 0.05). The expression levels of CD44v6, integrin-β1, CA199, and CEA in the patients 1 month post cryosurgery were significantly lower than those in the patients prior cryosurgery (P < 0.05). Interestingly, no significant difference was found for the CD44v6, integrin-β1, CA199, and CEA levels between the patients prior and post-chemotherapy (P > 0.05). The higher expression of CD44v6, integrin-β1, CA199, and CEA are closely related to the progression and metastasis of pancreatic cancer and may play a important role in the curative evaluation of cryosurgery of pancreatic cancer.

Abstract B145: The novel Hedgehog pathway inhibitor IPI-926 delays metastatic spread and provides a survival benefit in an experimental model of pancreatic liver metastasis.

Abstract Metastasis, or the spread of disseminated tumor cells to and colonization of distal sites, is a major cause of cancer mortality. The Hedgehog (Hh) signaling pathway is normally involved in embryogenesis; however recent evidence suggests a role for this pathway in cancer and metastasis. Thus, inhibitors of Hh signaling may have potential utility as novel targeted anti-tumor therapies. IPI-926 is a novel, selective, small molecule that antagonizes the Hh pathway by binding to the Smoothened receptor, the major upstream facilitator of Hh signal transduction, and is currently in phase 2 clinical trials for pancreatic cancer, chondrosarcoma and primary myelofibrosis. Administration of IPI-926 in combination with gemcitabine inhibits incidence of metastases in a KRAS/p53 transgenic model of pancreatic cancer (Olive et al., 2009), supporting an integral role for the Hh pathway in this process. To further explore the activity of IPI-926 in the setting of metastasis, we developed an experimental model of pancreatic cancer liver metastasis where luciferase-tagged tumor cells were implanted in the liver via an intra-splenic injection, followed by splenectomy, resulting in liver metastases. Tumor burden, as assessed by bioluminescence and survival were employed to evaluate IPI-926 activity utilizing different dosing regimens. While treatment of tumor-bearing animals with established disease provided no survival benefit, IPI-926 administered on the day of cell implant provided modest but reproducible increase in survival. However, prophylactic administration of IPI-926 beginning 14 days (d) prior to cell implant resulted in a significant decrease in disease burden and enhanced survival. The activity was dependent on the schedule of IPI-926 administration. Treatment initiated 14d prior to cell implant was more effective than 7d before cell implant, and treatment started only 2d prior to cell implantation showed no difference compared to treatment initiated on the day of cell implant. Histological analysis of pre-treated vs control livers confirmed decreased metastatic nodule formation. In addition, analysis of serum harvested from IPI-926 14d pre-treated vs control animals showed down-regulation of components of the VEGF, PDGF and MMP pathways, suggesting that IPI-926 modulation of these pathways may be involved in the observed effects on metastatic spread. The activity of IPI-926 was also evaluated in the setting of sunitinib enhancement of metastases (Ebos et al., 2010) in the above model. As has been shown previously, short, high-dose sunitinib administration resulted in striking increases in disease burden and shortening of survival compared to control animals. However, when IPI-926 was administered 14d prior to cell implant and sunitinib treatment, disease burden and survival were returned to the levels observed in the control group. In summary, IPI-926 administration delays metastasis in an experimental model of pancreatic liver metastasis and in response to high-dose anti-angiogenic therapy. These results provide rationale for the evaluation of IPI-926 as adjuvant/neoadjuvant therapy to help control metastatic spread post surgical resection in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B145.

Liver Metastases of Pancreatic Cancer

To evaluate the effect of chemoembolization on pancreatic cancer liver metastases. Thirty-two patients with pancreatic cancer liver metastases retrospectively underwent chemoembolization (4- to 8-week intervals). Size-based evaluation (response evaluation criteria in solid tumors [RECIST]) and survival indexes were assessed overall and for sex and number of lesions. Of the patients, 71.87% showed stable disease, 9.37% partial response (PR) and 18.75% progressive disease (PD). Survival rate for 1, 3, and 5 years from first TACE was 60%, 25%, and 11%, respectively. Median survival time was 16 months and for stable disease group was 20 months. Progression-free survival for 6 months and 1, 3, and 5 years was 84%, 57.3%, 20%, and 10%, respectively. There was significant difference between men and women in response. Survival rates for 1 and 5 years for the men were 80% and 14% and for the women were 47% and 0%. There was no significant difference between oligonodular liver lesion (n < 5) and multinodular (n > 5) groups. Survival rates for 1 and 5 years for oligonodular were 84% and 14%, and for multinodular was 50% and 0%. Repetitive TACE resulted in a relevant response for the control of liver metastases of pancreatic cancer with respectable median survival time. Interestingly, the number of lesions, statistically, was not an effective factor.

High Level of PTEN Expression Is Associated with Low-grade Liver Metastasis and Satisfactory Patient Survival in Pancreatic Cancer

We investigated PTEN expression in primary pancreatic cancer and pancreatic cancer liver metastasis in order to evaluate the interrelationship between PTEN expression and clinicopathological characteristics of pancreatic cancer patients with and without liver metastasis. Eighty five primary pancreatic cancer specimens without liver metastasis were analyzed as controls. Eighty seven pancreatic cancer specimens and homologous liver metastasis specimens were investigated immunohistochemically, and the correlation between immunohistochemical findings and clinicopathological factors was investigated. A strong PTEN expression was observed in 52 (61.2%) specimens from patients without liver metastasis. In contrast, only 26 (29.9%) specimens were observed in patients with liver metastasis. A strong PTEN expression was apparently associated with low-grade lymph node metastasis (p <0.05) and TNM stage (p <0.05). PTEN expression in patients without liver metastasis was apparently stronger than that with liver metastasis. In addition, among patients with liver metastasis, the 5-year survival rate was markedly higher in patients with strong PTEN expression compared to those with weak PTEN expression. Our results suggest that a high level of PTEN expression is associated with low-grade liver metastasis and satisfactory patient survival in pancreatic cancer. The diagnostic evaluation of PTEN expression may provide valuable prognostic information to aid treatment strategies for pancreatic cancer patients.

Submillimeter-Resolution Fluorescence Laparoscopy of Pancreatic Cancer in a Carcinomatosis Mouse Model Visualizes Metastases Not Seen with Standard Laparoscopy

Staging laparoscopy can visualize peritoneal and liver metastases in pancreatic cancer otherwise undetectable by preoperative imaging. However, false-negative rates may be as high as 18%-26%. The aim of the present study was to improve detection of metastatic pancreatic cancer with the use of fluorescence laparoscopy (FL) in a nude-mouse model with the tumors expressing green fluorescent protein (GFP). The carcinomatosis mouse model of human pancreatic cancer was established by intraperitoneal injections of green fluorescent protein-expressing MiaPaca-2 human pancreatic cancer cells into 6-week-old female athymic mice. Two weeks later, mice underwent diagnostic laparoscopy. Laparoscopy was performed first under standard brightfield lighting, followed by fluorescent lighting. The number of metastatic foci identified within the four quadrants of the peritoneal cavity was recorded. After laparoscopy, the animals were sacrificed, opened, and imaged with the OV-100 Small Animal Imaging system as a positive control to identify metastasis. Tumors were collected and processed for histologic review. FL enabled visualization of pancreatic cancer metastatic foci not visualized with standard brightfield laparoscopy (BL). Under FL, in 1 representative mouse, 26 separate micrometastatic lesions were identified. In contrast, only very large tumors were seen using BL. Use of the OV-100 images, as positive controls, confirmed the presence of tumor foci. FL thus allowed identification and exact localization of submillimeter tumor foci. Such small-sized tumor foci were not distinguished from surrounding tissue under BL. All malignant lesions were histologically confirmed. The use of FL enables the identification of tumor foci that cannot be seen with standard laparoscopy. The technology described in this report has important potential for the clinical development of FL.