Human Pancreatic Tumor Cells Research Articles

Targeting a Novel Onco-glycoprotein Antigen at Tumoral Pancreatic Cell Surface by mAb16D10 Induces Cell Death

The mAb16D10 was raised against a pathological onco-glycoform of bile salt-dependent lipase isolated from the pancreatic juice of a patient suffering from a pancreatic adenocarcinoma. We previously showed that mAb16D10 specifically discriminates human pancreatic tumor tissues from other cancer and nontumor tissues. In this study, we report that mAb16D10 inhibited the proliferation of only human pancreatic tumor cells expressing 16D10 plasma membrane Ag. Interaction of mAb16D10 with its cognate surface Ag on pancreatic cells promoted cell death by activation of the p53- and caspase-dependent apoptotic pathway, and silencing of p53 decreased cell death. The decreased proliferation was also partly due to cell cycle arrest in G1/S phase, mAb16D10 triggering of glycogen synthase kinase-3β (GSK-3β) activation, degradation of β-catenin, and decreased expression of cyclin D1. GSK-3β positively affected p53 expression in pancreatic tumor cells after mAb16D10 binding. Inhibition of GSK-3β activity reversed the effects induced by mAb16D10 in SOJ-6 cells, supporting the pivotal role of GSK-3β signaling in the mechanisms of action induced by mAb16D10. Also, mAb16D10 cell treatment led to membrane overexpression of E-cadherin. Both E-cadherin and tumor Ag were localized in membrane lipid cholesterol-rich microdomains and are thought to belong to signaling platforms involved in the induction of cell cycle arrest and cell death. Overall, this study reveals that mAb16D10 holds great potential to prevent pancreatic tumor proliferation by apoptotic cell death, thus promising therapeutic prospects for treatment of pancreatic adenocarcinoma, a highly lethal disease.

Sensing dynamic cytoplasm refractive index changes of adherent cells with quantitative phase microscopy using incorporated microspheres as optical probes

The intracellular refractive index is an important parameter that describes the optical density of the cytoplasm and the concentration of the intracellular solutes. The refractive index of adherently grown cells is difficult to access. We present a method in which silica microspheres in living cells are used to determine the cytoplasm refractive index with quantitative phase microscopy. The reliability of our approach for refractive index retrieval is shown by data from a comparative study on osmotically stimulated adherent and suspended human pancreatic tumor cells. Results from adherent human fibro sarcoma cells demonstrate the capability of the method for sensing of dynamic refractive index changes and its usage with microfluidics.

EGF Receptor Signaling Is Essential for K-Ras Oncogene-Driven Pancreatic Ductal Adenocarcinoma

Clinical evidence indicates that mutation/activation of EGF receptors (EGFRs) is mutually exclusive with thepresence of K-RAS oncogenes in lung and colon tumors. We have validated these observations using genetically engineered mouse models. However, development of pancreatic ductal adenocarcinomas driven by K-Ras oncogenes are totally dependent on EGFR signaling. Similar results were obtained using human pancreatic tumor cell lines. EGFRs were also essential even in the context of pancreatic injury and absence of p16Ink4a/p19Arf. Only loss of p53 made pancreatic tumors independent of EGFR signaling. Additional inhibition of PI3K and STAT3 effectively prevented proliferation of explants derived from these p53-defective pancreatic tumors. These findings may provide the bases for more rational approaches to treat pancreatic tumors in the clinic.

Ready, Set, Go: The EGF Receptor at the Pancreatic Cancer Starting Line

Acinar-to-ductal metaplasia (ADM) results from pancreatic injury or KRAS activation, and is an early step in pancreatic cancer progression. In this issue of Cancer Cell, Ardito and colleagues and Navas and colleagues demonstrate that ADM- and KRAS-driven pancreatic cancer require EGFR signaling, revealing a mechanism for developmental reprogramming that primes tumorigenesis.

Evaluation of a Gene-Directed Enzyme-Product Therapy (GDEPT) in Human Pancreatic Tumor Cells and Their Use as In Vivo Models for Pancreatic Cancer

BackgroundGene-directed enzyme prodrug therapy (GDEPT) is a two-step treatment protocol for solid tumors that involves the transfer of a gene encoding a prodrug-activating enzyme followed by administration of the inactive prodrug that is subsequently activated by the enzyme to its tumor toxic form. However, the establishment of such novel treatment regimes to combat pancreatic cancer requires defined and robust animal model systems.MethodsHere, we comprehensively compared six human pancreatic cancer cell lines (PaCa-44, PANC-1, MIA PaCa-2, Hs-766T, Capan-2, and BxPc-3) in subcutaneous and orthotopical mouse models as well as in their susceptibility to different GDEPTs.ResultsTumor uptake was 83% to 100% in the subcutaneous model and 60% to 100% in the orthotopical mouse model, except for Hs-766T cells, which did not grow orthotopically. Pathohistological analyses of the orthotopical models revealed an infiltrative growth of almost all tumors into the pancreas; however, the different cell lines gave rise to tumors with different morphological characteristics. All of the resultant tumors were positive for MUC-1 staining indicating their origin from glandular or ductal epithelium, but revealed scattered pan-cytokeratin staining. Transfer of the cytochrome P450 and cytosine deaminase suicide gene, respectively, into the pancreatic cancer cell lines using retroviral vector technology revealed high level infectibility of these cell lines and allowed the analysis of the sensitivity of these cells to the chemotherapeutic drugs ifosfamide and 5-fluorocytosine, respectively.ConclusionThese data qualify the cell lines as part of valuable in vitro and in vivo models for the use in defined preclinical studies for pancreas tumor therapy.

Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer

AbstractCancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti–human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, < 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

Translational assessment of the efficacy of CPI-613 against pancreatic cancer in animal models versus patients with stage IV disease.

3075 Background: CPI-613 is a novel agent that selectively targets the altered mitochondrial enzymes of tumor cells, causing apoptosis, necrosis, and autophagia. Results assessing clinical efficacy of CPI-613 translated from animal tumor xenograft models to patients with stage IV pancreatic cancer are presented. Methods: Efficacy of CPI-613 was tested in mice with pancreatic tumor xenografts generated by inoculation of BxPC-3 human pancreatic tumor cells. The safety and efficacy of CPI-613 (70-320 mg/m2), when used in combination with gemcitabine (1,000 mg/m2), was assessed in patients with stage IV pancreatic cancer. Results: In the animal pancreatic tumor xenograft model (n=10/grp), CPI-613 (25 mg/kg, IV, 1x weekly for 4 weeks) suppressed tumor growth by ~100%, when compared to vehicle. The positive control, gemcitabine (50 mg/kg, IV, 1x weekly for 4 weeks), suppressed tumor growth by only ~50%. Median overall survival in tumor-bearing mice treated with CPI-613 was ~240 days, which was significantly longer than those with gemcitabine or vehicle treatments (~65 and ~50 days, respectively). In 6 humans with stage IV pancreatic cancer (Table), the CPI-613+gemcitabine combination was well-tolerated. In those without prior chemotherapy before participating in the clinical trial (first three patients in the table), the CPI-613+gemcitabine combination prolonged survival that correlated with the dose of CPI-613. Conclusions: CPI-613 exhibits efficacy against pancreatic cancer in animal models, which is translational to patients with stage IV disease. [Table: see text]

Abstract LB-239: ROCK1 inhibition reduces tumor collagen content in the KPC model for PDAC

Abstract ROCK1 is a multifunctional member of the AGC (protein kinase A/G/C) kinase family that has been implicated in the modulation of stress-fiber assembly, cell contraction, apoptosis, and in the migration and invasion of multiple cancer cell types. It has been reported that ROCK1 plays an important role in the activation and function of pancreatic stellate cells, including in the formation of desmoplasia in pancreatic ductal adenocarcinoma (PDAC)(Masamune, 2003, Br J Pharmacol, 140:1292). Desmoplasia and its effects are thought to contribute to poor drug delivery in PDAC treatment. We therefore hypothesized that ROCK1 inhibition may reduce desmoplasia and enhance drug efficacy in PDAC treatment. We previously reported that ROCK1 is overexpressed in human pancreatic tumor cells and amplified in a small subset of PDAC patient samples. In this study we report that the ROCK inhibitor, fasudil, reduced collagen mRNA levels by 3.9-fold in cultured pancreatic stellate cells derived from PDAC tissues treated at 100µM. We also observed a 3.0-fold reduction in collagen protein expression in co-cultured tumor and stellate cells at the same concentration of fasudil. Similar reductions were observed in immunofluorescence analysis of co-cultured tumor and stellate cells treated with fasudil. We next sought to determine if ROCK1 inhibition would result in a reduction in collagen expression in the the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mouse model for PDAC. KPC mice were treated orally, b.i.d., with 100mg/kg fasudil and by i.p. with gemcitabine (q3dx4) at 80mg/kg. Tumors in KPC mice treated with the combination of fasudil and gemcitabine showed a marked reduction in overall tumor collagen content when compared to vehicle-treated mice as measured by a pentachrome staining. Histochemical analysis of tumors also showed reductions in the overall proliferation of the pancreatic stellate cell compartment. Ongoing efforts are being made to assess whether this treatment regimen may enhance gemcitabine efficacy in the KPC mice. These results indicate that ROCK1 is a potential therapeutic target in pancreatic cancer, and may provide an effective means to enhance current treatment regimens. 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 LB-239. doi:1538-7445.AM2012-LB-239

Abstract 299: Modulation of the leptin receptor in pancreatic cancer cells mediates tumor growth

Abstract Background: Pancreatic cancer is the fourth leading cause of cancer death with a five year survival rate around 5%, which has not changed in 30 years. Obesity and increased abdominal adipose tissue independently correlate with an increased relative risk for the development of pancreatic cancer. These conditions have been associated with altered levels of adipokines, or adipose secreted cytokines. Circulating serum levels of the adipokine leptin are increasesddramatically in obese patients as well as in high fat diet induced obese mice. Leptin has been shown to induce oncogenic signaling in breast and prostate cancer. We have previously shown an increase in orthotopic pancreatic tumor size in high fat diet induced obese mice compared with regular diet control mice. We hypothesize that leptin signaling mediates pancreatic tumorigenesis. Methods: Leptin receptor status was determined in human as well as murine pancreatic cell lines. Leptin stimulated cell proliferation was determined using a modified BrdU assay. Leptin receptor levels were knocked down in human and murine pancreatic tumor cells using a shRNAmir approach. Leptin receptor shRNA Panc02 knockdown cells were injected orthotopically into the pancreas of C57/Bl6J mice on regular or high fat diet to determine the contribution of leptin to pancreatic tumor growth. Results: We have detected the long form of the leptin receptor in five human and four murine pancreatic cancer cell lines. In vitro administration of leptin stimulated proliferation of Panc1 and CFPAC1 cell lines, which was abrogated with co-incubation of a leptin antagonist. To better understand the mechanism of leptin-mediated signaling, we studied downstream targets and identified a significant increase in phosphorylation of STAT3 in Panc1, BXPC3 and CFPAC1 cell lines after leptin treatment. Orhtotopic injection of leptin receptor shRNA Panc02 cells into normal and obese mice showed a markedly diminished tumor growth in obese mice when compared to the nonsilencing control Panc02 cell growth in obese mice. Conclusion: These results implicate leptin as a mediator of pancreatic tumorigenesis and suggest that leptin activation is mediated in part through STAT3 signaling. Knockdown of the leptin receptor results in inhibition of high fat diet associated tumor growth in vivo. 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 299. doi:1538-7445.AM2012-299

Abstract 232: Protein kinase C b1 (PKCb1) inhibits the malignant phenotype of the human pancreatic tumor-derived cell line PANC1

Abstract Pancreatic cancer is a deadly disease with a 5% overall survival after five years. Different members of the PKC family have been described as modulators of pancreatic cancer progression but, till nowadays, the relevance of each PKC isoform in this process is unclear. The aim of the present work was to study the effect of PKCb1 overexpression, on several critical determinants of tumor progression using the human pancreatic tumor-derived cell line PANC1. Cells were stably transfected with an expression vector containing the full-length PKCb1 gene (PKCb1-PANC1) or with the empty vector, as control (pMTH-PANC1). While no morphologic alterations could be detected in vitro, PKCb1 overexpression induced a significant reduction in the proliferative capacity of PANC1 cells. The reduction in this capacity could be associated with a decrease in Cyclin D1, Cyclin E and phosphorylated ERK1/2 levels, and an increase in p21 expression levels, as measured by Western blot. In addition PKCb1-PANC1 cells showed an increased resistance to cell death induced by serum starvation (% viability: 58.54±4.1 vs 39.29±4.2 in pMTH-PANC1, p&amp;lt;0.05). Using flow cytometry we could determine that serum deprivation promoted PKCb1-PANC1 arrest in the G1 phase of the cell cycle (cells in G0/G1: 86.7±1.0% vs 45.5±0.8% in pMTH-PANC1, p&amp;lt;0.05). Moreover, the overexpression of PKCb1 reduced the in vitro invasive capacity using Matrigel coated transwells (cells x 10 microscopic x400 fields: 2.22±0.9 vs 10.10±2.6 in pMTH-PANC1, p&amp;lt;0.05). This behavior could be associated with a significant inhibition of secreted proteases activity (uPA: 4.11±0.8 vs 12.32±4.1 IU/mg/24h in pMTH-PANC1, p&amp;lt;0.05 and MMP-9: 15.24±0.03 vs 24.89±2.6 AU/mg in pMTH-PANC1, p&amp;lt;0.05). When control and PANC1-PKCb1 cells were s.c. inoculated into nude mice it was observed that PKCb1 cells were almost unable to grow and form tumors while vector-transfected cells retained this ability. In sum, our results suggest that PKCb1 overexpression in human pancreatic tumor cells PANC1 induced a less malignant phenotype both in vivo and in vitro. 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 232. doi:1538-7445.AM2012-232

Anti-tumour activity of afatinib, an irreversible ErbB family blocker, in human pancreatic tumour cells

Background:The combination of the reversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib with gemcitabine obtained FDA approval for treating patients with pancreatic cancer. However, duration of response is often limited and there is currently no reliable predictive marker.Methods:We determined the sensitivity of a panel of human pancreatic tumour cell lines to treatment with afatinib, erlotinib, monoclonal antibody (mAb) ICR62, and gemcitabine, using the Sulforhodamine B colorimetric assay. The effect of these agents on cell signalling and cell-cycle distribution was determined by western blot and flow cytometry, respectively.Results:At 200 nM, ICR62 had no effect on growth of these tumour cells with the exception of BxPC-3 cells. BxPC-3 cells were also sensitive to treatment with afatinib and erlotinib with respective IC50 values of 11 and 1200 nM. Compared with erlotinib, afatinib was also more effective in inhibiting the growth of the other human pancreatic tumour cell lines and in blocking the EGF-induced phosphorylation of tyrosine, EGFR, MAPK, and AKT. When tested in BxPC-3 xenografts, afatinib induced significant delay in tumour growth.Conclusion:The superiority of afatinib in this study encourages further investigation on the therapeutic potential of afatinib as a single agent or in combination with gemcitabine in pancreatic cancer.

Extracelluar matrix metalloproteinase as a novel target for pancreatic cancer therapy

The objective of this study was to evaluate extracellular matrix metalloproteinase (EMMPRIN) as a novel target in orthotopic pancreatic cancer murine models. MIA PaCa-2 human pancreatic tumor cells were implanted in groups 1 and 3-7, whereas MIA PaCa-2 EMMPRIN knockdown cells were implanted in group 2. Dosing with anti-EMMPRIN antibody started immediately after implantation for groups 1-3 (residual tumor model) and at 21 days after cell implantation for groups 4-7 (established tumor model). Groups 3, 5, and 7 were treated with anti-EMMRPIN antibody (0.2-1.0 mg) twice weekly for 2-3 weeks, whereas the other groups served as the control. In the residual tumor model, tumor growth of anti-EMMPRIN-treated group was successfully arrested for 21 days (15 ± 4 mm(3)), which was significantly lower than that of the EMMPRIN knockdown group (80 ± 15 mm(3); P=0.001) or the control group (240 ± 41 mm(3); P<0.001). In the established tumor model, anti-EMMPRIN therapy lowered tumor volume increase by approximately 40% compared with the control, regardless of the dose amount. Ki67-expressed cell density of group 5 was 939 ± 150 mm(-2), which was significantly lower than that of group 4 (1709 ± 145 mm(-2); P=0.006). Microvessel density of group 5 (30 ± 6 mm(-2)) was also significantly lower than that of group 4 (53 ± 5 mm(-2); P=0.014), whereas the microvessel size of group 5 (191 ± 22 μm(2)) was significantly larger than that of group 4 (113 ± 26 μm(2); P=0.049). These data show the high potential of anti-EMMPRIN therapy for pancreatic cancer and support its clinical translation.

Dickkopf-3 maintains the PANC-1 human pancreatic tumor cells in a dedifferentiated state

Pancreatic cancer (PaCa) is the fourth leading cause of cancer deaths in Western societies, with pancreatic ductal adenocarcinomas (PDACs) accounting for >90% of such cases. PDAC is a heterogeneous disease that includes a subset showing overexpression of the secreted glycoprotein Dickkopf-related protein 3 (Dkk-3), a protein shown to be downregulated in various cancers of different tissues. The biological function of Dkk-3 in this subset was studied using the Dkk-3 expressing PANC-1 cell line as a model for PDACs. The influence of Dkk-3 overexpression and knockdown on cellular differentiation and proliferation of PANC-1 was investigated. Confocal microscopy showed that Dkk-3 was expressed in a fraction of PANC-1 cells. While lentiviral-mediated overexpression of DKK3 did not alter cellular proliferation, knockdown of DKK3 resulted in significant reduction of cellular proliferation and concomitant induction of cell cycle inhibitors CDKN2B (p15INK4b), CDKN1A (p21CIP1) and CDKN1B (p27KIP1). In parallel, pancreatic epithelial cell differentiation markers AMY2A, CELA1, CTRB1, GCG, GLB1 and INS were significantly upregulated. PANC-1 cells differentiated using exendin-4 showed analogous induction of cell cycle inhibitors and differentiation markers. Thus, we conclude that Dkk-3 is required to maintain a highly dedifferentiated and consequently proliferative state in PANC-1, indicating a similar function in the Dkk-3 overexpressing subset of PDACs. Therefore, Dkk-3 represents a potential target for the treatment of Dkk-3-positive subtypes of PaCa to drive cells into cell cycle arrest and differentiation.

Increased expression of the large GTPase dynamin 2 potentiates metastatic migration and invasion of pancreatic ductal carcinoma

Pancreatic ductal tumors invade local parenchyma and metastasize to distant organs. Src-mediated tyrosine kinase signaling pathways promote pancreatic ductal adenocarcinoma (PDAC) metastasis, though the molecular mechanisms supporting this invasive process are poorly understood and represent important and novel therapeutic targets. The large GTPase Dynamin2 (Dyn2), a Src-kinase substrate, regulates membrane-cytoskeletal dynamics although it is yet to be defined if this mechanoenzyme contributes to tumor cell migration and invasion. Therefore the goal of this study was to test if Dyn2 is upregulated in human pancreatic tumors and to define its role in cell migration and metastatic invasion using in vitro assays and nude mouse models. Histological analysis showed that 81% of the 85 patients tested had elevated Dyn2 in PDAC tissue. To test if Dyn2 overexpression alters metastatic properties of human pancreatic tumor cells, stable clones of BxPC-3 cells overexpressing either wild-type Dyn2 or a phosphorylation-deficient mutant Dyn2Y(231/597)F known to attenuate Dyn2 function, were generated and analyzed for migratory capacity. Importantly, tumor cells expressing 2-3 fold levels of Dyn2 protruded lamellipodia at twice the rate, migrated faster (180%) and farther (2.5-fold greater net distance) on glass and through transwell chambers (2-3 fold more cells through the filter) compared to cells expressing Dyn2Y(231/597)F or vector alone. Further, siRNA-mediated depletion of Dyn2 and dynamin inhibitors MiTMAB and Dynasore significantly reduced cell migration (>66%), wound healing (>75%) and invasion in transwell assays (>95%) compared to DMSO treated cells. To test the metastatic potential conferred by increased Dyn2 expression, the BxPC-3 clonal cell lines were implanted orthotopically into the pancreas of nude mice. Cells expressing Dyn2-GFP exhibited a 3-fold increase in large distal tumors compared to cells expressing Dyn2Y(231/597)F or vector alone. Finally, histological analysis of pancreatic metastases from human patients revealed that Dyn2 is upregulated in 60% of metastatic tumors examined. These findings are the first to implicate dynamin in any neoplastic condition and to directly demonstrate a role for this mechanoenzyme in invasive cell migration.

In vitro cytotoxic activity of abietane diterpenes from Peltodon longipes as well as Salvia miltiorrhiza and Salvia sahendica

Phytochemical investigations of the n-hexane extract from the roots of Peltodon longipes (Lamiaceae) resulted in the isolation of 12 known abietane diterpenes (1–12). Structures were established on the basis of one and two dimensional nuclear magnetic resonance spectroscopic data (1H and 13C, COSY, HSQC and HMBC), electron ionization mass spectrometric analysis (EIMS) as well as comparison with data from literature. These compounds, as well as eight known diterpenes (13–19) from Salvia miltiorrhiza, and two from Salvia sahendica (20 and 21) were evaluated for their cytotoxic effects in human pancreatic (MIAPaCa-2) and melanoma (MV-3) tumor cell lines using the MTT assay. Tanshinone IIa (13), 7α-acetoxyroyleanone (1), 1,2-dihydrotanshinone (16) and cryptotanshinone (14) had the highest cytotoxic effects in MIAPaCa-2, displaying IC50 of 1.9, 4.7, 5.6, and 5.8μM, respectively. Structure–activity relationships of abietane diterpenoid quinones are discussed.

A phase I study of NPC-1C, a novel therapeutic antibody to treat pancreas and colorectal cancers.

TPS141 Background: NPC-1C (N: ensituximab) is a chimeric monoclonal antibody being developed as a novel agent for pancreatic and colorectal cancers. This antibody was selected from a panel of hybridomas generated from mice immunized with semi-purified membrane-associated proteins derived from biologically screened, pooled human allogeneic colon cancer tissues. N recognizes a unique immunogenic epitope with homology to a region of MUC5AC. This epitope is expressed by colon and pancreatic tumor tissues and cell lines. N does not cross-react with normal tissues (or MUC5AC from other types of cancers), except for occasional, weak binding to certain GI tract tissues. The N antibody shows ADCC activity against human colon and pancreatic tumor cells, but not control tumor cell lines. Human pancreatic tumor xenograft models showed significant, and reproducible, anti-tumor action, including some complete tumor regressions. Several examples of human tumor tissues stained with N showed a strong correlation of N staining against pancreatic and colon tumors (45% of tumors strongly positive). Methods: A Phase I open label, multi-center dose escalation clinical trial with N is currently accruing patients with advanced, refractory pancreatic and colorectal cancer. A standard 3+3 design with an expansion cohort of 10 pancreatic cancer patients is planned. Patients will be treated with N starting at 1 mg/kg IV q2wks; cohort 1 has been completed without DLT and cohort 2 is being accrued; patients are hospitalized for 48 hours for observation after the first dose of N. Eligibility criteria included good end organ function and performance status, >20% of tumor tissue staining positive for MUC5AC, and an exclusion for QTc prolongation or history of hemolytic episodes. The primary objectives of the trial are to determine the safety/tolerability of escalating doses of N therapy and to assess pharmacokinetics and immune responses to the antibody at each dose level. Secondary objectives are to evaluate evidence of clinical benefit, as measured by RECIST criteria, and to explore the immunologic correlates associated with administration of N therapy. Results from this trial will determine the dosage levels to be used in further trials. NCT01040000.

Development of a new stool biomarker ELISA for the early detection of colorectal cancer.

e14128 Background: An ELISA was developed using NPC-1C, a novel antibody that reacts with an antigen expressed specifically by human colorectal and pancreatic tumor tissues and cell lines. The target antigen that NPC-1C recognizes was shown to be related to MUC5AC, a member of the mucin family of glycoproteins. NPC-1C reacts with neither normal tissues from healthy donors (rare, weak binding to normal colon and esophagus), nor cell lines derived from other tumor types, thereby providing the basis for a proof of concept comparative evaluation with stool from normal healthy donors. Methods: An ELISA that uses NPC-1C antibody as both capture and detection reagent was developed. Samples of stool collected during colonoscopy from colorectal cancer patients (n=4), stool from people with small polyps (n=4), stool from people with multiple polyps (n=2), stool from people with large polyps (n=3), and stool from healthy adults (n=13) were applied to the ELISA. A soluble extract of stool was prepared by detergent lysis and centrifugation. The level of NPC-1C-specific MUC5AC antigen measured in this ELISA was compared among all groups. Results: Preliminary results demonstrated that healthy people did not express NPC-1C antigen in their stool. The signal in the assay was similar to background levels (average 723 units). In contrast, people with small polyps had higher levels (average 3,819 units); people with multiple polyps expressed higher levels (average 7,369 units); people with large polyps had even higher levels (average 10,189 units); and colon cancer patients had the highest levels (average 175,983 units), more than 240 times the level of MUC5AC-related antigen compared with healthy people. Conclusions: The proof of concept has been established to correlate the level of NPC-1C reactive antigen, measured by a novel stool-based ELISA, with colon cancer disease progression. The level of NPC-1C-specific MUC5AC detected increased concomitantly with the number and size of polyps observed during colonoscopy, and reached the highest levels in patients with colon cancer. Our goal is to apply this ELISA test for early non-invasive diagnostic screening for colorectal cancer, an area of still largely unmet need.

Retinoic Acid-Induced Stellate Cell Quiescence Results in Sfrp4 Secretion Which Abrogates Wnt-β-Catenin Signalling Pathway Modulating Human Pancreatic Tumour Cell Behaviour

Retinoic Acid-Induced Stellate Cell Quiescence Results in Sfrp4 Secretion Which Abrogates Wnt-β-Catenin Signalling Pathway Modulating Human Pancreatic Tumour Cell Behaviour

Abstract 4582: Characterization of a new antibody that reacts with a tumor-specific variant of CEACAM-5 and CEACAM-6

Abstract Introduction: h16C3 is a humanized monoclonal antibody being developed as a novel biological treatment for several types of solid tumor. By mass spectroscopy identification and target validation research, we determined that h16C3 appears to recognize a variant form of CEACAM-5 and CEACAM-6, molecules with a well-known association to cancer. Here we show the pre-clinical activities of this new mAb which shows great potential as a therapeutic agent to treat solid tumors. Methods: The 16C3 antibody was selected from hundreds of hybridoma clones generated from mice immunized with semi-purified membrane-associated proteins derived from biologically screened, pooled human allogeneic colon cancer tissues. In vitro assays and in vivo studies were performed to characterize the potential of h16C3 as a therapeutic modality for solid tumors that express the variant CEACAM antigens. Results: Here we show that unlike most modalities targeting the CEACAM molecules, h16C3 reacts with variant CEACAM-5/6 expressed specifically by human tumor tissues as follows: 98% of human colorectal tumor tissues, 80% of pancreatic tumor tissues, 76% of lung tumor tissues, 43% of uterus tumor tissues, and 29% of breast tumor tissues stained positively with h16C3. Importantly, h16C3 did not cross-react significantly with normal human tissues, except for occasional, weak binding to certain GI tract tissues, which may indicate a pre-malignant state. We showed further that the h16C3 antibody exhibits cell-specific binding and ADCC activity against human colorectal and pancreatic tumor cells, but not against antigen-negative control tumor cell lines. In vivo, the anti-tumor efficacy of h16C3 was tested using pre-established subcutaneous human tumor xenograft models. The data showed significant anti-tumor action compared to control treated mice, including some complete tumor regressions. The bio-distribution of radiolabeled h16C3 in mice with pre-established human tumors exhibited specific accumulation of h16C3 at the tumor site, with little or no binding or accumulation in several major organ systems. Conclusions: The pre-clinical studies of h16C3 to date indicate that it has significant, specific anti-tumor potency, and suggest that it may have clinical activity in cancer patients whose tumor expresses the variant CEACAM-5/6 epitope. The therapeutic humanized 16C3 antibody is being developed for testing in a Phase I clinical trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4582. doi:10.1158/1538-7445.AM2011-4582

Abstract 4584: Pre-clinical studies of a novel antibody to treat pancreatic and colorectal cancers

Abstract Introduction: NPC-1C is a chimeric monoclonal antibody being developed as a novel biological treatment for pancreatic and colorectal cancers. NPC-1C appears to recognize a variant form of MUC5AC expressed specifically by human pancreatic and colorectal tumor tissues and cell lines. Here we show pre-clinical studies in preparation for testing this therapeutic for a Phase I clinical trial. Methods: The NPC-1C antibody was selected from a panel of hybridomas generated from mice immunized with semi-purified membrane-associated proteins derived from biologically screened, pooled human allogeneic colon cancer tissues. In vitro assays and in vivo studies were performed to characterize the GMP-grade antibody. Results: Here we show that NPC-1C reacts with colorectal and pancreatic tumor tissues, but does not cross-react with normal human tissues, except for occasional, weak binding to certain GI tract tissues, which may indicate a pre-malignant state. The NPC-1C antibody exhibits cell-specific binding and ADCC activity against human colorectal and pancreatic tumor cells, but not against control tumor cell lines. In vivo, the anti-tumor efficacy of NPC-1C was tested using pre-established subcutaneous human tumor xenograft models. The data showed significant, and reproducible, anti-tumor action, including some complete tumor regressions. The pre-clinical toxicity profile of NPC-1C in normal BALB/c mice demonstrated little, if any, toxicity following single or multiple intravenous injections. The bio-distribution of radiolabeled NPC-1C in mice with pre-established human tumors revealed specific accumulation of NPC-1C at the tumor site, with little or no binding or accumulation in several major organ systems. Finally, the cytokine and antibody responses to NPC-1C were studied in the pre-clinical mouse model. Although anti-NPC-1C antibodies were detected (as expected) in a time-dependent fashion, there were no changes detect in Type I and Type II cytokines either 3 days or 14 days after intravenous administration. Conclusions: The pre-clinical development of NPC-1C indicates that it is safe and efficacious, and suggests that it may have clinical activity in patients whose tumor expresses the variant MUC5AC epitope. The potential clinical application for this antibody was bolstered by showing approximately 50-70% of human pancreatic and colon tumor tissues stained positively with NPC-1C. The therapeutic antibody is now being tested in a Phase I clinical trial in patients with advanced pancreatic and colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4584. doi:10.1158/1538-7445.AM2011-4584