Sylvia S.W. Ng

Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) overexpression in pancreatic ductal adenocarcinoma correlates with poor survival

BackgroundPancreatic ductal adenocarcinoma is a lethal disease with a 5-year survival rate of 4% and typically presents in an advanced stage. In this setting, prognostic markers identifying the more agrressive tumors could aid in managment decisions. Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3, also known as IMP3 or KOC) is an oncofetal RNA-binding protein that regulates targets such as insulin-like growth factor-2 (IGF-2) and ACTB (beta-actin).MethodsWe evaluated the expression of IGF2BP3 by immunohistochemistry using a tissue microarray of 127 pancreatic ductal adenocarcinomas with tumor grade 1, 2 and 3 according to WHO criteria, and the prognostic value of IGF2BP3 expression.ResultsIGF2BP3 was found to be selectively overexpressed in pancreatic ductal adenocarcinoma tissues but not in benign pancreatic tissues. Nine (38%) patient samples of tumor grade 1 (n = 24) and 27 (44%) of tumor grade 2 (n = 61) showed expression of IGF2BP3. The highest rate of expression was seen in poorly differentiated specimen (grade 3, n = 42) with 26 (62%) positive samples. Overall survival was found to be significantly shorter in patients with IGF2BP3 expressing tumors (P = 0.024; RR 2.3, 95% CI 1.2-4.8).ConclusionsOur data suggest that IGF2BP3 overexpression identifies a subset of pancreatic ductal adenocarcinomas with an extremely poor outcome and supports the rationale for developing therapies to target the IGF pathway in this cancer.

The Autophagy Inhibitor Verteporfin Moderately Enhances the Antitumor Activity of Gemcitabine in a Pancreatic Ductal Adenocarcinoma Model

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to chemotherapy. It has been described as requiring elevated autophagy for growth and inhibiting autophagy has been proposed as a treatment strategy. To date, all preclinical reports and clinical trials investigating pharmacological inhibition of autophagy have used chloroquine or hydroxychloroquine, which interfere with lysosomal function and block autophagy at a late stage. Verteporfin is a newly discovered autophagy inhibitor that blocks autophagy at an early stage by inhibiting autophagosome formation. Here we report that PDAC cell lines show variable sensitivity to verteporfin in vitro, suggesting cell-line specific autophagy dependence. Using image-based and molecular analyses, we show that verteporfin inhibits autophagy stimulated by gemcitabine, the current standard treatment for PDAC. Pharmacokinetic and efficacy studies in a BxPC-3 xenograft mouse model demonstrated that verteporfin accumulated in tumors at autophagy-inhibiting levels and inhibited autophagy in vivo, but did not reduce tumor volume or increase survival as a single agent. In combination with gemcitabine verteporfin moderately reduced tumor growth and enhanced survival compared to gemcitabine alone. While our results do not uphold the premise that autophagy inhibition might be widely effective against PDAC as a single-modality treatment, they do support autophagy inhibition as an approach to sensitize PDAC to gemcitabine.

Irinophore C™, a lipid nanoparticulate formulation of irinotecan, improves vascular function, increases the delivery of sequentially administered 5-FU in HT-29 tumors, and controls tumor growth in patient derived xenografts of colon cancer

PURPOSE A liposomal formulation of irinotecan, Irinophore C™ (IrC™) is efficacious in a panel of tumor models, normalizes tumor vasculature, and increases the accumulation of a second drug in the same tumor. We now show that Irinophore C™ is also effective against patient derived xenografts (PDX) of colon cancer, and examine the kinetics of vascular normalization in the HT-29 tumor model and assess how these changes might be used with 5-FU sequentially. MATERIALS AND METHODS Rag2M mice bearing HT-29 tumors were treated with IrC™ (25mg/kg; Q7D×3) for up to three weeks. Groups of tumors were harvested for analysis at 7, 14 and 21days after the start of treatment. Drug and lipid levels in the tumor were evaluated using HPLC and scintillation counts, respectively. Changes in tumor morphology (H&E), vasculature (CD31), perfusion (Hoechst 33342) and apoptosis (TUNEL) were quantified using microscopy. The accumulation of a second drug ([(14)C]-5-FU, 40mg/kg) given 3h before sacrifice was determined using liquid scintillation. The efficacy of IrC™ (Q7D×3) followed by 5-FU treatment (Q7D×3) was assessed in mice bearing established HT-29 tumors. The efficacy of IrC™ was also evaluated in primary human colorectal tumors grown orthotopically in NOD-SCID mice. RESULTS Following a single dose of IrC™ the active lactone forms of irinotecan and its metabolite SN-38 were measurable in HT-29 tumors after 7days. The treatment reduced tumor cell density and increased apoptosis. Hoechst 33342 perfusion and accumulation of [(14)C]-5-FU in the treated tumors increased significantly on days 7 and 14. This was accompanied by an increase in the number of endothelial cells relative to total nuclei in the tumor sections. Pre-treatment with IrC™ (Q7D×3) followed by 5-FU (Q7D×3) delayed the time taken for tumors to reach 1cm(3) by 9days (p<0.05). IrC™ was just as effective as free irinotecan when used on patient derived xenografts of colorectal cancer. CONCLUSIONS Treatment with IrC™ reduces tumor cell viability and appears to normalize the vascular function of the tumor after a single treatment cycle. A concomitant increase in the accumulation of a second drug (5-FU) in the tumor was observed in tumors from IrC™ treated animals and this was correlated with changes in vascular structure consistent with normalization. The treatment effects of sequential 5-FU dosing following IrC™ are additive with no additional toxicity in contrast to previous studies where concurrent 5-FU and IrC™ treatment exacerbated 5-FU toxicity. The studies with PDX tumors also indicate that IrC™ is just as effective as free irinotecan on PDX tumors even though the delivered dose is halved.

Evaluation of 111In labeled antibodies for SPECT imaging of mesothelin expressing tumors

INTRODUCTION Mesothelin is expressed in many cancers, especially in mesothelioma and lung, pancreatic and ovarian cancers. In the present study, we evaluate (111)In labeled antimesothelin antibodies as an imaging bioprobe for the SPECT imaging of mesothelin-expressing tumors. METHODS We radiolabeled the antimesothelin antibodies mAbMB and mAbK1 with (111)In using the p-SCN-bn-DTPA chelator. The immunoreactivity, affinity (K(d)) and internalization properties of the resulting two (111)In labeled antibodies were evaluated in vitro using mesothelin-expressing A431K5 cells. The biodistribution and microSPECT/CT imaging studies with (111)In labeled antibodies were performed in mice bearing both mesothelin positive (A431K5) and mesothelin negative (A431) tumors. RESULTS In vitro studies demonstrated that (111)In-mAbMB bound with a higher affinity (K(d)=3.6±1.7 nM) to the mesothelin-expressing A431K5 cells than did the (111)In-mAbK1 (K(d)=29.3±2.3 nM). (111)In-mAbMB was also internalized at a greater rate and extent into the A431K5 cells than was the (111)In-mAbK1. Biodistribution studies showed that (111)In-mAbMB was preferentially localized in A431K5 tumors when compared to A431 tumors. At the low dose, the peak A431K5 tumor uptake of 9.65±2.65% ID/g (injected dose per gram) occurred at 48 h, while at high dose tumor uptake peaked with 14.29±6.18% ID/g at 72 h. Non-specific localization of (111)In-mAbMB was mainly observed in spleen.(111)In-mAbK1 also showed superior localization in A431K5 tumors than in A431 tumors, but the peak uptake was only 3.04±0.68% ID/g at 24 h. MicroSPECT/CT studies confirmed better visualization of A431K5 tumors with (111)In-mAbMB, than with (111)In-mAbK1. CONCLUSION SPECT imaging of mesothelin expressing tumors was demonstrated successfully. Our findings indicate that the antimesothelin antibody mAbMB has the potential to be developed into a diagnostic agent for imaging mesothelin-expressing cancers.

B7-H4 expression in normal and diseased human islet β cells.

Objectives B7-H4 is a negative coregulatory molecule known to be involved in immune response. We study here B7-H4 expression and its possible role in diabetes and cancer development. Methods Formalin-fixed, paraffin-processed pancreas samples from patients with type 1 diabetes (T1D), insulinoma, pancreatic ductal adenocarcinoma (PDAC), and normal organ donors were studied by bright-field and multifluorescence immunohistochemistry to examine B7-H4 expression and its colocalization with islet endocrine hormones. Quantitative RT-PCR and Western blot assay were used to examine B7-H4 mRNA and protein expression in the islet and exocrine tissues from normal donors and pancreatic cancer cell lines. Results B7-H4 protein expression in islet &bgr; cells is decreased in T1D and PDAC, but increased in insulinoma patients when compared to normal controls; the changes in B7-H4 expression are concomitant with insulin expression on the islet &bgr; cells. The insulin/B7-H4 colocalization on the &bgr; cells, expressed in colocalization coefficient Pearson r, is also changed in these islets. Conclusions Our observation of altered B7-H4 expression, concomitant with insulin expression, in the pancreatic islets of T1D, PDAC, and insulinoma patients when compared to normal controls suggests that B7-H4 pathway might play an important role in maintenance of &bgr;-cell function, but its exact role remains to be explored.

Zaprinast, a type V phosphodiesterase inhibitor, dilates capacitance vessels in anaesthetised rats

The effects of zaprinast (a type V phosphodiesterase inhibitor) on mean arterial pressure, heart rate, cardiac output, mean circulatory filling pressure, arterial and venous resistances were compared to those of sodium nitroprusside in three groups, each of intact or ganglion-blocked, Inactin-anaesthetised rats. In intact rats, zaprinast (1.5, 3.0 mg kg(-1) min(-1)) and sodium nitroprusside (8.0, 64.0,microg kg(-1) min(-1)) dose-dependently reduced mean arterial pressure and arterial resistance, but did not alter cardiac output and venous resistance. Both increased heart rate, with the effect of zaprinast less than that of sodium nitroprusside. Mean circulatory filling pressure was elevated by both doses of zaprinast but only the high dose of sodium nitroprusside. In rats given mecamylamine (3.7 micromol kg(-1), i.v. bolus) and noradrenaline (7.3 nmol kg(-l) min(-1)), zaprinast and sodium nitroprusside elicited dose-dependent reductions in mean arterial pressure, arterial and venous resistances, and mean circulatory filling pressure. Both increased cardiac output, with the effect of zaprinast greater than that of sodium nitroprusside at the low dose. Zaprinast but not sodium nitroprusside reduced heart rate. Our results indicate that zaprinast, similar to sodium nitroprusside, dilates both resistance and capacitance vessels in ganglion-blocked rats infused with noradrenaline to restore vasomotor tone. Zaprinast but not sodium nitroprusside has a direct, negative chronotropic effect on the heart.

The involvement of insulin-like growth factor 2 binding protein 3 (IMP3) in pancreatic cancer cell migration, invasion, and adhesion

BackgroundOver-expression of insulin-like growth factor 2 mRNA binding protein 3 (IMP3) is correlated with poor prognosis in pancreatic ductal adenocarcinoma (PDAC). Previous studies examining other cancer types have implicated IMP3 in the regulation of several cellular functions that are characteristic of tumour cells. However, the role of this oncofetal protein in PDAC progression remained unclear.MethodsUsing siRNA, we examined the effect of IMP3 inhibition on the motility, invasive ability, and matrix adhesion of PDAC cells. In addition, we also evaluated the expression of cytoskeleton-associated genes following IMP depletion.ResultsKnockdown of IMP3 significantly decreased the motility, invasion, and extracellular matrix adhesion of select PDAC cells in vitro. In addition, IMP3-depleted cells exhibited lower levels of CD44 protein and KIF11 mRNA. Moreover, we also observed a reduction in downstream RhoA signaling following IMP3 knockdown, indicating that IMP3 modulates the levels of proteins involved in cytoskeletal organization.ConclusionsThese results suggest that IMP3 facilitates PDAC progression by enhancing the pro-metastatic behaviour of tumour cells.

Abstract 5261: Irinophore C, a liposomal formulation of irinotecan, has anti-vascular effects in primary tumors of colorectal cancer grown orthotopically in mice

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Colorectal cancer accounts for ∼10% of cancer deaths in North America. Our group has developed a series of primary tumors from human colorectal cancer tissue obtained during surgery. These tumors are passaged orthotopically in mice and maintain the complexity and heterogeneity of the original patient sample. We have used these tumors to examine the cytotoxic and anti-vascular effects of Irinophore CTM, a liposomal form of irinotecan, which is more efficacious and less toxic than the parent drug. Materials and Methods: Primary tumor tissues from colorectal cancer patients, were validated by a reference pathologist and implanted subcutaneously in SCID mice. Tumors that grew successfully were then passaged orthotopically on the ascending colon of new mice. When these tumors reached ∼200mm3, groups of mice were treated with saline, irinotecan (50mg/kg), or IrinophoreCTM (25mg/kg) once a week for 6 weeks. Separate groups of tumors, A, B and C were harvested on days 3, 21 and 42 after treatment started, respectively. Magnetic resonance imaging (MRI) was used to assess tumor perfusion in mice from group B. Treatment effects on tumor metabolism were assessed with 18F -fluorodeoxyglucose and positron emission tomography (FDG-PET) for groups A and C mice. Immunofluorescence staining was carried out on tumors from all treatment groups to determine levels of cell proliferation, apoptosis, hypoxia, and vessel density. Results: 4 of 14 samples were successfully propagated and maintain their original morphology. Irinophore CTM treatment reduced tumor volume by 54% to 92% compared to the untreated controls depending on the tumor line. No toxic effects were seen with Irinophore CTM. The aggregate data for cell proliferation (Ki67), necrosis (HE however, Irinophore CTM treatment did reduce vascular density in the tumors. The volume transfer coefficient, Ktrans, derived from MRI, decreased when tumors were treated with irinotecan, but increased with Irinophore CTM treatment. Differences in the metabolic activity of the tumors were also seen. Conclusion: Orthotopic models of colorectal cancer propagated from patient tumors were successfully developed. These models retain the characteristics of the original patient sample and are a good alternative to xenograft models grown from immortalized cell-lines. The anti-tumor activity of Irinophore CTM at lower doses is greater than irinotecans, and with fewer side effects. Treatment with Irinophore CTM also reduces tumor metabolism and appears to improve vascular function. The data imply that Irinophore CTM has sustained anti-tumor activity and multiple mechanisms of action compared to irinotecan. 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 5261. doi:1538-7445.AM2012-5261

Abstract B12: The effects of Irinophore C™ on the microenvironment and vasculature of a primary orthotopic model of colorectal cancer.

Introduction: Colorectal cancer is a common cancer that accounts for ∼10% of cancer cell deaths in North America. There are numerous in vivo xenograft models for colorectal cancer available, but in general, these are derived from immortalized cell lines and fail to capture the complexities and heterogeneity of tumors seen in the clinic. Our group has developed a series of orthotopic, primary tumors from samples of human colorectal cancer tissue obtained during surgical resection that maintain the characteristics of the original sample. The effects of Irinophore C™, (a liposomal formulation of irinotecan that is more efficacious and less toxic than the parent drug) on the microenvironment and vascular function of these primary colorectal tumors were examined in the present study. Materials and Methods: Primary tumor tissues, obtained from colorectal cancer patients, were validated by a reference pathologist and implanted subcutaneously in SCID mice. Small pieces of subcutaneous tumors that grew successfully were then passaged orthotopically on the ascending colon of new mice. When these tumors reached ∼200mm3, groups of mice were treated with vehicle control (0.9% saline), irinotecan (50mg/kg), or Irinophore C™ (25mg/kg) once a week for 6 weeks. Separate groups of tumors were harvested on days 3 and 42 after treatment started. Immunofluorescence staining was performed on tumor cryosections followed by computerized image analysis to determine levels of cell proliferation, apoptosis, hypoxia, and vessel density. Results: 4 of 14 samples were successfully propagated orthotopically and were characterized by a reference pathologist. The tumors maintain their original morphology, and one is a highly mucinous adenocarcinoma whereas the others are typical colorectal adenocarcinomas. Treatment with irinotecan and Irinophore C™ reduced tumor volumes by 54% and 92%, respectively, compared to the vehicle control. No toxic effects were seen with Irinophore C™. Immunostaining data showed that the distance between blood vessels remained the same for Irinotecan when compared to control, but increased with Irinophore C™ treatment (+30%; P Conclusion: An orthotopic model of colorectal cancer was successfully developed using patient tumor materials that retained the morphology of the primary tumor. Irinophore C™ was more effective in controlling tumor growth than irinotecan despite being delivered at a lower dose. In addition, treatment with Irinophore C™ appeared to improve overall vascular function in the tumor. Based on these data, it is clear that Irinophore C™ has different mechanisms of action and is more efficacious than irinotecan against primary models of colorectal 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 B12.

Abstract C235: Interleukin-6 secreted by cancer-associated fibroblasts mediates gemcitabine resistance in pancreatic cancer.

Introduction: Pancreatic ductal adenocarcinoma is characterized by a dense stroma containing an abundance of cancer-associated fibroblasts (CAFs). It is also notably resistant to gemcitabine. We hypothesize that the large population of CAFs in pancreatic tumors secrete elevated levels of multiple soluble factors, which promote survival of pancreatic cancer cells and reduce their sensitivity to gemcitabine. Previously, we found that growth of MiaPaCa-2 and BxPC-3 tumors was suppressed with gemcitabine treatment, but that of tumors from MiaPaCa-2 and BxPC-3 co-injected with primary pancreatic CAFs was not. This suggests that the presence of CAFs causes pancreatic cancer cells to become more resistant to gemcitabine. Subsequent Affymetrix analyses showed that interleukin-6 (IL-6) is one of 382 genes that are differentially upregulated by >2-fold (P Methods: Sixteen primary CAF lines were established in culture from resected pancreatic ductal adenocarcinoma tumor tissues. As well, eleven primary orthotopic tumor xenograft lines were established in SCID mice from the resected tissues. Subcutaneous tumors were also grown by injecting 5×10 6 MiaPaCa-2, BxPC-3, or Panc-1 pancreatic cancer cells into SCID mice. Using ELISA, the levels of IL-6, soluble IL-6 receptor (sIL-6R) and the soluble form of gp130 were evaluated in the primary CAFs and orthotopic tumor xenografts, as well as in several pancreatic cancer cell lines. Levels of mouse IL-6 in the serum of mice bearing orthotopic tumors and subcutaneous cell line xenografts were also assayed. Results: The levels of human IL-6 protein were 1.1- to 79-fold higher in the CAF lines than in normal human fibroblasts. However, human IL-6 protein was not detected in pancreatic cancer cell lines PK8, MiaPaCa-2, Panc-1, Capan-2, BxPC-3, or in normal human pancreatic ductal epithelial cells (HPDEs). The levels of mouse IL-6 protein were 1.2- to 94-fold higher in the serum of mice bearing orthotopic tumors as compared to the serum of normal healthy mice. Interestingly, no mouse IL-6 protein was detected in the serum of mice bearing subcutaneous cancer cell line tumors. Furthermore, high levels of mouse IL-6 (12–100 pg/mg) but not human IL-6 were evident in the lysates of the orthotopic tumors. Human sIL-6Rprotein levels were found to be high (24–65 pg/mg) in pancreatic cancer cell lines, but present only at low levels (∼12 pg/mg) in four of the sixteen CAF lines. It was also detected at 13–66 pg/mg in all of the orthotopic tumors. Soluble gp130 protein was observed at higher levels in the CAFs than in the pancreatic cancer cell lines. It was also detected in all of the orthotopic tumors. Conclusion: Our results demonstrate that CAFs are the major producers of IL-6 in pancreatic tumors. It is likely that IL-6 secreted by CAFs binds to IL-6 receptors on pancreatic cancer cells, resulting in the activation of downstream cell survival proteins. Co-culture experiments are underway to identify these proteins and to determine if inhibition of IL-6 signaling may increase the sensitivity of pancreatic cancer cells to gemcitabine. 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 C235.