Todd W. Bauer

Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells.

Vascular endothelial growth factor (VEGF) is associated with tumor angiogenesis and poor prognosis in human colorectal cancer (CRC). VEGF receptor-1 (VEGFR-1 or Flt-1) is a high-affinity receptor for VEGF and is typically considered specific to endothelial cells. Here we report the expression and function of VEGFR-1 in CRC cell lines. VEGFR-1 was expressed in all CRC cell lines studied as determined by RT–PCR, Western blot analysis, FACS, and ELISA. Treatment of the human CRC cell lines HT-29 and SW480 with VEGF-A (a ligand for both VEGFR-1 and -2) or VEGF-B (a ligand specific for VEGFR-1) led to activation of Erk-1/2, SAPK/JNK, and translocation of the p65 subunit of nuclear factor-κB into the nucleus. Both VEGF-A and -B led to significant induction of cell motility and invasiveness of CRC cells. Stimulation of cells with VEGF-A or -B also led to larger and more numerous colonies in soft agar. However, activation of VEGFR-1 did not increase CRC cell proliferation. In contrast to the previous paradigm that VEGFRs are not present on tumor cells of epithelial origin, we found that VEGFR-1 is present and functional on CRC cells, and activation by VEGF family ligands can activate processes involved in tumor progression and metastasis.

Vascular endothelial growth factor receptor-1 activation mediates epithelial to mesenchymal transition in human pancreatic carcinoma cells

Our laboratory has shown that vascular endothelial growth factor receptor-1 (VEGFR-1) expression on human pancreatic cancer cell lines mediates cell migration and invasion. Because epithelial to mesenchymal transition (EMT) also plays a role in cell motility by altering the cell phenotype and morphology, we hypothesized that VEGFR-1 activation induces molecular alterations that mediate EMT. Our treatment of the human pancreatic cancer cell line L3.6pl with the VEGFR-1 ligands VEGF-A and VEGF-B led to morphologic changes characteristic of EMT, including loss of polarity, increased intercellular separation, and the presence of pseudopodia. Immunofluorescent staining with antibodies to E-cadherin and beta-catenin showed that VEGFR-1 activation led to translocation of E-cadherin and beta-catenin from their usual cell membrane-bound location to the cytoplasm and nucleus, respectively. Western blotting showed that VEGFR-1 activation led to decreased expression of the epithelial markers E-cadherin and plakoglobin, increased expression of the mesenchymal markers vimentin and N-cadherin, and increased nuclear expression of beta-catenin. Pretreatment of tumor cells with a VEGFR-1 blocking antibody inhibited the VEGFR-1-induced immunohistochemical and molecular changes in E-cadherin. VEGFR-1 activation led to an increase in expression of the EMT-associated transcription factors Snail, Twist, and Slug. The changes mediated by VEGFR-1 in this pancreatic carcinoma cell line are highly consistent with the changes characteristic of EMT. Given our previous finding of VEGFR-1-mediated tumor cell invasion and migration in pancreatic carcinoma cells, we hypothesize that VEGFR-1 plays a role in tumor progression in pancreatic cancer through the induction of EMT.

Targeting of urokinase plasminogen activator receptor in human pancreatic carcinoma cells inhibits c-Met- and insulin-like growth factor-I receptor-mediated migration and invasion and orthotopic tumor growth in mice

Pancreatic carcinomas express high levels of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), both of which mediate cell migration and invasion. We investigated the hypotheses that (a) insulin-like growth factor-I (IGF-I)- and hepatocyte growth factor (HGF)-mediated migration and invasion of human pancreatic carcinoma cells require uPA and uPAR function and (b) inhibition of uPAR inhibits tumor growth, retroperitoneal invasion, and hepatic metastasis of human pancreatic carcinomas in mice. Using transwell assays, we investigated the effect of IGF-I and HGF on L3.6pl migration and invasion. We measured the induction of uPA and uPAR following treatment of cells with IGF-I and HGF using immunoprecipitation and Western blot analysis. The importance of uPA and uPAR on L3.6pl cell migration and invasion was studied by inhibiting their activities with amiloride and antibodies before cytokine treatment. In an orthotopic mouse model of human pancreatic carcinoma, we evaluated the effect of anti-uPAR monoclonal antibodies with and without gemcitabine on primary tumor growth, retroperitoneal invasion, and hepatic metastasis. IGF-I and HGF mediated cell migration and invasion in L3.6pl cells. In addition, IGF-I and HGF induced uPA and uPAR expression in L3.6pl cells. In vitro, blockade of uPA and uPAR activity inhibited IGF-I- and HGF-mediated cell migration and invasion. Treatment of mice with anti-uPAR monoclonal antibody significantly decreased pancreatic tumor growth and hepatic metastasis and completely inhibited retroperitoneal invasion. Our study shows the importance of the uPA/uPAR system in pancreatic carcinoma cell migration and invasion. These findings suggest that uPAR is a potential target for therapy in patients with pancreatic cancer.

Regulatory role of c-Met in insulin-like growth factor-I receptor–mediated migration and invasion of human pancreatic carcinoma cells

Pancreatic carcinoma cells overexpress the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) and the hepatocyte growth factor (HGF) receptor, c-Met, which are both known to mediate tumor cell migration and invasion. We hypothesized that IGF-IR and c-Met cooperate to induce migration and invasion of human pancreatic carcinoma cells and that IGF-I-mediated migration and invasion depend on c-Met. Migration and invasion assays were done with the human pancreatic cancer cell line L3.6pl treated with PBS, IGF-I, HGF, or IGF-I plus HGF. To determine if c-Met is necessary for IGF-IR-mediated migration and invasion, c-Met was down-regulated in L3.6pl cells via adenoviral infection with a c-Met ribozyme before IGF-I treatment. IGF-I and HGF increased cell migration and invasion. Furthermore, IGF-I plus HGF had a greater than additive effect on cell migration and invasion compared with either growth factor alone. Down-regulation of c-Met nearly completely inhibited IGF-I-mediated migration and invasion. Our findings suggest that IGF-IR and c-Met cooperate to induce migration and invasion of human pancreatic carcinoma cells. Furthermore, c-Met is required for both HGF- and IGF-I-mediated migration and invasion. Elucidation of the signaling pathways that contribute to tumor progression and metastasis should provide a foundation for the development of targeted therapies for pancreatic carcinoma. [Mol Cancer Ther 2006;5(7):1676–82]

Insulinlike Growth Factor-I–Mediated Migration and Invasion of Human Colon Carcinoma Cells Requires Activation of c-Met and Urokinase Plasminogen Activator Receptor

Objective:To determine whether insulinlike growth factor-I (IGF-I) and hepatocyte growth factor (HGF) cooperate to induce migration and invasion of human colorectal carcinoma (CRC) cells and whether the effects of IGF-I and/or HGF are mediated through activation of the urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system, a central mediator of tumor-cell migration and invasion. Summary Background Data:CRC cells must invade through the basement membrane of the colon and migrate to form metastases. CRC cells are known to overexpress IGF-I receptor (IGF-IR), c-Met, and uPAR, 3 cell-surface receptors known to mediate cell migration and invasion. We hypothesized that IGF-IR and c-Met cooperate to induce migration and invasion in CRC cells and that this signaling is dependent on uPAR. Methods:KM12L4 human CRC cells were treated with IGF-I, HGF, or IGF-I + HGF in transwell migration and invasion chambers; cells that had migrated or invaded were counted. To determine the role of c-Met in IGF-I-induced migration and invasion, c-Met was inhibited by infection of cells with an adenovirus containing a c-Met ribozyme; transwell assays were then repeated. To determine the role of the uPA/uPAR system in IGF-I-induced CRC cell migration and invasion, transwell assays were repeated after pretreating cells with the uPA inhibitor amiloride or with neutralizing antibodies to uPA and uPAR. Results:IGF-I and HGF, alone or in combination, increased cell migration and invasion. The c-Met ribozyme inhibited IGF-I- and HGF-mediated migration and invasion, indicating that c-Met is essential for these processes. uPA and uPAR inhibition blocked IGF-I- and HGF-mediated migration and invasion, suggesting that uPAR is downstream of IGF/IGF-IR and HGF/c-Met in the signaling pathways that mediate cell migration and invasion. Conclusions:IGF-I and HGF cooperate to induce migration and invasion of CRC cells, and c-Met and uPA/uPAR are required for IGF-I-mediated migration and invasion. In our in vitro model of CRC migration and invasion, uPA and uPAR appear to be downstream of IGF-IR and c-Met and are required for migration and invasion. Elucidation of the pathways that contribute to tumor progression and metastasis should provide a foundation for the rational development and use of targeted therapies for CRC.

Use of a falciform ligament pedicle flap to decrease pancreatic fistula after distal pancreatectomy.

Objectives: Postoperative pancreatic fistula (POPF) remains a significant source of morbidity after distal pancreatectomy (DP). We describe a technique for coverage of the pancreatic stump after DP using a pedicled falciform ligament flap with a low POPF rate. Methods: A retrospective review of clinical, radiographic, and pathologic variables of patients undergoing open DP between November 2005 and August 2009 was performed. After standardized DP, the pancreatic stump was closed using a pedicled falciform ligament flap. Postoperative pancreatic fistula was defined using the International Study Group classification for pancreatic fistula definition. Results: Twenty-three consecutive patients underwent open DP and splenectomy with closure of the pancreatic stump using a pedicled falciform ligament flap. Pancreatic transection and stump closure was performed in a uniform fashion in all patients. Eight patients (35%) had additional organs resected. Two patients (8.7%) developed grade C POPFs, which were successfully managed with percutaneous drain placement. No additional patients developed a POPF or abdominal abscess. The median length of stay was 5 days. There were no perioperative mortalities. Conclusions: We conclude that use of a pedicled falciform ligament flap for coverage of the pancreatic stump is associated with a low incidence of POPF. Continued investigation of this technique is warranted.

Management of Biliary Cystic Tumors: A Multi-institutional Analysis of a Rare Liver Tumor

OBJECTIVE To characterize clinical and radiological features associated with biliary cystic tumors (BCTs) of the liver, and to define recurrence-free and overall survival. BACKGROUND Biliary cystadenoma (BCA) and biliary cystadenocarcinoma (BCAC) are rare tumors that arise in the liver. METHODS Between 1984 and 2013, 248 patients who underwent surgical resection of BCA or BCAC were identified. Clinical and outcome data were analyzed. RESULTS Median total bilirubin, CA19-9, and carcinoembryonic antigen (CEA) levels were 0.6 mg/dL, 15.0 U/mL, and 2.7 ng/mL, respectively. Preoperative imaging included computed tomography only (62.5%), magnetic resonance imaging only (6.9%), or CT + MRI (18.5%). Features on cross-sectional imaging included multiloculation (56.9%), mural nodularity (16.5%), and biliary ductal dilatation (17.7%). The presence of these factors did not reliably predict BCAC versus BCA (sensitivity, 81%; specificity, 21%). Median biliary cyst size was 10.0 cm (interquartile range, 7-13 cm). Operative interventions included unroofing/partial excision of the lesion (14.1%), less than hemihepatectomy (48.8%), or hemi-/extended hepatectomy (36.3%). On pathology most lesions were BCA (89.1%), whereas 27 (10.9%) were BCAC. At last follow-up, there were 46 (18.3%) recurrences; 2 patients who initially had BCA recurred with BCAC. Median overall survival was 18.1 years; 1-year, 3-year, and 5-year survival was 95.0%, 86.8%, and 84.2%, respectively. Long-term outcomes were associated with BCAC versus BCA, as well as the presence of spindle cell/ovarian stroma (both P < 0.05). CONCLUSIONS Among patients undergoing surgery for BCT, associated malignancy was uncommon (10%) and no preoperative findings reliably predicted underlying BCAC. After excision of BCA, long-term outcomes were good; however, patients with BCAC had a worse long-term prognosis.

A Long Gastrojejunostomy Is Associated With Decreased Incidence and Severity of Delayed Gastric Emptying After Pancreaticoduodenectomy.

Objectives Delayed gastric emptying (DGE) after pancreaticoduodenectomy (PD) is associated with increased hospital length of stay (LOS) and health care costs. We hypothesized that a long gastrojejunostomy for PD (LGPD) is associated with decreased incidence of DGE. Methods Data were reviewed from patients who underwent standard PD (SPD), pylorus-preserving PD (PPPD), or LGPD with a 9-cm-long anastomosis between August 2000 and July 2010. Primary outcomes included presence and grade of DGE and LOS. The International Study Group of Pancreatic Surgery definition was used to define DGE. Results A total of 194 PDs (28 SPDs, 82 PPPDs, and 84 LGPDs) were performed. The rates of DGE were 46.4%, 37.8%, and 16.7%, respectively (P = 0.001). The LGPD was associated with fewer grades B/C DGE (2.4%) compared to SPD (10.7%) and PPPD (17.5%). Rates of postoperative abdominal fluid collection and abscess were similar among the groups. Patients with DGE had significantly longer LOS (14.0 vs 7.0 days, P < 0.001). Conclusions This is the first study evaluating the effect of a long gastrojejunostomy on the incidence of DGE after PD. The LGPD is associated with significantly decreased DGE compared to SPD and PPPD and warrants further exploration as a means to improve outcome for patients who undergo PD.

Pancreatic duct size and gland texture are associated with pancreatic fistula after pancreaticoduodenectomy but not after distal pancreatectomy

Background Pancreatic fistula remains a morbid complication after pancreatectomy. Since the proposed mechanism of pancreatic fistula is different between pancreaticoduodenectomy and distal pancreatectomy, we hypothesized that pancreatic gland texture and duct size are not associated with pancreatic fistula after distal pancreatectomy. Methods All patients ≥18 years in the 2014–15 American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) targeted pancreatectomy dataset were linked with the ACS NSQIP Public Use File (PUF). Pancreatic duct size (<3 mm, 3–6 mm, >6 mm) and pancreatic gland texture (hard, intermediate, soft) were categorized. Separate multivariable analyses were performed to evaluate associations between pancreatic duct size and gland texture after pancreaticoduodenectomy and distal pancreatectomy. Results A total of 9366 patients underwent pancreaticoduodenectomy or distal pancreatectomy during the study period. Proportion of pancreatic fistula was similar after distal pancreatectomy (606 of 3132, 19.4%) and pancreaticoduodenectomy (1163 of 6335, 18.4%, p = 0.245). Both pancreatic gland texture and duct size were significantly associated with pancreatic fistula after pancreaticoduodenectomy (p<0.001). However, there was no association between pancreatic fistula and gland texture or duct size (all p≥0.169) after distal pancreatectomy. Operative approach (minimally invasive versus open) was not associated with pancreatic fistula after distal pancreatectomy (p = 0.626). Patients with pancreatic fistula after distal pancreatectomy had increased rate of postoperative complications including longer length of stay, higher rates of readmission and reoperation compared to patients who did not have a pancreatic fistula (all p<0.001). Conclusions Unlike among patients who had pancreaticoduodenectomy, pancreatic gland texture and duct size are not associated with development of pancreatic fistula following distal pancreatectomy. Other clinical factors should be considered in this patient population.

Evaluation of SAS1B as a target for antibody-drug conjugate therapy in the treatment of pancreatic cancer

Successful therapeutic options remain elusive for pancreatic cancer. The exquisite sensitivity and specificity of humoral and cellular immunity may provide therapeutic approaches if antigens specific for pancreatic cancer cells can be identified. Here we characterize SAS1B (ovastacin, ASTL, astacin-like), a cancer-oocyte antigen, as an attractive immunotoxin target expressed at the surface of human pancreatic cancer cells, with limited expression among normal tissues. Immunohistochemistry shows that most pancreatic cancers are SAS1Bpos (68%), while normal pancreatic ductal epithelium is SAS1Bneg. Pancreatic cancer cell lines developed from patient-derived xenograft models display SAS1B cell surface localization, in addition to cytoplasmic expression, suggesting utility for SAS1B in multiple immunotherapeutic approaches. When pancreatic cancer cells were treated with an anti-SAS1B antibody-drug conjugate, significant cell death was observed at 0.01-0.1 μg/mL, while SAS1Bneg human keratinocytes were resistant. Cytotoxicity was correlated with SAS1B cell surface expression; substantial killing was observed for tumors with low steady state SAS1B expression, suggesting a substantial proportion of SAS1Bpos tumors can be targeted in this manner. These results demonstrate SAS1B is a surface target in pancreatic cancer cells capable of binding monoclonal antibodies, internalization, and delivering cytotoxic drug payloads, supporting further development of SAS1B as a novel target for pancreatic cancer.