Ryan M. Thomas

Transport properties of pancreatic cancer describe gemcitabine delivery and response

BACKGROUND The therapeutic resistance of pancreatic ductal adenocarcinoma (PDAC) is partly ascribed to ineffective delivery of chemotherapy to cancer cells. We hypothesized that physical properties at vascular, extracellular, and cellular scales influence delivery of and response to gemcitabine-based therapy. METHODS We developed a method to measure mass transport properties during routine contrast-enhanced CT scans of individual human PDAC tumors. Additionally, we evaluated gemcitabine infusion during PDAC resection in 12 patients, measuring gemcitabine incorporation into tumor DNA and correlating its uptake with human equilibrative nucleoside transporter (hENT1) levels, stromal reaction, and CT-derived mass transport properties. We also studied associations between CT-derived transport properties and clinical outcomes in patients who received preoperative gemcitabine-based chemoradiotherapy for resectable PDAC. RESULTS Transport modeling of 176 CT scans illustrated striking differences in transport properties between normal pancreas and tumor, with a wide array of enhancement profiles. Reflecting the interpatient differences in contrast enhancement, resected tumors exhibited dramatic differences in gemcitabine DNA incorporation, despite similar intravascular pharmacokinetics. Gemcitabine incorporation into tumor DNA was inversely related to CT-derived transport parameters and PDAC stromal score, after accounting for hENT1 levels. Moreover, stromal score directly correlated with CT-derived parameters. Among 110 patients who received preoperative gemcitabine-based chemoradiotherapy, CT-derived parameters correlated with pathological response and survival. CONCLUSION Gemcitabine incorporation into tumor DNA is highly variable and correlates with multiscale transport properties that can be derived from routine CT scans. Furthermore, pretherapy CT-derived properties correlate with clinically relevant endpoints. TRIAL REGISTRATION Clinicaltrials.gov NCT01276613. FUNDING Lustgarten Foundation (989161), Department of Defense (W81XWH-09-1-0212), NIH (U54CA151668, KCA088084).

Silencing of RON Receptor Signaling Promotes Apoptosis and Gemcitabine Sensitivity in Pancreatic Cancers

The RON receptor tyrosine kinase is overexpressed in premalignant pancreatic intraepithelial neoplasia (PanIN) and in the majority of pancreatic cancers. In pancreatic cells, RON is an important K-Ras effector and RON ligand can enhance migration/invasion and apoptotic resistance. However, the pathobiological significance of RON overexpression in pancreatic cancers has yet to be fully established. In this study, we demonstrate that RON signaling mediates a unique transcriptional program that is conserved between cultured cells derived from murine PanIN or human pancreatic cancer cells grown as subcutaneous tumor xenografts. In both systems, RON signaling regulates expression of genes implicated in cancer-cell survival, including Bcl-2 and the transcription factors signal transducer and activator of transcription 3 (STAT 3) and c-Jun. shRNA-mediated silencing of RON in pancreatic cancer xenografts inhibited their growth, primarily by increasing susceptibility to apoptosis and by sensitizing them to gemcitabine treatment. Escape from RON silencing was associated with re-expression of RON and/or expression of phosphorylated forms of the related c-Met or epidermal growth factor receptors. These findings indicate that RON signaling mediates cell survival and in vivo resistance to gemcitabine in pancreatic cancer, and they reveal mechanisms through which pancreatic cancer cells may circumvent RON-directed therapies.

The chemokine receptor CXCR4 is expressed in pancreatic intraepithelial neoplasia

Objective: The chemokine CXCL12, together with its specific receptor, CXCR4, have been shown to mediate invasiveness and metastatic behaviour in pancreatic cancer cells. The expression of CXC12/CXCR4 has not been previously examined in pancreatic intraepithelial neoplasias (PanIN), the accepted precursor lesions to pancreatic duct cancer. Design: In this study we sought to characterise the expression of CXCL12 and CXCR4 during the progression of PanIN using both a murine model and human tissues. Results: These studies reveal that both CXCL12 and CXCR4 are expressed in PanIN and that the frequency increases during PanIN progression (0% CXCR4 expression in normal mouse and human ducts vs 100% in mouse PanIN 3 and 77% in human PanIN 3). Next we demonstrate a dose-dependent increase in the proliferation of murine PanIN cells when exposed to CXCL12. Finally, we show that expression of CXCR4 in murine PanIN cells is partially dependent on mitogen-activated protein kinase (MAPK) signalling and that the effect of CXCL12 on PanIN proliferation can be abrogated by an MAPK inhibitor. Conclusions: Together these results demonstrate that CXCL12/CXCR4 expression begins in the pre-invasive stages of pancreatic neoplasia, and suggest that the presence of an autocrine loop that is at least partially regulated by MAPK signalling. Further studies that define the role of CXCR4 signalling in PanIN progression will determine if CXCR4 could serve as a novel target for chemoprevention and early stage therapy in pancreatic cancer.

Multi-color palette of fluorescent proteins for imaging the tumor microenvironment of orthotopic tumorgraft mouse models of clinical pancreatic cancer specimens

Pancreatic‐cancer‐patient tumor specimens were initially established subcutaneously in NOD/SCID mice immediately after surgery. The patient tumors were then harvested from NOD/SCID mice and passaged orthotopically in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary patient tumors acquired RFP‐expressing stroma. The RFP‐expressing stroma included cancer‐associated fibroblasts (CAFs) and tumor‐associated macrophages (TAMs). Further passage to transgenic nude mice ubiquitously expressing green fluorescent protein (GFP) resulted in tumors that acquired GFP stroma in addition to their RFP stroma, including CAFs and TAMs as well as blood vessels. The RFP stroma persisted in the tumors growing in the GFP mice. Further passage to transgenic nude mice ubiquitously expressing cyan fluorescent protein (CFP) resulted in tumors acquiring CFP stroma in addition to persisting RFP and GFP stroma, including RFP‐ and GFP‐expressing CAFs, TAMs and blood vessels. This model can be used to image progression of patient pancreatic tumors and to visually target stroma as well as cancer cells and to individualize patient therapy. J. Cell. Biochem. 113: 2290–2295, 2012.

Chemokine receptor CXCR4 enhances proliferation in pancreatic cancer cells through AKT and ERK dependent pathways.

Objectives: We previously detected CXCR4 expression in pancreatic intraepithelial neoplasia (PanIN) tissues and demonstrated CXCR4-enhanced proliferation of PanIN cells. Our objective was to determine if the CXCR4 targets AKT and ERK mediate CXCR4-dependent PanIN and pancreatic cancer proliferation. Methods: We exposed cultured murine-derived PanIN, invasive pancreatic cancer (5143PDA) and liver metastasis (5143LM) cells, and human pancreatic cancer PANC-1 cells to CXCL12, the specific CXCR4 ligand, and measured phosphorylation of AKT and ERK1/2. The roles of AKT and ERK1/2 in CXCR4-dependent cell proliferation were assessed by the PI/3K-AKT small molecular inhibitor LY294002 and the ERK signaling inhibitor UO126. Results: We discovered increases in phosphorylation of AKT in PanIN, 5143PDA, and PANC-1 cells but no increase in 5143LM cells after exposure to CXCL12. We also observed that exposure to CXCL12 over varying periods phosphorylated ERK1/2 in an oscillatory pattern for all cell lines. Administration of LY294002 resulted in complete abrogation of CXCL12-induced proliferation in PanIN, 5143LM, and PANC-1 cells but not 5143PDA cells, whereas UO126 resulted in complete abrogation of CXCR4-enhanced proliferation in all cell lines. Conclusions: Our studies show that CXCR4-induced proliferation is mediated by both AKT and ERK signaling in both murine and human pancreatic cancer cells.

Management of Acute Post-operative Portal Venous Thrombosis

BackgroundPortal vein thrombosis can be a devastating, but often overlooked, complication of hepatobiliary procedures. Symptoms of acute portal vein thrombosis range from nondescript abdominal pain to septic shock secondary to mesenteric ischemia.DiscussionThe surgeon must be cognizant of these symptoms and the potential for portal vein thrombosis after any hepatobiliary procedures as an expedient diagnosis and treatment is necessary in order to prevent thrombus propagation, bowel ischemia, and death. This report outlines the symptoms, diagnosis, and a review of the literature on the treatment of acute portal vein thrombosis after hepatobiliary surgery with a special note made regarding a case of portal vein thrombosis after pancreatectomy and autologous islet cell transplantation.

Extracellular lumican inhibits pancreatic cancer cell growth and is associated with prolonged survival after surgery.

Purpose: To evaluate the relevance between lumican expression patterns and the clinical course of patients with pancreatic ductal adenocarcinoma (PDAC), and to investigate the role of lumican in PDAC progression. Experimental Design: One hundred thirty-one patient tumors were chosen for tissue microarray staining, and Cox regression analysis was used to test the associations between lumican expression and clinical, pathologic, and oncologic outcomes in all patients. Primary PDAC cells and recombinant human lumican protein were used to establish a working model to mimic the in vivo interactions between stromal lumican and PDAC cells. Using this model, we tested the effects of lumican on EGFR signaling via Akt and hypoxia-inducible factor-1α (HIF1α) and its subsequent influence on glucose consumption, lactate production, intracellular ATP, and apoptotic cell death. Results: Lumican was present in the stroma surrounding PDAC cells in roughly one-half of primary tumors and the direct xenografts. Patients with stromal lumican were associated with a profound reduction in metastatic recurrence after surgery and 3-fold longer survival than patients without stromal lumican. In PDAC cells, extracellular lumican reduced EGFR expression and phosphorylation through enhanced dimerization and internalization of EGFR and the resultant inhibition of Akt kinase activity. Lumican also reduced HIF1α expression and activity via Akt. PDAC cells with enhanced HIF1α activity were resistant to lumican-induced inhibition of glucose consumption, lactate production, intracellular ATP, and apoptosis. Conclusions: There is a positive association between stromal lumican in primary PDAC tumors and prolonged survival after tumor resection. Lumican plays a restrictive role in EGFR-expressing pancreatic cancer progression. Clin Cancer Res; 20(24); 6529–40. ©2014 AACR.

Patient-Derived Xenograft Models for Pancreatic Adenocarcinoma Demonstrate Retention of Tumor Morphology through Incorporation of Murine Stromal Elements

Direct implantation of viable surgical specimens provides a representative preclinical platform in pancreatic adenocarcinoma. Patient-derived xenografts consistently demonstrate retained tumor morphology and genetic stability. However, the evolution of the tumor microenvironment over time remains poorly characterized in these models. This work specifically addresses the recruitment and incorporation of murine stromal elements into expanding patient-derived pancreatic adenocarcinoma xenografts, establishing the integration of murine cells into networks of invading cancer cells. In addition, we provide methods and observations in the establishment and maintenance of a patient-derived pancreatic adenocarcinoma xenograft model. A total of 25 histologically confirmed pancreatic adenocarcinoma specimens were implanted subcutaneously into nonobese diabetic severe combined immunodeficiency mice. Patient demographics, staging, pathological analysis, and outcomes were analyzed. After successful engraftment of tumors, histological and immunofluorescence analyses were performed on explanted tumors. Pancreatic adenocarcinoma specimens were successfully engrafted in 15 (60%) of 25 attempts. Successful engraftment does not appear to correlate with clinicopathologic factors or patient survival. Tumor morphology is conserved through multiple passages, and tumors retain metastatic potential. Interestingly, despite morphological similarity between passages, human stromal elements do not appear to expand with invading cancer cells. Rather, desmoplastic murine stroma dominates the xenograft microenvironment after the initial implantation. Recruitment of stromal elements in this manner to support and maintain tumor growth represents a novel avenue for investigation into tumor-stromal interactions.

Multimodality therapy offers a chance for cure in patients with pancreatic adenocarcinoma deemed unresectable at first operative exploration.

BACKGROUND Patients identified at surgical exploration with unresectable pancreatic ductal adenocarcinoma receive palliative, noncurative therapy. We hypothesized that accurate radiographic restaging, multimodality treatment, and advanced surgical technique can offer patients deemed unresectable at previous exploration the possibility for curative salvage pancreatectomy. STUDY DESIGN Review of a prospectively maintained pancreatic ductal adenocarcinoma database identified all patients (1990 to 2010) evaluated after being deemed unresectable at first exploration elsewhere. Referring hospitals were categorized per National Cancer Data Base criteria as academic, community, or international. Patients were restaged using objective imaging (CT) criteria and classified based on anatomic resectability. Clinicopathologic factors and cancer-related outcomes were assessed. RESULTS We evaluated 88 patients who underwent previously unsuccessful resection attempts at academic (n = 50), community (n = 25), and international (n = 13) centers. Radiographic restaging confirmed that 7 (8%) patient tumors were locally advanced and unresectable, but 81 (92%) were resectable (n = 61) or borderline resectable (n = 20). Using a surgery first (9%) or preoperative chemoradiation (91%) approach, successful reoperative pancreatectomy was performed in 66 (81%) patients, with 94% receiving R0 resections. Vascular resection/reconstruction was required in 30 (46%) patients and 50 (76%) required complex revision of previously created biliary/gastrointestinal bypass. The major complication rate was 20% and 3 (4.5%) patients died perioperatively. Median overall survival was 29.6 months for successfully resected patients vs 10.6 and 5.1 months (p < 0.0001) for those patients with locally advanced unresectable disease at initial referral or in whom metastatic disease developed before resection, respectively. CONCLUSIONS In this very selected cohort of high-risk patients, the majority had anatomically resectable tumors on restaging. Accurate radiographic restaging, a multimodality treatment strategy, and advanced surgical techniques can provide an opportunity for cure in a substantial proportion of select patients who were deemed unresectable at exploration.

The RON tyrosine kinase receptor regulates vascular endothelial growth factor production in pancreatic cancer cells.

Objectives: The RON receptor mediates tumorigenic phenotypes in pancreatic cancer (PC), but no investigations currently have implicated RON signaling as a regulator of angiogenesis in PC. Angiogenesis is vital to oncogenesis, and vascular endothelial growth factor (VEGF) is the most well-characterized angiogenic protein. This study sought to determine the effect of RON stimulation on in vitro angiogenesis and VEGF production in PC cell lines. Methods: Vascular endothelial growth factor levels from conditioned media of hepatocyte growth factor-like protein-stimulated BxPC-3 and FG cells were quantitated via enzyme-linked immunosorbent assay and likewise interrogated in the presence and absence of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase/AKT inhibitors. To determine in vitro angiogenesis, human microvascular endothelial cells were subsequently exposed to the same conditioned media to assay for microtubule formation. Results: RON signaling resulted in a 52% and 34% increase in VEGF levels in BxPC-3 and FG cells, respectively. Vascular endothelial growth factor secretion was inhibited with MAPK or phosphatidylinositol-3-kinase blockade in BxPC-3 cells, but only MAPK inhibition resulted in decreased VEGF production in FG cells. BxPC-3 conditioned media induced tubule formation in human microvascular endothelial cells, which was abrogated by RON inhibition. Conclusions: RON signaling results in MAPK-mediated VEGF secretion by PC cells and promotion of microtubule formation. These findings suggest another mechanism by which RON signaling may promote PC progression.