Daniel Delitto

c-Met signaling in the development of tumorigenesis and chemoresistance: Potential applications in pancreatic cancer

Pancreatic ductal adenocarcinoma is the 4(th) leading cause of cancer deaths in the United States. The majority of patients are candidates only for palliative chemotherapy, which has proven largely ineffective in halting tumor progression. One proposed mechanism of chemoresistance involves signaling via the mesenchymal-epithelial transition factor protein (MET), a previously established pathway critical to cell proliferation and migration. Here, we review the literature to characterize the role of MET in the development of tumorigenesis, metastasis and chemoresistance, highlighting the potential of MET as a therapeutic target in pancreatic cancer. In this review, we characterize the role of c-Met in the development of tumorigenesis, metastasis and chemoresistance, highlighting the potential of c-Met as a therapeutic target in pancreatic cancer.

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.

Primary outgrowth cultures are a reliable source of human pancreatic stellate cells.

Recent advances demonstrate a critical yet poorly understood role for the pancreatic stellate cell (PSC) in the pathogenesis of chronic pancreatitis (CP) and pancreatic cancer (PC). Progress in this area has been hampered by the availability, fidelity, and/or reliability of in vitro models of PSCs. We examined whether outgrowth cultures from human surgical specimens exhibited reproducible phenotypic and functional characteristics of PSCs. PSCs were cultured from surgical specimens of healthy pancreas, CP and PC. Growth dynamics, phenotypic characteristics, soluble mediator secretion profiles and co-culture with PC cells both in vitro and in vivo were assessed. Forty-seven primary cultures were established from 52 attempts, demonstrating universal α-smooth muscle actin and glial fibrillary acidic protein but negligible epithelial surface antigen expression. Modification of culture conditions consistently led to cytoplasmic lipid accumulation, suggesting induction of a quiescent phenotype. Secretion of growth factors, chemokines and cytokines did not significantly differ between donor pathologies, but did evolve over time in culture. Co-culture of PSCs with established PC cell lines resulted in significant changes in levels of multiple secreted mediators. Primary PSCs co-inoculated with PC cells in a xenograft model led to augmented tumor growth and metastasis. Therefore, regardless of donor pathology, outgrowth cultures produce PSCs that demonstrate consistent growth and protein secretion properties. Primary cultures from pancreatic surgical specimens, including malignancies, may represent a reliable source of human PSCs.

Nicotine Reduces Survival via Augmentation of Paracrine HGF–MET Signaling in the Pancreatic Cancer Microenvironment

Purpose: The relationship between smoking and pancreatic cancer biology, particularly in the context of the heterogeneous microenvironment, remains incompletely defined. We hypothesized that nicotine exposure would lead to the augmentation of paracrine growth factor signaling between tumor-associated stroma (TAS) and pancreatic cancer cells, ultimately resulting in accelerated tumor growth and metastasis. Experimental Design: The effect of tobacco use on overall survival was analyzed using a prospectively maintained database of surgically resected patients with pancreatic cancer. Nicotine exposure was evaluated in vitro using primary patient–derived TAS and pancreatic cancer cells independently and in coculture. Nicotine administration was then assessed in vivo using a patient-derived pancreatic cancer xenograft model. Results: Continued smoking was associated with reduced overall survival after surgical resection. In culture, nicotine-stimulated hepatocyte growth factor (HGF) secretion in primary patient-derived TAS and nicotine stimulation was required for persistent pancreatic cancer cell c-Met activation in a coculture model. c-Met activation in this manner led to the induction of inhibitor of differentiation-1 (Id1) in pancreatic cancer cells, previously established as a mediator of growth, invasion and chemoresistance. HGF-induced Id1 expression was abrogated by both epigenetic and pharmacologic c-Met inhibition. In patient-derived pancreatic cancer xenografts, nicotine treatment augmented tumor growth and metastasis; tumor lysates from nicotine-treated mice demonstrated elevated HGF expression by qRT-PCR and phospho-Met levels by ELISA. Similarly, elevated levels of phospho-Met in surgically resected pancreatic cancer specimens correlated with reduced overall survival. Conclusions: Taken together, these data demonstrate a novel, microenvironment-dependent paracrine signaling mechanism by which nicotine exposure promotes the growth and metastasis of pancreatic cancer. Clin Cancer Res; 22(7); 1787–99. ©2015 AACR.

Human Pancreatic Cancer Cells Induce a MyD88-Dependent Stromal Response to Promote a Tumor-Tolerant Immune Microenvironment

Cancer cells exert mastery over the local tumor-associated stroma (TAS) to configure protective immunity within the tumor microenvironment. The immunomodulatory character of pancreatic lysates of patients with cancer differs from those with pancreatitis. In this study, we evaluated the cross-talk between pancreatic cancer and its TAS in primary human cell culture models. Upon exposure of TAS to pancreatic cancer cell-conditioned media, we documented robust secretion of IL6 and IL8. This TAS response was MyD88-dependent and sufficient to directly suppress both CD4+ and CD8+ T-cell proliferation, inducing Th17 polarization at the expense of Th1. We found that patients possessed a similar shift in circulating effector memory Th17:Th1 ratios compared with healthy controls. The TAS response also directly suppressed CD8+ T-cell-mediated cytotoxicity. Overall, our results demonstrate how TAS contributes to the production of an immunosuppressive tumor microenvironment in pancreatic cancer. Cancer Res; 77(3); 672-83. ©2016 AACR.

Targeting tumor tolerance: A new hope for pancreatic cancer therapy?

With a 5-year survival rate of just 8%, pancreatic cancer (PC) is projected to be the second leading cause of cancer deaths by 2030. Most PC patients are not eligible for surgery with curative intent upon diagnosis, emphasizing a need for more effective therapies. However, PC is notoriously resistant to chemoradiation regimens. As an alternative, immune modulating strategies have recently achieved success in melanoma, prompting their application to other solid tumors. For such therapeutic approaches to succeed, a state of immunologic tolerance must be reversed in the tumor microenvironment and that has been especially challenging in PC. Nonetheless, knowledge of the PC immune microenvironment has advanced considerably over the past decade, yielding new insights and perspectives to guide multimodal therapies. In this review, we catalog the historical groundwork and discuss the evolution of the cancer immunology field to its present state with a specific focus on PC. Strategies currently employing immune modulation in PC are reviewed, specifically highlighting 66 clinical trials across the United States and Europe.

Isolation of Pancreatic Cancer Cells from a Patient-Derived Xenograft Model Allows for Practical Expansion and Preserved Heterogeneity in Culture

Commercially available, highly passaged pancreatic cancer (PC) cell lines are of limited translational value. Attempts to overcome this limitation have primarily consisted of cancer cell isolation and culture directly from human PC specimens. However, these techniques are associated with exceedingly low success rates. Here, we demonstrate a highly reproducible culture of primary PC cell lines (PPCLs) from patient-derived xenografts, which preserve, in part, the intratumoral heterogeneity known to exist in PC. PPCL expansion from patient-derived xenografts was successful in 100% of attempts (5 of 5). Phenotypic analysis was evaluated with flow cytometry, immunofluorescence microscopy, and short tandem repeat profiling. Importantly, tumorigenicity of PPCLs expanded from patient-derived xenografts was assessed by subcutaneous injection into nonobese diabeteic.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice. Morphologically, subcutaneous injection of all PPCLs into mice yielded tumors with similar characteristics to the parent xenograft. PPCLs uniformly expressed class I human leukocyte antigen, epithelial cell adhesion molecule, and cytokeratin-19. Heterogeneity within each PPCL persisted in culture for the frequency of cells expressing the cancer stem cell markers CD44, CD133, and c-Met and the immunologic markers human leukocyte antigen class II and programmed death ligand 1. This work therefore presents a reliable method for the rapid expansion of primary human PC cells and, thereby, provides a platform for translational investigation and, importantly, potential personalized therapeutic approaches.

Improved Breast Cancer Care Quality Metrics After Implementation of a Standardized Tumor Board Documentation Template

PURPOSE Cancer treatment requires a coordinated multidisciplinary treatment approach, which led to the development of the Rapid Quality Reporting System by the Commission on Cancer. However, the lack of immediate availability of documented treatment plans and the inefficiency of global medical record reviews represent significant barriers to adherence reporting and the timely implementation of quality improvement measures. METHODS Adherence to national guidelines in the areas of radiation treatment, chemotherapy, and hormone therapy was assessed after breast conservation surgery (BCS). Adherence rates within 1 year of BCS were analyzed 10 weeks before and after the implementation of a standardized documentation template at weekly multidisciplinary breast cancer conferences. RESULTS Documented adherence rates increased postimplementation in patients undergoing consideration for both radiation treatment and hormone therapy within 1 year of BCS (89% v 65%; P = .045% and 85% v 62%; P = .002, respectively). No change was observed in patients undergoing evaluation for cytotoxic chemotherapy (80% v 85%; P = 1.00). CONCLUSION The addition of a documentation template to multidisciplinary breast cancer conferences resulted in increased recorded adherence rates to national guidelines. This template provided a means of both accurate and efficient documentation of evidence-based practice, which represents a concept with broad application in quality improvement. Although evaluation of the project was not continued beyond the pilot stage, current quality measure scores remain within the same range.

Orthotopic Patient-Derived Pancreatic Cancer Xenografts Engraft Into the Pancreatic Parenchyma, Metastasize, and Induce Muscle Wasting to Recapitulate the Human Disease

OBJECTIVE Limitations associated with current animal models serve as a major obstacle to reliable preclinical evaluation of therapies in pancreatic cancer (PC). In an effort to develop more reliable preclinical models, we have recently established a subcutaneous patient-derived xenograft (PDX) model. However, critical aspects of PC responsible for its highly lethal nature, such as the development of distant metastasis and cancer cachexia, remain underrepresented in the flank PDX model. The purpose of this study was to evaluate the degree to which an orthotopic PDX model of PC recapitulates these aspects of the human disease. METHODS Human PDX-derived PC tumors were implanted directly into the pancreas of NOD.Cg-Prkdc Il2rg/SzJ mice. Tumor growth, metastasis, and muscle wasting were then evaluated. RESULTS Orthotopically implanted PDX-derived tumors consistently incorporated into the murine pancreatic parenchyma metastasized to both the liver and lungs and induced muscle wasting directly proportional to the size of the tumor, consistent of the cancer cachexia syndrome. CONCLUSIONS Through the orthotopic implantation technique described, we demonstrate a highly reproducible model that recapitulates both local and systemic aspects of human PC.Objective Limitations associated with current animal models serve as a major obstacle to reliable preclinical evaluation of therapies in pancreatic cancer (PC). In an effort to develop more reliable preclinical models, we have recently established a subcutaneous patient-derived xenograft (PDX) model. However, critical aspects of PC responsible for its highly lethal nature, such as the development of distant metastasis and cancer cachexia, remain underrepresented in the flank PDX model. The purpose of this study was to evaluate the degree to which an orthotopic PDX model of PC recapitulates these aspects of the human disease. Methods Human PDX-derived PC tumors were implanted directly into the pancreas of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Tumor growth, metastasis, and muscle wasting were then evaluated. Results Orthotopically implanted PDX-derived tumors consistently incorporated into the murine pancreatic parenchyma, metastasized to both the liver and lungs and induced muscle wasting directly proportional to the size of the tumor, consistent of the cancer cachexia syndrome. Conclusions Through the orthotopic implantation technique described, we demonstrate a highly reproducible model that recapitulates both local and systemic aspects of human PC.

Viscoelastic properties of human pancreatic tumors and in vitro constructs to mimic mechanical properties

Pancreatic ductal adenocarcinoma (PDAC) is almost universally fatal, in large part due to a protective fibrotic barrier generated by tumor-associated stromal (TAS) cells. This barrier is thought to promote cancer cell survival and confounds attempts to develop effective therapies. We present a 3D in vitro system that replicates the mechanical properties of the PDAC microenvironment, representing an invaluable tool for understanding the biology of the disease. Mesoscale indentation quantified viscoelastic metrics of resected malignant tumors, inflamed chronic pancreatitis regions, and histologically normal tissue. Both pancreatitis (2.15 ± 0.41 kPa, Mean ± SD) and tumors (5.46 ± 3.18 kPa) exhibit higher Steady-State Modulus (SSM) than normal tissue (1.06 ± 0.25 kPa; p < .005). The average viscosity of pancreatitis samples (63.2 ± 26.7 kPa·s) is significantly lower than that of both normal tissue (252 ± 134 kPa·s) and tumors (349 ± 222 kPa·s; p < .005). To mimic this remodeling behavior, PDAC and TAS cells were isolated from human PDAC tumors. Conditioned medium from PDAC cells was used to culture TAS-embedded collagen hydrogels. After 7 days, TAS-embedded gels in control medium reached SSM (1.45 ± 0.12 kPa) near normal pancreas, while gels maintained with conditioned medium achieved higher SSM (3.38 ± 0.146 kPa) consistent with tumors. Taken together, we have demonstrated an in vitro system that recapitulates in vivo stiffening of PDAC tumors. In addition, our quantification of viscoelastic properties suggests that elastography algorithms incorporating viscosity may be able to more accurately distinguish between pancreatic cancer and pancreatitis. STATEMENT OF SIGNIFICANCE Understanding tumor-stroma crosstalk in pancreatic ductal adenocarcinoma (PDAC) is challenged by a lack of stroma-mimicking model systems. To design appropriate models, pancreatic tissue must be characterized with a method capable of evaluating in vitro models as well. Our indentation-based characterization tool quantified the distinct viscoelastic signatures of inflamed resections from pancreatitis, tumors from PDAC, and otherwise normal tissue to inform development of mechanically appropriate engineered tissues and scaffolds. We also made progress toward a 3D in vitro system that recapitulates mechanical properties of tumors. Our in vitro model of stromal cells in collagen and complementary characterization system can be used to investigate mechanisms of cancer-stroma crosstalk in PDAC and to propose and test innovative therapies.