Final Report Summary - EPC-TM-NET (Targeting the tumour microenvironment to improve pancreatic cancer prognosis)
Executive Summary:
Pancreatic cancer is one of the most lethal human cancers with a five-year survival rate of less than 5%. Late presentation and a high level of resistance to chemotherapeutic drugs are among the major reasons for this dismal prognosis. The presence of the highest degree of desmoplasia among all solid tumours and the fact that chronic inflammatory pancreatic disease is associated with an increased risk for pancreatic cancer indicate that the tumour microenvironment is of particular importance for carcinogenesis in the pancreas. The long-term objective of this project is to increase survival of pancreatic cancer patients by exploring the contribution of the tumour microenvironment to the failure of presently available oncological treatments.
EPC-TM-Net represents a coordinated effort to structure and integrate the Europe-wide research into the pancreatic cancer microenvironment. The programme was specifically designed to systematically elucidate the impact of the tumour microenvironment on tumour progression and translate it into clinical applications and to furnish a suite of new compounds and therapeutic strategies targeting different components of the tumour microenvironment, which will directly benefit the patient, advance the knowledge on the molecular basis of the disease and has the potential to foster health economy in Europe.
Overall we have succeeded in:
• Establishing a structured and comprehensive research platform to study the impact of the microenvironment on the dismal prognosis and drug resistance of pancreatic cancer clearly requires pan-European collaboration and is unprecedented so far, and has achiever world-wide recognition.
• The knowledge gain in this programme has increased the competitiveness of the European Research Area on the role of tumor microenvironment in pancreatic cancer.
• The programme has stimulated innovation by ensuring close and productive interactions between groups with expertise in a range of areas that we consider central for progress in cancer research and medicine.
• The combination of multiple levels of expertise from the participating groups has fostered reverse-translation of clinical findings in the genetically engineered mouse models for the development of novel therapeutic strategies targeting the tumour microenvironment.
• EPC-TM-Net has generated and characterized in-vitro and in-vivo models to study tumour-microenvironment interactions that will be of enormous value for the scientific community and industry as models for cross-sectional and longitudinal studies of carcinogenesis in the pancreas, and to test or validate diagnostic and therapeutic approaches.
• One further benefit of the breadth of expertise among the partners brought together in this programme was a wide dissemination of radically new technologies for the study of tumour-microenvironment interactions within the consortium.
• Novel compounds targeting the tumour microenvironment used alone or in combination with standard cytotoxic drugs such as gemcitabine have been tested in preclinical models and have partly entered clinical trials such as hyaluronidase.
• Clinical studies conducted in EPC-TM-Net have established surrogate serum protein profiles for tumour stroma, in order to predict those patients who are likely to respond to conventional treatment, and those for whom entry to clinical trials of novel compounds may be a more appropriate option. EPC-TM-Net has thus provided additional benefit for pancreatic cancer patients by providing the means for personalised treatment schedules, that will be tested in future clinical trials.
• As contribution to generate opportunities for education and training this collaborative project provided opportunities for personnel exchange and training. This mobility contributed to elevate the educational and training standards of involved personnel, which has fostered their career opportunities.
Project Context and Objectives:
Pancreatic cancer belongs to the most lethal human cancers with a 5-year survival rate of less than 5%. Late presentation and a high level of resistance to chemotherapeutic drugs are among the major reasons for this dismal prognosis.
To date, most efforts to identify new therapeutic targets for pancreatic cancer have focused on tumour cells themselves or on the tumour as a whole. Histologically, however, pancreatic cancer consists of a heterogeneous population of tumour cells including cancer stem cells that are highly resistant to standard chemotherapy and an extremely high proportion of desmoplastic stromal tissue surrounding the tumour cells, which accounts for up to 90% of the tumour area.
Evidence accumulated during recent years for multiple tumours has clearly demonstrated that the tumour microenvironment comprising stroma, blood vessels, infiltrating inflammatory cells and a variety of associated tissue cells are of paramount importance in regulating proliferating tumour cells including a subpopulation of cancer stem cells and thus determine tumour aggression and chemoresistance.
The presence of the highest degree of desmoplasia among all solid tumours and the fact that chronic inflammatory pancreatic disease is associated with an increased risk for pancreatic cancer in both human and mouse models indicate that the tumour microenvironment seems to be of particular importance for carcinogenesis in the pancreas.
The overall objective of EPC-TM-Net therefore was to improve survival of pancreatic cancer patients by exploring the contribution of the tumour microenvironment to the failure of presently available oncological treatments.
The limited knowledge, coupled with the likelihood that stroma plays a fundamental role in tumour progression and therapeutic responsiveness, demands concerted and integrated efforts to dissect the molecular and biological evolution and characteristics of PDAC stroma and its functional relationship to the cancer cell compartment. In view of the many scientific advances in the understanding of PDAC that have occurred over the past decade, the field appears poised to exploit many of the genomic technologies and model systems used to advance our understanding of the PDAC cancer cell in the area of stromal biology. This is a daunting challenge in light of stroma’s many components and multiple heterotypic interactions among tumour fibroblasts/mesenchymal cells, blood vessels, innate and adaptive immunocytes, and the ECM. Thus an interdisciplinary, multicentric and Pan-European academic and industrial approach as concentrated in EPC-TM-Net was necessary to tackle this enormous challenge.
Based on the eminent role of the tumour stroma, an intriguing novel approach beyond current therapeutic concepts is to primarily target the stromal components and directly or indirectly the cancer stem cell compartment, rather than the tumour cells as a whole. This could abolish the growth advantage for the tumour cells originating from stromal components and possibly facilitate the accessibility of the tumour cells for chemotherapeutic agents after tackling the stromal “belt” surrounding the tumour cells. The first evidence that targeting tumour stroma does indeed reduce tumour progression by enhancing the local accessibility of chemotherapeutic drugs was provided by a member of this consortium prior to the start of the consortium. In a mouse model of pancreatic cancer elimination of the tumour stroma by inhibition of hedgehog signalling significantly enhanced delivery and efficacy of chemotherapeutic treatment (Olive et al 2009).
Many questions relating to the revolutionary new concept of targeting stromal components to improve survival of pancreatic cancer patients are still to be addressed and were the focus of the EPC-TM Net consortium:
1) On a molecular level, the role of the individual stroma-related cell types and components including cancer stem cells and the networking between each other remain to be defined.
2) The interplay and functional relevance of each stromal cell component has to be verified in appropriate mouse models.
3) New drug targets affecting the functional integrity of the stromal networks have to be identified by candidate approaches or high-throughput screens or newly generated hypotheses and validated in-vivo.
4) Specific drugs for the identified targets have to be developed and validated preclinically in-vitro and in-vivo in mouse models of pancreatic cancer.
5) Cell type specific drug delivery needs to be established to specifically modulate the crosstalk between stroma and cancer cells.
Project Results:
Summary of the main results/foreground of EPC-TM-Net:
The long-term objective of this proposal was to increase survival of pancreatic cancer patients by exploring the contribution of the tumour microenvironment to the failure of presently available oncological treatments. For this purpose we reverse-translated this clinical observation into innovative in-vitro and mouse models closely mimicking the human disease. Work was done in the context of 5 scientific work packages that focused on different compartments or components of the microenvironment.
Within work package 1 (WP01) several groups focused on the role and the targeting of fibroblastic cells in the stroma. One of the groups at the CNIO in Madrid (Real et al) has newly generated mouse strains to allow for the selective killing of pancreatic stellate cells, regardless of their tissue distribution. This mouse strain will give the unique possibility to study the influence of the stellate cells in the PDAC microenvironment on processes such as tumor progression and chemotherapy resistance. The group at CNIO has furthermore established the completely new concept that two different transcription factors (Gata6 and Nr5a2) have direct links between epithelial differentiation and suppression of inflammation.
The group at TUM in Munich studied histological patterns and molecular signatures of different cellular compartments in wild type and KrasG12D-transgenic mice during acute pancreatitis and found that the regeneration of the adult pancreas from acute pancreatitis is a default process that is divided into three distinctive phases: inflammation, organ regeneration and refinement. In contrast to the wild type KrasG12D, pancreata were remodelled by pre-neoplastic lesions showing extended phase of inflammation with impaired acinar cell proliferation (e.g. organ regeneration). Using sophisticated bioinformatics approaches, they have furthermore identified molecular signatures associated with the pro-tumorigenic effect of inflammatory stroma in mice and validated them in humans.
The group at KI in Stockholm developed a 3D heterospheroid model comprising both human pancreatic cancer cells and the organ-residing mesenchymal cells, the pancreatic stellate cell (PSC) into one avascular minitumor, to study the effect of the different cells on chemoresistance. The assay was adapted to the 384-well format to prepare for high-throughput drug screening, allowing fully automated screening approach of libraries with more than 100.000 compounds.
Within work package 2 (WP02) several groups aimed at targeting inflammatory cells in pancreatic cancer stroma that comprise tumour-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), lymphocytes and other immune or immunomodulatory cells that are essential for cancer growth. The group in Marburg analysed the in-vitro interaction of TAMs and cancer cells in organotypic cultures and saw that pancreatic cell lines secrete a distinct panel of proinflammatory cytokines in a K-Ras dependent manner. This cytokines modulated macrophage infiltration and phenotypic polarisation towards a tumor-promoting M2-action. Analysis of the phenotypical and functional properties of isolated TAMs in genetically-engineered mouse models of pancreatic cancer and human tissues revealed TAM polarisation towards a tumor-promoting M2 phenotype already in PanIN pancreata. This was accompanied by distinct miRNA alterations in TAM that were shown to enhance secretion of proinvasive and proangiogenic cytokines, thereby creating a positive feedback loop and enhancing tumor progression. Finally the group in Marburg studied the effect of smoking, a strong risk factor for pancreatic cancer in humans, on development of inflammatory stroma, preneoplastic lesions and invasive cancer in genetically-engineered mouse models, and found that the incidence and progression of preinvasive lesions was markedly accelerated after smoke exposure, whereas progression to invasive tumors did not increase. Study of miRNA-signature changes in the mouse model identified a panel of miRNAs which are differentially regulated during tumor progression and influence NFkB and STAT signalling thereby defining macrophage action. Moreover we validated a significant MDSC-subset accumulation (MDSC1, MDSC2, MDSC4) in pancreatic patients compared the control (sex and age-matched healthy donors) that confirm MDSC presence as a potential prognostic marker in pancreatic cancer. Finally the group developed a promising preclinical therapeutic intervention targeting TAMs in KPC-mice using liposomal clodronate which led to a marked decrease in metastasis formation.
The groups CERMS-UniTO, UniVR, NatiMab, ULIV validated DNA-based vaccination strategies in KC and KPC-mice. ENO1 vaccine was able to significantly prolong mouse survival and decreased the percentage of the transformed ducts compared to normal ducts as well the size of transformed ducts, and thus represent a promising candidate target for DNA vaccination in pancreatic cancer.
In work package 3 (WP03) aimed at targeting the stromal components in the microenvironment and cancer stem cells residing therein. To this end, the groups in Marburg and Cambridge aimed to target inert and soluble components of the tumor stroma in order to break down the ECM scaffold relieve vessel compression and increase drug delivery to hypovascular pancreatic tumors. One interesting example was the use of human recombinant PEGylated hyaluronidase (PEGPH20), n murine pancreas tumors resulted in the re-expansion of blood vessels and improved drug delivery accompanied by slowing of tumor growth and prolonged survival in genetically engineered mice (GEMM) (Jacobetz et al. Gut 2013). Several preclinical studies on secreted protein acidic and rich in cysteine (SPARC) which is overexpressed by cancer associated fibroblasts (CAFs) were done. Furthermore, they studied the efficacy and mechanism of action of nab-paclitaxel, a novel, albumin-formulated chemotherapy that holds great promise for PDA patients, and found a remarkable therapeutic efficacy for nab-paclitaxel in highly treatment resistant GEMM (Frese et al. Cancer Discovery 2012). Therapeutic inhibition of Connective tissue growth factor (CTGF) using a monoclonal human antibody FG-3019 resulted in a significantly increased induction of tumor cell apoptosis in GEMMs when combined with gemcitabine (Neesse et al. PNAS 2013). In addition to these novel experimental therapeutic avenues they also further characterized the stromal-targeting mechanisms of approved multi-targeting agents such as regorafenib to elucidate the molecular alterations in the tumor cell – stroma crosstalk following targeted therapy approaches. The group of C. Heeschen at CNIO in Madrid identified a strong bilateral interaction between CSCs and tumour-associated macrophages. Targeting this cross-talk between the PDAC stroma (e.g. TAMs) and PDAC CSC is possible and bears significant therapeutic value.
For tumour cells to physically invade into external environment, proteolytic degradation is required. With work package 4 (WP=4) the group in Greifswald studied the possibility to target proteases, in particular Cathepsins, involved in tumour-stroma cross-talk. Cathepsins, chemically cysteine- and aspartic proteases which may be implicated in processes important for tumour development and progression of pancreatic ductal adenocarcinoma. Cathepsin B, L and D are upregulated in solid tumours but exert distinct roles in multistage tumourigenesis. They found that cathepsin D expression and serum cathepsin D activity in tumour resected patients is negatively correlated with the poor post-resection survival in patients with pancreatic ductal adenocarcinoma. Furthermore, loss of cathepsin B confers a survival advantage to mice with PDAC. This benefit is associated with decreased tumour proliferation and liver metastasis in tumour-bearing mice. Suggesting that cathepsin B is a logical target for future drug development process. The use of siRNAs that allow specific intervention at single molecular stages of the disease may provide a potential therapeutic agent to target the expression of genes such as proteases. The group has developed a novel formulation that not only delivers the siRNAs adequately and specifically to the tumour site but also allows non-invasive imaging of the uptake of our therapeutic agent, based on dextran coated superparamagnetic nanoparticles. Administration of these nanoparticles in tumor-bearing mice allowed monitoring of delivery of the agent to the tumour and metastasis by MRI imaging and results in efficient silencing of the target genes. This approach can significantly advance the therapeutic potential of siRNAs by providing a way not only to effectively shuttle siRNA to target sites but also to noninvasively access the bioavailability and efficacy of the siRNA at tumour site.
In work package 5 (WP05) aimed at identifying serum markers of stromal biology, since currently there is no effective mechanism to qualitatively assess the nature of the tumoral stroma in a patient without surgically removing the tumour. Since the majority of pancreatic cancer patients present with advanced cancer, removal of the tumour is not an option. Therefore, personalised treatment, based on an assessment of an individual’s stroma is not possible, but would be of paramount importance in view of the findings that stromal components have a major role in mediating resistance to therapy. Markers of stromal biology that can be readily assayed are required as surrogate markers, to predict those patients who are likely to respond to conventional treatment, and those for whom entry to clinical trials of novel compounds may be more a more appropriate option. Using the Luminex Platform they observed many associations between features in the stroma and circulating levels of serum cytokines. However all of these associations were weak, suggesting that serum cytokines are not likely to provide a good surrogate for the type of stroma present in pancreatic tumours. Interesting additional observations include the finding that the presence of large blood vessels within the tumour correlates with poor patient outcome in two independent patient cohorts. Our data from these analyses will be published shortly.
Main results/foreground of the different work packages:
WP01: Targeting the stroma: fibroblastic cells
Here, we have newly generated mouse strains to allow for the selective killing of pancreatic stellate cells, regardless of their tissue distribution. Firstly, we have generated through BAC transgenesis a line expressing Flp recombinase in the Sm22a locus; we have shown the specific activity of the recombinase using a reporter Alkphos strain. Secondly, we have generated a knockin construct and mouse strain expressing HBEGF (DT receptor) in the GFAP locus, to be conditionally expressed upon deletion of a Frt-STOP-Frt cassette. The murine embryo fibroblasts derived from this line can be infected with adenovirus-flipase to delete the STOP cassette and allow DTR expression which, in turn, renders the cells sensitive to DT. These proofs of concept experiments have been successfully completed in vitro. The two strains have been crossed and stellate cells from the compound strains have been placed in culture; the same experiments of recombination are ongoing (delayed due to the need to generate the BAC transgene). Crosses with mutant KrasG12V established and experiments will be performed to first establish stellate cell activation and then test the concept.
As of inflammation, we have established for two different transcription factors (Gata6 and Nr5a2) direct links between epithelial differentiation and suppression of inflammation, a completely new concept. The relevance of this link is shown by the fact that inactivation of either of these genes (in the case of Nr5a2, haploinsufficiency) reveals a tumor suppressor effect of these transcription factors in the context of the KrasG12V model.
In this study, we furthermore found that the regeneration of the adult pancreas from acute pancreatitis is a default process that is divided into three distinctive phases: inflammation, organ regeneration and refinement. The inflammatory phase is characterized by immune cell infiltration, proliferation of pancreatic progenitors and mesenchymal cells whereas the organ regeneration phase is predominantly characterized by acinar cell proliferation. In contrast, KrasG12D pancreata were remodelled by pre-neoplastic lesions showing extended phase of inflammation with impaired acinar cell proliferation (e.g. organ regeneration). An extensive bioinformatical analysis of expression profiles confirmed these histological findings. In an inducible mouse system, we further demonstrated that induction of KrasG12D and p53 inactivation during the inflammatory phase caused formation of pre-neoplastic cells whereas the induction of genetic events during the regenerating phase had no such effect. Using sophisticated bioinformatics approaches, we have isolated molecular signatures associated with the pro-tumorigenic effect of inflammatory stroma. These molecular signatures consist of activation of Notch pathways within pancreatic epithelial compartment, NF-κB-mediated cytokine networks within inflammatory compartment and proteinase cascades and retinoid acid signals within mesenchymal compartment. The relevance of these molecular signatures has been validated using human samples.
With the hypothesis being that the stroma plays an active role in chemoresistance, we further developed our 3D monspheroid model into a 3D heterospheroid model comprising both human pancreatic cancer cells and the organ-residing mesenchymal cells, the pancreatic stellate cell (PSC) into one avascular minitumor. In contrast to the hypothesis, these heterospheroids did not demonstrate a significantly increased chemoresistance when compared with the 3D monospehroids for the vast majority of compounds. Some few drugs showed lower efficacy, though (allicin and derivatives). For this drug, the addition of macrophages further increased chemoresistance. A possible explanation for these findings could be that the stromal contribution to chemoresistance is made up by several other factors not represented in our model such as physical tissue pressure leading to compressed non-functional vessels and the lack of fibrillous collagen in vitro (despite expression of lysine-2-monooxygenase and addition of vit. C).
To prepare for high-throughput drug screening, the existing 96-well assay was adapted to the 384-well format allowing fully automated screening approach. We have libraries totalling more than 100,000 compounds at hand. We tested a library with some 3,000 natural compounds (TimTec).
WP02: Targeting the stroma: inflammatory components
Inflammatory cells are prevalent in pancreatic cancer stroma and include tumour-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), lymphocytes and other immune or immunomodulatory cells that are essential for cancer growth. The molecular crosstalk between inflammatory cells and tumour cells remains to be elucidated, which was one of the major aims of this work package.
The main results of WP2 are:
1. Analysis of the in-vitro interaction of TAMs and cancer cells in organotypic cultures: To this extent, pancreatic preneoplasic and neoplastic cell lines were co-cultured with primary human and murine macrophages as well as macrophage cell lines in organotypic cultures. We saw that pancreatic cell lines secrete a distinct panel of proinflammatory cytokines in a K-Ras dependent manner. This cytokines modulated macrophage infiltration and phenotypic polarisation towards a tumor-promoting M2-action. This was accompanied by transcriptomic changes, in particular miRNA regulations. This miRNA changes also enhanced the protumoral phenotype of the macrophage.
2. Analysis of the phenotypical and functional properties of isolated TAMs in genetically-engineered mouse models of pancreatic cancer and human tissues: TAMs and macrophages from normal pancreas, pancreata with preneoplastic PanIN lesions and invasive pancreatic cancers in the KC (LSL-KrasG12D/+, Pdx-1-Cre) and KPC mouse models (LSL-KrasG12D/+, LSL-TP53R172H/+;Pdx-1-Cre) were isolated by flow cytometry at defined time points. Transcriptional alterations compared to macrophages from normal pancreas were evaluated. TAM polarisation was altered towards a tumor-promoting M2 phenotype already in PanIN pancreata. This was accompanied by distinct miRNA alterations. Several of these miRNAs in TAM were shown to enhance secretion of proinvasive and proangiogenic cytokines, thereby creating a positive feedback loop and enhancing tumor progression.
3. Effect of smoking on development of inflammatory stroma, preneoplastic lesions and invasive cancer in genetically-engineered mouse models: Smoking is a strong risk factor for pancreatic cancer, and thought to be mediated at least in part via chronic inflammation. However, the exact mechanisms remain to be elucidated. We investigated the effect of prolonged exposure of KC mice to cigarette smoke in whole-body chambers. The incidence and progression of preinvasive lesions was markedly accelerated after smoke exposure. However, progression to invasive tumors did not increase, suggesting that more genetic hits are required irrespective of smoke exposure. No differences were detected in stroma composition or infiltration of inflammatory cells.
4. Identification of signalling pathways involved in tumor-stroma interactions in human pancreatic cancer by transcriptional profiling of human myeloid cells: This task was performed to identify crucial signalling nodes defining macrophage phenotype. We focussed on miRNA changes in the mouse model and identified a panel of miRNAs which are differentially regulated during tumor progression and influence NFkB and STAT signalling thereby defining macrophage action. Moreover we validated a significant MDSC-subset accumulation (MDSC1, MDSC2, MDSC4) in pancreatic patients compared the control (sex and age-matched healthy donors) that confirm MDSC presence as a potential prognostic marker in pancreatic cancer.
5. Development of preclinical therapeutic interventions targeting TAMs in KPC: The value of the modulation of TAMs effectors circuitries as a strategy to impair their tumor-promoting behaviour was evaluated in KPC mice. We depleted macrophages by liposomal clodronate which resulted in a marked decrease in metastasis formation and angiogenic response, suggesting that pharmacological modulation of macrophages is a promising novel therapeutic avenue.
6. Validation of DNA-based vaccination strategies in KPC-mice and development of therapeutic monoclonal antibodies (responsible CERMS-UniTO, UniVR, NatiMab, ULIV): We have used pVAX plasmid coding for ENO1 to vaccinate KC and KPC mice with both prophylactic and therapeutical setting protocol. ENO1 vaccine was able to significantly prolong mouse survival and decreased the percentage of the transformed ducts compared to normal ducts as well the size of transformed ducts. ENO1 vaccine elicited an integrated humoral and cellular response specific to ENO and significantly decreased the percentage of myeloid derived suppressor cells as well as of regulatory T cells. Thus, ENO1 is a promising candidate target for DNA vaccination in pancreatic cancer.
WP03: Targeting the stroma and cancer stem cells to improve treatment response
Pancreatic ductal adenocarcinoma (PDA) is a stroma-rich and highly complicated cancer to treat. Over the last decades it has become increasingly clear that the complex network extracellular matrix (ECM) components collaboratively interact within the tumor microenvironment sustaining and driving cancer cell proliferation, invasion and early metastasis. Histologically, PDA represents an extremely stroma-rich and hypovascular tumor, and indeed, most of the pancreatic tumor mass consists of activated (myo-) fibroblasts, immune cells and ECM components such as collagen, desmin, fibronectin and hyaluronic acid.
More recently, the tumor microenvironment has also been appreciated to mediate therapeutic resistance in PDA thus opening numerous avenues for novel therapeutic explorations. In WP3, we have focused to develop novel therapeutic strategies for pancreatic cancer targeting the tumor stroma. To this end, we aimed to target inert and soluble components of the tumor stroma in order to break down the ECM scaffold relieve vessel compression and increase drug delivery to hypovascular pancreatic tumors.
One interesting example is the enzymatic depletion of the glycosaminoglycan hyaluronic acid (HA) by human recombinant PEGylated hyaluronidase (PEGPH20). Pancreatic cancers are rich in the megadalton form of HA, and the solvation of water by HA is thought to be responsible for the extremely high interstitial fluid pressure in PDA that results in the compression of intratumoral blood vessels. In WP3 we could show that treatment with PEGPH20 in murine pancreas tumors results in the re-expansion of blood vessels and improved drug delivery accompanied by slowing of tumor growth and prolonged survival in genetically engineered mice (GEMM) (Jacobetz et al. Gut 2013).
Secreted protein acidic and rich in cysteine (SPARC) is overexpressed by cancer associated fibroblasts (CAFs) and represents another intriguing target in PDA. In WP3 we have conducted several preclinical studies regarding SPARC and the efficacy and mechanism of action of nab-paclitaxel, a novel, albumin-formulated chemotherapy that holds great promise for PDA patients. We found a remarkable therapeutic efficacy for nab-paclitaxel in highly treatment resistant GEMMs. Moreover, we identified a synergistic effect of nab-paclitaxel through the reactive oxygen species (ROS-) mediated degradation of the primary gemcitabine metabolising enzyme, cytidine deaminase without disrupting the tumor stroma (Frese et al. Cancer Discovery 2012).
Connective tissue growth factor (CTGF/CCN2) is a pleiotropic growth factor that is overexpressed in human and murine pancreas tumors by CAFs. Therapeutic inhibition of CTGF using a monoclonal human antibody FG-3019 resulted in a significantly increased induction of tumor cell apoptosis in GEMMs when combined with gemcitabine. Notably, neither stromal depletion nor increased drug delivery occurred. Rather, stromal derived CTGF impinged on the anti-apoptotic machinery in tumor cells, and the X-linked inhibitor of apoptosis protein (XIAP) was down-regulated upon treatment with FG-019. Also, co-treatment with FG-019 and gemcitabine resulted in slowing of murine tumors and extended survival in GEMM) (Neesse et al. PNAS 2013).
In addition to these novel experimental therapeutic avenues we also aim to further characterize the stromal-targeting mechanisms of approved multi-targeting agents such as regorafenib and to elucidate the molecular alterations in the tumor cell – stroma crosstalk following targeted therapy approaches.
Targeting the tumor microenvironment provides a novel, much needed avenue for novel treatment approaches. Given the long list of failed clinical trials in the past, it remains speculative which ECM targets and compounds will make the difference for patients with pancreatic cancer, however, we are hopeful that the continuous effort to translate these findings to the clinic will eventually benefit our patients.
Furthermore, we have identified a strong bilateral interaction between CSCs and tumour-associated macrophages. In response to secretion of TGFbeta family members (i.e. TGFbeta, Nodal and Activin) macrophages acquire an M2 phenotype including the secretion of several factors that in return enhance the phenotypes of cancer stem cells. Specifically, LL-37 and ISG15 are both strongly enhancing self-renewal of cancer stem cells and equip them with marked invasive capabilities translating into a highly metastatic phenotype. Targeting this cross-talk between the PDAC stroma (e.g. TAMs) and PDAC CSC is possible and bears significant therapeutic value.
WP04: Targeting proteases involved in tumour-stroma cross-talk
Despite the knowledge and insights into properties and genetic aberrations of malignant cells, today's therapy of pancreatic ductal adenocarcinoma remains incompletely effective and transitory. The hallmarks of cancer is the diversity of the different types of cells, accompanying to cancer cells forming intrinsic barriers and protective functions (Hanahan et al., 2000). It is now widely accepted that cancer cells do not manifest the disease alone, but rather with the help of recruit and corrupt resident normal cell types associated with cancer cells contribute in sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, reprogramming energy metabolism, and evading immune destruction. This tumour associated cells (stromal cells), namely Infiltrating immune cells, endothelial cells, cancer associated fibroblastic cells, pericytes etc., constitutes the "Tumour microenvironment" (TME) (Hanahan et al., 2012). Involvement of TME in remodelled extracellular matrix (ECM) and tumour angiogenesis has been an important aspect in resilience of cancer cells to therapy.
In order to physically invade into external environment, proteolytic degradation is required. Range of proteases produced by cancer cells as well as stromal cells contributes to the invasion and ECM remodelling (Joyce et al., 2009). The tissue-stroma reaction in the pancreas is one of the most prominent known in pathology (Gress et al., 1995; Lerch et al., 1999; Mayerle et al., 2003). The stromal cell derived proteases includes specific matrix metallo-proteases, cysteine-, aspartic- and serine proteases (Gocheva et al., 2007; Gocheva et al., 2006; Lynch et al., 2002).
Cathepsins, chemically cysteine- and aspartic proteases which may be implicated in processes important for tumor development and progression of pancreatic ductal adenocarcinoma. Cathepsin B, L and D are upregulated in solid tumours but exert distinct roles in multistage tumourigenesis. The exocrine pancreas secretes more proteins per cell than any other exocrine organ and much of these secreted proteins consist of these cathepsins. The biology of pancreatic ductal adenocarcinoma might therefore be more dependent on protease activity than solid tumours.
The traditional method of studying cancer cell performance in vitro has been two dimensional monolayer cultures. Although this has resulted in an enormous amount of information regarding genetic, biochemical and cell-biological processes involved in tumour formation, cells grown on flat 2D tissue culture systems differ considerably in their morphology, differentiation as well as their cell-cell and cell-matrix interactions compared to cells in biological system. In order to study transformed cell performance, and interactions with other cell types as well as with the stroma, in a model that is still relatively easily agreeable for experimental manipulation, more physiologically relevant 3D tissue culture models can provide a solution and bridge the gap between traditional 2D monolayer culture and the more complex animal models. We developed the three dimensional organotypic co-culture model which consists of cancer cells spheroids embed in collagen matrix along with pancreatic cancer stellate cells. This model enabled us to study interaction of stromal compartment with tumour cells and influence of cathepsin inhibition or knocked-out on modulation of stroma. We found out that cathepsin D expression and serum cathepsin D activity in tumour resected patients is negatively correlated with the poor post-resection survival in patients with pancreatic ductal adenocarcinoma. We found that loss of cathepsin B confers a survival advantage to mice with PDAC. This benefit is associated with decreased tumour proliferation and liver metastasis in tumour-bearing mice. This is the first report of extended survival due to a genetic deficiency in KPC model of advanced pancreatic cancer. Our observation that early morbidity is absent in cathepsin B null mice indicates that optimal tumor development requires the activity of cathepsin B. We therefore conclude that cathepsin B is a logical target for future drug development process.
Despite the knowledge and insights into biological properties and genetic aberrations of malignant cells, therapeutic options of pancreatic ductal adenocarcinoma remain incompletely effective and transitory. One plausible explanation for this futile response is insufficient delivery of anticancer drugs to the tumour site. The use of siRNAs that allow specific intervention at single molecular stages of the disease may provide a potential therapeutic agent. siRNAs virtually can be used to inhibit the expression of any gene. Major hurdles of In vivo delivery of siRNAs are the adequate and specific to site of interest delivery, stability of siRNAs in serum and non-invasive monitoring of the influence of siRNAs.
Figure 1, see upload: Schematic diagram of siPLK1 coupled streptavidin conjugated dextran coated magnetic nanoparticles (siPLK1-StAv-MNPs) conjugated to membrane translocation peptides (MPAP), underglycosylated MUC-1 specific peptide(EPPT-1) and siRNA molecules targeting PLK1 (siPLK1).
Keeping the limitations of classical pancreatic ductal adenocarcinoma therapy and In vivo delivery of siRNA in mind, we have developed a novel formulation that not only delivers the siRNAs adequately and specifically to the tumour site but also allows non-invasive imaging of the uptake of our therapeutic agent. In order to accomplish this, we designed dual purpose functional probes comprising of dextran coated superparamagnetic nanoparticles as a backbone which can be detected by magnetic resonance imaging (MRI) and which are further conjugated with streptavidin. Reflecting the fact of streptavidin and biotin as the strongest covalent binding, we coupled biotin conjugated myristoylated polyarginine peptides (MPAPs) for translocation of the complex into the cytosol by electrostatic interaction as well biotin conjugated EPPT-1, a non-immunogenic underglycosylated MUC-1 antigen highly specific for MUC-1 protein, (a trans-membrane protein, highly expressed in pancreatic ductal adenocarcinoma) which allows tumour specific intake. Furthermore, we linked biotin conjugated siRNA to the streptavidin anker to be used as a therapeutic target. Administration of these nanoparticles in tumor-bearing mice allowed monitoring of delivery of the agent to the tumour and metastasis by MRI imaging and results in efficient silencing of the target genes. This approach can significantly advance the therapeutic potential of siRNAs by providing a way not only to effectively shuttle siRNA to target sites but also to noninvasively access the bioavailability and efficacy of the siRNA at tumour site.
WP05: Serum markers of stromal biology
Pancreatic ductal adenocarcinoma (PDAC) has an exceptionally poor prognosis [1]. Pancreatic cancer treatment has been dominated for many years by two chemotherapeutic agents, 5-fluorouracil (5-FU) and gemcitabine, and although new regimens have been successful in selected patients, 5-FU and gemcitabine remains the mainstay for the vast majority of patients. Trials undertaken by the European Study Group for Pancreatic Cancer (ESPAC), co-ordinated by the Liverpool Clinical Trials Unit, Liverpool, UK have systematically evaluated the efficacy of clinically relevant adjuvant chemotherapy for pancreatic cancer patients [2-4]. ESPAC-1 reported a significant survival benefit for 5-FU chemotherapy, over observation only [2]. A subsequent trial, ESPAC-3(v2), compared adjuvant 5-FU plus folinic acid (551 patients) and gemcitabine (537 patients) [4]. This trial showed no significant difference in overall survival between gemcitabine and 5-FU-treated patients (median survival~ 23.0 months in both arms). The ESPAC-4 trial is already underway, exploring whether the addition of an oral fluoropyrimidine (capecitabine) to gemcitabine (GEM-CAP) enhances overall survival compared to gemcitabine alone. Novel therapeutic approaches are being actively sought to improve outcomes for patients with pancreatic cancer. Equally important however is improving the capacity to predict the patients that are likely to respond to existing therapies.
Tumoral stroma has emerged as a key factor in the resistance of pancreatic cancers to treatment. While this new found understanding offers many opportunities it also presents challenges: There is no effective mechanism to qualitatively assess the nature of the tumoral stroma in a patient without surgically removing the tumour. Since the majority of pancreatic cancer patients present with advanced cancer, removal of the tumour is not an option. Therefore, personalised treatment, based on an assessment of an individual’s stroma is not possible. Even when the primary tumour, along with its stroma, can be removed, this provides information on the nature of the stroma in the primary tumour, but does not provide direct data on the stroma at sites of recurrence.
Markers of stromal biology that can be readily assayed are required. We set out to find surrogate serum protein (including cytokine) profiles for tumour stroma, in order to predict those patients who are likely to response to conventional treatment, and those for whom entry to clinical trials of novel compounds may be more a more appropriate option.
Therefore, as part of EPC-TM-NET, a systematic analysis of stroma from two independent patient cohorts was undertaken. This was carried out to establish and categorise stroma in pancreatic cancer, regardless of treatment in the first instance. Markers examined are summarised in Table 1.
Table 1, sea upload. List of immunohistochemical markers used to assess stromal content.
Matched serum samples for these patient sets were then assessed for their cytokine levels using the Bio-Rad Bio-Plex 27 Human Cytokine, Chemokine and Growth Factor Kit (Luminex Platform). These serum protein levels then underwent Pearson ‘s rank correlation with stroma measures.
We observed many associations between features in the stroma and circulating levels of serum cytokines. For example, MIP-1b levels were significantly negatively correlated with CD68+ macrophage levels in the stromal compartment of the tumour in both discovery and validation patient cohorts. In addition, IL-6 levels were significantly correlated with the levels of of large lymphatic vessels in the tumour in both the discovery and validation patient cohorts. However all of these associations were weak, suggesting that serum cytokines are on likely to provide a good surrogate for the type of stroma present in pancreatic tumours.
Interesting additional observations include the finding that the presence of large blood vessels within the tumour correlates with poor patient outcome in two independent patient cohorts.
A major aim of WP5 was to establish surrogate serum protein profiles for tumour stroma, in order to predict patients’ response to treatment. This project analysed serum samples from clinical pancreatic cancer cases recruited in Europe’s largest pancreatic cancer clinical trials (ESPAC and Telovac). Our data from these analyses will be published shortly.
References:
1. Costello E, Greenhalf W, Neoptolemos JP. New biomarkers and targets in pancreatic cancer and their application to treatment. Nat Rev Gastroenterol Hepatol 2012.
2. Neoptolemos JP, Stocken DD, Friess H, Bassi C, Dunn JA, Hickey H, Beger H, Fernandez-Cruz L, Dervenis C, Lacaine F, Falconi M, Pederzoli P, Pap A, Spooner D, Kerr DJ, Buchler MW. A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer. The New England journal of medicine 2004; 350: 1200-10.
3. Neoptolemos JP, Stocken DD, Tudur Smith C, Bassi C, Ghaneh P, Owen E, Moore M, Padbury R, Doi R, Smith D, Buchler MW. Adjuvant 5-fluorouracil and folinic acid vs observation for pancreatic cancer: composite data from the ESPAC-1 and -3(v1) trials. British journal of cancer 2009; 100: 246-50.
4. Neoptolemos JP, Stocken DD, Bassi C, Ghaneh P, Cunningham D, Goldstein D, Padbury R, Moore MJ, Gallinger S, Mariette C, Wente MN, Izbicki JR, Friess H, Lerch MM, Dervenis C, Olah A, Butturini G, Doi R, Lind PA, Smith D, Valle JW, Palmer DH, Buckels JA, Thompson J, McKay CJ, Rawcliffe CL, Buchler MW. Adjuvant chemotherapy with fluorouracil plus folinic acid vs gemcitabine following pancreatic cancer resection: a randomized controlled trial. JAMA 2010; 304: 1073-81.
WP06: Project management
One work package was dedicated to project management to take care of all administrative and coordinating tasks. In order to support the coordinator in monitoring the compliance by beneficiaries with their obligations under the grant agreement, the project management office at concentris kept a close eye on the following partners` performance:
• making sure that tasks assigned to them were correctly and timely performed,
• that reports were submitted according to the guidelines and in time,
• that funds are used and claimed according to the rules,
• that the partners fulfilled their obligations regarding dissemination and funding acknowledgements,
• that any changes to the work plan were communicated to the EC swiftly,
• and compliance to ethical regulations.
The project office acted as a helpdesk for all participants; it was the central node of communication on a day-by-day basis and communicated with the European Commission on behalf of the Coordinator regarding administrative and managerial issues (i.e. contract, amendment, reporting etc.).
Potential Impact:
Socio-economic impact and the wider societal implications of EPC-TM-Net:
The tumour microenvironment has emerged as one of the most important determinants of drug resistance and patient survival in several cancer entities. This is particularly true for pancreatic cancer which is characterized by an extremely high content of stromal components defining the microenvironment surrounding the tumour cells. Given the appalling prognosis of pancreatic cancer patients, largely due to the tumour´s drug-resistance, identification of novel strategies to target the tumour stroma is of the utmost importance and will have great impact on the survival of pancreatic cancer patients.
EPC-TM-Net represents a coordinated effort to structure and integrate the Europe-wide research into the pancreatic cancer microenvironment, which will have a major impact on the development of future therapeutic strategies. The programme was specifically designed to systematically elucidate the impact of the tumour microenvironment on tumour progression and translate it into clinical applications and to furnish a suite of new compounds and therapeutic strategies targeting different components of the tumour microenvironment, which apart from the direct benefits outlined above for pancreatic cancer patients will impact a number of important areas.
Overall we have succeeded in:
• Establishing a structured and comprehensive research platform to study the impact of the microenvironment on the dismal prognosis and drug resistance of pancreatic cancer clearly requires pan-European collaboration and is unprecedented so far, and has achiever world wide recognition.
• The knowledge gain in this programme has increased the competitiveness of the European Research Area on the role of tumor microenvironment in pancreatic cancer.
• The programme has stimulated innovation by ensuring close and productive interactions between groups with expertise in a range of areas that we consider central for progress in cancer research and medicine.
• The combination of multiple levels of expertise from the participating groups has fostered reverse-translation of clinical findings in the genetically engineered mouse models for the development of novel therapeutic strategies targeting the tumour microenvironment.
• EPC-TM-Net has generated and characterized in-vitro and in-vivo models to study tumour-microenvironment interactions that will be of enormous value for the scientific community and industry as models for cross-sectional and longitudinal studies of carcinogenesis in the pancreas, and to test or validate diagnostic and therapeutic approaches.
• One further benefit of the breadth of expertise among the partners brought together in this programme was a wide dissemination of radically new technologies for the study of tumour-microenvironment interactions within the consortium.
• Novel compounds targeting the tumour microenvironment used alone or in combination with standard cytotoxic drugs such as gemcitabine have been tested in preclinical models and have partly entered clinical trials such as hyaluronidase.
• Clinical studies conducted in WP5 have established surrogate serum protein profiles for tumour stroma, in order to predict those patients who are likely to respond to conventional treatment, and those for whom entry to clinical trials of novel compounds may be a more appropriate option. EPC-TM-Net has thus provided additional benefit for pancreatic cancer patients by providing the means for personalised treatment schedules, that will be tested in future clinical trials.
• As contribution to generate opportunities for education and training this collaborative project provided opportunities for personnel exchange and training. This mobility contributed to elevate the educational and training standards of involved personnel, which has fostered their career opportunities.
To summarize the research programme of the individual WPs of EPC-TM-Net has effectively delivered:
1. novel experimental models to study the interplay between pancreatic tumor cells and the microenvironment
2. novel mechanisms representing the basis for the interaction of pancreatic tumor cell with the microenvironment
3. novel compounds for treatment targeting the interaction between tumor cells and microenvironment
4. novel ways of compound delivery specifically targeting cellular components forming tumor microenvironment
5. novel therapeutic strategies to overcome drug resistance by combined targeting of tumor cells and microenvironment.
6. novel opportunities for personalized therapy based on serum biomarkers specific for the tumor microenvironment
The main dissemination activities of EPC-TM-Net are:
Posters:
P1 UniMar: WNT5A mediates resistance to apoptosis in pancreatic cancer via NFATc2 (22/06/2011), EPC Magdeburg.
P1 UniMar: Identification of targets acting synergistically with erlotinib in pancreatic cancer using a kinome-wide loss-of-function screen (22/06/2011), EPC Magdeburg.
P1 UniMar: WNT5A - NFAT Signalkaskade reguliert die Therapieresistenz beim Pankreaskarzinom (14/09/2011), DGVS Leipzig.
P3 CNIO: LRH1/NR5A2, a link between pancreatitis and pancreatic cancer? (20/05/2012), Keystone Symposia “The Role of Inflammation during Carcinogenesis”, Dublin, Ireland.
P3 CNIO: Mnk1 kinase is part of the pancreatic acinar transcriptional programme required for tissue homeostasis upon stress (25/05/2012), The 3rd EMBO Conference Series on Cellular Signaling & Molecular Medicine, Dubrovnik, Croatia.
P3 CNIO: Mitochondrial metabolism, the Achilles heel of pancreatic cancer stem cells (18/05/2014), AACR special conference on pancreatic cancer, New Orleans, LA, USA.
P5 EMAU-G: Cathepsin B and D drives the fibrinogenic potential of pancreatic ductal adenocarcinoma (1/11/2013), Miami, USA.
P8 TUM-MED: Definition of the Kras-induced “switch-on” in malignant transformation to pancreatic cancer (20/10/2012), DPC Leipzig, Germany.
P13 CERMS-UniTO: Mouse to human autoantibody signature in pancreatic ductal adenocarcinoma (21/06/2011), 6th ItPA (Italian Proteomics Association) Annual National Congress, Torino, Italy.
P13 CERMS-UniTO: Mouse to human autoantibody signature in pancreatic ductal adenocarcinoma (04/09/2011), HUPO 10th Annual World Congress, Geneva, Switzerland.
P13 CERMS-UniTO: Characterization of circulating autoantibodies to post-translationally modified alpha-enolase in pancreatic ductal adenocarcinoma (04/09/2011), HUPO 10th Annual World Congress, Geneva, Switzerland.
P13 CERMS-UniTO: Autoantibodies against Ser-419-phosphorylated alpha-enolase are more frequent in DRB1*08 pancreatic ductal adenocarcinoma patients: can it improve overall survival? (31/05/2012), 16th International HLA and Immunogenetics Conference, Liverpool, UK.
P13 CERMS-UniTO: Does Th17 response be harnessed by the antigen DNA vaccination against pancreatic adenocarcinoma? (06/02/2012), Keystone Symposia: Th17 cells in health and disease. Keystone, Colorado, USA
.
P13 CERMS-UniTO: Autoantibody Signature in pancreatic Ductal Adenocarcinoma (18/06/2012), Pancreatic cancer: progress and challenges; Lake Tahoe NV; USA.
P13 CERMS-UniTO: Effects of alpha-enolase silencing in a pancreatic adenocarcinoma cell line (18/06/2012), Pancreatic cancer: progress and Challenges. Lake Tahoe NV, USA.
P17 FCSR: Survivin vaccine for pancreatic adenocarcinoma (29/11/2013), San Raffaele Scientific Institute, Milan, Italy.
Presentations:
P1 UniMar: Cux1 stimulates pancreatic cancer progression by modulating the nfκb-dependent cytokine expression in tumour-associated macrophages (20/10/2012), Ueg week Amsterdam, The Netherlands.
P1 UniMar: Identification of targets acting synergistically with erlotinib in pancreatic cancer using a kinome-wide loss-of-function screen (20/10/2012), Ueg week Amsterdam, The Netherlands.
P1 UniMar: Zell-basierte Hochdurchsatz-Analysen identifizieren ADRBK1 als neues Zielgen im Pankreaskarzinom (11/09/2013), DGVS Nürnberg, Germany.
P1 UniMar: Einfluss der pharmakologischen Makrophagendepletion auf die Pankreaskarzinogenese in vivo (11/09/2013), DGVS Nürnberg, Germany.
P1 UniMar: KRAS-dependent signalling modulates the activation of infiltrating macrophages associated with a distinct miRNA profile in pancreatic cancer (23/01/2014), DPC Mannheim, Germany.
P2 ULIV: Combined LC/MS-MS and iTRAQ Analysis of Serum and Pancreatic Juice for the Detection of Biomarkers for Pancreatic Cancer: Influence of Biliary Obstruction (05/11/2011), Chicago, USA.
P2 ULIV: Periampullary cancer ESPAC 3-three trial: a randomised control phase 3 trial of adjuvant chemotherapy versus observation in patients with Periampullary adenocarcinomas of the head of the pancreas (05/11/2011), Chicago, USA.
P2 ULIV: Quality control of Protein Biomarkers: ECMC Annual Network Meeting (01/05/2012), Wellcome Collection, London.
P2 ULIV: Nucleotide transporters and dCK1 as prognostic markers for patients with resected pancreatic ductal adenocarcinoma with or without adjuvant gemcitabine or 5FU hENT1 tested with samples from patients randomized in the ESPAC1/3 trials (21/06/2012), Prague, Czeck Republic.
P2 ULIV: Role of S100A8/A9 in the Cross-talk between Cancer Cells and Stroma-associated Cells (22/06/2012), Prague, Czeck Republic.
P2 ULIV: The EUROPAC Registry and the EUROPAC 2 clinical trial (23/06/2012), Prague, Czech Republic.
P2 ULIV: Quantification of Pancreatic Ductal Adenocarcinoma Stroma (01/10/2013), Society of Great Britain and Ireland (Pancsoc), Liverpool, UK.
P2 ULIV: iTRAQ reveals a Serum Marker significantly altered in Patients up to 24 months prior to diagnosis with Pancreatic Cancer (31/10/2013), American Pancreatic Association Conference, Miami, USA.
P2 ULIV: Biomarkers in pancreatic cancer (29/11/2013), 39th Annual Meeting of the Pancreatic Society of Great Britain and Ireland, Liverpool, UK.
P2 ULIV: Involvement of microRNAs in the activation of cancer-associated pancreatic stellate cells (22/11/2013), Pancreatic Cancer Research Fund Supporters Conference, London, UK.
P2 ULIV: Modelling of Pancreatic Cancer (21/05/2013), QuasiVivo meeting, Liverpool, UK.
P2 ULIV: High hENT1 Expression is Predictive of Response to Adjuvant Gemcitabine for Pancreatic Ductal Adenocarcinoma Patients Randomised to Adjuvant Therapies in the ESPAC Trials (03/11/2012), American Pancreatic Association, Miami, USA.
P2 ULIV: Development of a Novel Next Generation Sequencing (NGS) Method for Detection of Low Concentration Sequence Variants (29/11/2013), 39th Annual Meeting of the Pancreatic Society of Great Britain and Ireland, Liverpool, UK.
P3 CNIO: Metformin targets the metabolic Achilles heel of pancreatic cancer stem cells (19/06/2012), Lake Tahoe, California.
P3 CNIO: Prospective isolation and characterization of circulation cancer stem cells (19/06/2012), Lake Tahoe, California.
P3 CNIO: Pancreatic cancer molecular pathophysiology (21/06/2012), MDACC Madrid Conference, Madrid, Spain.
P3 CNIO: Exploiting Acute Experimental Pancreatitis Models to Identify Novel Genes Involved in Exocrine Physiology and Pancreatic Cancer (25/06/2012), National Institute for Digestive Diseases and Kidney (NIDDK) workshop, Bethesda, USA.
P3 CNIO: Advanced Avatar models for studying cancer stem cell dynamics in human pancreatic ductal adenocarcinoma (05/11/2013), AACR Translational Impact of Model Organisms in Cancer Program, San Diego, USA.
P3 CNIO: Stem cells: Understanding the origins of pancreatic cancer (02/02/2014), Keystone conference - Stem Cells and Cancer, Banff, Canada.
P5 EMAU-G: Amino-acid depletion and growth factor depletion induce autophagy via different signaling pathways (20/06/2012), EPC Prague, Czech Republic.
P5 EMAU-G: Die Autophagie im Pankreaskarzinom ist unabhängig vom klassischen Beclin1-abhängigen Signalweg (17/11/2011), DPC Marburg, Germany.
P5 EMAU-G: The influence of the inhibition of pancreatic and neutrophil elastase on tumor growth in a syngenic orthotopic tumor model (17/11/2011), DPC Marburg, Germany.
P5 EMAU-G: Superparamagnetic nanoparticles (MNPS) coupled to siPLK1 represents a novel treatment option for pancreatic ductal adenocarcinoma (14/10/2013), Berlin, Germany.
P5 EMAU-G: Cathepsin B and D drives the fibrinogenic potential of pancreatic ductal adenocarcinoma (24/06/2014), Southampton, UK.
P5 EMAU-G: Superparamagnetic nanoparticles (MNPS) coupled to siPLK1 represents a novel treatment option for pancreatic ductal adenocarcinoma (27/06/2013), Zurich, Switzerland.
P8 TUM-MED: Comparative analysis of the microenvironment switch in early stages of pancreatic carcinogenesis (20/10/2012), DPC Leipzig, Germany.
P8 TUM-MED: Molecular/phenotypic dissection of oncogenic mTOR signaling in pancreatic cancer (16/01/2014), DPC Mannheim, Germany.
P8 TUM-MED: Tumor-promoting microenvironment is featured by expansion of pancreatic progenitors, mesenchymal cells and inflammation (16/01/2014), DPC Mannheim, Germany.
P13 CERMS-UniTO: Autoantibodysignature in pancreaticductal adenocarcinoma (18/11/2011), Riunione scientifica della Scuola Dianzani. Sassari, Italy.
P13 CERMS-UniTO: Alla ricerca degli anticorpi che spiano i tumori (14/04/2014), Liceo Scientifico Galileo Ferraris, Turin, Italy.
P16 TUM: Tenascin-C induces functional changes on pancreatic cancer cell lines (23/11/2012), DPC (German Pancreatic Club) 2012, Leipzig, Germany.
P16 TUM: Collagen type V influences the tumor microenvironment in pancreatic cancer (24/05/2013), DGP (Annual Meeting of the German Society of Pathology) 2014, Heidelberg, Germany.
P16 TUM: Collagen type V supports the malignant phenotype of pancreatic cancer cell lines through the integrin signaling pathway (28/06/2013), EPC (European Pancreatic Club) 2013, Zurich, Switzerland.
P16 TUM: Collagen type V promotes the malignant phenotype of pancreatic cancer (24/01/2014), DPC (German Pancreatic Club) 2014, Mannheim, Germany.
Organisation of Workshops:
P1 UniMar: Biologie-Schwerpunkt-Kurs (21/12/2011), University of Marburg.
P1 UniMar: Biologie-Schwerpunkt-Kurs (28/01/2013), University of Marburg.
P2 ULIV: Biobanking and Bioinformatics (B3i) Workshop (15/06/2012), University of Liverpool, UK.
P10 CR-UK: Mouse Hospital – Palpations and Ultrasound imaging in pancreatic cancer mice (10/04/2011), Cambridge CRUK.
Articles published in the popular press:
P6 KI: More research money will improve the bad prognosis of pancreatic cancer (26/05/2012), Sweden.
P13 CERMS-UniTO: Tumore al pancreas, nel futuro c’è un vaccine (02/07/2014), La Stampa, Torino, Italy.
Websites/Applications:
P15 concentris: Project website: www.epcnet.eu (22/06/2011).
The main exploitable results of EPC-TM-Net are:
P3 CNIO: Novel treatment strategy for pancreatic cancer: Development of a novel treatment strategy with combined targeting of the cancer stem cell niche and the cancer stem cells directly. This results in dramatically increased treatment response in preclinical models (01/10/2011), sector: cancer, for 5 years, EP 11382337.1-2107.
P8 TUM-MED: General advancement of knowledge: The molecular characterization of early pancreatic carcinogenesis is instrumental to design combinational treatments for chemoprevention in high-risk individuals. These pharmaceutical interventions include inhibitions of mesenchymal cells and progenitors proliferations as well as anti-inflammation therapies: Understanding the underlying mechanisms of tumor-stroma interaction accomplished by expansion of mesenchymal cells might provide researchers with new therapeutic strategies against pancreatic cancer. Our generated data-set could serve as a basis for following studies explaining the role of tumor stroma more in detail where the detection of new therapeutic targets could be implemented. Additionally, the identified inflammatory signature from our data might be useful for high-risk patient stratification as well as therapy prediction (27/06/2014), sector: preclinical studies, until 2017.
P14 NatiMab: Diagnostic and prognostic marker, therapeutic target: NatiMab Therapeutics studied the anti-cancer immunity developed by patients with PDAC. It became evident that Ezrin (EZR) and the phosphorylated form of α-Enolase of isotype 1 and 2 (ENOA) are pancreas Tumor Associated Antigens (TAAs). EZR is over-expressed by pancreas tumor cells and stimulates the patient production of specific antibodies that are a very early tumor marker. The blood samples from a prospective study show that circulating antibodies to EZR are found in patients much before the clinical diagnosis is possible, when the tumor is still localized and potentially resectable, In mice that spontaneously develop pancreas tumor, EZR is overexpressed by tumor in situ and the production of antibodies to EZR is evident before the cancer is detectable and becomes metastatic. ENOA is over-expressed by pancreas tumor cells. The antibodies to ENOA are found in patient sera and their presence correlates with a slower disease progression upon anti-cancer treatment. The presence of the antibodies against EZR is a marker of the initial stage of PDAC, while concurrent blood positivity of the antibodies against EZR and ENOA is of general importance for diagnosis of PDAC. Further, the concurrent presence in the blood of these antibodies together with CA19.9 detects PDAC with 100% sensitivity and 93.7% specificity. NatiMab is considering the industrial development and validation of two novel immunodiagnostic assays based on the two markers cited above, recently identified as specific markers of early stage pancreatic cancer, EZR, and its treatment follow up, ENOA. The detection of circulating antibodies to Ezrin, and to ENOA will consent an unquestionable diagnosis of PDAC while the detection of antibodies to Ezrin shall help the screen of population associated to a major risk to develop PDAC such as people with a recent diagnosis of type II diabetes, people with chronic pancreatitis and people belonging to a family with cases of PDAC. (31/12/2014), sectors: oncology, pancreatic tumors, minimum 3 years, commercialization of kit, license of patent, license of kits’ commercial rights.
P16 TUM: General advancement of knowledge: The molecular characterization of collagen V-dependent pathways: The molecular characterization of collagen V-dependent pathways in early pancreatic carcinogenesis and advanced pancreatic cancer is instrumental to identify specific diagnostic markers for molecular imaging and for the design of combinational cancer treatments (01/01/2015), sector: preclinical studies, starting from 2018.
List of Websites:
The address of the public website is: www.epcnet.eu
The contact details of the scientific representative of the project's co-ordinator are:
Prof. Dr. Thomas Gress
Philipps Universitaet Marburg
Dept. of Gastroenterology
Baldingerstrasse
D-35043 Marburg
Germany
Tel: +49 (0) 6421 58 66459
Fax: +49 (0) 6421 58 68922
E-mail: gress@med.uni-marburg.de
Pancreatic cancer is one of the most lethal human cancers with a five-year survival rate of less than 5%. Late presentation and a high level of resistance to chemotherapeutic drugs are among the major reasons for this dismal prognosis. The presence of the highest degree of desmoplasia among all solid tumours and the fact that chronic inflammatory pancreatic disease is associated with an increased risk for pancreatic cancer indicate that the tumour microenvironment is of particular importance for carcinogenesis in the pancreas. The long-term objective of this project is to increase survival of pancreatic cancer patients by exploring the contribution of the tumour microenvironment to the failure of presently available oncological treatments.
EPC-TM-Net represents a coordinated effort to structure and integrate the Europe-wide research into the pancreatic cancer microenvironment. The programme was specifically designed to systematically elucidate the impact of the tumour microenvironment on tumour progression and translate it into clinical applications and to furnish a suite of new compounds and therapeutic strategies targeting different components of the tumour microenvironment, which will directly benefit the patient, advance the knowledge on the molecular basis of the disease and has the potential to foster health economy in Europe.
Overall we have succeeded in:
• Establishing a structured and comprehensive research platform to study the impact of the microenvironment on the dismal prognosis and drug resistance of pancreatic cancer clearly requires pan-European collaboration and is unprecedented so far, and has achiever world-wide recognition.
• The knowledge gain in this programme has increased the competitiveness of the European Research Area on the role of tumor microenvironment in pancreatic cancer.
• The programme has stimulated innovation by ensuring close and productive interactions between groups with expertise in a range of areas that we consider central for progress in cancer research and medicine.
• The combination of multiple levels of expertise from the participating groups has fostered reverse-translation of clinical findings in the genetically engineered mouse models for the development of novel therapeutic strategies targeting the tumour microenvironment.
• EPC-TM-Net has generated and characterized in-vitro and in-vivo models to study tumour-microenvironment interactions that will be of enormous value for the scientific community and industry as models for cross-sectional and longitudinal studies of carcinogenesis in the pancreas, and to test or validate diagnostic and therapeutic approaches.
• One further benefit of the breadth of expertise among the partners brought together in this programme was a wide dissemination of radically new technologies for the study of tumour-microenvironment interactions within the consortium.
• Novel compounds targeting the tumour microenvironment used alone or in combination with standard cytotoxic drugs such as gemcitabine have been tested in preclinical models and have partly entered clinical trials such as hyaluronidase.
• Clinical studies conducted in EPC-TM-Net have established surrogate serum protein profiles for tumour stroma, in order to predict those patients who are likely to respond to conventional treatment, and those for whom entry to clinical trials of novel compounds may be a more appropriate option. EPC-TM-Net has thus provided additional benefit for pancreatic cancer patients by providing the means for personalised treatment schedules, that will be tested in future clinical trials.
• As contribution to generate opportunities for education and training this collaborative project provided opportunities for personnel exchange and training. This mobility contributed to elevate the educational and training standards of involved personnel, which has fostered their career opportunities.
Project Context and Objectives:
Pancreatic cancer belongs to the most lethal human cancers with a 5-year survival rate of less than 5%. Late presentation and a high level of resistance to chemotherapeutic drugs are among the major reasons for this dismal prognosis.
To date, most efforts to identify new therapeutic targets for pancreatic cancer have focused on tumour cells themselves or on the tumour as a whole. Histologically, however, pancreatic cancer consists of a heterogeneous population of tumour cells including cancer stem cells that are highly resistant to standard chemotherapy and an extremely high proportion of desmoplastic stromal tissue surrounding the tumour cells, which accounts for up to 90% of the tumour area.
Evidence accumulated during recent years for multiple tumours has clearly demonstrated that the tumour microenvironment comprising stroma, blood vessels, infiltrating inflammatory cells and a variety of associated tissue cells are of paramount importance in regulating proliferating tumour cells including a subpopulation of cancer stem cells and thus determine tumour aggression and chemoresistance.
The presence of the highest degree of desmoplasia among all solid tumours and the fact that chronic inflammatory pancreatic disease is associated with an increased risk for pancreatic cancer in both human and mouse models indicate that the tumour microenvironment seems to be of particular importance for carcinogenesis in the pancreas.
The overall objective of EPC-TM-Net therefore was to improve survival of pancreatic cancer patients by exploring the contribution of the tumour microenvironment to the failure of presently available oncological treatments.
The limited knowledge, coupled with the likelihood that stroma plays a fundamental role in tumour progression and therapeutic responsiveness, demands concerted and integrated efforts to dissect the molecular and biological evolution and characteristics of PDAC stroma and its functional relationship to the cancer cell compartment. In view of the many scientific advances in the understanding of PDAC that have occurred over the past decade, the field appears poised to exploit many of the genomic technologies and model systems used to advance our understanding of the PDAC cancer cell in the area of stromal biology. This is a daunting challenge in light of stroma’s many components and multiple heterotypic interactions among tumour fibroblasts/mesenchymal cells, blood vessels, innate and adaptive immunocytes, and the ECM. Thus an interdisciplinary, multicentric and Pan-European academic and industrial approach as concentrated in EPC-TM-Net was necessary to tackle this enormous challenge.
Based on the eminent role of the tumour stroma, an intriguing novel approach beyond current therapeutic concepts is to primarily target the stromal components and directly or indirectly the cancer stem cell compartment, rather than the tumour cells as a whole. This could abolish the growth advantage for the tumour cells originating from stromal components and possibly facilitate the accessibility of the tumour cells for chemotherapeutic agents after tackling the stromal “belt” surrounding the tumour cells. The first evidence that targeting tumour stroma does indeed reduce tumour progression by enhancing the local accessibility of chemotherapeutic drugs was provided by a member of this consortium prior to the start of the consortium. In a mouse model of pancreatic cancer elimination of the tumour stroma by inhibition of hedgehog signalling significantly enhanced delivery and efficacy of chemotherapeutic treatment (Olive et al 2009).
Many questions relating to the revolutionary new concept of targeting stromal components to improve survival of pancreatic cancer patients are still to be addressed and were the focus of the EPC-TM Net consortium:
1) On a molecular level, the role of the individual stroma-related cell types and components including cancer stem cells and the networking between each other remain to be defined.
2) The interplay and functional relevance of each stromal cell component has to be verified in appropriate mouse models.
3) New drug targets affecting the functional integrity of the stromal networks have to be identified by candidate approaches or high-throughput screens or newly generated hypotheses and validated in-vivo.
4) Specific drugs for the identified targets have to be developed and validated preclinically in-vitro and in-vivo in mouse models of pancreatic cancer.
5) Cell type specific drug delivery needs to be established to specifically modulate the crosstalk between stroma and cancer cells.
Project Results:
Summary of the main results/foreground of EPC-TM-Net:
The long-term objective of this proposal was to increase survival of pancreatic cancer patients by exploring the contribution of the tumour microenvironment to the failure of presently available oncological treatments. For this purpose we reverse-translated this clinical observation into innovative in-vitro and mouse models closely mimicking the human disease. Work was done in the context of 5 scientific work packages that focused on different compartments or components of the microenvironment.
Within work package 1 (WP01) several groups focused on the role and the targeting of fibroblastic cells in the stroma. One of the groups at the CNIO in Madrid (Real et al) has newly generated mouse strains to allow for the selective killing of pancreatic stellate cells, regardless of their tissue distribution. This mouse strain will give the unique possibility to study the influence of the stellate cells in the PDAC microenvironment on processes such as tumor progression and chemotherapy resistance. The group at CNIO has furthermore established the completely new concept that two different transcription factors (Gata6 and Nr5a2) have direct links between epithelial differentiation and suppression of inflammation.
The group at TUM in Munich studied histological patterns and molecular signatures of different cellular compartments in wild type and KrasG12D-transgenic mice during acute pancreatitis and found that the regeneration of the adult pancreas from acute pancreatitis is a default process that is divided into three distinctive phases: inflammation, organ regeneration and refinement. In contrast to the wild type KrasG12D, pancreata were remodelled by pre-neoplastic lesions showing extended phase of inflammation with impaired acinar cell proliferation (e.g. organ regeneration). Using sophisticated bioinformatics approaches, they have furthermore identified molecular signatures associated with the pro-tumorigenic effect of inflammatory stroma in mice and validated them in humans.
The group at KI in Stockholm developed a 3D heterospheroid model comprising both human pancreatic cancer cells and the organ-residing mesenchymal cells, the pancreatic stellate cell (PSC) into one avascular minitumor, to study the effect of the different cells on chemoresistance. The assay was adapted to the 384-well format to prepare for high-throughput drug screening, allowing fully automated screening approach of libraries with more than 100.000 compounds.
Within work package 2 (WP02) several groups aimed at targeting inflammatory cells in pancreatic cancer stroma that comprise tumour-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), lymphocytes and other immune or immunomodulatory cells that are essential for cancer growth. The group in Marburg analysed the in-vitro interaction of TAMs and cancer cells in organotypic cultures and saw that pancreatic cell lines secrete a distinct panel of proinflammatory cytokines in a K-Ras dependent manner. This cytokines modulated macrophage infiltration and phenotypic polarisation towards a tumor-promoting M2-action. Analysis of the phenotypical and functional properties of isolated TAMs in genetically-engineered mouse models of pancreatic cancer and human tissues revealed TAM polarisation towards a tumor-promoting M2 phenotype already in PanIN pancreata. This was accompanied by distinct miRNA alterations in TAM that were shown to enhance secretion of proinvasive and proangiogenic cytokines, thereby creating a positive feedback loop and enhancing tumor progression. Finally the group in Marburg studied the effect of smoking, a strong risk factor for pancreatic cancer in humans, on development of inflammatory stroma, preneoplastic lesions and invasive cancer in genetically-engineered mouse models, and found that the incidence and progression of preinvasive lesions was markedly accelerated after smoke exposure, whereas progression to invasive tumors did not increase. Study of miRNA-signature changes in the mouse model identified a panel of miRNAs which are differentially regulated during tumor progression and influence NFkB and STAT signalling thereby defining macrophage action. Moreover we validated a significant MDSC-subset accumulation (MDSC1, MDSC2, MDSC4) in pancreatic patients compared the control (sex and age-matched healthy donors) that confirm MDSC presence as a potential prognostic marker in pancreatic cancer. Finally the group developed a promising preclinical therapeutic intervention targeting TAMs in KPC-mice using liposomal clodronate which led to a marked decrease in metastasis formation.
The groups CERMS-UniTO, UniVR, NatiMab, ULIV validated DNA-based vaccination strategies in KC and KPC-mice. ENO1 vaccine was able to significantly prolong mouse survival and decreased the percentage of the transformed ducts compared to normal ducts as well the size of transformed ducts, and thus represent a promising candidate target for DNA vaccination in pancreatic cancer.
In work package 3 (WP03) aimed at targeting the stromal components in the microenvironment and cancer stem cells residing therein. To this end, the groups in Marburg and Cambridge aimed to target inert and soluble components of the tumor stroma in order to break down the ECM scaffold relieve vessel compression and increase drug delivery to hypovascular pancreatic tumors. One interesting example was the use of human recombinant PEGylated hyaluronidase (PEGPH20), n murine pancreas tumors resulted in the re-expansion of blood vessels and improved drug delivery accompanied by slowing of tumor growth and prolonged survival in genetically engineered mice (GEMM) (Jacobetz et al. Gut 2013). Several preclinical studies on secreted protein acidic and rich in cysteine (SPARC) which is overexpressed by cancer associated fibroblasts (CAFs) were done. Furthermore, they studied the efficacy and mechanism of action of nab-paclitaxel, a novel, albumin-formulated chemotherapy that holds great promise for PDA patients, and found a remarkable therapeutic efficacy for nab-paclitaxel in highly treatment resistant GEMM (Frese et al. Cancer Discovery 2012). Therapeutic inhibition of Connective tissue growth factor (CTGF) using a monoclonal human antibody FG-3019 resulted in a significantly increased induction of tumor cell apoptosis in GEMMs when combined with gemcitabine (Neesse et al. PNAS 2013). In addition to these novel experimental therapeutic avenues they also further characterized the stromal-targeting mechanisms of approved multi-targeting agents such as regorafenib to elucidate the molecular alterations in the tumor cell – stroma crosstalk following targeted therapy approaches. The group of C. Heeschen at CNIO in Madrid identified a strong bilateral interaction between CSCs and tumour-associated macrophages. Targeting this cross-talk between the PDAC stroma (e.g. TAMs) and PDAC CSC is possible and bears significant therapeutic value.
For tumour cells to physically invade into external environment, proteolytic degradation is required. With work package 4 (WP=4) the group in Greifswald studied the possibility to target proteases, in particular Cathepsins, involved in tumour-stroma cross-talk. Cathepsins, chemically cysteine- and aspartic proteases which may be implicated in processes important for tumour development and progression of pancreatic ductal adenocarcinoma. Cathepsin B, L and D are upregulated in solid tumours but exert distinct roles in multistage tumourigenesis. They found that cathepsin D expression and serum cathepsin D activity in tumour resected patients is negatively correlated with the poor post-resection survival in patients with pancreatic ductal adenocarcinoma. Furthermore, loss of cathepsin B confers a survival advantage to mice with PDAC. This benefit is associated with decreased tumour proliferation and liver metastasis in tumour-bearing mice. Suggesting that cathepsin B is a logical target for future drug development process. The use of siRNAs that allow specific intervention at single molecular stages of the disease may provide a potential therapeutic agent to target the expression of genes such as proteases. The group has developed a novel formulation that not only delivers the siRNAs adequately and specifically to the tumour site but also allows non-invasive imaging of the uptake of our therapeutic agent, based on dextran coated superparamagnetic nanoparticles. Administration of these nanoparticles in tumor-bearing mice allowed monitoring of delivery of the agent to the tumour and metastasis by MRI imaging and results in efficient silencing of the target genes. This approach can significantly advance the therapeutic potential of siRNAs by providing a way not only to effectively shuttle siRNA to target sites but also to noninvasively access the bioavailability and efficacy of the siRNA at tumour site.
In work package 5 (WP05) aimed at identifying serum markers of stromal biology, since currently there is no effective mechanism to qualitatively assess the nature of the tumoral stroma in a patient without surgically removing the tumour. Since the majority of pancreatic cancer patients present with advanced cancer, removal of the tumour is not an option. Therefore, personalised treatment, based on an assessment of an individual’s stroma is not possible, but would be of paramount importance in view of the findings that stromal components have a major role in mediating resistance to therapy. Markers of stromal biology that can be readily assayed are required as surrogate markers, to predict those patients who are likely to respond to conventional treatment, and those for whom entry to clinical trials of novel compounds may be more a more appropriate option. Using the Luminex Platform they observed many associations between features in the stroma and circulating levels of serum cytokines. However all of these associations were weak, suggesting that serum cytokines are not likely to provide a good surrogate for the type of stroma present in pancreatic tumours. Interesting additional observations include the finding that the presence of large blood vessels within the tumour correlates with poor patient outcome in two independent patient cohorts. Our data from these analyses will be published shortly.
Main results/foreground of the different work packages:
WP01: Targeting the stroma: fibroblastic cells
Here, we have newly generated mouse strains to allow for the selective killing of pancreatic stellate cells, regardless of their tissue distribution. Firstly, we have generated through BAC transgenesis a line expressing Flp recombinase in the Sm22a locus; we have shown the specific activity of the recombinase using a reporter Alkphos strain. Secondly, we have generated a knockin construct and mouse strain expressing HBEGF (DT receptor) in the GFAP locus, to be conditionally expressed upon deletion of a Frt-STOP-Frt cassette. The murine embryo fibroblasts derived from this line can be infected with adenovirus-flipase to delete the STOP cassette and allow DTR expression which, in turn, renders the cells sensitive to DT. These proofs of concept experiments have been successfully completed in vitro. The two strains have been crossed and stellate cells from the compound strains have been placed in culture; the same experiments of recombination are ongoing (delayed due to the need to generate the BAC transgene). Crosses with mutant KrasG12V established and experiments will be performed to first establish stellate cell activation and then test the concept.
As of inflammation, we have established for two different transcription factors (Gata6 and Nr5a2) direct links between epithelial differentiation and suppression of inflammation, a completely new concept. The relevance of this link is shown by the fact that inactivation of either of these genes (in the case of Nr5a2, haploinsufficiency) reveals a tumor suppressor effect of these transcription factors in the context of the KrasG12V model.
In this study, we furthermore found that the regeneration of the adult pancreas from acute pancreatitis is a default process that is divided into three distinctive phases: inflammation, organ regeneration and refinement. The inflammatory phase is characterized by immune cell infiltration, proliferation of pancreatic progenitors and mesenchymal cells whereas the organ regeneration phase is predominantly characterized by acinar cell proliferation. In contrast, KrasG12D pancreata were remodelled by pre-neoplastic lesions showing extended phase of inflammation with impaired acinar cell proliferation (e.g. organ regeneration). An extensive bioinformatical analysis of expression profiles confirmed these histological findings. In an inducible mouse system, we further demonstrated that induction of KrasG12D and p53 inactivation during the inflammatory phase caused formation of pre-neoplastic cells whereas the induction of genetic events during the regenerating phase had no such effect. Using sophisticated bioinformatics approaches, we have isolated molecular signatures associated with the pro-tumorigenic effect of inflammatory stroma. These molecular signatures consist of activation of Notch pathways within pancreatic epithelial compartment, NF-κB-mediated cytokine networks within inflammatory compartment and proteinase cascades and retinoid acid signals within mesenchymal compartment. The relevance of these molecular signatures has been validated using human samples.
With the hypothesis being that the stroma plays an active role in chemoresistance, we further developed our 3D monspheroid model into a 3D heterospheroid model comprising both human pancreatic cancer cells and the organ-residing mesenchymal cells, the pancreatic stellate cell (PSC) into one avascular minitumor. In contrast to the hypothesis, these heterospheroids did not demonstrate a significantly increased chemoresistance when compared with the 3D monospehroids for the vast majority of compounds. Some few drugs showed lower efficacy, though (allicin and derivatives). For this drug, the addition of macrophages further increased chemoresistance. A possible explanation for these findings could be that the stromal contribution to chemoresistance is made up by several other factors not represented in our model such as physical tissue pressure leading to compressed non-functional vessels and the lack of fibrillous collagen in vitro (despite expression of lysine-2-monooxygenase and addition of vit. C).
To prepare for high-throughput drug screening, the existing 96-well assay was adapted to the 384-well format allowing fully automated screening approach. We have libraries totalling more than 100,000 compounds at hand. We tested a library with some 3,000 natural compounds (TimTec).
WP02: Targeting the stroma: inflammatory components
Inflammatory cells are prevalent in pancreatic cancer stroma and include tumour-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), lymphocytes and other immune or immunomodulatory cells that are essential for cancer growth. The molecular crosstalk between inflammatory cells and tumour cells remains to be elucidated, which was one of the major aims of this work package.
The main results of WP2 are:
1. Analysis of the in-vitro interaction of TAMs and cancer cells in organotypic cultures: To this extent, pancreatic preneoplasic and neoplastic cell lines were co-cultured with primary human and murine macrophages as well as macrophage cell lines in organotypic cultures. We saw that pancreatic cell lines secrete a distinct panel of proinflammatory cytokines in a K-Ras dependent manner. This cytokines modulated macrophage infiltration and phenotypic polarisation towards a tumor-promoting M2-action. This was accompanied by transcriptomic changes, in particular miRNA regulations. This miRNA changes also enhanced the protumoral phenotype of the macrophage.
2. Analysis of the phenotypical and functional properties of isolated TAMs in genetically-engineered mouse models of pancreatic cancer and human tissues: TAMs and macrophages from normal pancreas, pancreata with preneoplastic PanIN lesions and invasive pancreatic cancers in the KC (LSL-KrasG12D/+, Pdx-1-Cre) and KPC mouse models (LSL-KrasG12D/+, LSL-TP53R172H/+;Pdx-1-Cre) were isolated by flow cytometry at defined time points. Transcriptional alterations compared to macrophages from normal pancreas were evaluated. TAM polarisation was altered towards a tumor-promoting M2 phenotype already in PanIN pancreata. This was accompanied by distinct miRNA alterations. Several of these miRNAs in TAM were shown to enhance secretion of proinvasive and proangiogenic cytokines, thereby creating a positive feedback loop and enhancing tumor progression.
3. Effect of smoking on development of inflammatory stroma, preneoplastic lesions and invasive cancer in genetically-engineered mouse models: Smoking is a strong risk factor for pancreatic cancer, and thought to be mediated at least in part via chronic inflammation. However, the exact mechanisms remain to be elucidated. We investigated the effect of prolonged exposure of KC mice to cigarette smoke in whole-body chambers. The incidence and progression of preinvasive lesions was markedly accelerated after smoke exposure. However, progression to invasive tumors did not increase, suggesting that more genetic hits are required irrespective of smoke exposure. No differences were detected in stroma composition or infiltration of inflammatory cells.
4. Identification of signalling pathways involved in tumor-stroma interactions in human pancreatic cancer by transcriptional profiling of human myeloid cells: This task was performed to identify crucial signalling nodes defining macrophage phenotype. We focussed on miRNA changes in the mouse model and identified a panel of miRNAs which are differentially regulated during tumor progression and influence NFkB and STAT signalling thereby defining macrophage action. Moreover we validated a significant MDSC-subset accumulation (MDSC1, MDSC2, MDSC4) in pancreatic patients compared the control (sex and age-matched healthy donors) that confirm MDSC presence as a potential prognostic marker in pancreatic cancer.
5. Development of preclinical therapeutic interventions targeting TAMs in KPC: The value of the modulation of TAMs effectors circuitries as a strategy to impair their tumor-promoting behaviour was evaluated in KPC mice. We depleted macrophages by liposomal clodronate which resulted in a marked decrease in metastasis formation and angiogenic response, suggesting that pharmacological modulation of macrophages is a promising novel therapeutic avenue.
6. Validation of DNA-based vaccination strategies in KPC-mice and development of therapeutic monoclonal antibodies (responsible CERMS-UniTO, UniVR, NatiMab, ULIV): We have used pVAX plasmid coding for ENO1 to vaccinate KC and KPC mice with both prophylactic and therapeutical setting protocol. ENO1 vaccine was able to significantly prolong mouse survival and decreased the percentage of the transformed ducts compared to normal ducts as well the size of transformed ducts. ENO1 vaccine elicited an integrated humoral and cellular response specific to ENO and significantly decreased the percentage of myeloid derived suppressor cells as well as of regulatory T cells. Thus, ENO1 is a promising candidate target for DNA vaccination in pancreatic cancer.
WP03: Targeting the stroma and cancer stem cells to improve treatment response
Pancreatic ductal adenocarcinoma (PDA) is a stroma-rich and highly complicated cancer to treat. Over the last decades it has become increasingly clear that the complex network extracellular matrix (ECM) components collaboratively interact within the tumor microenvironment sustaining and driving cancer cell proliferation, invasion and early metastasis. Histologically, PDA represents an extremely stroma-rich and hypovascular tumor, and indeed, most of the pancreatic tumor mass consists of activated (myo-) fibroblasts, immune cells and ECM components such as collagen, desmin, fibronectin and hyaluronic acid.
More recently, the tumor microenvironment has also been appreciated to mediate therapeutic resistance in PDA thus opening numerous avenues for novel therapeutic explorations. In WP3, we have focused to develop novel therapeutic strategies for pancreatic cancer targeting the tumor stroma. To this end, we aimed to target inert and soluble components of the tumor stroma in order to break down the ECM scaffold relieve vessel compression and increase drug delivery to hypovascular pancreatic tumors.
One interesting example is the enzymatic depletion of the glycosaminoglycan hyaluronic acid (HA) by human recombinant PEGylated hyaluronidase (PEGPH20). Pancreatic cancers are rich in the megadalton form of HA, and the solvation of water by HA is thought to be responsible for the extremely high interstitial fluid pressure in PDA that results in the compression of intratumoral blood vessels. In WP3 we could show that treatment with PEGPH20 in murine pancreas tumors results in the re-expansion of blood vessels and improved drug delivery accompanied by slowing of tumor growth and prolonged survival in genetically engineered mice (GEMM) (Jacobetz et al. Gut 2013).
Secreted protein acidic and rich in cysteine (SPARC) is overexpressed by cancer associated fibroblasts (CAFs) and represents another intriguing target in PDA. In WP3 we have conducted several preclinical studies regarding SPARC and the efficacy and mechanism of action of nab-paclitaxel, a novel, albumin-formulated chemotherapy that holds great promise for PDA patients. We found a remarkable therapeutic efficacy for nab-paclitaxel in highly treatment resistant GEMMs. Moreover, we identified a synergistic effect of nab-paclitaxel through the reactive oxygen species (ROS-) mediated degradation of the primary gemcitabine metabolising enzyme, cytidine deaminase without disrupting the tumor stroma (Frese et al. Cancer Discovery 2012).
Connective tissue growth factor (CTGF/CCN2) is a pleiotropic growth factor that is overexpressed in human and murine pancreas tumors by CAFs. Therapeutic inhibition of CTGF using a monoclonal human antibody FG-3019 resulted in a significantly increased induction of tumor cell apoptosis in GEMMs when combined with gemcitabine. Notably, neither stromal depletion nor increased drug delivery occurred. Rather, stromal derived CTGF impinged on the anti-apoptotic machinery in tumor cells, and the X-linked inhibitor of apoptosis protein (XIAP) was down-regulated upon treatment with FG-019. Also, co-treatment with FG-019 and gemcitabine resulted in slowing of murine tumors and extended survival in GEMM) (Neesse et al. PNAS 2013).
In addition to these novel experimental therapeutic avenues we also aim to further characterize the stromal-targeting mechanisms of approved multi-targeting agents such as regorafenib and to elucidate the molecular alterations in the tumor cell – stroma crosstalk following targeted therapy approaches.
Targeting the tumor microenvironment provides a novel, much needed avenue for novel treatment approaches. Given the long list of failed clinical trials in the past, it remains speculative which ECM targets and compounds will make the difference for patients with pancreatic cancer, however, we are hopeful that the continuous effort to translate these findings to the clinic will eventually benefit our patients.
Furthermore, we have identified a strong bilateral interaction between CSCs and tumour-associated macrophages. In response to secretion of TGFbeta family members (i.e. TGFbeta, Nodal and Activin) macrophages acquire an M2 phenotype including the secretion of several factors that in return enhance the phenotypes of cancer stem cells. Specifically, LL-37 and ISG15 are both strongly enhancing self-renewal of cancer stem cells and equip them with marked invasive capabilities translating into a highly metastatic phenotype. Targeting this cross-talk between the PDAC stroma (e.g. TAMs) and PDAC CSC is possible and bears significant therapeutic value.
WP04: Targeting proteases involved in tumour-stroma cross-talk
Despite the knowledge and insights into properties and genetic aberrations of malignant cells, today's therapy of pancreatic ductal adenocarcinoma remains incompletely effective and transitory. The hallmarks of cancer is the diversity of the different types of cells, accompanying to cancer cells forming intrinsic barriers and protective functions (Hanahan et al., 2000). It is now widely accepted that cancer cells do not manifest the disease alone, but rather with the help of recruit and corrupt resident normal cell types associated with cancer cells contribute in sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, reprogramming energy metabolism, and evading immune destruction. This tumour associated cells (stromal cells), namely Infiltrating immune cells, endothelial cells, cancer associated fibroblastic cells, pericytes etc., constitutes the "Tumour microenvironment" (TME) (Hanahan et al., 2012). Involvement of TME in remodelled extracellular matrix (ECM) and tumour angiogenesis has been an important aspect in resilience of cancer cells to therapy.
In order to physically invade into external environment, proteolytic degradation is required. Range of proteases produced by cancer cells as well as stromal cells contributes to the invasion and ECM remodelling (Joyce et al., 2009). The tissue-stroma reaction in the pancreas is one of the most prominent known in pathology (Gress et al., 1995; Lerch et al., 1999; Mayerle et al., 2003). The stromal cell derived proteases includes specific matrix metallo-proteases, cysteine-, aspartic- and serine proteases (Gocheva et al., 2007; Gocheva et al., 2006; Lynch et al., 2002).
Cathepsins, chemically cysteine- and aspartic proteases which may be implicated in processes important for tumor development and progression of pancreatic ductal adenocarcinoma. Cathepsin B, L and D are upregulated in solid tumours but exert distinct roles in multistage tumourigenesis. The exocrine pancreas secretes more proteins per cell than any other exocrine organ and much of these secreted proteins consist of these cathepsins. The biology of pancreatic ductal adenocarcinoma might therefore be more dependent on protease activity than solid tumours.
The traditional method of studying cancer cell performance in vitro has been two dimensional monolayer cultures. Although this has resulted in an enormous amount of information regarding genetic, biochemical and cell-biological processes involved in tumour formation, cells grown on flat 2D tissue culture systems differ considerably in their morphology, differentiation as well as their cell-cell and cell-matrix interactions compared to cells in biological system. In order to study transformed cell performance, and interactions with other cell types as well as with the stroma, in a model that is still relatively easily agreeable for experimental manipulation, more physiologically relevant 3D tissue culture models can provide a solution and bridge the gap between traditional 2D monolayer culture and the more complex animal models. We developed the three dimensional organotypic co-culture model which consists of cancer cells spheroids embed in collagen matrix along with pancreatic cancer stellate cells. This model enabled us to study interaction of stromal compartment with tumour cells and influence of cathepsin inhibition or knocked-out on modulation of stroma. We found out that cathepsin D expression and serum cathepsin D activity in tumour resected patients is negatively correlated with the poor post-resection survival in patients with pancreatic ductal adenocarcinoma. We found that loss of cathepsin B confers a survival advantage to mice with PDAC. This benefit is associated with decreased tumour proliferation and liver metastasis in tumour-bearing mice. This is the first report of extended survival due to a genetic deficiency in KPC model of advanced pancreatic cancer. Our observation that early morbidity is absent in cathepsin B null mice indicates that optimal tumor development requires the activity of cathepsin B. We therefore conclude that cathepsin B is a logical target for future drug development process.
Despite the knowledge and insights into biological properties and genetic aberrations of malignant cells, therapeutic options of pancreatic ductal adenocarcinoma remain incompletely effective and transitory. One plausible explanation for this futile response is insufficient delivery of anticancer drugs to the tumour site. The use of siRNAs that allow specific intervention at single molecular stages of the disease may provide a potential therapeutic agent. siRNAs virtually can be used to inhibit the expression of any gene. Major hurdles of In vivo delivery of siRNAs are the adequate and specific to site of interest delivery, stability of siRNAs in serum and non-invasive monitoring of the influence of siRNAs.
Figure 1, see upload: Schematic diagram of siPLK1 coupled streptavidin conjugated dextran coated magnetic nanoparticles (siPLK1-StAv-MNPs) conjugated to membrane translocation peptides (MPAP), underglycosylated MUC-1 specific peptide(EPPT-1) and siRNA molecules targeting PLK1 (siPLK1).
Keeping the limitations of classical pancreatic ductal adenocarcinoma therapy and In vivo delivery of siRNA in mind, we have developed a novel formulation that not only delivers the siRNAs adequately and specifically to the tumour site but also allows non-invasive imaging of the uptake of our therapeutic agent. In order to accomplish this, we designed dual purpose functional probes comprising of dextran coated superparamagnetic nanoparticles as a backbone which can be detected by magnetic resonance imaging (MRI) and which are further conjugated with streptavidin. Reflecting the fact of streptavidin and biotin as the strongest covalent binding, we coupled biotin conjugated myristoylated polyarginine peptides (MPAPs) for translocation of the complex into the cytosol by electrostatic interaction as well biotin conjugated EPPT-1, a non-immunogenic underglycosylated MUC-1 antigen highly specific for MUC-1 protein, (a trans-membrane protein, highly expressed in pancreatic ductal adenocarcinoma) which allows tumour specific intake. Furthermore, we linked biotin conjugated siRNA to the streptavidin anker to be used as a therapeutic target. Administration of these nanoparticles in tumor-bearing mice allowed monitoring of delivery of the agent to the tumour and metastasis by MRI imaging and results in efficient silencing of the target genes. This approach can significantly advance the therapeutic potential of siRNAs by providing a way not only to effectively shuttle siRNA to target sites but also to noninvasively access the bioavailability and efficacy of the siRNA at tumour site.
WP05: Serum markers of stromal biology
Pancreatic ductal adenocarcinoma (PDAC) has an exceptionally poor prognosis [1]. Pancreatic cancer treatment has been dominated for many years by two chemotherapeutic agents, 5-fluorouracil (5-FU) and gemcitabine, and although new regimens have been successful in selected patients, 5-FU and gemcitabine remains the mainstay for the vast majority of patients. Trials undertaken by the European Study Group for Pancreatic Cancer (ESPAC), co-ordinated by the Liverpool Clinical Trials Unit, Liverpool, UK have systematically evaluated the efficacy of clinically relevant adjuvant chemotherapy for pancreatic cancer patients [2-4]. ESPAC-1 reported a significant survival benefit for 5-FU chemotherapy, over observation only [2]. A subsequent trial, ESPAC-3(v2), compared adjuvant 5-FU plus folinic acid (551 patients) and gemcitabine (537 patients) [4]. This trial showed no significant difference in overall survival between gemcitabine and 5-FU-treated patients (median survival~ 23.0 months in both arms). The ESPAC-4 trial is already underway, exploring whether the addition of an oral fluoropyrimidine (capecitabine) to gemcitabine (GEM-CAP) enhances overall survival compared to gemcitabine alone. Novel therapeutic approaches are being actively sought to improve outcomes for patients with pancreatic cancer. Equally important however is improving the capacity to predict the patients that are likely to respond to existing therapies.
Tumoral stroma has emerged as a key factor in the resistance of pancreatic cancers to treatment. While this new found understanding offers many opportunities it also presents challenges: There is no effective mechanism to qualitatively assess the nature of the tumoral stroma in a patient without surgically removing the tumour. Since the majority of pancreatic cancer patients present with advanced cancer, removal of the tumour is not an option. Therefore, personalised treatment, based on an assessment of an individual’s stroma is not possible. Even when the primary tumour, along with its stroma, can be removed, this provides information on the nature of the stroma in the primary tumour, but does not provide direct data on the stroma at sites of recurrence.
Markers of stromal biology that can be readily assayed are required. We set out to find surrogate serum protein (including cytokine) profiles for tumour stroma, in order to predict those patients who are likely to response to conventional treatment, and those for whom entry to clinical trials of novel compounds may be more a more appropriate option.
Therefore, as part of EPC-TM-NET, a systematic analysis of stroma from two independent patient cohorts was undertaken. This was carried out to establish and categorise stroma in pancreatic cancer, regardless of treatment in the first instance. Markers examined are summarised in Table 1.
Table 1, sea upload. List of immunohistochemical markers used to assess stromal content.
Matched serum samples for these patient sets were then assessed for their cytokine levels using the Bio-Rad Bio-Plex 27 Human Cytokine, Chemokine and Growth Factor Kit (Luminex Platform). These serum protein levels then underwent Pearson ‘s rank correlation with stroma measures.
We observed many associations between features in the stroma and circulating levels of serum cytokines. For example, MIP-1b levels were significantly negatively correlated with CD68+ macrophage levels in the stromal compartment of the tumour in both discovery and validation patient cohorts. In addition, IL-6 levels were significantly correlated with the levels of of large lymphatic vessels in the tumour in both the discovery and validation patient cohorts. However all of these associations were weak, suggesting that serum cytokines are on likely to provide a good surrogate for the type of stroma present in pancreatic tumours.
Interesting additional observations include the finding that the presence of large blood vessels within the tumour correlates with poor patient outcome in two independent patient cohorts.
A major aim of WP5 was to establish surrogate serum protein profiles for tumour stroma, in order to predict patients’ response to treatment. This project analysed serum samples from clinical pancreatic cancer cases recruited in Europe’s largest pancreatic cancer clinical trials (ESPAC and Telovac). Our data from these analyses will be published shortly.
References:
1. Costello E, Greenhalf W, Neoptolemos JP. New biomarkers and targets in pancreatic cancer and their application to treatment. Nat Rev Gastroenterol Hepatol 2012.
2. Neoptolemos JP, Stocken DD, Friess H, Bassi C, Dunn JA, Hickey H, Beger H, Fernandez-Cruz L, Dervenis C, Lacaine F, Falconi M, Pederzoli P, Pap A, Spooner D, Kerr DJ, Buchler MW. A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer. The New England journal of medicine 2004; 350: 1200-10.
3. Neoptolemos JP, Stocken DD, Tudur Smith C, Bassi C, Ghaneh P, Owen E, Moore M, Padbury R, Doi R, Smith D, Buchler MW. Adjuvant 5-fluorouracil and folinic acid vs observation for pancreatic cancer: composite data from the ESPAC-1 and -3(v1) trials. British journal of cancer 2009; 100: 246-50.
4. Neoptolemos JP, Stocken DD, Bassi C, Ghaneh P, Cunningham D, Goldstein D, Padbury R, Moore MJ, Gallinger S, Mariette C, Wente MN, Izbicki JR, Friess H, Lerch MM, Dervenis C, Olah A, Butturini G, Doi R, Lind PA, Smith D, Valle JW, Palmer DH, Buckels JA, Thompson J, McKay CJ, Rawcliffe CL, Buchler MW. Adjuvant chemotherapy with fluorouracil plus folinic acid vs gemcitabine following pancreatic cancer resection: a randomized controlled trial. JAMA 2010; 304: 1073-81.
WP06: Project management
One work package was dedicated to project management to take care of all administrative and coordinating tasks. In order to support the coordinator in monitoring the compliance by beneficiaries with their obligations under the grant agreement, the project management office at concentris kept a close eye on the following partners` performance:
• making sure that tasks assigned to them were correctly and timely performed,
• that reports were submitted according to the guidelines and in time,
• that funds are used and claimed according to the rules,
• that the partners fulfilled their obligations regarding dissemination and funding acknowledgements,
• that any changes to the work plan were communicated to the EC swiftly,
• and compliance to ethical regulations.
The project office acted as a helpdesk for all participants; it was the central node of communication on a day-by-day basis and communicated with the European Commission on behalf of the Coordinator regarding administrative and managerial issues (i.e. contract, amendment, reporting etc.).
Potential Impact:
Socio-economic impact and the wider societal implications of EPC-TM-Net:
The tumour microenvironment has emerged as one of the most important determinants of drug resistance and patient survival in several cancer entities. This is particularly true for pancreatic cancer which is characterized by an extremely high content of stromal components defining the microenvironment surrounding the tumour cells. Given the appalling prognosis of pancreatic cancer patients, largely due to the tumour´s drug-resistance, identification of novel strategies to target the tumour stroma is of the utmost importance and will have great impact on the survival of pancreatic cancer patients.
EPC-TM-Net represents a coordinated effort to structure and integrate the Europe-wide research into the pancreatic cancer microenvironment, which will have a major impact on the development of future therapeutic strategies. The programme was specifically designed to systematically elucidate the impact of the tumour microenvironment on tumour progression and translate it into clinical applications and to furnish a suite of new compounds and therapeutic strategies targeting different components of the tumour microenvironment, which apart from the direct benefits outlined above for pancreatic cancer patients will impact a number of important areas.
Overall we have succeeded in:
• Establishing a structured and comprehensive research platform to study the impact of the microenvironment on the dismal prognosis and drug resistance of pancreatic cancer clearly requires pan-European collaboration and is unprecedented so far, and has achiever world wide recognition.
• The knowledge gain in this programme has increased the competitiveness of the European Research Area on the role of tumor microenvironment in pancreatic cancer.
• The programme has stimulated innovation by ensuring close and productive interactions between groups with expertise in a range of areas that we consider central for progress in cancer research and medicine.
• The combination of multiple levels of expertise from the participating groups has fostered reverse-translation of clinical findings in the genetically engineered mouse models for the development of novel therapeutic strategies targeting the tumour microenvironment.
• EPC-TM-Net has generated and characterized in-vitro and in-vivo models to study tumour-microenvironment interactions that will be of enormous value for the scientific community and industry as models for cross-sectional and longitudinal studies of carcinogenesis in the pancreas, and to test or validate diagnostic and therapeutic approaches.
• One further benefit of the breadth of expertise among the partners brought together in this programme was a wide dissemination of radically new technologies for the study of tumour-microenvironment interactions within the consortium.
• Novel compounds targeting the tumour microenvironment used alone or in combination with standard cytotoxic drugs such as gemcitabine have been tested in preclinical models and have partly entered clinical trials such as hyaluronidase.
• Clinical studies conducted in WP5 have established surrogate serum protein profiles for tumour stroma, in order to predict those patients who are likely to respond to conventional treatment, and those for whom entry to clinical trials of novel compounds may be a more appropriate option. EPC-TM-Net has thus provided additional benefit for pancreatic cancer patients by providing the means for personalised treatment schedules, that will be tested in future clinical trials.
• As contribution to generate opportunities for education and training this collaborative project provided opportunities for personnel exchange and training. This mobility contributed to elevate the educational and training standards of involved personnel, which has fostered their career opportunities.
To summarize the research programme of the individual WPs of EPC-TM-Net has effectively delivered:
1. novel experimental models to study the interplay between pancreatic tumor cells and the microenvironment
2. novel mechanisms representing the basis for the interaction of pancreatic tumor cell with the microenvironment
3. novel compounds for treatment targeting the interaction between tumor cells and microenvironment
4. novel ways of compound delivery specifically targeting cellular components forming tumor microenvironment
5. novel therapeutic strategies to overcome drug resistance by combined targeting of tumor cells and microenvironment.
6. novel opportunities for personalized therapy based on serum biomarkers specific for the tumor microenvironment
The main dissemination activities of EPC-TM-Net are:
Posters:
P1 UniMar: WNT5A mediates resistance to apoptosis in pancreatic cancer via NFATc2 (22/06/2011), EPC Magdeburg.
P1 UniMar: Identification of targets acting synergistically with erlotinib in pancreatic cancer using a kinome-wide loss-of-function screen (22/06/2011), EPC Magdeburg.
P1 UniMar: WNT5A - NFAT Signalkaskade reguliert die Therapieresistenz beim Pankreaskarzinom (14/09/2011), DGVS Leipzig.
P3 CNIO: LRH1/NR5A2, a link between pancreatitis and pancreatic cancer? (20/05/2012), Keystone Symposia “The Role of Inflammation during Carcinogenesis”, Dublin, Ireland.
P3 CNIO: Mnk1 kinase is part of the pancreatic acinar transcriptional programme required for tissue homeostasis upon stress (25/05/2012), The 3rd EMBO Conference Series on Cellular Signaling & Molecular Medicine, Dubrovnik, Croatia.
P3 CNIO: Mitochondrial metabolism, the Achilles heel of pancreatic cancer stem cells (18/05/2014), AACR special conference on pancreatic cancer, New Orleans, LA, USA.
P5 EMAU-G: Cathepsin B and D drives the fibrinogenic potential of pancreatic ductal adenocarcinoma (1/11/2013), Miami, USA.
P8 TUM-MED: Definition of the Kras-induced “switch-on” in malignant transformation to pancreatic cancer (20/10/2012), DPC Leipzig, Germany.
P13 CERMS-UniTO: Mouse to human autoantibody signature in pancreatic ductal adenocarcinoma (21/06/2011), 6th ItPA (Italian Proteomics Association) Annual National Congress, Torino, Italy.
P13 CERMS-UniTO: Mouse to human autoantibody signature in pancreatic ductal adenocarcinoma (04/09/2011), HUPO 10th Annual World Congress, Geneva, Switzerland.
P13 CERMS-UniTO: Characterization of circulating autoantibodies to post-translationally modified alpha-enolase in pancreatic ductal adenocarcinoma (04/09/2011), HUPO 10th Annual World Congress, Geneva, Switzerland.
P13 CERMS-UniTO: Autoantibodies against Ser-419-phosphorylated alpha-enolase are more frequent in DRB1*08 pancreatic ductal adenocarcinoma patients: can it improve overall survival? (31/05/2012), 16th International HLA and Immunogenetics Conference, Liverpool, UK.
P13 CERMS-UniTO: Does Th17 response be harnessed by the antigen DNA vaccination against pancreatic adenocarcinoma? (06/02/2012), Keystone Symposia: Th17 cells in health and disease. Keystone, Colorado, USA
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P13 CERMS-UniTO: Autoantibody Signature in pancreatic Ductal Adenocarcinoma (18/06/2012), Pancreatic cancer: progress and challenges; Lake Tahoe NV; USA.
P13 CERMS-UniTO: Effects of alpha-enolase silencing in a pancreatic adenocarcinoma cell line (18/06/2012), Pancreatic cancer: progress and Challenges. Lake Tahoe NV, USA.
P17 FCSR: Survivin vaccine for pancreatic adenocarcinoma (29/11/2013), San Raffaele Scientific Institute, Milan, Italy.
Presentations:
P1 UniMar: Cux1 stimulates pancreatic cancer progression by modulating the nfκb-dependent cytokine expression in tumour-associated macrophages (20/10/2012), Ueg week Amsterdam, The Netherlands.
P1 UniMar: Identification of targets acting synergistically with erlotinib in pancreatic cancer using a kinome-wide loss-of-function screen (20/10/2012), Ueg week Amsterdam, The Netherlands.
P1 UniMar: Zell-basierte Hochdurchsatz-Analysen identifizieren ADRBK1 als neues Zielgen im Pankreaskarzinom (11/09/2013), DGVS Nürnberg, Germany.
P1 UniMar: Einfluss der pharmakologischen Makrophagendepletion auf die Pankreaskarzinogenese in vivo (11/09/2013), DGVS Nürnberg, Germany.
P1 UniMar: KRAS-dependent signalling modulates the activation of infiltrating macrophages associated with a distinct miRNA profile in pancreatic cancer (23/01/2014), DPC Mannheim, Germany.
P2 ULIV: Combined LC/MS-MS and iTRAQ Analysis of Serum and Pancreatic Juice for the Detection of Biomarkers for Pancreatic Cancer: Influence of Biliary Obstruction (05/11/2011), Chicago, USA.
P2 ULIV: Periampullary cancer ESPAC 3-three trial: a randomised control phase 3 trial of adjuvant chemotherapy versus observation in patients with Periampullary adenocarcinomas of the head of the pancreas (05/11/2011), Chicago, USA.
P2 ULIV: Quality control of Protein Biomarkers: ECMC Annual Network Meeting (01/05/2012), Wellcome Collection, London.
P2 ULIV: Nucleotide transporters and dCK1 as prognostic markers for patients with resected pancreatic ductal adenocarcinoma with or without adjuvant gemcitabine or 5FU hENT1 tested with samples from patients randomized in the ESPAC1/3 trials (21/06/2012), Prague, Czeck Republic.
P2 ULIV: Role of S100A8/A9 in the Cross-talk between Cancer Cells and Stroma-associated Cells (22/06/2012), Prague, Czeck Republic.
P2 ULIV: The EUROPAC Registry and the EUROPAC 2 clinical trial (23/06/2012), Prague, Czech Republic.
P2 ULIV: Quantification of Pancreatic Ductal Adenocarcinoma Stroma (01/10/2013), Society of Great Britain and Ireland (Pancsoc), Liverpool, UK.
P2 ULIV: iTRAQ reveals a Serum Marker significantly altered in Patients up to 24 months prior to diagnosis with Pancreatic Cancer (31/10/2013), American Pancreatic Association Conference, Miami, USA.
P2 ULIV: Biomarkers in pancreatic cancer (29/11/2013), 39th Annual Meeting of the Pancreatic Society of Great Britain and Ireland, Liverpool, UK.
P2 ULIV: Involvement of microRNAs in the activation of cancer-associated pancreatic stellate cells (22/11/2013), Pancreatic Cancer Research Fund Supporters Conference, London, UK.
P2 ULIV: Modelling of Pancreatic Cancer (21/05/2013), QuasiVivo meeting, Liverpool, UK.
P2 ULIV: High hENT1 Expression is Predictive of Response to Adjuvant Gemcitabine for Pancreatic Ductal Adenocarcinoma Patients Randomised to Adjuvant Therapies in the ESPAC Trials (03/11/2012), American Pancreatic Association, Miami, USA.
P2 ULIV: Development of a Novel Next Generation Sequencing (NGS) Method for Detection of Low Concentration Sequence Variants (29/11/2013), 39th Annual Meeting of the Pancreatic Society of Great Britain and Ireland, Liverpool, UK.
P3 CNIO: Metformin targets the metabolic Achilles heel of pancreatic cancer stem cells (19/06/2012), Lake Tahoe, California.
P3 CNIO: Prospective isolation and characterization of circulation cancer stem cells (19/06/2012), Lake Tahoe, California.
P3 CNIO: Pancreatic cancer molecular pathophysiology (21/06/2012), MDACC Madrid Conference, Madrid, Spain.
P3 CNIO: Exploiting Acute Experimental Pancreatitis Models to Identify Novel Genes Involved in Exocrine Physiology and Pancreatic Cancer (25/06/2012), National Institute for Digestive Diseases and Kidney (NIDDK) workshop, Bethesda, USA.
P3 CNIO: Advanced Avatar models for studying cancer stem cell dynamics in human pancreatic ductal adenocarcinoma (05/11/2013), AACR Translational Impact of Model Organisms in Cancer Program, San Diego, USA.
P3 CNIO: Stem cells: Understanding the origins of pancreatic cancer (02/02/2014), Keystone conference - Stem Cells and Cancer, Banff, Canada.
P5 EMAU-G: Amino-acid depletion and growth factor depletion induce autophagy via different signaling pathways (20/06/2012), EPC Prague, Czech Republic.
P5 EMAU-G: Die Autophagie im Pankreaskarzinom ist unabhängig vom klassischen Beclin1-abhängigen Signalweg (17/11/2011), DPC Marburg, Germany.
P5 EMAU-G: The influence of the inhibition of pancreatic and neutrophil elastase on tumor growth in a syngenic orthotopic tumor model (17/11/2011), DPC Marburg, Germany.
P5 EMAU-G: Superparamagnetic nanoparticles (MNPS) coupled to siPLK1 represents a novel treatment option for pancreatic ductal adenocarcinoma (14/10/2013), Berlin, Germany.
P5 EMAU-G: Cathepsin B and D drives the fibrinogenic potential of pancreatic ductal adenocarcinoma (24/06/2014), Southampton, UK.
P5 EMAU-G: Superparamagnetic nanoparticles (MNPS) coupled to siPLK1 represents a novel treatment option for pancreatic ductal adenocarcinoma (27/06/2013), Zurich, Switzerland.
P8 TUM-MED: Comparative analysis of the microenvironment switch in early stages of pancreatic carcinogenesis (20/10/2012), DPC Leipzig, Germany.
P8 TUM-MED: Molecular/phenotypic dissection of oncogenic mTOR signaling in pancreatic cancer (16/01/2014), DPC Mannheim, Germany.
P8 TUM-MED: Tumor-promoting microenvironment is featured by expansion of pancreatic progenitors, mesenchymal cells and inflammation (16/01/2014), DPC Mannheim, Germany.
P13 CERMS-UniTO: Autoantibodysignature in pancreaticductal adenocarcinoma (18/11/2011), Riunione scientifica della Scuola Dianzani. Sassari, Italy.
P13 CERMS-UniTO: Alla ricerca degli anticorpi che spiano i tumori (14/04/2014), Liceo Scientifico Galileo Ferraris, Turin, Italy.
P16 TUM: Tenascin-C induces functional changes on pancreatic cancer cell lines (23/11/2012), DPC (German Pancreatic Club) 2012, Leipzig, Germany.
P16 TUM: Collagen type V influences the tumor microenvironment in pancreatic cancer (24/05/2013), DGP (Annual Meeting of the German Society of Pathology) 2014, Heidelberg, Germany.
P16 TUM: Collagen type V supports the malignant phenotype of pancreatic cancer cell lines through the integrin signaling pathway (28/06/2013), EPC (European Pancreatic Club) 2013, Zurich, Switzerland.
P16 TUM: Collagen type V promotes the malignant phenotype of pancreatic cancer (24/01/2014), DPC (German Pancreatic Club) 2014, Mannheim, Germany.
Organisation of Workshops:
P1 UniMar: Biologie-Schwerpunkt-Kurs (21/12/2011), University of Marburg.
P1 UniMar: Biologie-Schwerpunkt-Kurs (28/01/2013), University of Marburg.
P2 ULIV: Biobanking and Bioinformatics (B3i) Workshop (15/06/2012), University of Liverpool, UK.
P10 CR-UK: Mouse Hospital – Palpations and Ultrasound imaging in pancreatic cancer mice (10/04/2011), Cambridge CRUK.
Articles published in the popular press:
P6 KI: More research money will improve the bad prognosis of pancreatic cancer (26/05/2012), Sweden.
P13 CERMS-UniTO: Tumore al pancreas, nel futuro c’è un vaccine (02/07/2014), La Stampa, Torino, Italy.
Websites/Applications:
P15 concentris: Project website: www.epcnet.eu (22/06/2011).
The main exploitable results of EPC-TM-Net are:
P3 CNIO: Novel treatment strategy for pancreatic cancer: Development of a novel treatment strategy with combined targeting of the cancer stem cell niche and the cancer stem cells directly. This results in dramatically increased treatment response in preclinical models (01/10/2011), sector: cancer, for 5 years, EP 11382337.1-2107.
P8 TUM-MED: General advancement of knowledge: The molecular characterization of early pancreatic carcinogenesis is instrumental to design combinational treatments for chemoprevention in high-risk individuals. These pharmaceutical interventions include inhibitions of mesenchymal cells and progenitors proliferations as well as anti-inflammation therapies: Understanding the underlying mechanisms of tumor-stroma interaction accomplished by expansion of mesenchymal cells might provide researchers with new therapeutic strategies against pancreatic cancer. Our generated data-set could serve as a basis for following studies explaining the role of tumor stroma more in detail where the detection of new therapeutic targets could be implemented. Additionally, the identified inflammatory signature from our data might be useful for high-risk patient stratification as well as therapy prediction (27/06/2014), sector: preclinical studies, until 2017.
P14 NatiMab: Diagnostic and prognostic marker, therapeutic target: NatiMab Therapeutics studied the anti-cancer immunity developed by patients with PDAC. It became evident that Ezrin (EZR) and the phosphorylated form of α-Enolase of isotype 1 and 2 (ENOA) are pancreas Tumor Associated Antigens (TAAs). EZR is over-expressed by pancreas tumor cells and stimulates the patient production of specific antibodies that are a very early tumor marker. The blood samples from a prospective study show that circulating antibodies to EZR are found in patients much before the clinical diagnosis is possible, when the tumor is still localized and potentially resectable, In mice that spontaneously develop pancreas tumor, EZR is overexpressed by tumor in situ and the production of antibodies to EZR is evident before the cancer is detectable and becomes metastatic. ENOA is over-expressed by pancreas tumor cells. The antibodies to ENOA are found in patient sera and their presence correlates with a slower disease progression upon anti-cancer treatment. The presence of the antibodies against EZR is a marker of the initial stage of PDAC, while concurrent blood positivity of the antibodies against EZR and ENOA is of general importance for diagnosis of PDAC. Further, the concurrent presence in the blood of these antibodies together with CA19.9 detects PDAC with 100% sensitivity and 93.7% specificity. NatiMab is considering the industrial development and validation of two novel immunodiagnostic assays based on the two markers cited above, recently identified as specific markers of early stage pancreatic cancer, EZR, and its treatment follow up, ENOA. The detection of circulating antibodies to Ezrin, and to ENOA will consent an unquestionable diagnosis of PDAC while the detection of antibodies to Ezrin shall help the screen of population associated to a major risk to develop PDAC such as people with a recent diagnosis of type II diabetes, people with chronic pancreatitis and people belonging to a family with cases of PDAC. (31/12/2014), sectors: oncology, pancreatic tumors, minimum 3 years, commercialization of kit, license of patent, license of kits’ commercial rights.
P16 TUM: General advancement of knowledge: The molecular characterization of collagen V-dependent pathways: The molecular characterization of collagen V-dependent pathways in early pancreatic carcinogenesis and advanced pancreatic cancer is instrumental to identify specific diagnostic markers for molecular imaging and for the design of combinational cancer treatments (01/01/2015), sector: preclinical studies, starting from 2018.
List of Websites:
The address of the public website is: www.epcnet.eu
The contact details of the scientific representative of the project's co-ordinator are:
Prof. Dr. Thomas Gress
Philipps Universitaet Marburg
Dept. of Gastroenterology
Baldingerstrasse
D-35043 Marburg
Germany
Tel: +49 (0) 6421 58 66459
Fax: +49 (0) 6421 58 68922
E-mail: gress@med.uni-marburg.de
