Interrupting the nitrosative stress fuels tumor-specific cytotoxic T lymphocytes in pancreatic cancer

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors owing to its robust desmoplasia, low immunogenicity, and recruitment of cancer-conditioned, immunoregulatory myeloid cells. These features strongly limit the success of immunotherapy as a single agent, thereby suggesting the need for the development of a multitargeted approach. The goal is to foster T lymphocyte infiltration within the tumor landscape and neutralize cancer-triggered immune suppression, to enhance the therapeutic effectiveness of immune-based treatments, such as anticancer adoptive cell therapy (ACT). METHODS: We examined the contribution of immunosuppressive myeloid cells expressing arginase 1 and nitric oxide synthase 2 in building up a reactive nitrogen species (RNS)-dependent chemical barrier and shaping the PDAC immune landscape. We examined the impact of pharmacological RNS interference on overcoming the recruitment and immunosuppressive activity of tumor-expanded myeloid cells, which render pancreatic cancers resistant to immunotherapy. RESULTS: PDAC progression is marked by a stepwise infiltration of myeloid cells, which enforces a highly immunosuppressive microenvironment through the uncontrolled metabolism of L-arginine by arginase 1 and inducible nitric oxide synthase activity, resulting in the production of large amounts of reactive oxygen and nitrogen species. The extensive accumulation of myeloid suppressing cells and nitrated tyrosines (nitrotyrosine, N-Ty) establishes an RNS-dependent chemical barrier that impairs tumor infiltration by T lymphocytes and restricts the efficacy of adoptive immunotherapy. A pharmacological treatment with AT38 ([3-(aminocarbonyl)furoxan-4-yl]methyl salicylate) reprograms the tumor microenvironment from protumoral to antitumoral, which supports T lymphocyte entrance within the tumor core and aids the efficacy of ACT with telomerase-specific cytotoxic T lymphocytes. CONCLUSIONS: Tumor microenvironment reprogramming by ablating aberrant RNS production bypasses the current limits of immunotherapy in PDAC by overcoming immune resistance

RNA-based MAFA over-expression is sufficient to drive human pancreatic duct-derived cells toward a B-cell-like phenotype

RNA-based MAFA over-expression is sufficient to drive human pancreatic duct-derived cells toward a B-cell-like phenotype Elisa Corritore1, Erica Dugnani2, Valentina Pasquale2, Lorenzo Piemonti2, A. Vetere3,Susan Bonner-Weir4, Etienne M. Sokal1, Philippe A. Lysy1,5 1Institut de Recherche Expérimentale et Clinique, Pediatric Research Laboratory,Université Catholique de Louvain, Brussels, Belgium; 2San Raffaele Research Institute, Milan, Italy; 3Chemical Biology Program, Broad Institute of Harvard and MIT, Cambridge, USA ; 4Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Harvard Medical School, Boston, USA ; 5Pediatric Endocrinology Unit, Cliniques Universitaires Saint Luc, Université Catholique de Louvain, Brussels, Belgium Pancreatic epithelial cells represent an attractive cell source for replacement therapy of type 1 diabetes. Previously, we designed a protocol for expansion of human pancreatic duct-derived cells (HDDCs) and showed their β-cell engineering potential. In this study, we reprogrammed HDDCs into β-cell-like lineage by over-expressing mRNAs of key pancreatic transcription factors (TFs). Pancreatic duct cells (n=6) were purified and propagated into endothelial growth-promoting media. Synthetic modified (sm) RNAs were manufactured by unidirectional subcloning of PDX1, NGN3 and MAFA into a plasmid containing 5’ and 3’ UTR regions. The UTR-flanked inserts were excised and poly(A)-tailed. The final smRNAs were synthesized through in vitro transcription followed by phosphatase and DNase treatments, before being daily transfected in HDDCs. In all donors, transfection of PDX1, NGN3 and MAFA led to upregulation of endogenous target (ex: NGN3) and β-cell marker (ex: INS, synaptophysin, SLC2A2, GCK) genes with the highest expression levels being reached after MAFA transfection. Co-transfection protocols failed to show significant improvement of β-cell differentiation. Acceptable impact on innate immune response and cell viability was noticed after 7 consecutive daily smRNA transfections, based respectively on minimal IFNA and RIG-1 gene expression and on annexin-V/PI staining. After MAFA transfection, HDDCs stained positive for MAFA and insulin (19.3 ± 3.3 %) proteins, while ELISA assays showed detectable amounts of C-peptide content and release (21.45 ± 2.42 pg/mL/106 cells) under basal conditions. In conclusion, we showed that MAFA RNA over-expression is sufficient to efficiently reprogram HDDCs toward β-cell-like phenotype in a timely manner. Further research is mandatory to demonstrate a controlled insulin secretion capacity after differentiation

Fibronectin fragments generated by pancreatic trypsin act as endogenous inhibitors of pancreatic tumor growth

Abstract Background The pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations in the equilibrium between proteases and their inhibitors. The identification of proteolytic events, targets and pathways would set the basis for the design of new therapeutic approaches. Methods and results Here we demonstrate that spheroids isolated from human and murine healthy pancreas and co-transplanted orthotopically with pancreatic ductal adenocarcinoma (PDAC) in mouse pancreas inhibited tumor growth. The effect was mediated by trypsin-generated fibronectin (FN) fragments released by pancreatic spheroids. Tumor inhibition was observed also in a model of acute pancreatitis associated with trypsin activation. Mass spectrometry proteomic analysis of fragments and mAb against different FN epitopes identified the FN type III domain as responsible for the activity. By inhibiting integrin α5β1, FAK and FGFR1 signaling, the fragments induced tumor cell detachment and reduced cell proliferation. Consistent with the mutual relationship between the two pathways, FGF2 restored both FGFR1 and FAK signaling and promoted PDAC cell adhesion and proliferation. FAK and FGFR inhibitors additively inhibited PDAC growth in vitro and in orthotopic in vivo models. Conclusions This study identifies a novel role for pancreatic trypsin and fibronectin cleavage as a mechanism of protection against cancer by the pancreatic microenvironment. The finding of a FAK-FGFR cross-talk in PDAC support the combination of FAK and FGFR inhibitors for PDAC treatment to emulate the protective effect of the normal pancreas against cancer

Effect of Diabetes on Survival after Resection of Pancreatic Adenocarcinoma. A Prospective, Observational Study

<div><p>Aim</p><p>To investigate the effect of diabetes mellitus (DM) on disease-free and overall post-resection survival of patients with pancreatic ductal adenocarcinoma (PDAC)</p><p>Methods</p><p>Prospective observational study on patients admitted for pancreatic disease from January 2008 to October 2012. DM was classified as recent-onset (<48 months before PDAC diagnosis), longstanding (≥48 months before PDAC) or new onset (after surgery).</p><p>Results</p><p>Of 296 patients, 140 had a diagnosis of DM prior to surgery (26 longstanding, 99 recent-onset, 15 with unknown duration). Median follow-up time was 5.4 ± 0.22 years. Patients with recent onset DM had poorer postoperative survival than patients without DM: disease-free survival and overall survival were 1.14±0.13 years and 1.52±0.12 years in recent onset DM, versus 1.3±0.15 years and 1.87±0.15 years in non-diabetic patients (p = 0.013 and p = 0.025, respectively). Longstanding DM and postoperative new onset DM had no impact on prognosis. Compared to cases without DM, patients with recent onset DM were more likely to have residual disease after surgery and to develop liver metastases during follow-up. Multivariate analysis confirmed recent onset DM was independently associated with PDAC relapse (hazard ratio 1.45 [1.06–1.99]).</p><p>Conclusion</p><p>Preoperative recent onset DM has an impact on survival after the resection of PDAC.</p></div

Patients characteristics by postoperative diabetic status.

<p>Patients characteristics by postoperative diabetic status.</p

Patient characteristics by types of preoperative diabetes.

<p>Patient characteristics by types of preoperative diabetes.</p

Effect of pre-operative recent onset DM on PDAC relapse.

<p>Kaplan-Meier estimates of disease free survival and stratified according to seven main recurrence sites for 255 patients (156 no diabetes; 99 recent onset diabetes) with a diagnosis of pancreatic ductal carcinoma submitted to pancreatectomy with radical intent from the Pancreatic Surgery Unit of the S. Raffaele Scientific Institute (2008–2012). The <i>X-axis</i> shows the time since pancreatectomy and the <i>y-axis</i> the PDAC recurrence-free probability. P value of <i>log</i>-<i>rank test</i> vs no diabetes are reported.</p

Effect of pre-operative DM on PDAC outcome.

<p>Kaplan-Meier estimates of disease-free (left) and overall (right) survival stratified according to pre-operative DM for 281 patients with a diagnosis of pancreatic ductal carcinoma submitted to pancreatectomy with radical intent from the Pancreatic Surgery Unit of the S. Raffaele Scientific Institute (2008–2012). At the time of PDAC diagnosis participants were classified as having: long-standing diabetes if they had a documented diagnosis of DM for ≥48 months; recent onset DM if participants were diagnosed with DM at the time of the diagnosis of PDAC or had a documented diagnosis of DM for <48 months. The <i>X-axis</i> shows the time since pancreatectomy. P value of <i>log</i>-<i>rank test</i> vs no diabetes are reported.</p