IL-1ß+ macrophages fuel pathogenic inflammation in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with high resistance to therapies1. Inflammatory and immunomodulatory signals co-exist in the pancreatic tumour microenvironment, leading to dysregulated repair and cytotoxic responses. Tumour-associated macrophages (TAMs) have key roles in PDAC2, but their diversity has prevented therapeutic exploitation. Here we combined single-cell and spatial genomics with functional experiments to unravel macrophage functions in pancreatic cancer. We uncovered an inflammatory loop between tumour cells and interleukin-1ß (IL-1ß)-expressing TAMs, a subset of macrophages elicited by a local synergy between prostaglandin E2 (PGE2) and tumour necrosis factor (TNF). Physical proximity with IL-1ß+ TAMs was associated with inflammatory reprogramming and acquisition of pathogenic properties by a subset of PDAC cells. This occurrence was an early event in pancreatic tumorigenesis and led to persistent transcriptional changes associated with disease progression and poor outcomes for patients. Blocking PGE2 or IL-1ß activity elicited TAM reprogramming and antagonized tumour cell-intrinsic and -extrinsic inflammation, leading to PDAC control in vivo. Targeting the PGE2-IL-1ß axis may enable preventive or therapeutic strategies for reprogramming of immune dynamics in pancreatic cancer.

Establishment of a Transplantation Model of PDAC-Derived Liver Metastases.

BACKGROUND: The highly metastatic nature of pancreatic ductal adenocarcinoma (PDAC) and the difficulty to achieve favorable patient outcomes emphasize the need for novel therapeutic solutions. For preclinical evaluations, genetically engineered mouse models are often used to mimic human PDAC but frequently fail to replicate synchronous development and metastatic spread. This study aimed to develop a transplantation model to achieve synchronous and homogenous PDAC growth with controlled metastatic patterns in the liver. METHODS: To generate an orthotopic PDAC model, the DT6606 cell line was injected into the pancreas head of C57BL/6 mice, and their survival was monitored over time. To generate a heterotopic transplantation model, growing doses of three PDAC cell lines (DT6606, DT6606lm, and K8484) were injected into the portal vein of mice. Magnetic resonance imaging (MRI) was used to monitor metastatic progression, and histologic analysis was performed. RESULTS: Orthotopically injected mice succumbed to the tumor within an 11-week period (average survival time, 78.2 ± 4.45 days). Post-mortem examinations failed to identify liver metastasis. In the intraportal model, 2 × 105 DT6606 cells resulted in an absence of liver metastases by day 21, whereas 5 × 104 DT6606lm cells and 7 × 104 K8484 cells resulted in steady metastatic growth. Higher doses caused significant metastatic liver involvement. The use of K8484 cells ensured the growth of tumors closely resembling the histopathologic characteristics of human PDAC. CONCLUSIONS: This report details the authors' efforts to establish an "optimal" murine model for inducing metastatic PDAC, which is critical for advancing our understanding of the disease and developing more effective treatments.

A Comprehensive Characterization of Stemness in Cell Lines and Primary Cells of Pancreatic Ductal Adenocarcinoma.

The aim of this study is to provide a comprehensive characterization of stemness in pancreatic ductal adenocarcinoma (PDAC) cell lines. Seventeen cell lines were evaluated for the expression of cancer stem cell (CSC) markers. The two putative pancreatic CSC phenotypes were expressed heterogeneously ranging from 0 to 99.35% (median 3.46) for ESA+CD24+CD44+ and 0 to 1.94% (median 0.13) for CXCR4+CD133+. Cell lines were classified according to ESA+CD24+CD44+ expression as: Low-Stemness (LS; <5%, n = 9, median 0.31%); Medium-Stemness (MS; 6−20%, n = 4, median 12.4%); and High-Stemness (HS; >20%, n = 4, median 95.8%) cell lines. Higher degree of stemness was associated with in vivo tumorigenicity but not with in vitro growth kinetics, clonogenicity, and chemo-resistance. A wide characterization (chemokine receptors, factors involved in pancreatic organogenesis, markers of epithelial−mesenchymal transition, and secretome) revealed that the degree of stemness was associated with KRT19 and NKX2.2 mRNA expression, with CD49a and CA19.9/Tie2 protein expression, and with the secretion of VEGF, IL-7, IL-12p70, IL-6, CCL3, IL-10, and CXCL9. The expression of stem cell markers was also evaluated on primary tumor cells from 55 PDAC patients who underwent pancreatectomy with radical intent, revealing that CXCR4+/CD133+ and CD24+ cells, but not ESA+CD24+CD44+, are independent predictors of mortality.

CCL2/MCP-1 and CXCL12/SDF-1 blockade by L-aptamers improve pancreatic islet engraftment and survival in mouse.

The blockade of pro-inflammatory mediators is a successful approach to improve the engraftment after islet transplantation. L-aptamers are chemically synthesized, nonimmunogenic bio-stable oligonucleotides that bind and inhibit target molecules conceptually similar to antibodies. We aimed to evaluate if blockade-aptamer-based inhibitors of C-C Motif Chemokine Ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) and C-X-C Motif Chemokine Ligand 12/stromal cell-derived factor-1 (CXCL12/SDF-1) are able to favor islet survival in mouse models for islet transplantation and for type 1 diabetes. We evaluated the efficacy of the CCL2-specific mNOX-E36 and the CXCL12-specific NOX-A12 on islet survival in a syngeneic mouse model of intraportal islet transplantation and in a multiple low doses of streptozotocin (MLD-STZ) diabetes induction model. Moreover, we characterized intrahepatic infiltrated leukocytes by flow cytometry before and 3 days after islet infusion in presence or absence of these inhibitors. The administration for 14 days of mNOX-E36 and NOX-A12 significantly improved islet engraftment, either compound alone or in combination. Intrahepatic islet transplantation recruited CD45+ leucocytes and more specifically CD45+/CD11b+ mono/macrophages; mNOX-E36 and NOX-A12 treatments significantly decreased the recruitment of inflammatory monocytes, CD11b+ /Ly6Chigh /CCR2+ and CD11b+ /Ly6Chigh /CXCR4+ cells, respectively. Additionally, both L-aptamers significantly attenuated diabetes progression in the MLD-STZ model. In conclusion, CCL2/MCP-1 and CXCL12/SDF-1 blockade by L-aptamers is an efficient strategy to improve islet engraftment and survival.

Four-class tumor staging for early diagnosis and monitoring of murine pancreatic cancer using magnetic resonance and ultrasound.

Background: The widely used genetically engineered mouse LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre, termed KPC, spontaneously develops pancreatic cancer mirroring all phases of the carcinogenesis but in asynchronous manner. Preclinical studies need defined criteria for the enrollment of the KPC sharing the same stage of carcinogenesis. Aim: To define a tumor-staging criteria using magnetic resonance (MR) and ultrasound (US) and then to correlate the imaging stage with overall survival of KPC mice. Methods: Forty KPC (2- to 5-month-old mice) were imaged by axial fat-saturated T2-weighted sequences at MR and by brightness mode US to establish criteria for tumor staging. Immunohistopathology was used to validate imaging. A second cohort of 25 KPC was used to correlate imaging stage with survival by Kaplan-Meier analysis. Results: We defined a four-class tumor staging system ranking from stages 1 to 4. Stage 1 was described as radiologically healthy pancreas; precursor lesions were detectable in histology only. Cystic papillary neoplasms, besides other premalignant alterations, marked stage 2 in the absence of cancer nodules. Stages 3 and 4 identified mice affected by overt pancreatic cancer with size <5 or ≥5 mm, respectively. Regarding the prognosis, this staging system correlated with disease-related mortality whatever may be the KPC age when they staged. Conclusion: This imaging-based four-class tumor staging is an effective and safe method to stage pancreatic cancer development in KPC. As a result, regardless of their age, KPC mice can be synchronized based on prognosis or on a specific phase of tumorigenesis, such as the early but already radiologically detectable one (stage 2).

Gene expression analysis of embryonic pancreas development master regulators and terminal cell fate markers in resected pancreatic cancer: A correlation with clinical outcome.

BACKGROUND: Despite the recent introduction of new drugs and the development of innovative multi-target treatments, the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains very poor. Even when PDAC is resectable, the rate of local or widespread disease recurrence remains particularly high. Currently, reliable prognostic biomarkers of recurrence are lacking. We decided to explore the potential usefulness of pancreatic developmental regulators as biomarkers of PDAC relapse. METHODS: We analyzed by quantitative real-time PCR the mRNA of selected factors involved either in pancreatic organogenesis (ISL1, NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PAX6, PDX1 and PTF1α) or associated with terminally committed pancreatic cells (CHGA, CHGB, GAD2, GCG, HNF6α, INS, KRT19, SYP) in 17 PDAC cell lines and in frozen tumor samples from 41 PDAC patients. RESULTS: High baseline levels of the ISL1, KRT19, PAX6 and PDX1 mRNAs in PDAC cell lines, were risk factors for time-dependent xenograft appearance after subcutaneous injection in CD1-Nude mice. Consistently, in human PDAC samples, high levels of KRT19 mRNA were associated with reduced overall survival and earlier recurrence. Higher levels of PDX1 or PAX6 mRNAs were instead associated with a higher frequency of local recurrence. CONCLUSIONS: Our findings suggest that selected factors associated with pancreas development or its terminal differentiation might be implicated in mechanisms of PDAC progression and/or metastatic spread and that the measurement of their mRNA in tumors might be potentially used to improve patient prognostic stratification and prediction of the relapse site.

Establishment, characterization and long-term culture of human endocrine pancreas-derived microvascular endothelial cells.

BACKGROUND: In vitro primary cultures of microvascular endothelial cells from endocrine pancreas are difficult to obtain, but can be a very helpful tool for studies of islet biology, transplantation and regenerative medicine. METHODS: We applied a protocol recently described for the isolation and culture of brain microvascular endothelial cells (EC) on human pancreatic islets. EC obtained were characterized in terms of morphological (light and transmission electron microscopy), phenotypical (by immunofluorescence and flow cytometry) and functional (cord formation assay and protein secretion by multiplex bead-based assay) characteristics. RESULTS: EC were obtained from 25% of islet preparations processed. Two primary endothelial cell lines showed high proliferative potential and were deeply characterized: they presented endothelial cell morphology and expressed CD31, CD49a, CD49e, CD34, von Willebrand Factor (vWF), Vascular Endothelial CAdherin (VE-CAD), Tyrosine Kinase with Ig and EGF Homology Domains-2 (TIE2), Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), Ulex lectin and the endothelium endocrine-specific marker nephrin. Besides, they were able to form cordons in vitro and secreted factors involved in the process of angiogenesis such as Vascular Endothelial Growth Factor (VEGF), Monocyte Chemotactic Protein 1 (MCP-1), interleukin (IL)-8 and Melanoma Growth Stimulatory Activity Alpha (GROα). These cell lines were termed Human Islet Microvascular Endothelial Cells (HIMEC). DISCUSSION: This study establishes a simple and effective strategy for isolation and long-term culture of EC derived from human pancreatic islet. HIMEC in culture preserve phenotype and functional properties and are, therefore, a useful tool for future experiments of in vitro pancreas modelling, co-transplantation with pancreatic islets, re-vascularization of scaffold or matrix for regenerative medicine purposes.

Diabetes associated with pancreatic ductal adenocarcinoma is just diabetes: Results of a prospective observational study in surgical patients.

BACKGROUND: Identification of a specific diabetes signature associated to pancreatic ductal carcinoma (PDAC) could be a key to detect asymptomatic, early stage tumors. We aim to characterize the clinical signature and the pathogenetic factors of the different types of diabetes associated with PDAC, based on the time between diabetes and cancer diagnosis. METHODS: Prospective observational study on 364 PDAC patients admitted to a referral center for pancreatic disease. Hospital and/or outpatient medical records were reviewed. Blood biochemical values including fasting blood glucose, insulin and/or C-peptide, glycosylated hemoglobin and anti-islet antibodies were determined. Diabetes onset was assessed after surgery and during follow-up. RESULTS: The prevalence of diabetes in patients was 67%. Considering 174 patients (47.8%) already having diabetes when diagnosed with PDAC (long duration, short duration, concomitant), the clinical and biochemical profile was similar to that of patients with type 2 diabetes (T2D). Diabetes was associated with known risk factors (i.e., age, sex, family history for diabetes and increased BMI) and both beta-cell dysfunction and insulin resistance were present. Considering 70 patients (19.2%) with onset of diabetes after PDAC diagnosis (early and late onset), the strongest predictor was the loss of beta-cell mass following pancreatectomy in patients with risk factors for T2D. CONCLUSION: Different types of diabetes according to the time between diabetes and PDAC diagnosis are clinical entities widely overlapping with T2D. Therefore, the success of a strategy considering diabetes onset as a marker of asymptomatic PDAC will largely depend on our ability to identify new diabetes-unrelated biomarkers of PDAC.

Gemcitabine-releasing mesenchymal stromal cells inhibit in vitro proliferation of human pancreatic carcinoma cells.

BACKGROUND AIMS: Pancreatic cancer (pCa) is a tumor characterized by a fibrotic state and associated with a poor prognosis. The observation that mesenchymal stromal cells (MSCs) migrate toward inflammatory micro-environments and engraft into tumor stroma after systemic administration suggested new therapeutic approaches with the use of engineered MSCs to deliver and produce anti-cancer molecules directly within the tumor. Previously, we demonstrated that without any genetic modifications, MSCs are able to deliver anti-cancer drugs. MSCs loaded with paclitaxel by exposure to high concentrations release the drug both in vitro and in vivo, inhibiting tumor proliferation. On the basis of these observations, we evaluated the ability of MSCs (from bone marrow and pancreas) to uptake and release gemcitabine (GCB), a drug widely used in pCa treatment. METHODS: MSCs were primed by 24-h exposure to 2000 ng/mL of GCB. The anti-tumor potential of primed MSCs was then investigated by in vitro anti-proliferation assays with the use of CFPAC-1, a pancreatic tumor cell line sensitive to GCB. The uptake/release ability was confirmed by means of high-performance liquid chromatography analysis. A cell-cycle study and secretome evaluation were also conducted to better understand the characteristics of primed MSCs. RESULTS: GCB-releasing MSCs inhibit the growth of a human pCa cell line in vitro. CONCLUSIONS: The use of MSCs as a "trojan horse" can open the way to a new pCa therapeutic approach; GCB-loaded MSCs that integrate into the tumor mass could deliver much higher concentrations of the drug in situ than can be achieved by intravenous injection.

Diabetes after pancreatic surgery: novel issues.

In the developed world, pancreatic surgery is becoming more common, with an increasing number of patients developing diabetes because of either partial or total pancreatectomy, with a significant impact on quality of life and survival. Although these patients are expected to consume increasing health care resources in the near future, many aspects of diabetes after pancreatectomy are still not well defined. The treatment of diabetes in these patients takes advantage of the therapies used in type 1 and 2 diabetes; however, no specific guidelines for its management, both immediately after pancreatic surgery or in the long term, have been developed. In this article, on the basis of both the literature and our clinical experience, we address the open issues and discuss the most appropriate therapeutic options for patients with diabetes after pancreatectomy.

Rapamycin unbalances the polarization of human macrophages to M1.

Plasticity is a hallmark of macrophages, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic (M1) and alternative (M2). Rapamycin (RAPA) is crucial for survival and functions of myeloid phagocytes, but its effects on macrophage polarization are not yet studied. To address this issue, human macrophages obtained from six normal blood donors were polarized to M1 or M2 in vitro by lipopolysaccharide plus interferon-γ or interleukin-4 (IL-4), respectively. The presence of RAPA (10 ng/ml) induced macrophage apoptosis in M2 but not in M1. Beyond the impact on survival in M2, RAPA reduced CXCR4, CD206 and CD209 expression and stem cell growth factor-ß, CCL18 and CCL13 release. In contrast, in M1 RAPA increased CD86 and CCR7 expression and IL-6, tumour necrosis factor-α and IL-1ß release but reduced CD206 and CD209 expression and IL-10, vascular endothelial growth factor and CCL18 release. In view of the in vitro data, we examined the in vivo effect of RAPA monotherapy (0·1 mg/kg/day) in 12 patients who were treated for at least 1 month before islet transplant. Cytokine release by Toll-like receptor 4-stimulated peripheral blood mononuclear cells showed a clear shift to an M1-like profile. Moreover, macrophage polarization 21 days after treatment showed a significant quantitative shift to M1. These results suggest a role of mammalian target of rapamycin (mTOR) into the molecular mechanisms of macrophage polarization and propose new therapeutic strategies for human M2-related diseases through mTOR inhibitor treatment.

Mass spectrometric analysis reveals O-methylation of pyruvate kinase from pancreatic cancer cells.

Pyruvate kinase (PK) is an important glycolytic enzyme that catalyzes the dephosphorylation of phosphoenolpyruvate to pyruvate. Human PK isozyme M2 (PKM2), a splice variant of M1, is overexpressed in many cancer cells, and PKM2 has been investigated as a potential tumor marker for diagnostic assays and as a target for cancer therapy. To facilitate identification and characterization of PK, we studied the enzyme from pancreatic cancer cells and normal pancreatic duct cells by electrophoresis and mass spectrometry, and identified multiple O-methylated residues from PK. These findings advance our knowledge of the biochemical properties of PK and will be important in understanding its biological function in cells.

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

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 α5ß1, 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.

Directed self-assembly of a xenogeneic vascularized endocrine pancreas for type 1 diabetes.

Intrahepatic islet transplantation is the standard cell therapy for ß cell replacement. However, the shortage of organ donors and an unsatisfactory engraftment limit its application to a selected patients with type 1 diabetes. There is an urgent need to identify alternative strategies based on an unlimited source of insulin producing cells and innovative scaffolds to foster cell interaction and integration to orchestrate physiological endocrine function. We previously proposed the use of decellularized lung as a scaffold for ß cell replacement with the final goal of engineering a vascularized endocrine organ. Here, we prototyped this technology with the integration of neonatal porcine islet and healthy subject-derived blood outgrowth endothelial cells to engineer a xenogeneic vascularized endocrine pancreas. We validated ex vivo cell integration and function, its engraftment and performance in a preclinical model of diabetes. Results showed that this technology not only is able to foster neonatal pig islet maturation in vitro, but also to perform in vivo immediately upon transplantation and for over 18 weeks, compared to normal performance within 8 weeks in various state of the art preclinical models. Given the recent progress in donor pig genetic engineering, this technology may enable the assembly of immune-protected functional endocrine organs.

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.

Innate Immunity Mediated Inflammation and Beta Cell Function: Neighbors or Enemies?

Type 1 diabetes (T1D) is still considered a huge burden because the available treatments are not effective in preventing the onset or progression of the disease. Recently, the idea that diabetes is an autoimmune disease mediated exclusively by T cells has been reshaped. In fact, T cells are not the only players with an active role in beta cell destruction. Macrophages and neutrophils, which physiologically reside in pancreatic tissue, can also participate in tissue homeostasis and damage by promoting innate immune responses and modulating inflammation. During the development of the pancreatic islet inflammation there is a strong interplay of both adaptive and innate immune cells, and the presence of innate immune cells has been demonstrated both in exocrine and endocrine pancreatic compartments during the earliest stages of insulitis. Innate immune cell populations secrete cytokines, which must be considered both as physiological and pathological mediators. In fact, it has been demonstrated that cytokines could regulate directly and indirectly insulin secretion and, simultaneously, trigger inflammatory reaction. Indeed, cytokines pathways could represent targets both to improve glucose metabolism and to prevent autoimmune damage. Concordantly, the combination of immunomodulatory strategies against both innate and adaptive immunity should be tested in the next future, as they can be more efficient to prevent or delay islet damage and T1D onset.

Standardized GMP-compliant scalable production of human pancreas organoids.

BACKGROUND: Organoids are three-dimensional in vitro-grown cell clusters that recapitulate key features of native organs. In regenerative medicine, organoid technology represents a promising approach for the replacement of severely damaged organs, such as the pancreas in patients with type 1 diabetes. Isolation human pancreas organoids (hPOs) in chemically defined serum-free culture media would be a major milestone for this approach. METHODS: Starting from discarded pancreatic tissues, we developed a large-scale process for obtaining clinically relevant quantities of undifferentiated organoids, obviating enzymatic digestion and operator-dependent pancreatic ducts picking steps. hPO identity was characterized by molecular and flow cytometry analysis. RESULTS: This work demonstrates that it is possible to obtain a large-scale production of organoids. We introduced some innovations in the isolation, expansion, and freezing of hPOs from five donors. First of all, the choice of the starting material (islet-depleted pancreas) that allows obtaining a high quantity of hPOs at low passages. On the other hand, we introduced mechanical dissociation and we eliminated the picking step to exclude the operator-depending steps, without affecting the success of the culture (100% success rate). Another important improvement was to replace R-spondin-1 (Rspo1) conditioned medium with Rspo1 recombinant molecule to obtain a well-defined composition of the expansion medium. Finally, we implemented a GMP-compliant freezing protocol. hPOs showed exponential growth with diameter and area that increased three- and eight-fold in 7 days, respectively. Immunophenotypic profile and gene expression analysis revealed that hPOs were composed of ductal (82.33 ± 8.37%), acinar (2.80 ± 1.25%) cells, and pancreatic progenitors (5.81 ± 2.65%). CONCLUSION: This work represents a milestone for a GMP-compliance hPO production and, ultimately, their clinical application as a type 1 diabetes therapy.

Circulating Chromogranin A Is Cleaved Into Vasoregulatory Fragments in Patients With Pancreatic Ductal Adenocarcinoma.

Chromogranin A (CgA), a secretory protein released in the blood by the neuroendocrine system, consists of a mixture of full-length molecules and fragments endowed of vasoregulatory activity. The extent and the role of CgA fragmentation were investigated in patients with locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC, n=172). Multivariate analysis showed that full-length CgA was associated with better progression free and overall survival, whereas CgA C-terminal fragmentation was associated with worse prognosis. In vitro studies showed that PDAC cells can promote the cleavage of CgA C-terminal region by activating plasminogen to plasmin. Limited digestion of full-length CgA with plasmin abolished its anti-angiogenic activity and generated pro-angiogenic molecules. The fragmentation of CgA C-terminal region was increased also in murine models of PDAC. In these models, the inhibition of CgA fragmentation with aprotinin, an inhibitor of plasmin and other serine proteases, or the blockade of pro-angiogenic fragments with specific antibodies inhibited the growth of PDAC implanted subcutaneously in mice. Finally, administration of full-length CgA to mice bearing orthotopic PDAC reduced tumor perfusion, as measured by contrast-enhanced ultrasound. These findings suggest that PDAC can promote the cleavage of circulating CgA C-terminal region to generate fragments that regulate the tumor vascular biology and that may represent new potential therapeutic targets.

B lymphocytes contribute to stromal reaction in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent stromal reaction that has been variably implicated in both tumor growth and tumor suppression. B-lymphocytes have been recently implicated in PDAC progression but their contribution to the characteristic stromal desmoplasia has never been assessed before. In the present work, we aimed to verify whether B-lymphocytes contribute to stromal cell activation in PDAC. CD19+ B-lymphocytes purified from peripheral blood of patients with PDAC were cultivated in the presence of human pancreatic fibroblasts and cancer-associated fibroblasts. Released pro-fibrotic soluble factors and collagen production were assessed by ELISA and Luminex assays. Quantitative RT-PCR was used to assess fibroblast activation in the presence of B cells. The expression of selected pro-fibrotic and inflammatory molecules was confirmed on PDAC tissue sections by multi-color immunofluorescence studies. We herein demonstrate that B-cells from PDAC patients (i) produce the pro-fibrotic molecule PDGF-B and stimulate collagen production by fibroblasts; (ii) express enzymes implicated in extracellular matrix remodeling including LOXL2; and (iii) produce the chemotactic factors CCL-4, CCL-5, and CCL-11. In addition we demonstrate that circulating plasmablasts are expanded in the peripheral blood of patients with PDAC, stimulate collagen production by fibroblasts, and infiltrate pancreatic lesions. Our results indicate that PDAC is characterized by perturbations of the B-cell compartment with expansion of B-lymphocyte subsets that directly contribute to the stromal reaction observed at disease site. These findings provide an additional rationale for modulating B-cell activity in patients with pancreatic cancer.