Publications by authors named "Francesco Novelli"

89 Publications

IL17A critically shapes the transcriptional program of fibroblasts in pancreatic cancer and switches on their protumorigenic functions.

Proc Natl Acad Sci U S A 2021 Feb;118(6)

Laboratory of Tumor Immunology, Center for Experimental Research and Medical Studies, Città della Salute e della Scienza di Torino, University of Torino, 10126 Torino, Italy;

A hallmark of cancer, including pancreatic ductal adenocarcinoma (PDA), is a massive stromal and inflammatory reaction. Many efforts have been made to identify the anti- or protumoral role of cytokines and immune subpopulations within the stroma. Here, we investigated the role of interleukin-17A (IL17A) and its effect on tumor fibroblasts and the tumor microenvironment. We used a spontaneous PDA mouse model (KPC) crossed to IL17A knockout mice to show an extensive desmoplastic reaction, without impaired immune infiltration. Macrophages, especially CD80 and T cells, were more abundant at the earlier time point. In T cells, a decrease in FoxP3 cells and an increase in CD8 T cells were observed in KPC/IL17A mice. Fibroblasts isolated from IL17A and IL17A KPC mice revealed very different messenger RNA (mRNA) and protein profiles. IL17A fibroblasts displayed the ability to restrain tumor cell invasion by producing factors involved in extracellular matrix remodeling, increasing T cell recruitment, and producing higher levels of cytokines and chemokines favoring T helper 1 cell recruitment and activation and lower levels of those recruiting myeloid/granulocytic immune cells. Single-cell quantitative PCR on isolated fibroblasts confirmed a very divergent profile of IL17A-proficient and -deficient cells. All these features can be ascribed to increased levels of IL17F observed in the sera of IL17A mice, and to the higher expression of its cognate receptor (IL17RC) specifically in IL17A cancer-associated fibroblasts (CAFs). In addition to the known effects on neoplastic cell transformation, the IL17 cytokine family uniquely affects fibroblasts, representing a suitable candidate target for combinatorial immune-based therapies in PDA.
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http://dx.doi.org/10.1073/pnas.2020395118DOI Listing
February 2021

Computational modeling of the immune response in multiple sclerosis using epimod framework.

BMC Bioinformatics 2020 Dec 14;21(Suppl 17):550. Epub 2020 Dec 14.

Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy.

Background: Multiple Sclerosis (MS) represents nowadays in Europe the leading cause of non-traumatic disabilities in young adults, with more than 700,000 EU cases. Although huge strides have been made over the years, MS etiology remains partially unknown. Furthermore, the presence of various endogenous and exogenous factors can greatly influence the immune response of different individuals, making it difficult to study and understand the disease. This becomes more evident in a personalized-fashion when medical doctors have to choose the best therapy for patient well-being. In this optics, the use of stochastic models, capable of taking into consideration all the fluctuations due to unknown factors and individual variability, is highly advisable.

Results: We propose a new model to study the immune response in relapsing remitting MS (RRMS), the most common form of MS that is characterized by alternate episodes of symptom exacerbation (relapses) with periods of disease stability (remission). In this new model, both the peripheral lymph node/blood vessel and the central nervous system are explicitly represented. The model was created and analysed using Epimod, our recently developed general framework for modeling complex biological systems. Then the effectiveness of our model was shown by modeling the complex immunological mechanisms characterizing RRMS during its course and under the DAC administration.

Conclusions: Simulation results have proven the ability of the model to reproduce in silico the immune T cell balance characterizing RRMS course and the DAC effects. Furthermore, they confirmed the importance of a timely intervention on the disease course.
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http://dx.doi.org/10.1186/s12859-020-03823-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734848PMC
December 2020

In pancreatic cancer, chemotherapy increases antitumor responses to tumor-associated antigens and potentiates DNA vaccination.

J Immunother Cancer 2020 10;8(2)

Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy

Background: Pancreatic ductal adenocarcinoma (PDA) is an almost incurable tumor that is mostly resistant to chemotherapy (CT). Adaptive immune responses to tumor-associated antigens (TAA) have been reported, but immunotherapy (IT) clinical trials have not yet achieved any significant increase in survival, confirming the suppressive environment of PDA. As CT has immune-modulating properties, we investigated the effect of gemcitabine (GEM) in antitumor effector responses to TAA in patients with PDA.

Methods: The IgG antibody repertoire in patients with PDA before and after CT was profiled by serological proteome analysis and ELISA and their ability to activate complement-dependent cytotoxicity (CDC) was measured. Peripheral T cells were stimulated in vitro with recombinant TAA, and specific proliferation, IFN-γ/IL-10 and CD8/Treg ratios were measured. Mice that spontaneously developed PDA were treated with GEM and inoculated with an ENO1 (α-Enolase) DNA vaccine. In some experimental groups, the effect of depleting CD4, CD8 and B cells by specific antibodies was also evaluated.

Results: CT increased the number of TAA recognized by IgG and their ability to activate CDC. Evaluation of the IFN-γ/IL-10 ratio and CD8+/Treg ratios revealed that CT treatment shifted T cell responses to ENO1, G3P (glyceraldheyde-3-phosphate dehydrogenase), K2C8 (keratin, type II cytoskeletal 8) and FUBP1 (far upstream binding protein 1), four of the most recognized TAA, from regulatory to effector. In PDA mice models, treatment with GEM prior to ENO1 DNA vaccination unleashed CD4 antitumor activity and strongly impaired tumor progression compared with mice that were vaccinated or GEM-treated alone.

Conclusions: Overall, these data indicate that, in PDA, CT enhances immune responses to TAA and renders them suitable targets for IT.
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http://dx.doi.org/10.1136/jitc-2020-001071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594541PMC
October 2020

Diabetes promotes invasive pancreatic cancer by increasing systemic and tumour carbonyl stress in Kras mice.

J Exp Clin Cancer Res 2020 Aug 10;39(1):152. Epub 2020 Aug 10.

Department of Clinical and Molecular Medicine, "La Sapienza" University, Via di Grottarossa, 1035-1039 -, 00189, Rome, Italy.

Background: Type 1 and 2 diabetes confer an increased risk of pancreatic cancer (PaC) of similar magnitude, suggesting a common mechanism. The recent finding that PaC incidence increases linearly with increasing fasting glucose levels supports a central role for hyperglycaemia, which is known to cause carbonyl stress and advanced glycation end-product (AGE) accumulation through increased glycolytic activity and non-enzymatic reactions. This study investigated the impact of hyperglycaemia on invasive tumour development and the underlying mechanisms involved.

Methods: Pdx1-Cre;LSL-Kras mice were interbred with mitosis luciferase reporter mice, rendered diabetic with streptozotocin and treated or not with carnosinol (FL-926-16), a selective scavenger of reactive carbonyl species (RCS) and, as such, an inhibitor of AGE formation. Mice were monitored for tumour development by in vivo bioluminescence imaging. At the end of the study, pancreatic tissue was collected for histology/immunohistochemistry and molecular analyses. Mechanistic studies were performed in pancreatic ductal adenocarcinoma cell lines challenged with high glucose, glycolysis- and glycoxidation-derived RCS, their protein adducts AGEs and sera from diabetic patients.

Results: Cumulative incidence of invasive PaC at 22 weeks of age was 75% in untreated diabetic vs 25% in FL-926-16-gtreated diabetic and 8.3% in non-diabetic mice. FL-926-16 treatment suppressed systemic and pancreatic carbonyl stress, extracellular signal-regulated kinases (ERK) 1/2 activation, and nuclear translocation of Yes-associated protein (YAP) in pancreas. In vitro, RCS scavenging and AGE elimination completely inhibited cell proliferation stimulated by high glucose, and YAP proved essential in mediating the effects of both glucose-derived RCS and their protein adducts AGEs. However, RCS and AGEs induced YAP activity through distinct pathways, causing reduction of Large Tumour Suppressor Kinase 1 and activation of the Epidermal Growth Factor Receptor/ERK signalling pathway, respectively.

Conclusions: An RCS scavenger and AGE inhibitor prevented the accelerating effect of diabetes on PainINs progression to invasive PaC, showing that hyperglycaemia promotes PaC mainly through increased carbonyl stress. In vitro experiments demonstrated that both circulating RCS/AGEs and tumour cell-derived carbonyl stress generated by excess glucose metabolism induce proliferation by YAP activation, hence providing a molecular mechanism underlying the link between diabetes and PaC (and cancer in general).
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http://dx.doi.org/10.1186/s13046-020-01665-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418209PMC
August 2020

Immune-Complexome Analysis Identifies Immunoglobulin-Bound Biomarkers That Predict the Response to Chemotherapy of Pancreatic Cancer Patients.

Cancers (Basel) 2020 Mar 21;12(3). Epub 2020 Mar 21.

Department of Molecular Biotechnology and Health Sciences, University of Turin, 10126 Torino, Italy.

Pancreatic Ductal Adenocarcinoma (PDA) is an aggressive malignancy with a very poor outcome. Although chemotherapy (CT) treatment has poor efficacy, it can enhance tumor immunogenicity. Tumor-Associated Antigens (TAA) are self-proteins that are overexpressed in tumors that may induce antibody production and can be PDA theranostic targets. However, the prognostic value of TAA-antibody association as Circulating Immune Complexes (CIC) has not yet been elucidated, mainly due to the lack of techniques that lead to their identification. In this study, we show a novel method to separate IgG, IgM, and IgA CIC from sera to use them as prognostic biomarkers of CT response. The PDA Immune-Complexome (IC) was identified using a LTQ-Orbitrap mass spectrometer followed by computational analysis. The analysis of the IC of 37 PDA patients before and after CT revealed differential associated antigens (DAA) for each immunoglobulin class. Our method identified different PDA-specific CIC in patients that were associated with poor prognosis patients. Finally, CIC levels were significantly modified by CT suggesting that they can be used as effective prognostic biomarkers to follow CT response in PDA patients.
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http://dx.doi.org/10.3390/cancers12030746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140049PMC
March 2020

Metabolome of Pancreatic Juice Delineates Distinct Clinical Profiles of Pancreatic Cancer and Reveals a Link between Glucose Metabolism and PD-1 Cells.

Cancer Immunol Res 2020 04 4;8(4):493-505. Epub 2020 Feb 4.

Department of Immunology and Inflammation, Humanitas Clinical and Research Center-IRCCS, Rozzano, Milan, Italy.

Better understanding of pancreatic diseases, including pancreatic ductal adenocarcinoma (PDAC), is an urgent medical need, with little advances in preoperative differential diagnosis, preventing rational selection of therapeutic strategies. The clinical management of pancreatic cancer patients would benefit from the identification of variables distinctively associated with the multiplicity of pancreatic disorders. We investigated, by H nuclear magnetic resonance, the metabolomic fingerprint of pancreatic juice (the biofluid that collects pancreatic products) in 40 patients with different pancreatic diseases. Metabolic variables discriminated PDAC from other less aggressive pancreatic diseases and identified metabolic clusters of patients with distinct clinical behaviors. PDAC specimens were overtly glycolytic, with significant accumulation of lactate, which was probed as a disease-specific variable in pancreatic juice from a larger cohort of 106 patients. In human PDAC sections, high expression of the glucose transporter GLUT-1 correlated with tumor grade and a higher density of PD-1 T cells, suggesting their accumulation in glycolytic tumors. In a preclinical model, PD-1 CD8 tumor-infiltrating lymphocytes differentially infiltrated PDAC tumors obtained from cell lines with different metabolic consumption, and tumors metabolically rewired by knocking down the phosphofructokinase () gene displayed a decrease in PD-1 cell infiltration. Collectively, we introduced pancreatic juice as a valuable source of metabolic variables that could contribute to differential diagnosis. The correlation of metabolic markers with immune infiltration suggests that upfront evaluation of the metabolic profile of PDAC patients could foster the introduction of immunotherapeutic approaches for pancreatic cancer.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0403DOI Listing
April 2020

Proteomics-Based Evidence for a Pro-Oncogenic Role of ESRP1 in Human Colorectal Cancer Cells.

Int J Mol Sci 2020 Jan 16;21(2). Epub 2020 Jan 16.

Institute of Biostructure and Bioimaging, CNR c/o Molecular Biotechnology Centre, 10126 Turin, Italy.

The RNA-binding protein, Epithelial Splicing Regulatory Protein 1 (ESRP1) can promote or suppress tumorigenesis depending on the cell type and disease context. In colorectal cancer, we have previously shown that aberrantly high ESRP1 expression can drive tumor progression. In order to unveil the mechanisms by which ESRP1 can modulate cancer traits, we searched for proteins affected by modulation of in two human colorectal cancer cell lines, HCA24 and COLO320DM, by proteomics analysis. Proteins hosted by endogenous ESRP1 ribonucleoprotein complex in HCA24 cells were also analyzed following RNA-immunoprecipitation. Proteomics data were complemented with bioinformatics approach to exploit publicly available data on protein-protein interaction (PPI). Gene Ontology was analysed to identify a common molecular signature possibly explaining the pro-tumorigenic role of ESRP1. Interestingly, proteins identified herein support a role for ESRP1 in response to external stimulus, regulation of cell cycle and hypoxia. Our data provide further insights into factors affected by and entwined with ESRP1 in colorectal cancer.
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http://dx.doi.org/10.3390/ijms21020575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014300PMC
January 2020

Integrative Analysis of Novel Metabolic Subtypes in Pancreatic Cancer Fosters New Prognostic Biomarkers.

Front Oncol 2019 27;9:115. Epub 2019 Feb 27.

Department of Computer Sciences, University of Turin, Turin, Italy.

Most of the patients with Pancreatic Ductal Adenocarcinoma (PDA) are not eligible for a curative surgical resection. For this reason there is an urgent need for personalized therapies. PDA is the result of complex interactions between tumor molecular profile and metabolites produced by its microenvironment. Despite recent studies identified PDA molecular subtypes, its metabolic classification is still lacking. We applied an integrative analysis on transcriptomic and genomic data of glycolytic genes in PDA. Data were collected from public datasets and molecular glycolytic subtypes were defined using hierarchical clustering. The grade of purity of the cancer samples was assessed estimating the different amount of stromal and immunological infiltrate among the identified PDA subtypes. Analyses of metabolomic data from a subset of PDA cell lines allowed us to identify the different metabolites produced by the metabolic subtypes. Sera of a cohort of 31 PDA patients were analyzed using Q-TOF mass spectrometer to measure the amount of metabolic circulating proteins present before and after chemotherapy. Our integrative analysis of glycolytic genes identified two glycolytic and two non-glycolytic metabolic PDA subtypes. Glycolytic patients develop disease earlier, have poor prognosis, low immune-infiltrated tumors, and are characterized by a gain in chr12p13 genomic region. This gain results in the over-expression of , and . PDA cell lines with the gain of chr12p13 are characterized by an higher lipid uptake and sensitivity to drug targeting the fatty acid metabolism. Our sera proteomic analysis confirms that TPI1 serum levels increase in poor prognosis gemcitabine-treated patients. We identify four metabolic PDA subtypes with different prognosis outcomes which may have pivotal role in setting personalized treatments. Moreover, our data suggest TPI1 as putative prognostic PDA biomarker.
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http://dx.doi.org/10.3389/fonc.2019.00115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400843PMC
February 2019

Pregnancy Epigenetic Signature in T Helper 17 and T Regulatory Cells in Multiple Sclerosis.

Front Immunol 2018 8;9:3075. Epub 2019 Jan 8.

Department of Clinical and Biological Sciences, University of Turin, Turin, Italy.

Increasing evidence supports the anti-inflammatory role of estrogens in Multiple Sclerosis (MS), originating from the observation of reduction in relapse rates among women with MS during pregnancy, but the molecular mechanisms are still not completely understood. Using an integrative data analysis, we identified T helper (Th) 17 and T regulatory (Treg) cell-type-specific regulatory regions (CSR) regulated by estrogen receptor alpha (ERα). These CSRs were validated in polarized Th17 from healthy donors (HD) and in peripheral blood mononuclear cells, Th17 and Treg cells from relapsing remitting (RR) MS patients and HD during pregnancy. 17β-estradiol induces active histone marks enrichment at Forkhead Box P3 (FOXP3)-CSRs and repressive histone marks enrichment at RAR related orphan receptor C (RORC)-CSRs in polarized Th17 cells. A disease-associated epigenetic profile was found in RRMS patients during pregnancy, suggesting a FOXP3 positive regulation and a RORC negative regulation in the third trimester of pregnancy. Altogether, these data indicate that estrogens act as immunomodulatory factors on the epigenomes of CD4+ T cells in RRMS; the identified CSRs may represent potential biomarkers for monitoring disease progression or new potential therapeutic targets.
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http://dx.doi.org/10.3389/fimmu.2018.03075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331474PMC
October 2019

Stromal protein βig-h3 reprogrammes tumour microenvironment in pancreatic cancer.

Gut 2019 04 10;68(4):693-707. Epub 2018 Nov 10.

Cancer Research Center of Lyon, UMR INSERM 1052, Lyon, France.

Objective: Pancreatic cancer is associated with an abundant stromal reaction leading to immune escape and tumour growth. This massive stroma drives the immune escape in the tumour. We aimed to study the impact of βig-h3 stromal protein in the modulation of the antitumoural immune response in pancreatic cancer.

Design: We performed studies with Cre;, -Cre;/, -Cre; mice and tumour tissues from patients with pancreatic ductal adenocarcinoma (PDA). Some transgenic mice were given injections of anti-βig-h3, anti-CD8, anti-PD1 depleting antibodies. Tumour growth as well as modifications in the activation of local immune cells were analysed by flow cytometry, immunohistochemistry and immunofluorescence. Tissue stiffness was measured by atomic force microscopy.

Results: We identified βig-h3 stromal-derived protein as a key actor of the immune paracrine interaction mechanism that drives pancreatic cancer. We found that βig-h3 is highly produced by cancer-associated fibroblasts in the stroma of human and mouse. This protein acts directly on tumour-specific CD8 T cells and F4/80 macrophages. Depleting βig-h3 reduced tumour growth by enhancing the number of activated CD8 T cell within the tumour and subsequent apoptotic tumour cells. Furthermore, we found that targeting βig-h3 in established lesions released the tissue tension and functionally reprogrammed F4/80 macrophages in the tumour microenvironment.

Conclusions: Our data indicate that targeting stromal extracellular matrix protein βig-h3 improves the antitumoural response and consequently reduces tumour weight. Our findings present βig-h3 as a novel immunological target in pancreatic cancer.
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http://dx.doi.org/10.1136/gutjnl-2018-317570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580775PMC
April 2019

Soluble stroma-related biomarkers of pancreatic cancer.

EMBO Mol Med 2018 08;10(8)

Laboratory of Biology and Treatment of Metastasis, Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Bergamo and Milan, Italy

The clinical management of pancreatic ductal adenocarcinoma (PDAC) is hampered by the lack of reliable biomarkers. This study investigated the value of soluble stroma-related molecules as PDAC biomarkers. In the first exploratory phase, 12 out of 38 molecules were associated with PDAC in a cohort of 25 PDAC patients and 16 healthy subjects. A second confirmatory phase on an independent cohort of 131 PDAC patients, 30 chronic pancreatitis patients, and 131 healthy subjects confirmed the PDAC association for MMP7, CCN2, IGFBP2, TSP2, sICAM1, TIMP1, and PLG Multivariable logistic regression model identified biomarker panels discriminating respectively PDAC versus healthy subjects (MMP7 + CA19.9, AUC = 0.99, 99% CI = 0.98-1.00) (CCN2 + CA19.9, AUC = 0.96, 99% CI = 0.92-0.99) and PDAC versus chronic pancreatitis (CCN2 + PLG+FN+Col4 + CA19.9, AUC = 0.94, 99% CI = 0.88-0.99). Five molecules were associated with PanIN development in two GEM models of PDAC (PdxCre/LSL-Kras and PdxCre/LSL-Kras/LSL-Trp53), suggesting their potential for detecting early disease. These markers were also elevated in patient-derived orthotopic PDAC xenografts and associated with response to chemotherapy. The identified stroma-related soluble biomarkers represent potential tools for PDAC diagnosis and for monitoring treatment response of PDAC patients.
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http://dx.doi.org/10.15252/emmm.201708741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079536PMC
August 2018

The advanced glycation end-product N -carboxymethyllysine promotes progression of pancreatic cancer: implications for diabetes-associated risk and its prevention.

J Pathol 2018 06 4;245(2):197-208. Epub 2018 Apr 4.

Department of Clinical and Molecular Medicine, 'La Sapienza' University, Rome, Italy.

Diabetes is an established risk factor for pancreatic cancer (PaC), together with obesity, a Western diet, and tobacco smoking. The common mechanistic link might be the accumulation of advanced glycation end-products (AGEs), which characterizes all of the above disease conditions and unhealthy habits. Surprisingly, however, the role of AGEs in PaC has not been examined yet, despite the evidence of a tumour-promoting role of receptor for advanced glycation end-products (RAGE), the receptor for AGEs. Here, we tested the hypothesis that AGEs promote PaC through RAGE activation. To this end, we investigated the effects of the AGE N -carboxymethyllysine (CML) in human pancreatic ductal adenocarcinoma (PDA) cell lines and in a mouse model of Kras-driven PaC interbred with a bioluminescent model of proliferation. Tumour growth was monitored in vivo by bioluminescence imaging and confirmed by histology. CML promoted PDA cell growth and RAGE expression, in a concentration-dependent and time-dependent manner, and activated downstream tumourigenic signalling pathways. These effects were counteracted by RAGE antagonist peptide (RAP). Exogenous AGE administration to PaC-prone mice induced RAGE upregulation in pancreatic intraepithelial neoplasias (PanINs) and markedly accelerated progression to invasive PaC. At 11 weeks of age (6 weeks of CML treatment), PaC was observed in eight of 11 (72.7%) CML-treated versus one of 11 (9.1%) vehicle-treated [control (Ctr)] mice. RAP delayed PanIN development in Ctr mice but failed to prevent PaC promotion in CML-treated mice, probably because of competition with soluble RAGE for binding to AGEs and/or compensatory upregulation of the RAGE homologue CD166/ activated leukocyte cell adhesion molecule, which also favoured tumour spread. These findings indicate that AGEs modulate the development and progression of PaC through receptor-mediated mechanisms, and might be responsible for the additional risk conferred by diabetes and other conditions characterized by increased AGE accumulation. Finally, our data suggest that an AGE reduction strategy, instead of RAGE inhibition, might be suitable for the risk management and prevention of PaC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5072DOI Listing
June 2018

Next Generation Immunotherapy for Pancreatic Cancer: DNA Vaccination is Seeking New Combo Partners.

Cancers (Basel) 2018 Feb 16;10(2). Epub 2018 Feb 16.

Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin 10126, Italy.

Pancreatic Ductal Adenocarcinoma (PDA) is an almost incurable radio- and chemo-resistant tumor, and its microenvironment is characterized by a strong desmoplastic reaction associated with a significant infiltration of T regulatory lymphocytes and myeloid-derived suppressor cells (Tregs, MDSC). Investigating immunological targets has identified a number of metabolic and cytoskeletal related molecules, which are typically recognized by circulating antibodies. Among these molecules we have investigated alpha-enolase (ENO1), a glycolytic enzyme that also acts a plasminogen receptor. ENO1 is also recognized by T cells in PDA patients, so we developed a DNA vaccine that targets ENO1. This efficiently induces many immunological processes (antibody formation and complement-dependent cytotoxicity (CDC)-mediated tumor killing, infiltration of effector T cells, reduction of infiltration of myeloid and Treg suppressor cells), which significantly increase the survival of genetically engineered mice that spontaneously develop pancreatic cancer. Although promising, the ENO1 DNA vaccine does not completely eradicate the tumor, which, after an initial growth inhibition, returns to proliferate again, especially when Tregs and MDSC ensue in the tumor mass. This led us to develop possible strategies for combinatorial treatments aimed to broaden and sustain the antitumor immune response elicited by DNA vaccination. Based on the data we have obtained in recent years, this review will discuss the biological bases of possible combinatorial treatments (chemotherapy, PI3K inhibitors, tumor-associated macrophages, ENO1 inhibitors) that could be effective in amplifying the response induced by the immune vaccination in PDA.
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http://dx.doi.org/10.3390/cancers10020051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836083PMC
February 2018

Phosphoinositide 3-Kinase Gamma Inhibition Protects From Anthracycline Cardiotoxicity and Reduces Tumor Growth.

Circulation 2018 08;138(7):696-711

Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Italy (M.L., V.S., M.C.D.S., J.C., J.P.M., M.M., F.P., L.R., I.F., A.P., P.E.P., R.C.P.L.-J., E.H., A.G.).

Background: Anthracyclines, such as doxorubicin (DOX), are potent anticancer agents for the treatment of solid tumors and hematologic malignancies. However, their clinical use is hampered by cardiotoxicity. This study sought to investigate the role of phosphoinositide 3-kinase γ (PI3Kγ) in DOX-induced cardiotoxicity and the potential cardioprotective and anticancer effects of PI3Kγ inhibition.

Methods: Mice expressing a kinase-inactive PI3Kγ or receiving PI3Kγ-selective inhibitors were subjected to chronic DOX treatment. Cardiac function was analyzed by echocardiography, and DOX-mediated signaling was assessed in whole hearts or isolated cardiomyocytes. The dual cardioprotective and antitumor action of PI3Kγ inhibition was assessed in mouse mammary tumor models.

Results: PI3Kγ kinase-dead mice showed preserved cardiac function after chronic low-dose DOX treatment and were protected against DOX-induced cardiotoxicity. The beneficial effects of PI3Kγ inhibition were causally linked to enhanced autophagic disposal of DOX-damaged mitochondria. Consistently, either pharmacological or genetic blockade of autophagy in vivo abrogated the resistance of PI3Kγ kinase-dead mice to DOX cardiotoxicity. Mechanistically, PI3Kγ was triggered in DOX-treated hearts, downstream of Toll-like receptor 9, by the mitochondrial DNA released by injured organelles and contained in autolysosomes. This autolysosomal PI3Kγ/Akt/mTOR/Ulk1 signaling provided maladaptive feedback inhibition of autophagy. PI3Kγ blockade in models of mammary gland tumors prevented DOX-induced cardiac dysfunction and concomitantly synergized with the antitumor action of DOX by unleashing anticancer immunity.

Conclusions: Blockade of PI3Kγ may provide a dual therapeutic advantage in cancer therapy by simultaneously preventing anthracyclines cardiotoxicity and reducing tumor growth.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.117.030352DOI Listing
August 2018

Depletion of tumor-associated macrophages switches the epigenetic profile of pancreatic cancer infiltrating T cells and restores their anti-tumor phenotype.

Oncoimmunology 2018;7(2):e1393596. Epub 2017 Nov 13.

Dept. of Molecular Biotechnology and Health Sciences, University of Turin, via Nizza 52, Torino, Italy.

Pancreatic Ductal Adenocarcinoma (PDA) is characterized by a complex tumor microenvironment that supports its progression, aggressiveness and resistance to therapies. The delicate interplay between cancer and immune cells creates the conditions for PDA development, particularly due to the functional suppression of T cell anti-tumor effector activity. However, some of the mechanisms involved in this process are still poorly understood. In this study, we analyze whether the functional and epigenetic profile of T cells that infiltrate PDA is modulated by the microenvironment, and in particular by tumor-associated macrophages (TAMs). CD4 and CD8 T cells obtained from mice orthotopically injected with syngeneic PDA cells, and untreated or treated with Trabectedin, a cytotoxic drug that specifically targets TAMs, were sorted and analyzed by flow cytometry and characterized for their epigenetic profile. Assessment of cytokine production and the epigenetic profile of genes coding for IL10, T-bet and PD1 revealed that T cells that infiltrated PDA displayed activated promoter and repressed activity, in agreement with their regulatory phenotype (IL10/IFNγ, PD1). By contrast, in Trabectedin-treated mice, PDA-infiltrating T cells displayed repressed and and activated promoter activity, in accordance with their anti-tumor effector phenotype (IL10/IFNγ), indicating a key role of TAMs in orchestrating functions of PDA-infiltrating T cells by modulating their epigenetic profile towards a pro-tumoral phenotype. These results suggest the targeting of TAMs as an efficient strategy to obtain an appropriate T cell anti-tumor immune response and open new potential combinations for PDA treatment.
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http://dx.doi.org/10.1080/2162402X.2017.1393596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749621PMC
November 2017

Endogenous glutamine decrease is associated with pancreatic cancer progression.

Oncotarget 2017 Nov 24;8(56):95361-95376. Epub 2017 Aug 24.

Center for Experimental Research and Medical Studies, Città della Salute e della Scienza di Torino, 10126 Turin, Italy.

Pancreatic ductal adenocarcinoma (PDAC) is becoming the second leading cause of cancer-related death in the Western world. The mortality is very high, which emphasizes the need to identify biomarkers for early detection. As glutamine metabolism alteration is a feature of PDAC, its evaluation may provide a useful tool for biomarker identification. Our aim was to identify a handy method to evaluate blood glutamine consumption in mouse models of PDAC. We quantified the glutamine uptake by Mass Spectrometry (MS) in tumor cell supernatants and showed that it was higher in PDAC compared to non-PDAC tumor and pancreatic control human cells. The increased glutamine uptake was paralleled by higher activity of most glutamine pathway-related enzymes supporting nucleotide and ATP production. Free glutamine blood levels were evaluated in orthotopic and spontaneous mouse models of PDAC and other pancreatic-related disorders by High-Performance Liquid Chromatography (HPLC) and/or MS. Notably we observed a reduction of blood glutamine as much as the tumor progressed from pancreatic intraepithelial lesions to invasive PDAC, but was not related to chronic pancreatitis-associated inflammation or diabetes. In parallel the increased levels of branched-chain amino acids (BCAA) were observed. By contrast blood glutamine levels were stable in non-tumor bearing mice. These findings demonstrated that glutamine uptake is measurable both and . The higher avidity of PDAC cells corresponded to a lower blood glutamine level as soon as the tumor mass grew. The reduction in circulating glutamine represents a novel tool exploitable to implement other diagnostic or prognostic PDAC biomarkers.
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http://dx.doi.org/10.18632/oncotarget.20545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707027PMC
November 2017

Lack of CD4+ T cell percent decrease in alemtuzumab-treated multiple sclerosis patients with persistent relapses.

J Neuroimmunol 2017 12 17;313:89-91. Epub 2017 Oct 17.

Clinical and Biological Sciences Department, University of Torino, San Luigi Gonzaga Hospital, Orbassano (TO), Italy. Electronic address:

Alemtuzumab, a highly effective treatment for relapsing remitting multiple sclerosis (RRMS), induces lymphopenia especially of CD4+ T cells. Here, we report the atypical CD4+ T population behaviour of two patients with persistent disease activity despite repeated alemtuzumab treatments. Whereas lymphocytes count decreased and fluctuated accordingly to alemtuzumab administration, their CD4+ cell percentage was not or just mildly affected and was slightly below the lowest normal limit already before alemtuzumab. These cases anticipate further studies aimed to investigate whether the evaluation of the CD4+ cell percentage could represent a helpful tool to address the individual clinical response to alemtuzumab.
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http://dx.doi.org/10.1016/j.jneuroim.2017.10.009DOI Listing
December 2017

Regulation of Human Macrophage M1-M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1.

Front Immunol 2017 7;8:1097. Epub 2017 Sep 7.

Laboratory of Molecular Biology, Giannina Gaslini Institute, Genoa, Italy.

Macrophages (Mf) are a heterogeneous population of tissue-resident professional phagocytes and a major component of the leukocyte infiltrate at sites of inflammation, infection, and tumor growth. They can undergo diverse forms of activation in response to environmental factors, polarizing into specialized functional subsets. A common hallmark of the pathologic environment is represented by hypoxia. The impact of hypoxia on human Mf polarization has not been fully established. The objective of this study was to elucidate the effects of a hypoxic environment reflecting that occurring in diseased tissues on the ability of human Mf to polarize into classically activated (proinflammatory M1) and alternatively activated (anti-inflammatory M2) subsets. We present data showing that hypoxia hinders Mf polarization toward the M1 phenotype by decreasing the expression of T cell costimulatory molecules and chemokine homing receptors and the production of proinflammatory, Th1-priming cytokines typical of classical activation, while promoting their acquisition of phenotypic and secretory features of alternative activation. Furthermore, we identify the triggering receptor expressed on myeloid cells (TREM)-1, a member of the Ig-like immunoregulatory receptor family, as a hypoxia-inducible gene in Mf and demonstrate that its engagement by an agonist Ab reverses the M2-polarizing effect of hypoxia imparting a M1-skewed phenotype to Mf. Finally, we provide evidence that Mf infiltrating the inflamed hypoxic joints of children affected by oligoarticular juvenile idiopatic arthritis express high surface levels of TREM-1 associated with predominant M1 polarization and suggest the potential of this molecule in driving M1 proinflammatory reprogramming in the hypoxic synovial environment.
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http://dx.doi.org/10.3389/fimmu.2017.01097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5594076PMC
September 2017

The ATP-binding cassette transporter A1 regulates phosphoantigen release and Vγ9Vδ2 T cell activation by dendritic cells.

Nat Commun 2017 06 5;8:15663. Epub 2017 Jun 5.

Dipartimento di Biotecnologie Molecolari e Scienze della Salute, Università degli Studi di Torino, Via Nizza 52, Torino 10126, Italy.

Vγ9Vδ2 T cells are activated by phosphoantigens, such as isopentenyl pyrophosphate (IPP), which is generated in the mevalonate pathway of antigen-presenting cells. IPP is released in the extracellular microenvironment via unknown mechanisms. Here we show that the ATP-binding cassette transporter A1 (ABCA1) mediates extracellular IPP release from dendritic cells (DC) in cooperation with apolipoprotein A-I (apoA-I) and butyrophilin-3A1. IPP concentrations in the supernatants are sufficient to induce Vγ9Vδ2 T cell proliferation after DC mevalonate pathway inhibition with zoledronic acid (ZA). ZA treatment increases ABCA1 and apoA-I expression via IPP-dependent LXRα nuclear translocation and PI3K/Akt/mTOR pathway inhibition. These results close the mechanistic gap in our understanding of extracellular IPP release from DC and provide a framework to fine-tune Vγ9Vδ2 T cell activation via mevalonate and PI3K/Akt/mTOR pathway modulation.
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http://dx.doi.org/10.1038/ncomms15663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465356PMC
June 2017

Next generation of cancer immunotherapy calls for combination.

Oncoscience 2017 Mar 31;4(3-4):19-20. Epub 2017 Mar 31.

Department of Molecular Biotechnology and Health Sciences, University of Turin; Center for Experimental Research and Medical Studies, AOU Città della Salute e della Scienza di Torino; Molecular Biotechnology Center, University of Turin, Turin, 10126, Italy.

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http://dx.doi.org/10.18632/oncoscience.343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441468PMC
March 2017

Alpha-enolase (ENO1) controls alpha v/beta 3 integrin expression and regulates pancreatic cancer adhesion, invasion, and metastasis.

J Hematol Oncol 2017 01 13;10(1):16. Epub 2017 Jan 13.

Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

Background: We have previously shown that in pancreatic ductal adenocarcinoma (PDA) cells, the glycolytic enzyme alpha-enolase (ENO1) also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability.

Methods: The membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1) PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM). The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model.

Results: We observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN) via urokinase plasminogen activator receptor (uPAR). Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell migration and invasion of shENO1 PDA cells was observed.

Conclusion: These data demonstrate that ENO1 promotes PDA survival, migration, and metastasis through cooperation with integrins and uPAR.
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http://dx.doi.org/10.1186/s13045-016-0385-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5237223PMC
January 2017

Adenosine A2a receptor stimulation blocks development of nonalcoholic steatohepatitis in mice by multilevel inhibition of signals that cause immunolipotoxicity.

Transl Res 2017 04 6;182:75-87. Epub 2016 Dec 6.

Department of Health Science, University of Piemonte Orientale, Novara, Italy. Electronic address:

Lipotoxicity and immunoinflammation are associated with the evolution of steatosis toward nonalcoholic steatohepatitis (NASH). This study reports the ability of adenosine A2a receptor (A2aR) activation to inhibit NASH development by modulating the responses of CD4 T-helper (Th) cells to avoid an immuno-mediated potentiation of lipotoxicity. The effect of the A2aR agonist CGS21680 on immunoinflammatory signals, CD4Th cell infiltration and immunolipotoxicity was analyzed in steatotic C57BL/6 mice fed with a methionine-choline-deficient (MCD) diet and in mouse hepatocytes exposed to palmitic acid (PA). CGS21680 inhibited NASH development in steatotic mice and decreased cytokines and chemokines involved in Th cell recruitment or polarization (namely CXCL10, CCL2, tumor necrosis factor alfa [TNFα], tumor growth factor [TGFβ], and IL-12). CGS21680 also reduced the expansion of Th17, Th22, and Th1 cells and increased the immunosuppressive activity of T regulatory cells. In PA-treated mice hepatocytes, CGS21680 inhibited the production of CXCL10, TNFα, TGFβ, IL-12, and CCL2; CGS21680 also prevented JNK-dependent lipotoxicity and its intensification by IL-17 or IL-17 plus IL-22 through Akt/PI3-kinase stimulation and inhibition of the negative regulator of PI3-kinase, (phosphatase and tensin homologue deleted from chromosome 10 (PTEN), which is upregulated by IL-17. In MCD livers, CGS21680 reduced JNK activation and PTEN expression and increased Akt phosphorylation. In conclusion, A2aR stimulation inhibited NASH development by reducing Th17 cell expansion and inhibiting the exacerbation of the IL-17-induced JNK-dependent lipotoxicity. These data promote the implementation of further studies to evaluate the potential clinical application of A2aR agonists that, by being able to function as both cytoprotective and immunomodulatory agents, could efficiently antagonize the multi-faced pathogenesis of NASH.
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http://dx.doi.org/10.1016/j.trsl.2016.11.009DOI Listing
April 2017

Beta-2-glycoprotein-1 and alpha-1-antitrypsin as urinary markers of renal cancer in von Hippel-Lindau patients.

Biomarkers 2018 Mar 22;23(2):123-130. Epub 2016 Dec 22.

e Department of Urology , Azienda Ospedaliera Città della Salute e Della Scienza di Torino - Molinette , Turin , Italy.

Context: Von Hippel-Lindau disease (VHLD) is a rare inherited neoplastic syndrome. Among all the VHLD-associated tumors, clear cell renal cell carcinoma (ccRCC) is the major cause of death.

Objective: The aim of this paper is the discovery of new non-invasive biomarker for the monitoring of VHLD patients.

Materials And Methods: We compared the urinary proteome of VHLD patients, ccRCC patients and healthy volunteers.

Results: Among all differentially expressed proteins, alpha-1-antitrypsin (A1AT) and APOH (beta-2-glycoprotein-1) are strongly over-abundant only in the urine of VHLD patients with a history of ccRCC.

Discussion And Conclusion: A1AT and APOH could be promising non-invasive biomarkers.
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http://dx.doi.org/10.1080/1354750X.2016.1269132DOI Listing
March 2018

Humoral immune responses toward tumor-derived antigens in previously untreated patients with chronic lymphocytic leukemia.

Oncotarget 2017 Jan;8(2):3274-3288

Division of Hematology, University of Torino, AOU Città della Salute e della Scienza di Torino, Torino, Italy.

In chronic lymphocytic leukemia (CLL) the occurrence and the impact of antibody responses toward tumor-derived antigens are largely unexplored. Our serological proteomic data show that antibodies toward 47 identified antigens are detectable in 29 out of 35 patients (83%) with untreated CLL. The glycolytic enzyme alpha-enolase (ENO1) is the most frequently recognized antigen (i.e. 54% of CLL sera). We show that ENO1 is upregulated in the proliferating B-cell fraction of CLL lymph nodes. In CLL cells of the peripheral blood, ENO1 is exclusively expressed at the intracellular level, whereas it is exposed on the surface of apoptotic leukemic cells.From the clinical standpoint, patients with progressive CLL show a higher number of antigen recognitions compared to patients with stable disease. Consistently, the anti-ENO1 antibodies are prevalent in sera from patients with progressive disease and their presence is predictive of a shorter time to first treatment. This clinical inefficacy associates with the inability of patients' sera to trigger complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity against leukemic cells.Together, these results indicate that antibody responses toward tumor-derived antigens are frequently detectable in sera from patients with CLL, but they are expression of a disrupted immune system and unable to hamper disease progression.
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http://dx.doi.org/10.18632/oncotarget.13712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5356881PMC
January 2017

Alpha-Enolase (), a potential target in novel immunotherapies.

Front Biosci (Landmark Ed) 2017 01 1;22:944-959. Epub 2017 Jan 1.

Laboratory of Tumor Immunology, Center for Experimental Research and Medical Studies, Azienda Universitaria Ospedaliera Città della Salute e della Scienza di Torino, via Santena 5, Torino 10126 Italy,

Alpha-enolase () is a metabolic enzyme involved in the synthesis of pyruvate. It also acts as a plasminogen receptor and mediates the activation of plasmin and extracellular matrix degradation. In tumor cells, is up-regulated and supports the Warburg effect; it is expressed at the cell surface, where it promotes cancer invasion, and is subjected to a specific array of post-translational modifications, namely acetylation, methylation and phosphorylation. overexpression and post-translational modifications could be of diagnostic and prognostic value in many cancer types. Information on the biochemical, proteomics and immunological characterization of , and particularly its ability to trigger a strong specific humoral and cellular immune response, make this ubiquitous protein an interesting tumor target; DNA vaccination with in preclinical models efficiently delays the development of very aggressive tumors such as pancreatic cancer. This review aims to analyze the main stages by which the tumor associated antigen (TAA) has become a promising target that opens potential avenues for cancer immunotherapy.
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http://dx.doi.org/10.2741/4526DOI Listing
January 2017

Cancer and Chemotherapy Contribute to Muscle Loss by Activating Common Signaling Pathways.

Front Physiol 2016 19;7:472. Epub 2016 Oct 19.

Department of Surgery, Indiana University School of MedicineIndianapolis, IN, USA; Simon Cancer Center, Indiana University School of MedicineIndianapolis, IN, USA; Center for Cachexia Research Innovation and Therapy, Indiana University - Purdue University IndianapolisIndianapolis, IN, USA; Department of Otolaryngology, Head and Neck Surgery, Indiana University School of MedicineIndianapolis, IN, USA.

Cachexia represents one of the primary complications of colorectal cancer due to its effects on depletion of muscle and fat. Evidence suggests that chemotherapeutic regimens, such as Folfiri, contribute to cachexia-related symptoms. The purpose of the present study was to investigate the cachexia signature in different conditions associated with severe muscle wasting, namely Colon-26 (C26) and Folfiri-associated cachexia. Using a quantitative LC-MS/MS approach, we identified significant changes in 386 proteins in the quadriceps muscle of Folfiri-treated mice, and 269 proteins differentially expressed in the C26 hosts ( < 0.05; -1.5 ≥ fold change ≥ +1.5). Comparative analysis isolated 240 proteins that were modulated in common, with a large majority (218) that were down-regulated in both experimental settings. Interestingly, metabolic (47.08%) and structural (21.25%) proteins were the most represented. Pathway analysis revealed mitochondrial dysfunctions in both experimental conditions, also consistent with reduced expression of mediators of mitochondrial fusion (OPA-1, mitofusin-2), fission (DRP-1) and biogenesis (Cytochrome C, PGC-1α). Alterations of oxidative phosphorylation within the TCA cycle, fatty acid metabolism, and Ca signaling were also detected. Overall, the proteomic signature in the presence of both chemotherapy and cancer suggests the activation of mechanisms associated with movement disorders, necrosis, muscle cell death, muscle weakness and muscle damage. Conversely, this is consistent with the inhibition of pathways that regulate nucleotide and fatty acid metabolism, synthesis of ATP, muscle and heart function, as well as ROS scavenging. Interestingly, strong up-regulation of pro-inflammatory acute-phase proteins and a more coordinated modulation of mitochondrial and lipidic metabolisms were observed in the muscle of the C26 hosts that were different from the Folfiri-treated animals. In conclusion, our results suggest that both cancer and chemotherapy contribute to muscle loss by activating common signaling pathways. These data support the undertaking of combination strategies that aim to both counteract tumor growth and reduce chemotherapy side effects.
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http://dx.doi.org/10.3389/fphys.2016.00472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5070123PMC
October 2016

Anti-α-enolase antibody limits the invasion of myeloid-derived suppressor cells and attenuates their restraining effector T cell response.

Oncoimmunology 2016 May 21;5(5):e1112940. Epub 2015 Dec 21.

Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy; Center for Experimental Research and Medical Studies, University Hospital Città della Salute e della Scienza di Torino, Torino, Italy; Molecular Biotechnology Center, via Nizza 52, Torino, Italy; Transplant Immunology Service, University Hospital Cità della Salute e della Scienza di Torino, Turin, Italy.

Pancreatic Ductal Adenocarcinoma (PDA) is a very aggressive tumor for which effective therapeutical strategies are still lacking. Globally, the 5 y survival rate is 5-7% and surgery is the only potentially curative treatment. Immunotherapy represents a novel possibility for treating PDA, and myeloid-derived suppressor cells (MDSC), which are increased in cancer patients and correlate with metastatic burden and cancer stage, offer a new target in cancer therapy. We have previously shown that antibodies against the PDA-associated antigen α-enolase (ENO1) are detected in more than 60% of PDA patients and correlate with a better prognosis. Furthermore, ENO1-DNA vaccination in mice induced anti-ENO1 antibodies that mediated antitumor activity. In this study, the effects of anti-ENO1 binding on MDSC functions and on the T cell response were evaluated. Here, we show that MDSC express ENO1 on their surface, which increased after LPS stimulation. Moreover, anti-ENO1 mAb inhibited adhesion to endothelial cells, as well as in vitro and in vivo migration. Similarly, after ENO1 mAb treatment of MDSC, arginase activity decreased, while the secretion of pro-inflammatory cytokines (particularly IL-6) increased, and co-stimulatory molecule expression and suppression functions were only partially affected. Finally, we found that activated T cells in the presence of anti-ENO1 mAb-treated MDSC increased IFNγ and IL-17 secretion and decreased IL-10 and TGFβ secretion compared to control MDSC. In conclusion, anti-ENO1 antibodies may inhibit in vivo the infiltration into the tumor microenvironment of MDSC, and attenuate their restraining of effector T cell response, opening a new perspective to render PDA immunotherapy more effective.
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http://dx.doi.org/10.1080/2162402X.2015.1112940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910736PMC
May 2016

Peripheral ENO1-specific T cells mirror the intratumoral immune response and their presence is a potential prognostic factor for pancreatic adenocarcinoma.

Int J Oncol 2016 Jul 16;49(1):393-401. Epub 2016 May 16.

Department of Experimental and Clinical Medicine, University of Florence, and Department of Biomedicine, Azienda Ospedaliera Universitaria Careggi (AOUC), I-50134 Florence, Italy.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with an average survival of 4-6 months following diagnosis. Surgical resection is the only treatment with curative intent, but resectable PDAC patients are in the minority. Also, unlike other neoplasms, PDAC is resistant to conventional and targeted chemotherapy. Innovative treatments, such as immunotherapy, can be very important and the study of the immune response is fundamental. We previously demonstrated that PDAC patients show tumor-infiltrating T cells specific to α-enolase (ENO1), a glycolytic enzyme over-expressed by pancreatic tumor cells, which plays an important role in promoting cell migration and cancer metastasis. In the present study, we evaluate the functional anticancer proprieties of ENO1-specific T cells isolated from the peripheral blood of PDAC patients. Furthermore, comparing the T cell receptor repertoire of ENO1-specific peripheral and infiltrating tumor T cells from the same patient suggests that ENO1-specific T cells, despite having a different functional profile, can recirculate from the tumor to the periphery. Finally, of clinical relevance, the presence of peripheral ENO1-specific T cells has a prognostic value and significantly correlates with a longer survival.
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http://dx.doi.org/10.3892/ijo.2016.3524DOI Listing
July 2016

Macrophage PI3Kγ Drives Pancreatic Ductal Adenocarcinoma Progression.

Cancer Discov 2016 08 13;6(8):870-85. Epub 2016 May 13.

Moores Cancer Center, University of California, San Diego, La Jolla, California. Department of Pathology, University of California, San Diego, La Jolla, California.

Unlabelled: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a low 5-year survival rate, yet new immunotherapeutic modalities may offer hope for this and other intractable cancers. Here, we report that inhibitory targeting of PI3Kγ, a key macrophage lipid kinase, stimulates antitumor immune responses, leading to improved survival and responsiveness to standard-of-care chemotherapy in animal models of PDAC. PI3Kγ selectively drives immunosuppressive transcriptional programming in macrophages that inhibits adaptive immune responses and promotes tumor cell invasion and desmoplasia in PDAC. Blockade of PI3Kγ in PDAC-bearing mice reprograms tumor-associated macrophages to stimulate CD8(+) T-cell-mediated tumor suppression and to inhibit tumor cell invasion, metastasis, and desmoplasia. These data indicate the central role that macrophage PI3Kγ plays in PDAC progression and demonstrate that pharmacologic inhibition of PI3Kγ represents a new therapeutic modality for this devastating tumor type.

Significance: We report here that PI3Kγ regulates macrophage transcriptional programming, leading to T-cell suppression, desmoplasia, and metastasis in pancreas adenocarcinoma. Genetic or pharmacologic inhibition of PI3Kγ restores antitumor immune responses and improves responsiveness to standard-of-care chemotherapy. PI3Kγ represents a new therapeutic immune target for pancreas cancer. Cancer Discov; 6(8); 870-85. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.
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http://dx.doi.org/10.1158/2159-8290.CD-15-1346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5091937PMC
August 2016

Spatial distribution of B cells predicts prognosis in human pancreatic adenocarcinoma.

Oncoimmunology 2016 Apr 11;5(4):e1085147. Epub 2015 Sep 11.

Department of Immunology and Inflammation, Humanitas Clinical and Research Center, Rozzano, Italy; Department of Medical Biotechnologies and Translational Medicine, University of Milan, Milano, Italy.

B-cell responses are emerging as critical regulators of cancer progression. In this study, we investigated the role of B lymphocytes in the microenvironment of human pancreatic ductal adenocarcinoma (PDAC), in a retrospective consecutive series of 104 PDAC patients and in PDAC preclinical models. Immunohistochemical analysis revealed that B cells occupy two histologically distinct compartments in human PDAC, either scatteringly infiltrating (CD20-TILs), or organized in tertiary lymphoid tissue (CD20-TLT). Only when retained within TLT, high density of B cells predicted longer survival (median survival 16.9 mo CD20-TLT vs. 10.7 mo CD20-TLT; = 0.0085). Presence of B cells within TLT associated to a germinal center (GC) immune signature, correlated with CD8-TIL infiltration, and empowered their favorable prognostic value. Immunotherapeutic vaccination of spontaneously developing PDAC (Kras-Pdx1-Cre) mice with α-enolase (ENO1) induced formation of TLT with active GCs and correlated with increased recruitment of T lymphocytes, suggesting induction of TLT as a strategy to favor mobilization of immune cells in PDAC. In contrast, in an implanted tumor model devoid of TLT, depletion of B cells with an anti-CD20 antibody reinstated an antitumor immune response. Our results highlight B cells as an essential element of the microenvironment of PDAC and identify their spatial organization as a key regulator of their antitumor function. A mindfully evaluation of B cells in human PDAC could represent a powerful prognostic tool to identify patients with distinct clinical behaviors and responses to immunotherapeutic strategies.
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http://dx.doi.org/10.1080/2162402X.2015.1085147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4839336PMC
April 2016