In pancreatic cancer patients, chemotherapy reshapes the gene expression profile and antigen receptor repertoire of T lymphocytes and enhances their effector response to tumor-associated antigens.

Introduction: Pancreatic Ductal Adenocarcinoma (PDA) is one of the most aggressive malignancies with a 5-year survival rate of 13%. Less than 20% of patients have a resectable tumor at diagnosis due to the lack of distinctive symptoms and reliable biomarkers. PDA is resistant to chemotherapy (CT) and understanding how to gain an anti-tumor effector response following stimulation is, therefore, critical for setting up an effective immunotherapy. Methods: Proliferation, and cytokine release and TCRB repertoire of from PDA patient peripheral T lymphocytes, before and after CT, were analyzed in vitro in response to four tumor-associated antigens (TAA), namely ENO1, FUBP1, GAPDH and K2C8. Transcriptional state of PDA patient PBMC was investigated using RNA-Seq before and after CT. Results: CT increased the number of TAA recognized by T lymphocytes, which positively correlated with patient survival, and high IFN-γ production TAA-induced responses were significantly increased after CT. We found that some ENO1-stimulated T cell clonotypes from CT-treated patients were expanded or de-novo induced, and that some clonotypes were reduced or even disappeared after CT. Patients that showed a higher number of effector responses to TAA (high IFN-γ/IL-10 ratio) after CT expressed increased fatty acid-related transcriptional signature. Conversely, patients that showed a higher number of regulatory responses to TAA (low IFN-γ/IL-10 ratio) after CT significantly expressed an increased IRAK1/IL1R axis-related transcriptional signature. Conclusion: These data suggest that the expression of fatty acid or IRAK1/IL1Rrelated genes predicts T lymphocyte effector or regulatory responses to TAA in patients that undergo CT. These findings are a springboard to set up precision immunotherapies in PDA based on the TAA vaccination in combination with CT.

Circulating autoantibodies to alpha-enolase (ENO1) and far upstream element-binding protein 1 (FUBP1) are negative prognostic factors for pancreatic cancer patient survival.

Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis due to a lack of early diagnostic markers and effective therapy. In PDA patients, the glycolytic enzyme and plasminogen receptor alpha-enolase (ENO1) and the transcription factor far upstream element-binding protein 1 (FUBP1) are upregulated and elicit the production of autoantibodies (aAb) that discriminate healthy subjects from PDA patients, with the latter mostly directed to post-translational phosphorylated isoforms. Here, the correlation of prognosis with circulating ENO1 and FUBP1aAb, and their protein tissue expression was analyzed in PDA patients. Circulating ENO1 and FUBP1 aAb was analyzed in two cohorts of PDA patients by ELISA (n = 470), while tissues expression was observed by immunohistochemistry (n = 45). Overall survival (OS) was estimated using the Kaplan-Meier method, while the Cox model was used to estimate the hazard ratios (HR) adjusted for the main prognostic factors. Logistic models were applied to assess associations between death and its risk indicators. All statistical analyses were performed with Stata version 15. Unlike ENO1 aAb, there was a significant correlation between FUBP1 aAb and FUBP1 expression in tumors (p = 0.0268). In addition, we found that high ENO1 (p = 0.016) and intermediate FUBP1 aAb levels (p = 0.013) were unfavorable prognostic factors. Notably, it was found that high anti-FUBP1 aAb level is a good prognostic marker for tail-body PDA (p = 0.016). Our results suggest that different levels of circulating aAb to ENO1 and FUBP1 predict a poor outcome in PDA patients and can be used to improve therapeutic strategies.

Dendritic crystallization in hydrous basaltic magmas controls magma mobility within the Earth's crust.

The majority of basaltic magmas stall in the Earth's crust as a result of the rheological evolution caused by crystallization during transport. However, the relationships between crystallinity, rheology and eruptibility remain uncertain because it is difficult to observe dynamic magma crystallization in real time. Here, we present in-situ 4D data for crystal growth kinetics and the textural evolution of pyroxene during crystallization of trachybasaltic magmas in high-temperature experiments under water-saturated conditions at crustal pressures. We observe dendritic growth of pyroxene on initially euhedral cores, and a surprisingly rapid increase in crystal fraction and aspect ratio at undercooling ≥30 °C. Rapid dendritic crystallization favours a rheological transition from Newtonian to non-Newtonian behaviour within minutes. We use a numerical model to quantify the impact of rapid dendritic crystallization on basaltic dike propagation, and demonstrate its dramatic effect on magma mobility and eruptibility. Our results provide insights into the processes that control whether intrusions lead to eruption or not.

Inflated pyroclasts in proximal fallout deposits reveal abrupt transitions in eruption behaviour.

During explosive eruption of low viscosity magmas, pyroclasts are cooled predominantly by forced convection. Depending on the cooling efficiency relative to other timescales, a spectrum of deposits can be formed. Deposition of hot clasts, above their glass transition temperature, can form spatter mounds, ramparts and clastogenic lava flows. Clasts may also be deposited cold, producing tephra cones and blankets. Thus, the deposit and pyroclast type can provide information about eruption dynamics and magma properties. Here we examine pyroclasts from Tseax volcano, British Columbia, Canada. These newly identified inflated pyroclasts, are fluidal in form, have undergone post-depositional expansion, and are found juxtaposed with scoria. Detailed field, chemical and textural observations, coupled with high temperature rheometry and thermal modelling, reveal that abrupt transitions in eruptive behaviour - from lava fountaining to low-energy bubble bursts - created these pyroclastic deposits. These findings should help identify transitions in eruptive behaviour at other mafic volcanoes worldwide.

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

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.

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

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.

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

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.

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

Background: 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. Methods: 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. Results: 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 GAPDH, TPI1, and FOXM1. 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. Conclusions: 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.

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

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 TGFß 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.

Intra-tumoral IFN-γ-producing Th22 cells correlate with TNM staging and the worst outcomes in pancreatic cancer.

PDAC (pancreatic ductal adenocarcinoma) is the fifth leading cause of cancer-related death. The causes of this cancer remain unknown, but increasing evidence indicates a key role of the host immune response and cytokines in human carcinogenesis. Intra-tumoral IL (interleukin)-22 levels have been shown to be elevated in PDAC patients. However, little is known regarding the expression and clinical relevance of Th22 cells in human PDAC and, furthermore, which TILs (tumour-infiltrating lymphocytes) are the main producers of IL-22 is unknown. In the present study, we characterized the functional proprieties of the different subsets of IL-22-producing TILs and analysed their relationship with the TNM staging system and patient survival. We have demonstrated for the first time that, in PDAC patients, the T-cells co-producing IFN-γ (interferon γ) and exerting perforin-mediated cytotoxicity are the major intra-tumoral source of IL-22. In addition, isolated Th22 cells were able to induce apoptosis, which was antagonized by IL-22. Finally, we observed that the IL-22-producing T-cells were significantly increased in tumour tissue and that this increase was positively correlated with TNM staging of PDAC and poorer patient survival. These novel findings support the dual role of the anti-tumour immune system and that IL-22-producing cells may participate in PDAC pathogenesis. Therefore monitoring Th22 levels could be a good diagnostic parameter, and blocking IL-22 signalling may represent a viable method for anti-PDAC therapies.