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Expression of CCL24/eotaxin-2 in CAF supernatant-conditioned monocytes and in TDLNs.

Multivariate Cox regression analysis of the influence of the percentage of basophils in TDLNs on the survival of 35 stage IB-III pancreatic cancer patients after surgery.

In pancreatic ductal adenocarcinomas (PDAC), lymphoid infiltrates, comprised mainly of Th2 cells, predict a poor survival outcome in patients. IL4 signaling has been suggested to stabilize the Th2 phenotype in this setting, but the cellular source of IL4 in PDAC is unclear. Here, we show that basophils expressing IL4 are enriched in tumor-draining lymph nodes (TDLN) of PDAC patients. Basophils present in TDLNs correlated significantly with the Th2/Th1 cell ratio in tumors, where they served as an independent prognostic biomarker of patient survival after surgery. Investigations in mouse models of pancreatic cancer confirmed a functional role for basophils during tumor progression. The recruitment of basophils into TDLN relied partly upon the release of chemokine CCL7/MCP3 by "alternatively activated" monocytes, whereas basophil activation was induced by T-cell–derived IL3. Our results show how basophils recruited and activated in TDLNs under the influence of the tumor microenvironment regulate tumor-promoting Th2 inflammation in PDAC, helping in illuminating a key element of the immune milieu of pancreatic cancer. Cancer Res; 76(7); 1792–803. ©2016 AACR.

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.

Background The thymic stromal lymphopoietin (TSLP), a key cytokine for development of Th2 immunity, is produced by cancer associated fibroblasts (CAFs) in pancreatic cancer where predominant tumor infiltrating Th2 over Th1 cells correlates with reduced patients’ survival. Which cells and molecules are mostly relevant in driving TSLP secretion by CAFs in pancreatic cancer is not defined. Methods We performed in vitro, in vivo and ex-vivo analyses. For in vitro studies we used pancreatic cancer cell lines, primary CAFs cultures, and THP1 cells. TSLP secretion by CAFs was used as a read-out system to identify in vitro relevant tumor-derived inflammatory cytokines and molecules. For in vivo studies human pancreatic cancer cells and CAFs were orthotopically injected in immunodeficient mice. For ex-vivo studies immunohistochemistry was performed to detect ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) expression in surgical samples. Bioinformatics was applied to interrogate published data sets. Results We show in vitro that IL-1α and IL-1β released by pancreatic cancer cells and tumor cell-conditioned macrophages are crucial for TSLP secretion by CAFs. Treatment of immunodeficient mice orthotopically injected with human IL-1 positive pancreatic cancer cells plus CAFs using the IL-1R antagonist anakinra significantly reduced TSLP expression in the tumor. Importantly, we found that pancreatic cancer cells release alarmins, among which ASC, able to induce IL-1β secretion in macrophages. The relevance of ASC was confirmed ex-vivo by its expression in both tumor cells and tumor associated macrophages in pancreatic cancer surgical samples and survival data analyses showing statistically significant inverse correlation between ASC expression and survival in pancreatic cancer patients. Conclusions Our findings indicate that tumor released IL-1α and IL-1β and ASC are key regulators of TSLP secretion by CAFs and their targeting should ultimately dampen Th2 inflammation and improve overall survival in pancreatic cancer. Electronic supplementary material The online version of this article (10.1186/s40425-019-0521-4) contains supplementary material, which is available to authorized users.

CD4(+) T cells comprise a large fraction of tumor infiltrating lymphocytes and it is now established that they may exert an important role in tumor immune-surveillance. Several CD4(+) T cell subsets [i.e. T helper (Th)1, Th2, T regulatory (Treg), Th17, Th22 and follicular T helper (Tfh)] have been described and differentiation of each subset depends on both the antigen presenting cells responsible for its activation and the cytokine environment present at the site of priming. Tumor antigen-specific CD4(+) T cells with different functional activity have been found in the blood of cancer patients and different CD4(+) T cell subsets have been identified at the tumor site by the expression of specific transcription factors and the profile of secreted cytokines. Importantly, depending on the subset, CD4(+) T cells may exert antitumor versus pro-tumor functions. Here we review the studies that first identified the presence of tumor-specific CD4(+) T cells in cancer patients, the techniques used to identify the tumor antigens recognized, the role of the different CD4(+) T cell subsets in tumor immunity and in cancer prognosis and the development of therapeutic strategies aimed at activating efficient antitumor CD4(+) T cell effectors.

Natural autoantibodies raised by humoral immune response to cancer can be exploited to identify potential tumor-associated antigens (TAAs), and might constitute new putative prognostic and/or diagnostic biomarkers. Here we describe how sera from tumor patients can be used to identify TAAs by screening antibody immunoreactivity against the cancer proteome resolved by two-dimensional gel electrophoresis.

CD4(+) T cells comprise a significant portion of tumor-infiltrating lymphocytes. Different subsets of CD4(+) T cells exist and they exert different effector functions in tumor immunity depending on the cytokines produced going from antitumor to pro-tumor. Methods that use small aliquots of cells to identify ex vivo the frequency and functional orientation of tumor-specific CD4(+) T cells in the blood and visualization of the presence of different CD4(+) T cell subsets and their localization at the tumor site are valuable tools to determine their clinical impact in neoplastic diseases.

Amyotrophic lateral sclerosis (ALS) a fatal degenerative disease that selectively affects motor neurons, likely results from a complex interplay among oxidative injury, excitotoxic stimulation, protein aggregation and genetic factors. Ceruloplasmin (Cp) protein is a ferroxidase that oxidizes toxic ferrous iron to its nontoxic ferric form, protecting the central nervous system (CNS) from iron deposition. Cp is thus considered as one of the main systems dedicated to the protection of the CNS from oxidative stress damage. We investigated Cp protein behaviour in the cerebrospinal fluid (CSF) of ALS patients of recent onset. An increased expression of Cp was observed in ALS (n = 16) compared to two control groups (healthy subjects, n = 11 and peripheral neuropathy patients, n = 10). 2-DE analysis revealed a differential expression of Cp isoforms in ALS patients compared to controls. ALS samples showed an increase in the relative abundance of more basic Cp forms, corresponding to the nonsialylated proteins. Despite the increase in protein expression, ferroxidase activity evaluated in the CSF of ALS patients was comparable to that of the controls, indicating a Cp functional impairment. Ceruloplasmin isoforms profile may be proposed as disease feature that could provide insight into the molecular mechanisms of ALS pathogenesis.

Natural autoantibodies raised by humoral immune response to cancer can be exploited to identify potential tumor-associated antigens (TAAs), and might constitute new putative prognostic and/or diagnostic biomarkers. Here we describe how sera from tumor patients can be used to identify TAAs by screening antibody immunoreactivity against the cancer proteome resolved by two-dimensional gel electrophoresis.

In pancreatic ductal adenocarcinomas (PDAC), lymphoid infiltrates, comprised mainly of Th2 cells, predict a poor survival outcome in patients. IL4 signaling has been suggested to stabilize the Th2 phenotype in this setting, but the cellular source of IL4 in PDAC is unclear. Here, we show that basophils expressing IL4 are enriched in tumor-draining lymph nodes (TDLN) of PDAC patients. Basophils present in TDLNs correlated significantly with the Th2/Th1 cell ratio in tumors, where they served as an independent prognostic biomarker of patient survival after surgery. Investigations in mouse models of pancreatic cancer confirmed a functional role for basophils during tumor progression. The recruitment of basophils into TDLN relied partly upon the release of chemokine CCL7/MCP3 by "alternatively activated" monocytes, whereas basophil activation was induced by T-cell–derived IL3. Our results show how basophils recruited and activated in TDLNs under the influence of the tumor microenvironment regulate tumor-promoting Th2 inflammation in PDAC, helping in illuminating a key element of the immune milieu of pancreatic cancer. Cancer Res; 76(7); 1792–803. ©2016 AACR.

CD4(+) T cells comprise a significant portion of tumor-infiltrating lymphocytes. Different subsets of CD4(+) T cells exist and they exert different effector functions in tumor immunity depending on the cytokines produced going from antitumor to pro-tumor. Methods that use small aliquots of cells to identify ex vivo the frequency and functional orientation of tumor-specific CD4(+) T cells in the blood and visualization of the presence of different CD4(+) T cell subsets and their localization at the tumor site are valuable tools to determine their clinical impact in neoplastic diseases.

SUMMARY CD36 is a membrane glycoprotein and a putative scavenger receptor expressed by several cell types. In capillary endothelial cells, it mediates the adherence of erythrocytes infected with Plasmodium falciparum. The CD36 sequence contains two hydrophobic domains located at the amino-and carboxyl-termini of the protein, but the topology of this protein and the functional significance of these domains are still not clearly defined. We generated soluble CD36‐IgG chimeric molecules by fusion of the extracellular domains of CD36 with human immunoglobulin domains. The construct containing the N-terminal hydrophobic domain of CD36 was completely retained intracellularly as membrane-associated molecule, suggesting that the N-terminal hydrophobic domain of the CD36 is a real transmembrane domain and that CD36 has hairpin topology. A small amount of the CD36‐IgG chimeric construct lacking both transmembrane domains escaped retention, was correctly processed, and accumulated in the extracellular medium as a soluble molecule. This CD36‐IgG construct failed to bind Plasmodium falciparum-infected erythrocytes. Using monoclonal antibodies specific for either conformational or structural epitopes, we demonstrate that failure of this CD36‐IgG construct to bind infected erythrocytes was due to incorrect folding of the soluble chimeric molecule.

Pancreatic cancer is a devastating disease with dismal prognosis. The tumor microenvironment is composed by multiple cell types, molecular factors, and extracellular matrix forming a strong desmoplastic reaction, which is a hallmark of the disease. A complex cross-talk between tumor cells and the stroma exists with reciprocal influence that dictates tumor progression and ultimately the clinical outcome. In this context, tumor infiltrating immune cells through secretion of chemokine and cytokines exert an important regulatory role. Here we review the correlation between the immune infiltrates, evaluated on tumor samples of pancreatic cancer patients underwent surgical resection, and disease free and/or overall survival after surgery. Specifically, we focus on tumor infiltrating lymphocytes (TILs), mast cells (MCs) and macrophages that all contribute to a Th2-type inflammatory and immunosuppressive microenvironment. In these patients tumor immune infiltrates not only do not contribute to disease eradication but rather the features of Th2-type inflammation and immunosuppression is significantly associated with more rapid disease progression and reduced survival.

Th2-type inflammation has been proposed to facilitate tumor growth. In De Monte et al. (J Exp Med 208:469-478, 2011) we identify in pancreatic cancer a complex cytokine/chemokine cross-talk within the tumor microenvironment mediating Th2 immune-deviation and show that the ratio of Th2/Th1 tumor infiltrating lymphocytes is an independent predictive marker of patients survival.

Pancreatic cancer is a very aggressive disease characterized by a marked desmoplasia with a predominant Th2 (GATA-3(+)) over Th1 (T-bet(+)) lymphoid infiltrate. We found that the ratio of GATA-3(+)/T-bet(+) tumor-infiltrating lymphoid cells is an independent predictive marker of patient survival. Patients surgically treated for stage IB/III disease with a ratio inferior to the median value had a statistically significant prolonged overall survival, implying an active role for Th2 responses in disease progression. Thymic stromal lymphopoietin (TSLP), which favors Th2 cell polarization through myeloid dendritic cell (DC) conditioning, was secreted by cancer-associated fibroblasts (CAFs) after activation with tumor-derived tumor necrosis factor a and interleukin 1 beta. TSLP-containing supernatants from activated CAFs induced in vitro myeloid DCs to up-regulate the TSLP receptor (TSLPR), secrete Th2-attracting chemokines, and acquire TSLP-dependent Th2-polarizing capability in vitro. In vivo, Th2 chemoattractants were expressed in the tumor and in the stroma, and TSLPR-expressing DCs were present in the tumor stroma and in tumor-draining but not in nondraining lymph nodes. Collectively, this study identifies in pancreatic cancer a cross talk between tumor cells and CAFs, resulting in a TSLP-dependent induction of Th2-type inflammation which associates with reduced patient survival. Thus, blocking TSLP production by CAFs might help to improve prognosis in pancreatic cancer. "Pancreatic cancer is a very aggressive disease characterized by a marked desmoplasia with a predominant Th2 (GATA-3+) over Th1 (T-bet+) lymphoid infiltrate. We found that the ratio of GATA-3+\/T-bet+ tumor-infiltrating lymphoid cells is an independent predictive marker of patient survival. Patients surgically treated for stage IB\/III disease with a ratio inferior to the median value had a statistically significant prolonged overall survival, implying an active role for Th2 responses in disease progression. Thymic stromal lymphopoietin (TSLP), which favors Th2 cell polarization through myeloid dendritic cell (DC) conditioning, was secreted by cancer-associated fibroblasts (CAFs) after activation with tumor-derived tumor necrosis factor α and interleukin 1β. TSLP-containing supernatants from activated CAFs induced in vitro myeloid DCs to up-regulate the TSLP receptor (TSLPR), secrete Th2-attracting chemokines, and acquire TSLP-dependent Th2-polarizing capability in vitro. In vivo, Th2 chemoattractants were expressed in the tumor and in the stroma, and TSLPR-expressing DCs were present in the tumor stroma and in tumor-draining but not in nondraining lymph nodes. Collectively, this study identifies in pancreatic cancer a cross talk between tumor cells and CAFs, resulting in a TSLP-dependent induction of Th2-type inflammation which associates with reduced patient survival. Thus, blocking TSLP production by CAFs might help to improve prognosis in pancreatic cancer"

Sera from colon carcinoma patients were used to identify tumor-associated antigens (TAAs) by screening tumor proteome resolved by 2D electrophoresis. A panel of six TAAs eliciting a serological immune response in colorectal cancer was identified, showing a modification in antigen recognition by B cells in patients as a function of colon cancer progression. The expression of these proteins was either confined or increased in tumor as compared to normal mucosa.

Pancreatic Ductal AdenoCarcinoma (PDAC) is a very aggressive disease, characterized by desmoplasia and Th2-inflammation. Th2-inflammation correlates with poor prognosis and it is driven by the release of the Thymic Stromal LymphoPoietin (TSLP) from Cancer Associated Fibroblasts (CAFs) activated by tumor-derived pro-inflammatory cytokines. The aims of our study are: i) to verify which tumor-derived cytokines are responsible for CAFs activation, ii) to understand the stimuli/mechanisms of tumor-derived cytokine production in pancreatic cancer cells. Analyzing a series of 9 PDAC cell lines by both Real-time PCR and immunofluorescence, we found heterogeneous expression of IL-1α, IL-1β and TNF-α. Furthermore, ELISA tests revealed that some PDAC cell lines could either actively secrete or release during necrosis these pro-inflammatory cytokines. Next, we treated CAFs obtained from surgical patient samples with rhIL-1α, rhIL-1β, rhTNF-α and PDAC cell supernatants. Interestingly, we observed an induction in TSLP secretion that was significantly reduced by anti-IL1α and Anakinra (the recombinant form of IL-1Ra), suggesting a predominant role of the IL-1/IL-1R axis. It is known that IL-1α and TLRs ligands can activate NF-kB that in turn induces the synthesis of proIL-1β that is cleaved to matureIL-1β by caspase-1, upon inflammasome activation. In line with this, we performed Western Blot analysis and we observed constitutive activation of both NF-kB and inflammasome. We also demonstrated that IL-1α is able to increase IL-1β production in PDAC cells, with an autocrine loop. Furthermore, treating THP1 cells (a monocytic cell line that secrete IL-1β only upon stimulation) with PDAC cell supernatants we found that both IL-1-expressing and non-IL-1-expressing cells can release inflammasome-activating factors. Since pancreatic cancer is highly hypoxic, we cultured PDAC cell lines in hypoxic conditions and we demonstrated that prolonged severe hypoxia, in combination with starvation, induces an increase in both IL-1α and IL-1β secretion. In conclusion, our study demonstrates that PDAC cells can constitutively express IL-1α, IL-1β and TNF-α, thus inducing TSLP secretion from CAFs and eventually Th2-inflammation. PDAC cells secrete inflammasome-activating factors that sustain cytokine production with an autocrine loop, and that could induce cytokine release from infiltrating macrophages. Finally, the hypoxic environment of pancreatic cancer can further increase IL-1α and IL-1β secretion. Our findings support the development of clinical studies combining the use of Anakinra with conventional chemotherapy, in order to interfere with the IL-1/IL-1R axis and therefore Th2-inflammation, thus improving the prognosis of pancreatic cancer patients. Citation Format: Emanuela Brunetto, Lucia De Monte, Silvia Heltai, Maria Pia Protti. Mechanisms of IL-1 production and release in pancreatic cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 443. doi:10.1158/1538-7445.AM2015-443