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Apoptosis-associated Speck-like protein containing a CARD (caspase activation and recruitment domain) (ASC), also called PYCARD/Target of Methylation-induced Silencing-1 (TMS1), was originally discovered as a protein that forms aggregates (“specks”) in human leukemia cells treated with chemotherapeutic agents. Its expression was found to be silenced by methylation in many human tumors, preventing tumor cells from undergoing apoptosis and supporting its role as a tumor suppressor. Subsequently, ASC was also identified as a central adaptor molecule of the inflammasome complex, which mediates the secretion of inflammatory cytokines (i.e., IL-1β and IL-18). Inflammatory cytokines have been shown to mediate tumor-promoting functions. Thus, in the context of cancer development and progression, ASC may exert opposing functions, i.e., be either tumor-suppressing by inducing tumor cell apoptosis, or tumor-promoting by favoring secretion of inflammatory cytokines (by tumor cells and/or tumor infiltrating myeloid cells) within the tumor microenvironment. Here, we report and discuss this dual role of ASC by also considering the final contribution of each of its two main functions in several cancer types, taking into consideration the correlation between ASC expression, clinical correlates, and patients’ survival. ASC and inflammasome targeting strategies are being developed. However, before the use of such treatments in clinical practice, it is fundamental to better dissect the role of ASC in different tumors, in order to privilege or avoid their use in those tumors in which ASC exerts an anti-tumor or pro-tumor function, respectively.

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.

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.

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.

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