The NAIP/NLRC4 inflammasome is classically associated with the detection of bacterial invasion to the cytosol. However, recent studies have demonstrated that NAIP/NLRC4 is also activated in non-bacterial infections, and in sterile inflammation. Moreover, in addition to the well-established model for the detection of bacterial proteins by NAIP proteins, the participation of other cytosolic pathways in the regulation of NAIP/NLRC4-mediated responses has been reported in distinct contexts. Using pharmacological inhibition and genetic deletion, we demonstrate here that cathepsins, well known for their involvement in NLRP3 activation, also regulate NAIP/NLRC4 responses to cytosolic flagellin in murine and human macrophages. In contrast to that observed for NLRP3 agonists, cathepsins inhibition did not reduce ASC speck formation or caspase-1 maturation in response to flagellin, ruling out their participation in the effector phase of NAIP/NLRC4 activation. Moreover, cathepsins had no impact on NF-κB-mediated priming of pro-IL-1β, thus suggesting these proteases act downstream of the NAIP/NLRC4 inflammasome activation. IL-1β levels secreted in response to flagellin were reduced in the absence of either cathepsins or Gasdermin-D (GSDMD), a molecule involved in the induction of pyroptosis and cytokines release. Notably, IL-1β secretion was abrogated in the absence of both GSDMD and cathepsins, demonstrating their non-redundant roles for the optimal IL-1β release in response to cytosolic flagellin. Given the central role of NAIP/NLRC4 inflammasomes in controlling infection and, also, induction of inflammatory pathologies, many efforts have been made to uncover novel molecules involved in their regulation. Thus, our findings bring together a relevant contribution by describing the role of cathepsins as players in the NAIP/NLRC4-mediated responses.

Studies have indicated that some genes involved in carcinogenesis are highly methylated in their promoter regions but nevertheless strongly transcribed. It has been proposed that transcription factors could bind specifically to methylated promoters and trigger transcription. We looked at this rather comprehensively for pancreatic ductal adenocarcinoma (PDAC) and studied some cases in more detail. Some 2% of regulated genes in PDAC exhibited higher transcription coupled to promoter hypermethylation in comparison to healthy tissue. Screening 661 transcription factors, several were found to bind specifically to methylated promoters, in particular molecules of the NFAT family. One of them—NFATc1—was substantially more strongly expressed in PDAC than control tissue and exhibited a strong oncogenic role. Functional studies combined with computational analyses allowed determining affected genes. A prominent one was gene ALDH1A3, which accelerates PDAC metastasis and correlates with a bad prognosis. Further studies confirmed the direct up-regulation of ALDH1A3 transcription by NFATc1 promoter binding in a methylation-dependent process, providing insights into the oncogenic role of transcription activation in PDAC that is promoted by DNA methylation.

Tumor-associated macrophages (TAM) exhibit a dual role in tumor progression and antitumor immunity. However, understanding the functional states and molecular mechanisms of antitumor TAMs remains a challenge. Herein, we show that intratumoral administration of a combination of agonists against TLR3 and CD40 [hereafter termed myeloid cell treatment (MCT)] reprogrammed TAMs in situ to adopt a protective antitumor phenotype in an orthotopic mouse breast cancer model, and that this led to tumor regression. Single-cell RNA sequencing of TAMs from different tumor stages and after MCT revealed a transient antitumor TAM phenotype, present at 12 hours after MCT and characterized by markers such as inducible nitric oxide synthase and CD38, which was replaced by TAMs coexpressing tumor-limiting and tumor-promoting features by 72 hours after MCT. Maintenance of antitumor TAMs required repeated MCT administration, and this promoted the activation of CD8+ T cells and long-term tumor eradication. Mechanistically, reactive oxygen species and TNF-α were pivotal in TAM-mediated tumor control. Our findings uncover the vulnerability of transient TAM reprogramming and show that it can be overcome by repeated MCT administrations to sustain efficient antitumor immune responses.

(1) Background: A reliable non-invasive distinction between low- and high-risk pancreatic intraductal papillary mucinous neoplasms (IPMN) is needed to effectively detect IPMN with malignant potential. This would improve preventative care and reduce the risk of developing pancreatic cancer and overtreatment. The present study aimed at exploring the presence of autoreactive antibodies in the blood of patients with IPMN of various grades of dysplasia. (2) Methods: A single-center cohort was studied composed of 378 serum samples from patients with low-grade IPMN (n = 91), high-grade IPMN (n = 66), IPMN with associated invasive cancer (n = 30), pancreatic ductal adenocarcinoma (PDAC) stages T1 (n = 24) and T2 (n = 113), and healthy controls (n = 54). A 249 full-length recombinant human protein microarray was used for profiling the serum samples. (3) Results: 14 proteins were identified as potential biomarkers for grade distinction in IPMN, yielding high specificity but mediocre sensitivity. (4) Conclusions: The identified autoantibodies are potential biomarkers that may assist in the detection of malignancy in IPMN patients.

Tumor-associated autoreactive antibodies could represent a moderately sensitive but highly specific type of biomarker. While autoantibodies in the blood of patients with pancreatic cancer have been reported, the consensus among studies is low and data validation is lacking. We profiled the abundance of circulating autoantibodies in patients with pancreatic ductal adenocarcinoma (PDAC) in a multi-step approach. For this purpose, microarrays with in situ expressed proteins were produced that contained 3060 non-mutated human proteins; one emphasis was on cell surface proteins. The microarrays were utilized for autoantibody screening of sera collected from patients of PDAC stages I, IIA, IIB/III and IV. In addition, samples were studied that represented the different grades of the precursor lesion intraductal pupillary mucinous neoplasm (IPMN) – low-grade, high-grade dysplasia and IPMN with associated carcinoma. Third, sera from patients diseased with the PDAC risk factor chronic pancreatitis (CP) as well as healthy donors served as control groups. The analysis revealed a significant increase in the overall proportion of immunoreactive proteins in late grades of IPMN and stages of PDAC; within each group, some sera displayed higher reactivity than others. The screening phase led to the identification of about 100 proteins, which showed a relatively high immunoreactivity associated with PDAC in comparison to an age- and sex-matched healthy control group. The characteristics of PDAC-associated autoantibodies were confirmed for the 100 proteins by analyzing sera from a total of 1200 PDAC, IPMN and CP patients as well as healthy donors. The patient groups were age- and sex-matched among each other. This confirmation phase validated the PDAC-related immunoreactivity of 17 proteins, showing high specificity of samples from PDAC patients in comparison to the healthy control group but low sensitivity of less than 10%. The clinically defined PDAC stages displayed different autoantibody profiles. However, no stage-related autoantibodies could be identified. Further, these PDAC-related antigens were also immunoreactive in smaller proportions of CP and IPMN patients, demonstrating the biological relation between PDAC and its precursors CP and IPMN. The majority of the other antigens showed a high immunoreactivity without correlation of autoantibody abundance and PDAC diagnosis, highlighting the importance of validation in large cohorts. The presence of autoantibodies specific for the 17 identified PDAC-related proteins is currently technically cross-validated by Luminex multiplex serology. In conclusion, the study has identified novel PDAC-related autoantibodies towards non-modified human proteins. While the specificity of some detected autoantibodies was high, the low sensitivity and the immunoreactivity of related precursor lesions make the definition of a broad applicable biomarker panel unlikely or too complex. However, the antigens that are specific for PDAC indicate target molecules, which could offer new therapeutic options in a personalized approach. Citation Format: Henning Boekhoff, Laura Hendricks, Elena Cairo, Andrea Bauer, Mari Hambardzumyan, Markus W. Büchler, Silvia Calderazzo, Vivienn Weru, Anette Kopp-Schneider, Hermann Brenner, Thilo Hackert, Oliver Strobel, Tim Waterboer, Nathalia Giese, Joerg D. Hoheisel. Profiling of circulating autoreactive antibodies in some 1200 patients with pancreatic ductal adenocarcinoma and progressive precursor lesions [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A035.