A disintegrin and a metalloprotease (ADAM)‐9 is a metzincin cell‐surface protease with strongly elevated expression in solid tumors, including pancreatic ductal adenocarcinoma (PDAC). In this study, we performed immunohistochemistry (IHC) of a tissue microarray (TMA) to examine the expression of ADAM9 in a cohort of >100 clinically annotated PDAC cases. We report that ADAM9 is prominently expressed by PDAC tumor cells, and increased ADAM9 expression levels correlate with poor tumor grading (P = 0.027) and the presence of vasculature invasion (P = 0.017). We employed gene expression silencing to generate a loss‐of‐function system for ADAM9 in two established PDAC cell lines. In vitro analysis showed that loss of ADAM9 does not impede cellular proliferation and invasiveness in basement membrane. However, ADAM9 plays a crucial role in mediating cell migration and adhesion to extracellular matrix substrates such as fibronectin, tenascin, and vitronectin. This effect appears to depend on its catalytic activity. In addition, ADAM9 facilitates anchorage‐independent growth. In AsPC1 cells, but not in MiaPaCa‐2 cells, we noted a pronounced yet heterogeneous impact of ADAM9 on the abundance of various integrins, a process that we characterized as post‐translational regulation. Sprout formation of human umbilical vein endothelial cells (HUVECs) is promoted by ADAM9, as examined by transfer of cancer cell conditioned medium; this finding further supports a pro‐angiogenic role of ADAM9 expressed by PDAC cancer cells. Immunoblotting analysis of cancer cell conditioned medium highlighted that ADAM9 regulates the levels of angiogenic factors, including shed heparin‐binding EGF‐like growth factor (HB‐EGF). Finally, we carried out orthotopic seeding of either wild‐type AsPC‐1 cells or AsPC‐1 cells with silenced ADAM9 expression into murine pancreas. In this in vivo setting, ADAM9 was also found to foster angiogenesis without an impact on tumor cell proliferation. In summary, our results characterize ADAM9 as an important regulator in PDAC tumor biology with a strong pro‐angiogenic impact.

Proteomic analyses of clinical specimens often rely on human tissues preserved through formalin-fixation and paraffin embedding (FFPE). Minimal sample consumption is the key to preserve the integrity of pathological archives but also to deal with minimal invasive core biopsies. This has been achieved by using the acid-labile surfactant RapiGest in combination with a direct trypsinization (DTR) strategy. A critical comparison of the DTR protocol with the most commonly used filter aided sample preparation (FASP) protocol is lacking. Furthermore, it is unknown how common histological stainings influence the outcome of the DTR protocol. Four single consecutive murine kidney tissue specimens were prepared with the DTR approach or with the FASP protocol using both 10 and 30 k filter devices and analyzed by label-free, quantitative liquid chromatography–tandem mass spectrometry (LC–MS/MS). We compared the different protocols in terms of proteome coverage, relative label-free quantitation, missed cleavages, physicochemical properties and gene ontology term annotations of the proteins. Additionally, we probed compatibility of the DTR protocol for the analysis of common used histological stainings, namely hematoxylin & eosin (H&E), hematoxylin and hemalaun. These were proteomically compared to an unstained control by analyzing four human tonsil FFPE tissue specimens per condition. On average, the DTR protocol identified 1841 ± 22 proteins in a single, non-fractionated LC–MS/MS analysis, whereas these numbers were 1857 ± 120 and 1970 ± 28 proteins for the FASP 10 and 30 k protocol. The DTR protocol showed 15% more missed cleavages, which did not adversely affect quantitation and intersample comparability. Hematoxylin or hemalaun staining did not adversely impact the performance of the DTR protocol. A minor perturbation was observed for H&E staining, decreasing overall protein identification by 13%. In essence, the DTR protocol can keep up with the FASP protocol in terms of qualitative and quantitative reproducibility and performed almost as well in terms of proteome coverage and missed cleavages. We highlight the suitability of the DTR protocol as a viable and straightforward alternative to the FASP protocol for proteomics-based clinical research.

The concentration of thiol and thioether metabolites in plasma has diagnostic value in genetic diseases of B-vitamin metabolism linked to methionine utilization. Among these, cysteine/cystine (Cys/CSSC) and glutathione/oxidized glutathione (GSH/GSSG) act as cellular redox buffers. A new LC-MS/MS method was developed for the simultaneous detection of cystathionine (Cysta), methionine (Met), methionine sulfoxide (MSO), creatinine and the reduced and oxidized pairs of homocysteine (Hcy/HSSH), cysteine (Cys/CSSC) and glutathione (GSH/GSSG). A one-step thiol-blocking protocol with minimal sample preparation was established to determine redox thiol pairs in plasma and cells. The concentrations of diagnostic biomarkers Hcy, Met, Cysta, and Cys in a cohort of healthy adults (n = 53) agreed with reference ranges and published values. Metabolite concentrations were also validated in commercial samples of human, mouse, rat and Beagle dog plasma and by the use of a standardized ERNDIM quality control. Analysis of fibroblasts, endothelial and epithelial cells, human embryonic stem cells, and cancer cell lines showed cell specificity for both the speciation and concentration of thiol and thioether metabolites. This LC-MS/MS platform permits the fast and simultaneous quantification of 10 thiol and thioether metabolites and creatinine using 40 µL plasma, urine or culture medium, or 500,000 cells. The sample preparation protocols are directly transferable to automated metabolomic platforms.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with frequent post-surgical local recurrence. The combination of adjuvant chemotherapy with radiotherapy is under consideration to achieve a prolonged progression-free survival (PFS). To date, few studies have determined the proteome profiles associated with response to adjuvant chemoradiation. We herein analyzed the proteomes of primary PDAC tumors subjected to additive chemoradiation after surgical resection and achieving short PFS (median 6 months) versus prolonged PFS (median 28 months). Proteomic analysis revealed the overexpression of Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) and Monoamine Oxidase A (MAOA) in the short PFS cohort, which were corroborated by immunohistochemistry. In vitro, specific inhibition of ALDH1A1 by A37 in combination with gemcitabine, radiation, and chemoradiation lowered cell viability and augmented cell death in MiaPaCa-2 and Panc 05.04 cells. ALDH1A1 silencing in both cell lines dampened cell proliferation, cell metabolism, and colony formation. In MiaPaCa-2 cells, ALDH1A1 silencing sensitized cells towards treatment with gemcitabine, radiation or chemoradiation. In Panc 05.04, increased cell death was observed upon gemcitabine treatment only. These findings are in line with previous studies that have suggested a role of ALDH1A1 chemoradiation resistance, e.g., in esophageal cancer. In summary, we present one of the first proteome studies to investigate the responsiveness of PDAC to chemoradiation and provide further evidence for a role of ALDH1A1 in therapy resistance.

Little is known about the subcellular localization and function of programmed cell death 4 (PDCD4) in melanoma. Our past studies suggest PDCD4 interacts with Pleckstrin Homology Domain Containing A5 (PLEKHA5) to influence melanoma brain metastasis outcomes, as high intracranial PDCD4 expression leads to improved survival. We aimed to define the subcellular distribution of PDCD4 in melanoma and in the tumor microenvironment during neoplastic progression and its impact on clinical outcomes. We analyzed multiple tissue microarrays with well-annotated clinicopathological variables using quantitative immunofluorescence and evaluated single-cell RNA-sequencing on a brain metastasis sample to characterize PDCD4+ immune cell subsets. We demonstrate differences in PDCD4 expression during neoplastic progression, with high tumor and stromal PDCD4 levels associated with improved survival in primary melanomas and in intracranial metastases, but not in extracranial metastatic disease. While the expression of PDCD4 is well-documented on CD8+ T cells and natural killer cells, we show that it is also found on B cells and mast cells. PDCD4 expression in the tumor microenvironment is associated with increased immune cell infiltration. Further studies are needed to define the interaction of PDCD4 and PLEKHA5 and to evaluate the utility of this pathway as a therapeutic target in melanoma brain metastasis.