Nathalia A. Giese

Consensus guidelines for the detection of immunogenic cell death

Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named “immunogenic cell death” (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

VEGF expression by mesenchymal stem cells contributes to angiogenesis in pancreatic carcinoma.

Little is known about the factors that enable the mobilisation of human mesenchymal stem cells (MSC) from the bone marrow into the blood stream and their recruitment to and retention in the tumour. We found specific migration of MSC towards growth factors present in pancreatic tumours, such as PDGF, EGF, VEGF and specific inhibitors Glivec, Erbitux and Avastin interfered with migration. Within a few hours, MSC migrated into spheroids consisting of pancreatic cancer cells, fibroblasts and endothelial cells as measured by time-lapse microscopy. Supernatant from subconfluent MSC increased sprouting of HUVEC due to VEGF production by MSC itself as demonstrated by RT-PCR and ELISA. Only few MSCs were differentiated into endothelial cells in vitro, whereas in vivo differentiation was not observed. Lentiviral GFP-marked MSCs, injected in nude mice xenografted with orthotopic pancreatic tumours, preferentially migrated into the tumours as observed by FACS analysis of green fluorescent cells. By immunofluorescence and intravital microscopic studies, we found the interaction of MSC with the endothelium of blood vessels. Mesenchymal stem cells supported tumour angiogenesis in vivo, that is CD31+ vessel density was increased after the transfer of MSC compared with siVEGF-MSC. Our data demonstrate the migration of MSC toward tumour vessels and suggest a supportive role in angiogenesis.

Pancreatic Neuropathy and Neuropathic Pain—A Comprehensive Pathomorphological Study of 546 Cases

BACKGROUND & AIMS Chronic pancreatitis (CP) and pancreatic adenocarcinoma (PCa) are characterized by intrapancreatic neural alterations and pain. Our aims were to: (a) Investigate whether neuropathic changes like pancreatic neuritis, increased neural density, and hypertrophy are phenomena only in CP or whether they are also evident in other pancreatic disorders as well, (b) study possible variations in neural cancer cell invasion among malignant pancreatic tumors, and (c) explore whether these neuropathic changes contribute to pain sensation. METHODS Neuropathic changes were studied in PCa (n=149), in CP (n=141), in pancreatic tumors (PTm) including serous/mucinous cystadenomas, invasive/noninvasive intraductal papillary mucinous neoplasias, benign/malignant neuroendocrine tumors, ampullary cancers (n=196), and in normal pancreas (n=60). The results were correlated with GAP-43 expression, tissue inflammation, pancreatic neuritis, neural invasion, fibrosis, desmoplasia, pain, and patient survival. RESULTS Increased neural density and hypertrophy were only detected in PCa and CP and were strongly associated with GAP-43 over expression and abdominal pain. The severity of pancreatic neuritis was strongest in PCa and was closely linked to changes in neural density and hypertrophy. The aggressiveness of neural cancer cell invasion was most prominent in PCa and was related to neuropathic changes, desmoplasia, and pain. Severe and enduring pain were strongly associated with poor prognosis in PCa patients. CONCLUSIONS Enhanced neural density and hypertrophy are only typical features of CP and PCa among all investigated pancreatic disorders. Such neuropathic changes, including damage to nerves by inflammatory and/or cancer cells, seem to enhance and generate pancreatic neuropathic pain.

The Activated Stroma Index Is a Novel and Independent Prognostic Marker in Pancreatic Ductal Adenocarcinoma

BACKGROUND AND AIMS Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic tumor with an innate resistance to therapy. Pancreatic stellate cells (PSCs) produce this excessively desmoplastic microenvironment. The impact of PSC activity on PDAC behavior in vivo is analyzed. METHODS 233 patients who underwent surgery for PDAC were evaluated by immunohistochemistry using antibodies against alpha-smooth muscle actin as a marker of PSC activity. Aniline was used to stain collagen deposition. The ratio of alpha-smooth muscle actin-stained area to collagen-stained area was defined as the activated stroma index (ASI). Survival analysis was performed using the Kaplan-Meier method. Prognostic factors were determined in a multivariable analysis using a Cox proportional hazards model. RESULTS Four major patterns of collagen deposition were defined with regard to PSC activity. The combination of high stromal activity and low collagen deposition was associated with a worse prognosis, whereas the combination of high collagen deposition and low stromal activity indicated a better prognosis. Patients with the lowest ASI had the best median survival rate (25.7 mo). The highest ASI was found in patients with the worst median survival rate (16.1 mo; P = .007; lowest vs highest ASI: hazard ratio, 1.61; 95% confidence interval, 1.014-2.562). ASI was an independent prognostic marker in multivariable survival analysis comparable with the nodal status of cancer. CONCLUSIONS The activated stroma index is a novel independent prognostic marker in PDAC in cases undergoing surgery. This finding highlights the impact of the microenvironment in cancer progression and on patient survival.

Inhibition of Heme Oxygenase-1 Increases Responsiveness of Pancreatic Cancer Cells to Anticancer Treatment

Heme oxygenase-1 (HO-1) is believed to represent a key enzyme for the protection of cells against “stress.” Its overexpression in different types of human cancers supports the notion that HO-1 provides a growth advantage and contributes to cellular resistance against chemotherapy and radiotherapy. Given the poor survival rates of patients with pancreatic cancer due to its aggressive growth behavior and its exceptional resistance to all known forms of anticancer treatment, we have investigated the expression of HO-1 in human pancreatic cancer cells growth behavior and prognosis. Expression of HO-1 was analyzed in human pancreatic cancer samples in comparison with normal pancreas by quantitative PCR, Western blot, and confocal microscopy. The influence of radiotherapy and chemotherapy on HO-1 expression in pancreatic cancer cell lines was evaluated. Furthermore, HO-1 expression was specifically suppressed by small interfering RNA transfection and subsequently the alterations of growth behavior and resistance to anticancer treatment were tested. Human pancreatic cancer showed a 6-fold and 3.5-fold HO-1 up-regulation in comparison to normal pancreas based on mRNA and protein level, respectively (P < 0.05). Cancer tissues revealed marked HO-1 immunoreactivity in tumor cells and in tumor associated immunocytes. Treatment of the pancreatic cancer cell lines with gemcitabine or radiation strongly induced HO-1 expression. Targeted knockdown of HO-1 expression led to pronounced growth inhibition of the pancreatic cancer cells and made tumor cells significantly more sensitive to radiotherapy and chemotherapy. Therefore, specific inhibition of HO-1 expression may be a new option in pancreatic cancer therapy and may be used as sensitizer to chemotherapy and radiotherapy.

Seromic profiling of ovarian and pancreatic cancer

Autoantibodies, a hallmark of both autoimmunity and cancer, represent an easily accessible surrogate for measuring adaptive immune responses to cancer. Sera can now be assayed for reactivity against thousands of proteins using microarrays, but there is no agreed-upon standard to analyze results. We developed a set of tailored quality control and normalization procedures based on ELISA validation to allow patient comparisons and determination of individual cutoffs for specificity and sensitivity. Sera from 60 patients with pancreatic cancer, 51 patients with ovarian cancer, and 53 age-matched healthy donors were used to assess the binding of IgG antibodies against a panel of >8000 human antigens using protein microarrays and fluorescence detection. The resulting data interpretation led to the definition and ranking of proteins with preferred recognition by the sera from cancer patients in comparison with healthy donors, both by frequency and strength of signal. We found that 202 proteins were preferentially immunogenic in ovarian cancer sera compared to 29 in pancreatic cancer, with few overlaps. Correlates of autoantibody signatures with known tumor expression of corresponding antigens, functional pathways, clinical stage, and outcome were examined. Serological analysis of arrays displaying the complete human proteome (seromics) represents a new era in cancer immunology, opening the way to defining the repertoire of the humoral immune response to cancer.

Tenascin C and annexin II expression in the process of pancreatic carcinogenesis

Tenascin C (TNC) is a component of the provisional extracellular matrix (ECM) that characterizes solid tumours. Cell surface annexin II is a high‐affinity receptor for large TNC splice variants. The aim of this study was to analyse whether TNC and annexin II play a role in the development of pancreatic ductal adenocarcinoma (PDAC). PDAC is characterized by a rich ECM populated by pancreatic stellate cells, which play a crucial role in pancreatic desmoplasia. The mRNA and protein levels of TNC and of annexin II were analysed in pancreatic tissues by DNA array, quantitative reverse transcriptase‐polymerase chain reaction (QRT‐PCR) and immunohistochemistry. TNC large splice variants were detected by RT‐PCR. Enzyme linked immunosorbent assay (ELISA) was used to measure TNC levels in serum and culture supernatants. TNC and annexin II mRNA levels were significantly higher in pancreatic cancer tissues than in the normal pancreas. TNC expression was detected with increased frequency in the progression from PanIN‐1 lesions to PDAC, and a parallel switch from cytoplasmic to cell surface expression of annexin II was observed. Large TNC transcripts were found in pancreatic cancer and in chronic pancreatitis, but not in the normal pancreas. TNC expression was demonstrated in pancreatic stellate cells, where it could be induced by tumour necrosis factor α (TNFα), transforming growth factor β1 (TGF‐β1) and by cancer cell supernatants supplemented with TGF‐β1. In conclusion, the expression of TNC and cell surface annexin II increases in the progression from low‐grade PanIN lesions to pancreatic cancer. Pancreatic stellate cells are identified as a source of TNC in pancreatic tissues, possibly under the influence of soluble factors released by the tumour cells. Copyright

Role of Mononuclear Cells and Inflammatory Cytokines in Pancreatic Cancer-Related Cachexia

Background and Purpose: The mechanism behind aggressive development of cachexia in patients suffering from pancreatic cancer is not well understood. In this study, we investigated which factors are associated with the cachectic status of the patients and evaluated cachexia-promoting capacity of cancer and inflammatory cells. Experimental Design: DNA microarray analysis and quantitative reverse transcription-PCR were used to screen for cachexia-associated factors in pancreatic specimens obtained from noncachectic and cachetic patients diagnosed with pancreatic ductal adenocarcinoma. The expression pattern of the most prominently altered cachexia-associated factor, interleukin-6 (IL-6), was further analyzed in patients sera by ELISA, in pancreatic specimens by immunohistochemistry, and in a coculture system by quantitative reverse transcription-PCR using pancreatic cancer cell lines T3M4 (IL-6 positive) and Panc-1 (IL-6 negative) and peripheral blood mononuclear cells (PBMC) obtained from donors and noncachectic and cachectic patients. Results: Among numerous analyzed factors, IL-6 was significantly overexpressed in pancreatic specimens and elevated in serum of cachectic patients. The coculture system revealed that pancreatic cancer T3M4 cells but not Panc-1 cells were able to stimulate IL-6 exclusively in cachectic PBMC (by 14-fold) and this triggering was reduced by half in the presence of IL-6-neutralizing antibodies. Conclusion: IL-6 represents a prominent cachexia-associated factor in pancreatic cancer. IL-6 overexpression in cachectic patients is related to the ability of certain tumors to sensitize PBMC and induce cytokine expression in cachectic PBMC.

The neurotrophic factor artemin promotes pancreatic cancer invasion

Objective:To analyze the role of Artemin in pancreatic ductal adenocarcinoma (PDAC) in terms of expression, influence on cancer cell behavior, and pain correlation. Summary Background Data:PDAC is characterized by prominent local nerve alterations and perineural invasion, which frequently affects the extrapancreatic nerve plexus, causing severe pain and precluding curative resection. Artemin, a neurotrophic protein controlling growth, regeneration, and survival of neurons was analyzed to highlight the neuro-cancer interactions in PDAC. Methods:Artemin and its receptors (GFRα3/RET) were studied in PDAC tissues and normal pancreas by Western blot analysis and immunohistochemistry. RNA expression was analyzed in pancreatic tissues (normal, cancer) and pancreatic cancer cell lines by QRT-PCR. To evaluate whether Artemin influences cancer cell proliferation and invasion, MTT-growth and Matrigel-invasion assays were used. In addition, the tissue expression of Artemin was correlated with pain in PDAC. Results:Artemin and GFRα3/RET were both detected at enhanced levels in PDAC compared with normal pancreas, localizing predominantly in hypertrophic nerves and arterial walls, as well as in cancer cells of primary and metastatic lesions. The levels of Artemin and GFRα3 did not correlate with pain in PDAC patients. However, Artemin promoted pancreatic cancer cell invasion up to 5-fold, without affecting cancer cell proliferation. Conclusion:Artemin expression was not associated with pain in PDAC. However by increasing cancer cell invasion, Artemin seems to influence neural invasion and thereby contribute to cancer cell spreading along pancreatic nerves.

Indian hedgehog signaling pathway: Expression and regulation in pancreatic cancer

Pancreatic cancer is an aggressive malignancy that exhibits a number of genetic and epigenetic alterations. Indian hedgehog (Ihh) and its 2 signaling receptors, patched (Ptc) and smoothened (Smo), are involved in pancreatic development and regulation of β‐cell function as well as in certain human tumors. In the current study, we analyzed the expression, distribution and function of Ihh and its receptors in pancreatic cancer. Quantitative RT‐PCR and immunohistochemistry were utilized to analyze the expression, localization and transcriptional regulation of Ihh, Ptc and Smo. The effects of inhibition and stimulation of the hedgehog signaling pathway on pancreatic cancer cell growth were examined by the MTT cell growth assay. By quantitative RT‐PCR, Ihh, Ptc and Smo mRNA levels were increased 35‐, 1.2‐ and 1.6‐fold, respectively, in pancreatic cancer tissues in comparison to normal pancreatic tissues. By immunohistochemistry, Ihh, Ptc and Smo were expressed in the islet cells of normal and cancerous tissues and in pancreatic cancer cells. The growth of pancreatic cancer cells was dose‐dependently inhibited by the hedgehog antagonist cyclopamine through G0/G1 arrest. In contrast, Ihh agonists exhibited no significant effect on pancreatic cancer cell growth. TGF‐β1 repressed Ihh transcription in a TGF‐β1‐responsive pancreatic cancer cell line, but had no effect on the other tested cell lines. In conclusion, Ihh and its receptors Ptc and Smo are expressed in pancreatic cancer, and blockage of hedgehog signaling results in inhibition of pancreatic cancer cell growth, suggesting that aberrant activation of the Ihh signaling pathway contributes to tumor development in this malignancy.