KRAS mutations characterize pancreatic cell transformation from the earliest stages of carcinogenesis, and are present in >95% of pancreatic ductal adenocarcinoma (PDAC) cases. In search of novel biomarkers for the early diagnosis of PDAC, we identified the proteins secreted by the normal human pancreatic cell line (HPDE) recently transformed by inducing the overexpression of the KRASG12V oncogene. We report a proteomic signature of KRAS-induced secreted proteins, which was confirmed in surgical tumor samples from resected PDAC patients. The putative diagnostic performance of three candidates, Laminin-C2 (LAMC2), Tenascin-C (TNC) and Pentraxin-3 (PTX3), was investigated by ELISA quantification in two cohorts of PDAC patients (n = 200) eligible for surgery. Circulating levels of LAMC2, TNC and PTX3 were significantly higher in PDAC patients compared to the healthy individuals (p < 0.0001). The Receiver Operating Characteristics (ROC) curve showed good sensitivity (1) and specificity (0.63 and 0.85) for LAMC2 and PTX3, respectively, but not for TNC, and patients with high levels of LAMC2 had significantly shorter overall survival (p = 0.0007). High levels of LAMC2 and PTX3 were detected at early stages (I–IIB) and in CA19-9-low PDAC patients. In conclusion, pancreatic tumors release LAMC2 and PTX3, which can be quantified in the systemic circulation, and may be useful in selecting patients for further diagnostic imaging.
Malignant Pleural Mesothelioma (MPM) is an aggressive tumor of the pleural lining that is usually identified at advanced stages and resistant to current therapies. Appropriate pre-clinical mouse tumor models are of pivotal importance to study its biology. Usually, tumor cells have been injected intraperitoneally or subcutaneously. Using three available murine mesothelioma cell lines with different histotypes (sarcomatoid, biphasic, epithelioid), we have set up a simplified model of in vivo growth orthotopically by inoculating tumor cells directly in the thorax with a minimally invasive procedure. Mesothelioma tumors grew along the pleura and spread on the superficial areas of the lungs, but no masses were found outside the thoracic cavity. As observed in human MPM, tumors were highly infiltrated by macrophages and T cells. The luciferase-expressing cells can be visualized in vivo by bioluminescent optical imaging to precisely quantify tumor growth over time. Notably, the bioluminescence signal detected in vivo correctly matched the tumor burden quantified with classical histology. In contrast, the subcutaneous or intraperitoneal growth of these mesothelioma cells was considered either non-representative of the human disease or unreliable to precisely quantify tumor load. Our non-invasive in vivo model of mesothelioma is simple and reproducible, and it reliably recapitulates the human disease.
Background. Development of pre-clinical models of long-lasting inflammation is needed for the study of inflammatory diseases. Osteoarthritis (OA), the most common inflammatory joint pathology, is characterized by an abundance of M1-like macrophages producing several pro-inflammatory cytokines and proteolytic enzymes damaging the local tissues. Here we developed a novel in vitro model of long-lasting inflammation using primary human monocyte-derived macrophages continuously cultured for 15 days. Results. We first screened synovial fluids from 11 OA patients using multiplex Ella® and we found measurable levels of IL-6, CCL2, CCL5, CXCL8, CXCL10, TNFa, IL-1b, using them as read-outs of the following experiments. We then found an increased survival of macrophages differentiated with both M-CSF and GM-CSF (M-/GM-Mf) compared either with macrophages differentiated with M-CSF alone (M-Mf) or macrophages differentiated with GM-CSF alone (GM-Mf) and, consequently, decided to use them to set up the in vitro model of long-lasting inflammation. Consistently with the increased survival, the repeated stimulation of M-/GM-Mfs either with lipopolysaccharide + interferon-g (LPS+IFN-g) or Pam3CysSerLys4 (Pam3CSK4) led to the sustained production of IL-6, CCL2 and CXCL8 over time. Finally, to investigate the usefulness of our model for OA research, we repeatedly exposed stimulated macrophages to dexamethasone (DEX) and/or celecoxib (CEL), two anti-inflammatory drugs commonly used for OA therapy. We found an excellent anti-inflammatory activity but significant toxicity of DEX for concentrations over 10 nM, while CEL was not toxic and efficiently inhibited PGE2 secretion. Of interest, the inflammatory profile of macrophages differentiated with M-CSF or GM-CSF and activated with the two pro-inflammatory stimuli (LPS+IFN-g vs Pam3CSK4) was not identical, overall showing production of more inflammatory mediators with GM-CSF-macrophages stimulated with Pam3CSK4. Conclusions. We introduce here a novel easy-to-use in vitro culture model of long-lasting inflammation using primary human macrophages, that could be useful for the screening of new compounds and drug delivery systems, to improve the therapy of inflammatory disorders.
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