Search

Additional file 2: Supplementary Fig. S1. Altered miR-146a expression influences BRAF/MEKi sensitivity and apoptosis in melanoma. A) Boxplots representing IC50 values of 6 matched PLX4032-resistant (R) and sensitive (S) melanoma cell lines to PLX4032 (vemurafenib), BRAFi (dabrafenib), MEKi (trametinib), to the combined treatment BRAF/MEKi, and to sTRAIL-induced apoptosis. B) Inverse correlation between miR-146a expression levels and IC50 values of PLX4032, BRAFi and MEKi in melanoma cell lines (Spearman analysis). C) Forced expression of miR-146a (+m-miR-146a) in LM16R cell line and in LM69 and LM70 short term cultures increased the effects of PLX4032 treatment as shown by reduced cell growth and increased cell cytotoxicity and apoptosis, evaluated by CCK8, LDH and caspase 8 and 3/7 activity. D) Inhibition of miR-146a expression (i-miR-146a) in LM16 cells increased cell proliferation and decreased the release of LDH and the apoptosis rate as evaluated by CCK8, LDH and caspase 3/7 activity. E) Overexpression of miR-146a (m-miR-146a) upon PLX4032, BRAF/MEKi and sTRAIL treatments in LM47R cells increased cell cytotoxicity and apoptosis, as evaluated by LDH and caspase 8 and 3/7 activity. Data are plotted compared to scrambled control. *: p < 0.05, **: p < 0.01, ***: p < 0.0001 by Student’s unpaired t test. Supplementary Fig. S2. COX2 inhibition increases sensitivity to PLX4032 and to BRAF/MEKi and reduces PGE2 release. A) Relative luciferase activity after co-transfection of PTGS2 3’UTR luciferase reporter vector or control vector with miR-146a mimic or mimic negative control. Experiment was performed in LM16R cells. B) Inverse correlation between miR-146a expression levels and IC50 values of PLX4032 in melanoma cell lines (Spearman analysis). C) Western blot analyses showing downregulation of COX2 after transfection with siRNA against COX2 (siCOX2) compared to scrambled control (S). COX2 protein levels were downregulated to 15%, as determined by quantification of the signal and expressed as the ratio of COX2/Actin intensity. Experiment was performed with LM47R cells. D) Silencing of COX2 by siRNA transfection enhanced cell growth inhibition and increased cell cytotoxicity and apoptosis upon treatment with PLX4032 compared to scrambled control. E) PGE2 release in culture supernatants following 24 h treatment with the COX2 inhibitors celecoxib and NS398, alone or in combination with BRAF/MEKi. F) Combined treatment with celecoxib increased cell growth inhibition and cytotoxicity by PLX4032. ●: interaction index = 1. G) Dose-dependent effect of celecoxib alone (5, 10, 20, 40, 50 and 80 μM) and combined with PLX4032 (3 μM) on cell growth. ●: interaction index = 1 at the dose of 50 μM of celecoxib combined with PLX4032. P values were calculated by Student’s unpaired t test in A, D and F, by one-way ANOVA followed by Bonferroni correction in E, and by two-way ANOVA followed by Bonferroni correction in G. *: p < 0.05, **: p < 0.01, ***: p < 0.0001. Supplementary Fig. S3. miR-146a expression levels are inversely correlated with levels of target genes. Analysis of correlation between miR-146a expression and its target genes EGFR, CCL2 and BCL2L1 in metastatic melanoma specimens. The rp and p values resulting from Pearson analysis are shown. Supplementary Fig. S4. Dynamic culture in bioreactor preserves TME architecture of 3D tumor explants. Representative histological images of H&E-stained 3D tumor sections after 3 days culture in bioreactor. The marked areas in the left panels are shown at higher magnification in the right panels to display the preserved cellularity and tumor tissue histo-architecture. Scale bar 80 μM. Supplementary Fig. S5. miR-146a overexpression increases drug sensitivity in 3D cultures of tumor explants. A) Modulation of direct and indirect miR-146a target genes upon transfection of miR-146a mimic (m-miR-146a) compared to scrambled-transfected control (S) in 3D cultures of BRAF-mutated melanoma tumors from naïve patients. AU: arbitrary units. B) Decreased staining for Ki67 and pERK proliferating cells and increased Caspase 3 positive cells in 3D cultures upon treatment with BRAF/MEKi and transfection with miR-146a (m-miR-146a) or scrambled control (S). Scale bar 10 um.

C-arm cone-beam computed tomography (CT)-guided transthoracic lung core needle biopsy (CNB) is a safe and accurate procedure for the evaluation of patients with pulmonary nodules. This article will focus on the clinical features related to CNB in terms of diagnostic performance and complication rate. Moreover, the concept of categorizing pathological diagnosis into 4 categories, which could be used for clinical management, follow-up, and quality assurance is also introduced. We retrospectively collected data regarding 375 C-arm cone-beam CT-guided CNBs from January 2010 and June 2014. Clinical and radiological variables were evaluated in terms of success or failure rate. Pathological reports were inserted in 4 homogenous groups (nondiagnostic-L1, benign-L2, malignant not otherwise specified-L3, and malignant with specific histotype-L4), defining for each category a hierarchy of suggested actions. The sensitivity, specificity, and positive and negative predictive value and accuracy for patients subjected to CNBs were of 96.8%, 100%, 100%, 100%, and 97.2%, respectively. Roughly 75% of our samples were diagnosed as malignant, with 60% lung adenocarcinoma diagnoses. Molecular analyses were performed on 85 malignant samples to verify applicability of targeted therapy. The rate of “nondiagnostic” samples was 12%. C-arm cone-beam CT-guided transthoracic lung CNB can represent the gold standard for the diagnostic evaluation of pulmonary nodules. A clinical and pathological multidisciplinary evaluation of CNBs was needed in terms of integration of radiological, histological, and oncological data. This approach provided exceptional performances in terms of specificity, positive and negative predictive values; sensitivity in our series was lower compared with other large studies, probably due to the application of strong criteria of adequacy for CNBs (L1 class rate). The satisfactory rate of collected material was evaluated not only in terms of merely diagnostic performances but also for predictive results by molecular analysis.

Phosphaturic mesenchymal tumors that cause the paraneoplastic syndrome known as oncogenic osteomalacia are rare. The authors report on the case of a 57-year-old man with a history of osteomalacia and in whom was diagnosed a thoracic spine tumor at the T-4 level. Complete tumor resection was accomplished. The histological diagnosis was phosphaturic mesenchymal tumor (mixed connective tissue variant). After lesion removal, the paraneoplastic syndrome resolved. At the 24-month follow-up, no recurrence of the disease was observed. The clinical presentation, surgical technique, and follow-up in this case were reviewed in detail.

We evaluated the response to mild hypoxia exposure of A549 alveolar human cells and of a continuous alveolar cell line from human excised lungs (A30) exposed to 5% O2 for 5 and 24 h. No signs of increased peroxidation and of early apoptosis were detected. After 24 h of hypoxia total cell proteins/DNA ratio decreased significantly by about 20%. Similarly, we found a decrease in membrane phospholipid and cholesterol content. The membrane fluidity assessed by fluorescence anisotropy measurements was unchanged. We also prepared the detergent resistant membrane fraction (DRM) to analyze the distribution of the two types of lipid microdomains, caveolae and lipid rafts. The DRM content of Cav-1, marker of caveolae, was decreased, while CD55, marker of lipid rafts, increased in both cell lines. Total content of these markers in the membranes was unchanged indicating remodelling of their distribution between detergent-resistant and detergent-soluble fraction of the cellular membrane. The changes in protein markers distribution did not imply changes in the corresponding mRNA, except in the case of Cav-1 for A30 line. In the latter case we found a parallel decrease in Cav-1 and in the corresponding mRNA. We conclude that an exposure to a mild degree of hypoxia triggers a significant remodelling of the lipid microdomains expression, confirming that they are highly dynamic structures providing a prompt signalling platform to changes of the pericellular microenvironment. J. Cell. Biochem. 105: 503–513, 2008. © 2008 Wiley-Liss, Inc.

Several studies have shown that there is a paucity of immune cells within the stroma of pancreatic adenocarcinoma, a very aggressive cancer with a median survival of about 18 months. A 65-year-old man presented with jaundice. Abdominal ultrasound revealed intra- and extrahepatic bile duct dilatation and a 45-mm diameter hypoechoic solid mass within the pancreatic head; a computed tomography scan excluded vascular infiltration and metastatic lesions. The patient received immunotherapy consisting of 6,000,000 IU human recombinant interleukin-2 administered subcutaneously twice a day for 3 consecutive days. Thirty-six hours after the last dose, he underwent a pylorus-preserving pancreatoduodenectomy. Because of the presence of high-grade dysplasia detected by intraoperative histological examination of a distal section, a spleen preserving total pancreatectomy was performed. The postoperative course was uneventful. The patient died 32 months after surgery because of local recurrence. Histopathology showed G3 pancreatic ductal adenocarcinoma infiltrating the anterior and posterior peripancreatic tissue, duodenal wall and intrapancreatic common bile duct, with sarcoma-like foci and a component of intraductal tumor involving the common bile duct. In the distal pancreas, widespread foci of pancreatic intraepithelial neoplasia (PanIN2-3) were found. The Ki-67 proliferation index was 16%. TNM staging was pT3 pN1 R1. Sections were immunostained for the T-lymphocyte marker CD3 and for the dendritic cell marker CD 1a. Intratumoral infiltration was high for CD1a+ cells and mild for CD3+ cells. Preoperative immunotherapy with interleukin-2 may contribute to massive stromal infiltration of immune cells in pancreatic adenocarcinoma. This may prolong the survival even in the presence of negative prognostic factors (age >65 years, tumor diameter >20 mm, Rl, tumor grade G3).

RATIONALE AND OBJECTIVES: Our goal was to prospectively determine the value of perfusion computed tomography (CT) in the quantitative assessment of tumor-related angiogenesis in cirrhotic patients with hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Forty-seven patients met all the following inclusion criteria: 1) Child-Pugh class A or B liver cirrhosis; 2) presence of a single lesion suspected as HCC at screening ultrasound examination; and 3) lesion diameter between 1 and 3 cm. All patients underwent contrast-enhanced ultrasound, pre- and post-contrast triple-phase CT, and perfusion computed tomographic study using multidetector 16-slice CT. Six parameters related to the blood microcirculation and tissue perfusion were measured for the focal liver lesion and cirrhotic parenchyma: perfusion (P), tissue blood volume (BV), hepatic perfusion index (HPI), arterial perfusion (AP), portal perfusion (PP), and time to peak (TTP). Perfusion parameters were described with quartile values of their distribution; univariate paired and unpaired Wilcoxon signed rank tests were used for statistical analysis. RESULTS: HCC was diagnosed in 21 of the 47 patients; in the remaining 26, HCC was not found at contrast-enhanced ultrasound and multidetector 16-slice computed tomographic study. The values of perfusion parameters measured within tumor tissue were: P (ml/s/100 g): median = 47.0 (first quartile = 36.0, third quartile = 61.4); BV (ml/100 mg): median = 24.0 (first quartile = 18.7, third quartile = 29.3); HPI (%): median = 78.4 (first quartile = 62.9, third quartile = 100); AP (ml/min): median = 45.9 (first quartile = 39.0, third quartile = 60.1); PP (ml/min): median = 9.0 (first quartile = 0.0, third quartile = 24.5); and TTP (seconds): median = 18.7 (first quartile = 16.3, third quartile = 26.5). The corresponding values calculated in cirrhotic surrounding parenchyma were P (ml/s/100 g): median = 11.5 (first quartile = 9.4, third quartile = 13.9); BV (ml/100 mg): median = 10.7 (first quartile = 7.1, third quartile = 14.2); HPI (%): median = 10.6 (first quartile = 8.7, third quartile = 11.9); AP (ml/min): median = 13.2 (first quartile = 10.1, third quartile = 15.5); PP (ml/min) median = 55.2 (first quartile = 40.1, third quartile = 79.5); and TTP (seconds): median = 41.7 (first quartile = 38.9, third quartile = 44.6). P, BV, HPI, and AP values were higher (P < .001), whereas PP and TTP were lower (P < .001) in HCC relative to the surrounding liver. Values of perfusion parameters in the cirrhotic liver of patients with and without HCC were not significantly different (P > .001). CONCLUSION: In cirrhotic patients with HCC, perfusion computed tomographic technique can provide quantitative information about tumor-related angiogenesis.

TGF-β pathway is generally associated with the processes of metastasis, angiogenesis and EMT in cancer. Very little is known, however, about the role of TGF-β in cancer drug resistance. In this work, we show a specific activation of the TGF-β pathway in consequence of chemotherapeutic treatment in in vivo and in vitro models of colorectal carcinoma. 5-Fluorouracil (5FU) was able to stimulate the activation of SMAD3 and the transcription of specific genes such as ACVRL1, FN1 and TGFB1. On the other hand, the specific inhibition of TGF-βRI was able to repress the 5FU-induced genes transcription and to restore the sensitivity of chemoresistant cells to the toxic action of the drug, by decreasing the expression of BCL2L1 and ID1 genes. The role of the TGF-β molecule in the chemoresistant colon carcinoma cells' response to 5FU was further demonstrated by conditioned medium (CM) experiments: CM from 5FUtreated chemoresistant cells was able to protect chemosensitive cells against the toxic action of 5FU. In conclusion, these findings showed the pivotal role of TGF-β pathway in colon cancer mechanisms of drug resistance suggesting new possible approaches in diagnosis and treatment of colon cancer patients.